Genetic Engineering:

 

Application-Animal Biotechnology:

Transgenic animals:

Molecular Medicine:

 

Ever since the modern human specie arrived on the surface of this planet and started walking around and started living in groups, he collected animals and plants of his liking for his own use.  He also reared them and stock piled them under his care.  This is the beginning of animal and plant farming.   He also observed variation among the same animal and plants cross bred them to develop new varieties, this is yet another technology innovated by him called reproductive breeding, where he used transfer of an entire genome from one form to another.  This is nothing short of “Transgenic” methodology.  In 20th century one uses techniques where one or two genes are transferred into animal cells and obtain a complete animal called transgenic animals; so also transgenic plants are obtained.

 

In fact animal cloning experiments started early 1960s and by sixties experimenters were able to get a large number of amphibian clones.  The technique was simple; frogs lay eggs outside their body.  The eggs were large enough to be seen by naked eye.  One can obtain such eggs in large numbers by injecting frogs with reproductive hormones.  Enucleation of eggs was a simple technique, which can be perfected with time.  Then 2n nucleus sucked from somatic tissues such as intestine, and injected the same into enucleated eggs and prodded the cells to grow; and actually they grew into normal frogs.  Several hundreds of such amphibians were grown for experimental purposes.  In fact fishes were also used for the same purpose.  Some people have perfected culture medium for regrowth of organs such as frog palm or limbs by using special medium containing Vitamin-A. 

 

The first transgenic animal produced was from Ralph Brienster, in Lippincott, Univ.of Penn, Philadelphia, in 1980-82 (the author of this website was a witness).  They introduced human growth hormone gene into mice eggs.

 

Donor strain mice were reared and females were injected with pregnant mare serum, or one can use porcine follicle stimulating hormone. After 48 hrs human Gonadotrophin was injected.  Hybrid mice are better than inbreed mice?

 

Such females were mated with selected male studs.  Each of the female will produce 18-20 fertilized eggs. After 12 hrs of post coitum, oviducts were taken out and the eggs squeezed out into specific nutrient M16 media.

 

 

 

M16 Media:

NaCl,

KCl,

KH2PO4,

MgSO4,

Glucose,

NaHCO3,

Na Pyruvate,

CaCl2,

HEPES, pH 7.5,

Phenol Red.

 

http://bsoha.com/

Microinjection-http://www.en.wikipedia.org

 

Diagram of the intracytoplasmic sperm injection of a human egg. Micromanipulator on the left holds egg in position while microinjector on the right delivers a single sperm cellhttp://www.wikipedia.org

 

Eggs can be injected with Constructed Recombinant DNA; http://www.hwdsb.onca: www.biotecharticles.com

 

Gene gun ued to bombard DNA into plant cells

The instrument used for microinjection of DNA into the egg; http://www.bio.davidson.edu/

 

Natural News  Blog; http ; //naturalnewsblog.blogspot.com/

 

Wash eggs in M16 media and then they are transferred to M2 media micro drops.

Use powerful dissecting microscope equipped with microinjecting facility having very thin and narrow glass tips which can hold only few μl of liquid.

Inject linearized plasmid DNA containing Growth hormone gene into the nucleus of the egg.

 

 

Trangenic Mices with flouescent gene products expressed; http://www.whatisbiotechnology.org/

 

 Transgenic RNAi not only allows the study of biological processes not present in cultured cells but also offers chronic therapeutic potentials. In this review, we will summarize the developments in the generation of transgenic RNAi mice.

Transgenic RNA Interference in Mice; http://physiologyonline.physiology.org/

The amount injected is about 2picoliters containing at least 200 copies.

Prepare a set of mice by injecting hormones and mated with vesectomiced males when the females are in estrous stage or what is called animal in “Heat”. These animals behave or rather feel as pseudo pregnant and act as foster mothers.

 

All these operations have to be done in sterile condition and the job requires skill and deft hands. Under anesthetic conditions these females are operated and the oviduct and uterus is pulled out. Then eggs injected with a gene are transferred into the uterus via infundibulum. Stitch the animal and inject with antibiotics (heavy dose).

In about 19-22 days pups will be delivered.

When they are 10 to 15 days old one can peal off tail skin, for it grows back, and homogenize and isolate DNA and do southern blot, or isolate RNA and do northern blot or isolate proteins and do western blot and find out whether or not the injected genes are integrated and expressed their gene products.

 

Dr. Brinster receives National Medal of Honor from President Obama

Dr. Ralph Brienster produced first transgenic mices, in the lab (1980-81), Univ. Penn, Philadelphia;

I, Dr. G.R.kantharaj, the author of this website a witness when these mice were born in the Univ. Penn, downtown campus. Dr. Ralph Brienster is great friend of mine https://www.avma.org

 

 

Animal Cloning:

 

Transgenic animals can also be obtained by another method where blastocysts derived stem cells are transfected with the desirable gene placed under specific promoter to express under either induced condition or expressed in tissue specific manner.  They are selected against an antibiotic. 

 

They are further amplified and the same are transfused into freshly isolated blastocysts, which are then transplanted into uterus for further development.  Many mice cell lines have been created in this manner.  One such cell line is used for gene knock out experiment.

Molly and Polly 1977, were obtained as transgenic animals with the gene Factor-IX under the promoter beta lactoglobin for it expression in the mammary tissue during milking period, they used fibroblast cells.  A thirty day old fetal cell was used to produce “Transgenic Bull” in 1997 by ABC-Global at Wisconsin.  In Honolulu scientists obtained cow.

 

 

Cloning word has larger implications in terms of technology, such as DNA cloning, Reproductive Animal cloning and therapeutical cloning. DNA cloning involves cut the DNA and select a specific sequence and ligate it to a vector.  Animal cloning involves taking 2n fertilized egg, remove the nucleus and fuse with 2n embryonic cells or adult cells.  Therapeutical cloning uses blastocysts or stem cells from different tissues and regenerate the tissue or the organ for transplantation.

 

Animal cloning was new technique for it was already achieved in 1960s; frogs were the first to be cloned animals.  In later years scientists have shown new techniques employed in developing animal cloning.  In fact any cell having the full complement of genome is endowed with potentiality to regrow into a complete animal if the cells are provided with proper media and the required vitamins and hormones and stimulus.  This is what is called as totipotency of cells.  This phenomenon is easily demonstrated plants, where the plants are grown from one or two nucleated cells.  This technique of Micropropogation is now employed in agriculture and horticulture.

Professor Sir Ian Wilmut, Edinburgh, the man that led the team in the creation of Dolly the Sheep, She was the most celebrated animal that was obtained, in 1996-97, by modified cloning technique was a sheep called Dolly named after popular singer Dolly Parteon.  British scientists (Keith Campbell) from Rosalyn institute had field days of glory and rejoiced in spite of some diehard critics.  Dolly started her life, in a test tube, as with all other cloned animals lived. Once normal development was confirmed at 6 days, the embryo, that was eventually to become Dolly, was transferred into a surrogate mother.  British scientists (Keith Campbell) from Rosalyn institute countered the knighthood awarded to Sir Ian Wilmut. When Sir Ian was asked directly he replied “I did not create Dolly" was true, Sir Ian replied "yes". He also said that Professor Campbell deserves 66 per cent of the credit for Dolly, although Sir Ian insisted on putting his own name at the beginning of the scientific paper published in the journal Nature in February 1997.

Dolly was born on 5th July, 1996 died at the age of six.

Pregnancy was confirmed by an ultrasound scan at about 45 days of gestation and the pregnancy was monitored closely for the remaining 100 days. The pregnancy went without a problem and Dolly was born on the 5th July 1996. Unlike many cloned animals, which often have neonatal problems at birth, Dolly was a normal vigorous lamb and was standing and sucking unaided within minutes. The animal technicians were aware that this was an important lamb and critical to the research team that had produced her but they were completely unaware of the impact she would finally have.  Another scientist to be honored is Debby Reynolds, the UK government's former chief veterinary officer.

Cloning an Epic Historical Mammoth; Animals Whose Genes Have Been Tinkered with by scientists;  http://www.iceflux.com/;http://xfinity.comcast.net/

 

Americans were not far behind for they have also achieved animal cloning especially monkeys.  They used different techniques.

 

Fusing enucleated egg cell with cultured udder epithelial cell from six-year-old female sheep at Go stage, with an electric shock produced dolly.  They have made 227 attempts before they were able to get this cloned sheep.   Dolly has the distinction of becoming a mother.  There are claims that they have used fetal stem cells

There were some claims that the Dolly was obtained from fetal cells. Unfortunately, after all these years of glory, Dolly was believed to be suffered from arthritis.   The disease Arthritis may be due to other reasons, not because it is a cloned animal.  It has died but left a progeny. Dolly's Family

 

Dolly and Bonny

Dolly and her first lamb, Bonnie, born April 13th 1998 and fathered naturally by David, a welsh mountain Ram; Father of Dolly- awarded knighthood ; Dolly and Bonny;  http://www.coldmeadow.com/

Dolly and Bonny

Dolly and her first lamb, called Bonny

In an attempt to allow Dolly to have as normal  life as possible, it was decided that she should be allowed to breed.

A small welsh mountain ram was selected as her mate and between them they successfully produced 6 lambs. Their first, Bonny, was born in the spring of 1998. Twins followed the next year and triplets the year after that.

Dolly at the National Museum of Scotland

Dolly was made immortalized by duplicating in the wax. www.roslin.ed.ac.uk

 

Goat /sheep called Dolly? http://www.slideplayer.com/slide

 

Monkeys were developed by growing an embryo to 8-16 cells and then the cells were separated and grown individually into embryos, which were then implanted into pseudo pregnant mother. 

Human cloning was attempted by culturing embryos to 8-cell stage under in vitro conditions and for apparent reasons the embryonic cells are frozen for posterity. Who knows that human clones are not produced???

 

The clonal sheep Meg, Morgan and Tracy were obtained from embryonic cells.  Tracy has genetically manipulated to produce sodium channel proteins involved in cystic fibrosis. 

Honolulu people in 1997 also obtained a clone by implanting 2n nucleus into an enucleated egg of cumulina mice.  Since then many animals have been obtained as clonal animals. In Japan many cows have been developed as clones.  Scientists have attempted to produce pig clones without surface antigens belonging to MHC class.

People have claimed that they have cloned humans and their birth has been heralded all over the world, but no genetic proof for the said claims.  Cloning of human beings does not require great technological protocols; in fact with the existing methodologies one can do this in any labs, which have the facility for in human in vitro fertilization and implantation.

 

The diagram shows how to develop reconstructed embryo clone;

Robert A. Godke, Richard S. Denniston and Brett Reggio http://www.lsuagcenter.com/

 

 

Transgenic Bull Double Muscled Bull Belgian Blue ? https://en.wikipedia.org/

 

Cloning of a Dog

 

 

 

 

Cloning of Frogs

 

Ken (left) and Henry were created using DNA plucked from a skin cell of Melvin, the beloved pet of Paula and Phillip Dupont of Lafayette, La.;

Cloned Dogs, http://www.npr.org/

 

 

Fluorescent Cats obtained through Genetic Engineering- green and red- transgenic animals- from South Korea. http://news.softpedia.com/

 

 

Human cells can be grown and differentiated into specific cell types, then they are implanted.  No problem with graft rejection. http://therapeuticcloningprocessandethics.blogspot.in/

 

 

 

 

 

Macdonald farming-Transgenic Cows. https://www.scq.ubc.ca

 

The New Macdonald Pharm; Animal cloning: Cows; Australian scientists have claimed a world first, cloning a cow using a new technique that produces a healthier embryo. https://www.scq.ubc.ca

Old McDonald had a pharm

Genetically modified goats and chickens to produce drugs for humans. But hold on. http://www.salon.com/ 

 

 

Goat n Dog; Dog n Eagle; https://animal-mix.blogspot.in/2007

 

 

 

 

Each blastocyte cell can be implanted in the uterus and grow the embryo.

 

Image result for Human in vitro fertilization

IVF-In vitro fertilization; https://gullalaii.wordpress.com/

Image result for Human in vitro fertilization

In-Vitro fertilization; http://dphx.org/respect-life

 

Gene ‘Knock Out’ pig ; First knockout pig: alpha 1, 3 galactosyltransferase; http://arbg.missouri.edu/

 

 

 

 

 

 

             Lovely cloned cat; but the first cat cloned was from South Korea.  www.slideshare.net

 

Cloned cow; http://blogs.discovermagazine.com/

                       

 

 

Animal Farm Catch-;Cloned cow-calf- http://www.ibtimes.com/

 

Fourth Generation of Pig clones born Cloned piglets, Oh my god! they are so lovely and loving; http://pinktentacle.com/2007/08/fourth-generation-pig-clones-born/

 

 

 

I love these kids; are they cloned Kids?

 

 

13.  Hybridoma Technology:

 

This ingenious technique was developed around 1975-1976.  The production of monoclonal antibodies was invented by Cesar Milstein and Georges J. F. Köhler in 1975. Monoclonal antibodies were once considered as magic bullets that can target a cell, a tissue, molecule and or an infectious agent.

 

Polyclonal antibodies are produced by B-lymphocytes.  When a specific antigen comes in contact with specific receptors of B-lymphocytes, the genes within the cells get activated and start producing antibodies against the specific epitope that are presented to the lymphocytes.  Epitopes are the antigenic determinants of antigen. 

 

Antigens consists of different motifs, different domains derived from amino acid sequences which generate different secondary structural features.  Each of these specific domains is a distinct motif.  They are recognized by different B. Cells, for they carry specific receptors for a specific epitopes.   If an antigen has twenty different epitopes twenty different species of B-cells get activated and twenty different antibodies are produced in one to one manner.  Production of antibodies to each of the epitopes is not equal, some elicit weak response and some elicit strong response. Thus the antibodies produced are Polyclonal in nature.

 

 

Summary of Hybridoma Method of mAb production; http://php.med.unsw.edu.au/

 

 

(1) Immunization of a mouse; (2) Isolation of B cells from the spleen; (3) Cultivation of myeloma cells; (4) Fusion of myeloma and B cells; (5) Separation of cell lines; (6) Screening of suitable cell lines ;(7) in vitro (a) or in vivo (b) multiplication; (8) Harvesting; https://en.wikipedia.org

 

http://www.getdomainvids.com/

 

 

Many a time’s Polyclonal antibodies are not useful for specific purpose.  Yet Polyclonal antibodies are used on large scale for it is easy to obtain such antibodies and cost of production is relatively low.

 

Monoclonal antibodies are for a specific epitope.  Under certain situation one wants to target only one of the epitopes and in such circumstances monoclonal antibodies are very useful; that is why they are called as magic bullets. Production of monoclonal antibodies is possible by Hybridoma technology.

 

B-Lymphocytes:

B-Lymphocytes are capable of producing antibodies in response to specific antigens. 

These cells cannot grow and multiply in an in vitro culture medium. They can survive for some time and use salvage pathway if they are grown in the presence of Hypoxanthine, Aminopterin and Thymidine (HAT) medium.  Cells cannot multiply in culture conditions.  These cells have HGPRT and Thymidine kinase genes.

 

They can use hypoxanthine for purine synthesis even though Aminopterin blocks DHFR, they have salvage pathway.  The reason is the cells have enzymes called Hypoxanthine Guanosyl Phosphor Ribosyl Transferase (HGPRT).

 

HGPRT defective Myeloma cells:

They are incapable of producing antibodies. They are the transformed B-cells.

They have the ability to multiply under culture conditions by cell division.

Mutant cell lines, cannot utilize Hypoxanthine and Thymine in the presence of Aminopterin for they lacking in HGPRT and Thymidine kinase (TK).   

 

Protocol: Inject the required antigen subcutaneously to a rabbit skin.  After three such injections (second and third are called boosters), sacrifice the animal and take out spleen tissue and isolate B-lymphocytes.  These lymphocytes are activated to the injected antigen.  The cells are capable of producing Polyclonal antibodies.

B-lymphocytes are then fused to HGPRT (-) Myeloma cells.  The hybrid cells now can grow and multiply in culture medium even in the presence of Aminopterin.  At the same time they also secrete respective antibodies to specific epitopes. Such cells are called Hybridoma cells.

 

Image result for hybridoma technology

A general representation of the hybridoma method used to produce monoclonal antibodies;  César Milstein and Georges J. F. Köhler in 1975. The term hybridoma was coined by Leonard Herzenberg during his sabbatical in César Milstein's laboratory in 1976–1977 ;

Hybridoma Technology; Protocol. https://en.wikipedia.org

 

Such cells are diluted and dispensed into multi titer plates in such a way that each well contains one such Hybridoma cell.  Such cell lines divide and redivide, grow and produce antibodies to one specific epitope.

 

Medscape.com

 

Using a specific epitope from the antigen one can screen cells from each well and pick whatever clone from the titer plate one desires.  The same cell line can be propagated and maintained.

 

 

Monoclonal antibodies are very useful tools in research.

One can trace the position of an antigen.

Scarce proteins can be purified.

Used for disease diagnosis.

Such cells can be used to treat patients infected with a particular infectious agent.

It is possible to target a specific malignant tumor cell or cells and kill them by directing T-killer cells to them.

 

21.   Molecular diagnostics:

 

Infectious diseases are generally identified by symptoms, culturing and microscopic examination.  And many of the investigations are also done by biochemical analysis.  These methods are still in vogue.  But certain infectious diseases take a long time to manifest the symptoms and often-early diagnosis leads to miss interpretation.  Example malarial infection takes a month or so to fully blown manifestation.  On the contrary HIV infection may take a year or so.  By the time theses are diagnosed, treatment will be delayed and it can be disastrous.  Of late molecular diagnostic tools have been evolved. One such technique is ELISA (Enzyme Linked Immuno Sorbent) Assay) technique has been developed, either using Polyclonal antibodies or monoclonal antibodies.  This is certainly very effective, but not all diseases can be identified by this technique and expensive. Yet ELISA is a powerful and highly sensitive diagnostic method, which is used in medicine, research and industry.  Definitely a monoclonal antibody technique is very very costly.  Along with ELISA protocols, another technique called PCR has been developed to identify not only diagnosis of infectious agents but also to detect any suspected genetic diseases.  For example HIV infection can be positively diagnosed by PCR method.  Combined with ELISA the diagnosis is 100% efficient.

 

Antibodies as diagnostic tools:

Antibodies have to be raised for every antigen bearing infectious agent.  Most of the antibodies are polyclonal.  Some times Polyclonal antibodies cannot distinguish between pathogenic and nonpathogenic germs.  Hence people started using monoclonal antibodies, which was found be costly and not within the reach of common folks. But now it is possible and in reach of common man.

 

A List of antibodies employed in diagnosis (just few examples):

 

IgGs for Polypeptide hormones:

 

Chorionic gonadotrophin.

Growth hormones.

Leutinizing hormones.

Follicle stimulating hormone.

Thyroid stimulating hormone.

Prolactin.

 

Cytokines:

Interleukin 1-8.

Colony stimulating factor.

Tumor markers:

Carcinoma embryonic antigen,

Prostrate specific antigen,

Interlekin 2 receptor,

Epidermal growth factor.

 

Drug monitoring:

Theophylline,

Gentamycin,

Cyclosporin.

 

Others:

Thyroxine,

Vit-B12,

Ferritin,

Fibrin degrading factor,

 

Few examples IgGs against disease causing organisms:

Chlamydia,

Herpes simplex,

Rubella,

Hepatitis-B,

Hepatitis A,

Hepatitis-C,

Schigella,

Mycobacterium,

HIV,

HPTLV,

Cholera toxins,

Botulins,

Polio,

Nematodes,

Malarial antigens,

 

A large number of viral antigens and bacterial antigens and protozoan surface antigens have been used for developing diagnostic kits.  The above list is to give an idea what are few diagnostic IgGs.

 

In recent years instead of preparing IgGs for every antigen, scientists have used total human lymphocytes and prepared a composite but combinatorial cDNA library for both Heavy chains and light chains.  Lambda DNA has been used to prepare such library.  This method can generate 10^6 to 10^8 clones, each distinct from the other.  Proteins expressed can be screened with diagnostic antigens.  Now days the antibodies are generated using genetic manipulation in such a way the antibodies when injected to human, they don’t elicit immune response and such antibodies are called humanized antibodies.

 

DNA as the diagnostic material:

This has become a sine quo non technology when everything has failed.  This technique has undergone lot modification and sophistication.  The method is simple and fast.  This technique can be used to detect not only disease causing pathogens but also to detect parental pattern, if you are in doubt.

 

Genetic defects.

Most important requirement for PCR based diagnostic methods is knowledge of the pathogen’s DNA and its sequence.  From the sequence one can generate a set of primers for 5’ and 3’ ends.  Known primers can employed to identify.  The source of material required is tissue sample, fecal material, urine, blood sample, throat smears or throat wash, a hair follicle.  The amount DNA required can be as small as few picograms.  In order to combat disease all over the world, WHO has a program to develop 300- 500 diagnostic kits.

 

 

Diagnosis of Malarial parasites:

Full-blown disease manifestation takes 15 days to one month.   Malarial disease is caused by plasmodium falciparum.  Elisa is effectively used, but the parasite often camouflages by withdrawing its surface antigen and projecting another antigen.  Here the probe used is highly repetitive DNA sequences.  Such sequences when used they are distinguishable from P.falciform with other P.vivax, P.cyanomoglia and other related parasites.  Blood sample is more than enough diagnosis of the disease.

Similar highly repetitive sequences have been used to detect disease-causing parasite such as Trypanosome cruzi (chagas disease).  Amplification of the said DNA using specific primers it is possible identify this disease with accuracy and other related forms are not detected.  It is very specific.  This disease has devastated South American population.

 

Diagnosis of DMD:

Duchene’s muscular dystrophy sex linked disease, generally appears at greater frequency in male children than the female for the females are the carriers.  Here the diseased children suffer from muscular wastage for the surface protein Dystrophin is non functional.  This protein is found in the myelin sheath at the inner surface covering the axons.  The DMD gene is located in the XP21 arm.

The fully processed DMD mRNA is 14 000 nucleotides long.  The pre mRNA consists of `60 Exons.  The whole gene extends to an area of 20000kbp.  The protein has a molecular mass of 600 KD; perhaps it is one of the largest proteins known in eukaryotic organisms.  In fact the largest protein is ‘Titin’.

When the DMD gene is cut with Taq-I enzyme, it generates seven fragments.  Sites for the Taq-I enzyme is found in few introns.  Scientists have developed primers for each of the fragments and the same can be used for PCR amplification.  Control DNA generates 7 fragments of the size 10KB, 7.8 kbp, 4kbp, 3.8kbp, 3 kbp, 1.8kbp and 0.6 kbp and they can be discerned on an Agarose gel.  If a suspected patient’s DNA is used one will find some segments of the Gene are missing, using multiplex PCR it is possible to diagnose the disease in a short time; and that too with certainty.

The same technique can be employed in identifying phenyl ketoneuria, Sickle cell anemia, Hemophilia, Cystic fibrosis and others.

 

Diagnosis of Hemophilia:

Hemophilia polymorphism is due to change in restriction sites such as Xba-I and Bcl-I.  This is due to mutation in the sequence of these sites.  Hemophilia X factor has 26 Exons.  Abnormality was found in the 18 Th introns.  A pair of primers were developed for amplifying a 142 bp long DNA involving the introns.  After PCR amplification of the segment it was cut with Bcl-I and separated on a gel.  A patient, whether he or she is heterozygous and homozygous can be discerned from the gel pattern.

 

Control

(Homozygous)

Control

(Heterozygous)

Patient-1

Patient-2

 

       

     -----

     ------

      -----

(-) Bcl-I 142 bp

    ------

     -----

     ------

 

(+) Bcl-I 99bp

    ------

     -----

      -----

 

(+) Bcl-I 43bp

 

 

 

 

 

 

 

Multiplex PCR is used to detect mutations in b-Globin genes.  Primers are used are fluorescent labeled.  Thereby they were able to detect two mutations in b-Globin genes, ands one mutation in alpha Globin gene.

 

Human Genetic Diseases:

It has been estimated that human genetic disorder and their manifestations in the form of diseases is about 3000 and odd.  The diseases can be autosomal or X-linked.  Mutations can be dominant, negative dominant, recessive, can be incomplete penetrance, may show variability in expression, show heterogeneity, and or exist in alternate forms of the same gene.  Genes are pleotropic.

 

 

 Therapeutical Agents.

 

                                                                             

      Any chemical or biochemical compound designed to combat disease fall into the category called Therapeutical agent.  Since the days of Edward Jenner, early 200 years ago, scientists are in hot pursuit of discovering drugs against pathogen.  Vaccination against pathogens worldwide is the most effective preventive therapeutical agent next only to antibiotics.  Discovery of Antibiotics is a great event in the history of mankind, but over use and abuse of antibiotics resulted in the pathogens have become or becoming resistance to drugs.  It is constant and eve going struggle against pathogens and drug discoverers, who dominates that is the question is like survival of the fittest.

 

Vaccines:

Edward Jenner was an incredible country doctor, using folklore knowledge; he injected liquid from the cowpox pustule into an eight-year-old boy called James Philips.  It was a history unsurpassed of any discovery the mankind knows in centuries.

Most of the infectious agents infection spread of some of them in epidemic, rarely in pandemic proportions can be prevented by vaccination.  Any immunogenic injected into human body, elicits immune response against that antigen.  The efficiency of response depends upon the epitopes found on the presenting surface of the antigen.

According to ‘WHO’ the number of communicable disease against vaccine to be developed by 2000 was 400.  And the target now is much more.  Conventionally vaccines were prepared by using heat killed or formal in killed or inactivated organisms as inoculants.  Though maintenance of them and obtain them as pure forms endowed with some difficulties, it is still the cheapest for these have to be prepared in large quantities for global market.

With the advent of Molecular and gene engineering techniques, recent trend is to develop recombinant vaccines and many such products are made available for large-scale vaccination, ex. Vaccination against Hepatitis-B virus.  The modern methods have been developed to overcome drawbacks of traditional methods.

 

Methods employed for developing recombinant vaccines:

  1. Virulent genes of an infectious agent is deleted or made nonfunctional or dysfunctional and such organisms can be used as live vaccines.  It has to be made certain that the organism does not regain that gene or function.
  2.   Live nonpathogenic organism can be used as carriers.  These organism can genetically manipulated in such a way an antigenic determinant gene is introduced into the organism where it presents the antigen to immune system, thus such organisms can be used as vaccines.  But the antigenic determinant used should elicit strong response.
  3. Develop alternate host or forms for those organisms which cannot be grown and maintained on large scale, ex. Mycobacterium lepri. These have to be grown and sustained in the sub-epidermal tissues of Armadillo, which itself is rare to find in nature.  In such cases the target gene has to be cloned into either bacteria or into yeast cells so large-scale preparation are made easy.
  4. Some times infectious agents do not kill host cells, instead host immune system attacks infected cells and kills them.  It is now possible to create targeted and cell specific killer systems so as to destroy the infected cells.

 

Subunit Vaccine:

Instead of using the whole virus or bacterial cell or pathogenic animal cells, a specific component of infectious organism is used, such as viral capsid protein, surface glycoprotein, cell wall antigenic protein or glycoprotein can be effectively employed for eliciting strong immuno-response.  The said genes can be cloned into expression system and the same can be purified and can be used vaccines.  Such vaccines are stable, safe and the antigens are precisely defined and free from extra cellular contaminants.  But the process is costly and many a times immune response is good as expected, but that can be overcome by certain modifications.

 

Subunit vaccine against Herpes simplex virus:

The viruses cause deadly sexual diseases not only in males but also in females, where they act as reservoirs and males as transmitters.  Great many people belonging to higher social status have succumbed to these viruses and died with remorse.  The virus is an enveloped type and the protein used is protein-D (gD) is a glycoprotein; it elicits good immune response per se.  The said protein does not cause any disease or any other side effects.

The gene for this protein has been cloned into transfer vectors or episomal vectors and transfected Chinese Hamster Ovarian cell lines (CHO) produced properly folded and glycosylated proteins which are as good as the typical viral protein.  When the gene was cloned in secretory mode the proteins are secreted out of the cell into the media.

 

Foot and Mouth Disease Virus (FMDV):

This virus has the ability spread across the continents and cause very severe damage to live stock especially Cattles and Swine.  This is very severe in South America, even Indian animal population also suffers but Indian medicine has taken care of the problem. The medicine is a concocted extract of Curcumin, Garlic, Oscimum, Gingiber offcianale and Leucas aspera leaves and a little bit fresh capsicum.  If this given a week or so the disease is cured.  However the world over requirement of vaccine against this virus is not less than more than 2 billion doses.

The surface protein that is very antigenic is the coat protein called VP1.  This gene has been cloned but the protein per se was found to be very poor antigenically.  The virus possesses ~8000ntds long RNA genome and the cloned was expressed as fused protein with MS2 Replicase as a carrier protein.  Even this was not effective in eliciting a good response.

 

Scientist looked into different motifs of the protein and the same were selected and cloned; such as- from C-terminal 141-160=21aa, 151-150=11, 200-213=13aa, from the N-terminal sequences such as 9-24aa, 17-32aa, 25-41. These subunits were used with a carrier protein and all of them were found to be very immunogenically.  However fused segments from141-158 to 200-213 was found to be stimulatory but 1000x weaker than killed viruses.  But when the subunit from 142-160 was fused, Hb-surface antigen was found it was highly immunogenic.

 

Live recombinant Vaccines:

Methods employed- one is use nonpathogenic organism and genetically modify so at express the desired antigenic protein in proper perspective.  The second alternative is use pathogenic organism and delete the virulent gene responsible for causing the disease and retains its antigenic characters.

 

Live cholera vaccines:

Live vaccines are believed to be more advantageous than subunit vaccines.  Cholera is a deadly disease recurs periodically in tropical countries and kills loot of people.  Symptoms are fever, abdominal pain, vomiting, diarrhea, which if not take care of will result in death.  In many part of the world it is an endemic disease caused by bacteria called Vibrio cholerae, for on infection it colonizes small intestine, which is the target tissue and the bacteria secretes lot of hexameric enterotoxins.

This is the causative agent consists of subunits-A and the other 5 subunits called B.  The sub unit-A has ADP-ribosylation activity and stimulates Adenyl cyclase.  The A-subunit has A1 domain, which contains functional domain the other part help in joining A to B.  They bind to intestinal mucosal receptor and activate intestinal problem.

First deleting the A1 subunit part of the gene by homologous recombination method, in this process tetracycline gene is introduced in the place of A1 subunit segment has developed recombination live vaccine.

Plasmid containing homologous segments and a tetracycline gene id transferred to bacteria Cholera Vibrio.  This leads to recombination and deletes the A1 subunit.  But this cannot used for it has tetracycline gene.  So the enterotoxin gene was isolated and the A1 subunit deleted and plasmid is created containing the deleted enterotoxin gene with homologous segments at both flanking regions.  This plasmid is transferred to bacteria with deleted enterotoxin gene.  By recombination method the defective enterotoxin gene incorporated and the Tetracycline gene is removed.  Such genetically manipulated bacteria are used as live vaccine.  It is incredible feet indeed

 

HB-sAg vaccine:

HB-Ag gene form hepatitis was cloned into a plasmid contain Vaccinia strong promoter.  The flanking region of this construct contained homologous segments of Thymidine Kinase gene.  When this plasmid and wild Vaccinia viral DNA is co-infected into desired cell lines, by homologous recombination process the Hb-Sag gene is incorporated in the place of Thymidine kinase.  This can be screened by Glycover for the absence of Thymidine kinase.  This results in the expression surface antigen of Hepatitis, which can be used for vaccination purpose.

 

Employing the above method a wide variety of genes have been cloned to produce vaccines, ex. Rabies viral G-protein, Sindbis surface antigen, Influenza Np and HA protein, HSV glycoprotein, Vesicular stomatitis N & G protein and many others and used them for vaccination purpose.

 

Similarly adenoviruses can be used as live viruses.  This achieved by deleting replicative function of viruses and desired gene is cloned under the control of early genes.  Living viruses is generated using helper viruses.  Such viruses have been created for curing cystic fibrosis, a deadly disease among Greek and Cypriot population, where diseased children die before 20 yrs.  When recombinant virus id produced with a cystic fibrosis, channel protein gene with flanking regions such as LTR sequences, on delivery the Gene gets integrated into host cell genome.  This virus can be delivered into host by nasal spray.

 

Genetic Immunization:

A cloned gene construct which is capable of producing an antigenically active protein, when biolistically injected into the ears of a mice, the Gene with its borders (LTR) gets integrated and expressed a protein.  The protein now acts as an antigen and animal’s immune system responds and mounts antibody production against the protein.  This technique has a promising future.  When humans are very young as old as 2-3 years it is possible to inject such cloned DNA into body cells to make the child immune to a given antigen.  However continuous production of such antigens with in the body will have repercussion effects and how the body can tolerate such continuous stimulation is another question to be answered.  Another problem is the quantity of the antigen produced, there is no regulation.  I f the production of antigen is made to be regulated with specific stimulation, probably serves the system good.

 

Use of antibodies as drugs:

Coryneybacterium diphtheriae causes a severe disease faster and kill the patient fast.  It first shows infected symptoms in the throat and tonsils, where it generates exotoxins, which on circulation can severely damage organs, which are distant from the site of infection.

 

People use passive immunity against such disease causing agents.  By raising antibody in horse against such bacteria and then injecting the serum into humans, results in developing passive immunity.  However second time injection with horse serum will be so serious; it may give anaphylactic shock and cause death.  Yet the use of antibodies against a variety of disease causing agents and curing the disease is prevalent.  New methods and new approaches to the problems have been in vogue.  Monoclonal antibodies are once considered as magic bullets, but because of immune reactions to animal antibodies it became imperative develop antibodies, which are human compatible, called, humanized antibodies.  Though monoclonal antibodies are expensive, they are still used in diagnostics; tissue imaging and some times they are used as immuno-suppressants.

 

Among lymphocytes, T cells which differentiate in Thymus cells act as immunological helper cells and also effecter cells.  They are involved in rejection tissue grafting. If grafting to be successful T-cell mediated immune response to foreign tissue should be suppressed, thus one can save lives of a large number of human beings.  Researches developed antibodies against T cell receptors called CD3.  The first monoclonal antibody developed against CD3 in mice is called OKT3.  When injected it binds to the receptor and prevents full scale mounting of immune response to tissue grafting.  This kind of treatment was first approved by United States food and drug administration.  Without the approval no one can try to use trial on human beings at least in the US of A.

Such monoclonal antibodies are still in use in hospitals where bacteremia is endemic and kills lot of patients in USA.  Monoclonal antibodies were used to suppress proliferation of breast cancer cells by raising antibodies against specific cell surface receptors on tumor cells.  When such antibodies were used the cell surface receptors were blocked and growth of tumor has become stagnant.  Further more once the growth of tumor cell is halted, some factor should be added to cause apoptosis of cancer cells only.

 

Monoclonal antibodies (mAb or moAb) are monospecific antibodies: Targeting mAbs to specific protein/glycoprotein sites:

 

Blood clotting in vessels leading to brain and heart can lead to stroke and heart attack.  The blood clot in vessels is called Thrombus; it consists of a network of fibrin, one of the primary blood-clotting agents.  A defect in the endothelial layers of blood vessel is the cause. Thrombus kills millions of people all over the world, especially in developed world.

 

Plasminogen (86KD), a precursor protein for plasmin, is a serine protease.  The precursor Plasminogen is activated by Plasminogen activating factor (70KD). Once activated the plasmin acts on fibrinogen and fibrin and degrades the clot.

 

Raise antibodies to human fibrin and conjugate it with Plasminogen activating factor in such a way the enzyme’s active site is not disturbed.  When such conjugated composite factor is injected, it homes to the site wherever fibrin is present.  As most of the clots contain fibrin, the plasmin a serine protease acts and hydrolyses these proteins using serine sites in the protein.  Plasminogen is also activated by Urokinase (54KD).  Even Streptokinase takes part in dissolving blood clots. It is important to note that using monoclonal antibodies are used to deliver the required enzyme to specific site in the body.  This has great implications in curing several diseases.  While treating patients additional factors are also added with t-PA to inhibit t-PA activity, other wise it can lead to internal bleeding.

 

A general representation of the methods used to produce monoclonal antibodies; http://en.wikipedia.org/

 

https://www.google.co.in

 

 

Monoclonal antibodies for cancer. ADEPT, antibody directed enzyme prodrug therapy; ADCC, antibody dependent cell-mediated cytotoxicity; CDC, complement dependent cytotoxicity; MAb, monoclonal antibody; scFv, single-chain Fv fragment (WIKIPEDIA).

 

The first FDA-approved therapeutic monoclonal antibody was a murine IgG2a CD3 specific transplant rejection drug, OKT3 (also called muromonab), in 1986. This drug found use in solid organ transplant recipients who became steroid resistant.  Hundreds of therapies are undergoing clinical trials. Most are concerned with immunological and oncological targets (Wikipedia).

Example FDA approved therapeutic monoclonal antibodies[1]

Antibody

Brand name

Approval date

Type

Target

Indication
(What it's approved to treat)

Abciximab

ReoPro

1994

chimeric

inhibition of glycoprotein IIb/IIIa

Cardiovascular disease

Adalimumab

Humira

2002

human

inhibition of TNF-α signaling

Several auto-immune disorders

Alemtuzumab

Campath

2001

humanized

CD52

Chronic lymphocytic leukemia

Basiliximab

Simulect

1998

chimeric

IL-2Rα receptor (CD25)

Transplant rejection

Belimumab

Benlysta

2011

human

inihibition of B- cell activating factor

SLE[disambiguation needed ]

Bevacizumab

Avastin

2004

humanized

Vascular endothelial growth factor (VEGF)

Colorectal cancer, Age related macular degeneration (off-label)

Brentuximab vedotin

Adcetris

2011

Chimeric

CD30

Anaplastic large cell lymphoma (ALCL) and Hodgkin lymphoma

Canakinumab

Ilaris

2009

Human

IL-1β

Cryopyrin-associated periodic syndromes (CAPS)

Cetuximab

Erbitux

2004

chimeric

epidermal growth factor receptor

Colorectal cancer, Head and neck cancer

Certolizumab pegol[19]

Cimzia

2008

humanized

inhibition of TNF-α signaling

Crohn's disease

Daclizumab

Zenapax

1997

humanized

IL-2Rα receptor (CD25)

Transplant rejection

Denosumab

Prolia , Xgeva

2010

Human

RANK Ligand inhibitor

Postmenopausal osteoporosis , Solid tumor`s bony metasteses

Eculizumab

Soliris

2007

humanized

Complement system protein C5

Paroxysmal nocturnal hemoglobinuria

Efalizumab

Raptiva

2002

humanized

CD11a

Psoriasis

Gemtuzumab

Mylotarg

2000

humanized

CD33

Acute myelogenous leukemia (with calicheamicin)

Golimumab

Simponi

2009

Human

TNF-alpha inihibitor

Rheumatoid arthritis, Psoriatic arthritis, and Ankylosing spondylitis

Ibritumomab tiuxetan

Zevalin

2002

murine

CD20

Non-Hodgkin lymphoma (with yttrium-90 or indium-111)

Infliximab

Remicade

1998

chimeric

inhibition of TNF-α signaling

Several autoimmune disorders

Ipilimumab ( MDX-101 )

Yervoy

2011

Human

blocks CTLA-4

Melanoma

Muromonab-CD3

Orthoclone OKT3

1986

murine

T cell CD3 Receptor

Transplant rejection

Natalizumab

Tysabri

2006

humanized

alpha-4 (α4) integrin,

Multiple sclerosis and Crohn's disease

Ofatumumab

Arzerra

2009

Human

CD20

Chronic lymphocytic leukemia

Omalizumab

Xolair

2004

humanized

immunoglobulin E (IgE)

mainly allergy-related asthma

Palivizumab

Synagis

1998

humanized

an epitope of the RSV F protein

Respiratory Syncytial Virus

Panitumumab

Vectibix

2006

human

epidermal growth factor receptor

Colorectal cancer

Ranibizumab

Lucentis

2006

humanized

Vascular endothelial growth factor A (VEGF-A)

Macular degeneration

Rituximab

Rituxan, Mabthera

1997

chimeric

CD20

Non-Hodgkin lymphoma

Tocilizumab ( or Atlizumab )

Actemra and RoActemra

2010

Humanised

Anti- IL-6R

Rheumatoid arthritis

Tositumomab

Bexxar

2003

murine

CD20

Non-Hodgkin lymphoma

Trastuzumab

Herceptin

1998

humanized

ErbB2

Breast cancer

Recently, the bispecific antibodies, a novel class of therapeutic antibodies, have yielded promising results in clinical trials. In April 2009, the bispecific antibody catumaxomab was approved in the European Union.

 

Abzymes:

By genetic manipulations it is possible to convert antigen-binding site into an active site of the enzyme’.  This product has the ability to recognize the specific substrate as well as to perform enzyme activity.

 

Use of mABs in treatment has created problems for they themselves act as antigens, so they have modified the IgG in such way they act and behave like human antibodies (humanized).  In most of the cases monoclonal antibodies were obtained from mice, and mice antibodies are not human antibodies and they develop resistance to the use of antibodies as therapeutic agents.  So scientist started to look for human cell lines to generate good amount specific kind of IgGs against a specific antigen.

 

Developing Humanized IgGs (HIgGs):

Several strategies were employed. First they isolated human B-lymphocytes by tagging fluorescent antigen and then they are separated by flow cytometry and got reasonably enriched population of specific B-lymphocyte. Then human antibody producing lymphocytes are fused with human myeloma cells. Unfortunately the hybrid cell lines were found to be unstable.  Instead they transformed lymphocytes with Epstein Barr viruses.  Such transformed cells were viable, they divided and secreted IgGs, but alas!   The yield was very very poor.

 

The other strategy is to clone a human IgG gene, both L and H chains, and transform embryonic cells of the mouse and obtain the transgenic mouse and stimulate mouse for specific antigen and get specific antibodies.

 

In another method they obtained specific antibody gene for a specific antigen.  By genetic manipulation it is possible to replace human Fc fragment in the place of mice Fc fragment.  Similarly even the antigen binding hyper variable region can also be replaced. Such genes were transfected to known mammalian cell lines and expressed IgG were found to be human compatible.  But it is yet to be realized to produce such Abs on large scale.

 

Use of Bispecific Antibodies:

Produce two different but specific antibodies; say one for tumor cell surface receptor and the other for specific T-cell receptor.  When such antibodies were denatured and mixed to renature appropriately, one obtains antibodies containing two specificities called Bispecific antibodies.

 

Another way to generate Bispecific antibodies is to genetically manipulate hyper variable sites to the designed purpose.  When such Bispecific antibodies are employed one can target two systems simultaneously.  Bispecific antibodies, say one for specific T- killer cells and another for tumor cell, T-killer cells can be targeted to tumor cells.

 

In the case of malignant Hodgkin’s melanoma there are a large number of tumors infiltrating T-cells.  And tumor cells have their own specific antigenic receptors.  Raise antibodies separately for these two types of cells.  Generate hybrid IgGs.  Collect TIls (Tumor Infiltrating Lymphocytes) as much possible from the tumor and treat with specific Interleukin to activate the T-cells into killer cells.  Inject T-killer cells and Bispecific antibodies.  They bind to T-cell and bring them to tumor cells and bind.  The activated Tills destroy tumors and clinically found such tumors were regressed.

 

Bispecific Monoclonal Antibodies: It is symbolized as BsMAB, BsAB,. It is lab made antibody.  It can simultaneously bind to two different types of antigens. Current application is targeted to cancer immunotherapy and drug delivery. There are many format , but two of them are, one like IgG and another non-IgG like.

 

Three types of bispecific antibodies: trifunctional antibody, chemically linked Fab and bi-specific T-cell engager (bottom row). Blue and yellow parts distinguish parts from separate monoclonal antibodies. https://en.wikipedia.org

IgG like: It is like monoclonal antibody with two Fab  arms and one Fc region. The most common and prevalent one is tri functional antibodies.  These are produced with            quadroma or hybrid hybridoma method.

DVD-Ig Knob in Hole

The "knobs into holes" approach for manufacturing IgG-like bsMabs is shown on the left, while a diagram depicting the DVD-Ig format is on the right. The red dot indicates a possible site for introducing mutations in the heavy chain. Blue and yellow correspond to separate monoclonal antibodies. https://en.wikipedia.org

Non IgG like: There are other bsMabs that lack an Fc region completely. These are fusion proteins. there are various types of bivalent and trivalent single-chain variable fragments (scFvs).  They have vartiable domains. The new formats are bi-specific T-cell engagers (BITEs).

How they act: they are exemplified by catumaxomab, representing the first approved bispecific- trifunctional antibody. Thery are very efficient used for cancer therapy.

The mechanism of action of a BsMAb, exemplified by catumaxomab, representing the first approved bispecific trifunctional antibody. https://en.wikipedia.org

Micromet - Biting Cancer (Part I):

 

In the context of cancer therapy, Bispecific antibodies are usually designed to redirect immune cells to tumor cells, in a way that will lead to the attack of the tumor. One arm is directed against a target on a cancer cell while the other arm is directed against a target on the immune cell. In most cases, bispecific antibodies must not only bring the immune cell closer to cancer cells but also activate it to attack the tumor. In order to achieve both of these tasks, there is a need to identify specific targets on immune cells that can be activated by antibody binding, or find supplementary ways to achieve this activation. Naturally, there is a large number of options when designing a bsAb. One variable is the target on the cancer cells, another variable is the type of immune cell to be recruited, while a third variable is which structural element (antigen) on that specific immune cell should be targeted. This is much more complicated than designing mono-specific antibodies, in which only the target on the cancer cells should be chosen.

 

Genemab- Next Generation Technology:

 

Genmab is creating knowledge and intellectual property around Fab-arm exchange and the generation of bispecific antibodies, thereby establishing a strong basis for the DuoBody platform.

 

Developing fully human antibody therapeutics for the potential treatment of cancer; Gene Mabs; https://www.pharmatching.com

 

 

UniBodyTechnology:

UniBody is a proprietary antibody technology that creates a stable, smaller antibody format with an anticipated longer therapeutic window than current small antibody formats, based on pre-clinical studies to date. A UniBody molecule is about half the size of a regular type of inert antibody called IgG4 and binds with only one antibody arm to a therapeutic target. UniBody molecules are expected to be cleared from the body at a lower rate than other antibody fragments based on the preclinical studies to date. Unlike other antibodies which primarily work by killing targeted cells, a UniBody molecule will only inhibit or silence cells, which could be an advantage in the treatment of diseases such as asthma or allergies.

New antibody delivers a double blow ; 10:15 13 December 2003 by Andy Coghlan

 

 

http://www.newscientist.com/

Normal antibodies have two identical arms, each made of two chains, that both bind to the same target (see graphic). But in 1999 Rob Aalberse, of the University of Amsterdam, reported that the body also produces dual-target antibodies, in which each arm locks onto a different target (New Scientist print edition, 18 September 1999).

Cancer cells, for instance, could be better targeted by antibodies that recognize two distinguishing features instead of just one. "With two targets, you'd enhance specificity, so sparing more healthy cells," says Tomlinson, who unveiled the method last week at an antibody conference in San Diego, California.

Epigenetically inducement of vigor in older people;

Transfusion of younger blood into older people has been subjected to studies, but it works in Mice.  Where only blood without cells are pumped into older people particularly it is very helpful to older people with Alzheimer disease or for that matter anyone who is old wants to be young.  Based on these trails certain people opened a company Alkahest to treat Alzheimer’s disease. Astonishingly this company is funded by the participants.  Is this technology works or still in trails.  If it works, how what the younger blood plasma contains’ these have to be worked out. Whether Dr.J. Karmazin’s Ambrosia’ trail works or not, there is a possibility and it should be tested.  As transfusion of younger plasma does not cause any problem in the patient.  As with experiments on mice has worked, why not in humans.

Application of Biotechnology and Gene Engineering in Medicine:

Gene engineering is the part of biotechnology, but specialization in gene isolation, sequencing, modifying and introducing into living system and disease diagnosis at biochemistry  and molecular level.

 

Molecular Medicine 2017

 

Molecular medicine is a broad field, where physical, chemical, biological and medical techniques are used to describe molecular structures, mechanisms and identify fundamental molecular and genetic errors of disease, and to develop molecular interventions to correct them (Only few of them are discussed).  It is a medical field that diagnoses the causes and provides treatment with medicines, surgery and often with gene therapy. Understanding of biochemistry of medicines and how the chemicals act at biochemical and molecular level is important (?) for Medical Doctors who treat patients.  Typical applications of molecular medicines include gene therapy, molecular structural analysis, genetic epidemiology, and molecular and clinical pharmacology.  This understanding of it requires good education in these areas in medical colleges by Doctors who have trained in such areas who have such knowledge; unfortunately this is deficient in most of our medical colleges who teach medicine (in India).  Majority of doctors in our land have learnt disease diagnosis by symptoms and using stethoscope, prescribe medicines which are essentially remembered what medicine to what symptoms.  Any practicing doctor who does not have pharmaceutical understanding of the drug is a sin!.

 

Typical applications in molecular medicine include gene therapy, molecular structural analysis, genetic epidemiology, and molecular and clinical pharmacology. In November 1949, with the seminal paper, "Sickle Cell Anemia, a Molecular Disease", in Science magazine, Linus Pauling, Harvey Itano and their collaborators laid the groundwork for establishing the field of molecular medicine. In 1956, Roger J. Williams wrote Biochemical Individuality, a prescient book about genetics, prevention and treatment of disease on a molecular basis, and nutrition which is now variously referred to as individualized medicine and orthomolecular medicine.  Another paper in Science by Pauling in 1968, introduced and defined this view of molecular medicine that focuses on natural and nutritional substances used for treatment and prevention. Molecular medicine encompasses Biochemistry, Clinical Chemistry, Life Sciences, Medical Biology, Medical Chemistry, Medical Physics, Metabolomics, Molecular Biology, Molecular diagnostics and Molecular pathology are important components. www.wikipedia.org

 

Molecular Medicine accomplishments in research have been recognized by the 2006 Nobel Prize in Physiology or Medicine awarded to Craig Mello (shared with Andrew Fire of Stanford University), the 2008 Lasker Basic Medical Research Award to Victor Ambros (shared with Gary Ruvkun of Harvard and David Baulcombe of Cambridge University), and the 2007 Medical Foundation Basic Science Award to David Lambright.  Howard Hughes Medical Institute Investigators have done appointments to Michael Green, Roger Davis, and Craig Mello, and membership in the National Academy of Sciences (Mello and Ambros) and the Royal Society of London (Davis). Many other Molecular Medicine faculty have been recognized by awards for outstanding contributions in their fields of specialty, for example, a 2012 NIDA Avant Garde  award to Jeremy Luban, the 2000 Banting Award to Michael Czech and the Elizabeth Glaser Scientist Award to Katherine Luzuriaga. Pew Scholar awards have been bestowed upon Tom Fazzio, Bert van den Berg and David Guertin. www.wikipedia.org, The Program in Molecular Medicine offers within its building a broad spectrum of state-of-the-art methodologies to its laboratory groups including deep sequencing, ultrafast 3D digital imaging microscopy (wide field and TIRF) of live cells, spinning disc confocal microscopy, x-ray crystallography, mouse metabolic phenotyping, mouse knockout technology and RNAi-based gene silencing in vitro and in vivo.   Medical School Core facilities also make available a large number of additional technologies such as FACS analysis, gene profiling using microarrays, proteomics and both shRNA and small molecule screening. Expertise in chemistry, structural biology, biochemistry, cell and developmental biology, molecular biology, cell signaling and regulation, genomics and proteomics, bioinformatics, genetics, immunology and virology is strongly represented in the Program in Molecular Medicine. Program faculty members are also active in the teaching of these disciplines in both core and advanced courses for graduate and medical students. Structural biology at the UMass Medical School is supported by state-of-the-art X-ray and NMR core facilities housed in the Program in Molecular Medicine and the Department of Biochemistry and Molecular Pharmacology. Diffraction instrumentation includes three rotating anode X-ray generators equipped with R-axis IV, Mar 300 and Mar 350 image plates detectors, Osmic focusing mirrors, and nitrogen cryostreams. NMR instrumentation includes 400 MHz and 600 MHz Varian spectrometers equipped for multidimensional homonuclear and heteronuclear experiments. Computational resources include graphics workstations and multiprocessor Beowulf clusters for data processing, image reconstruction, 3D visualization, model building, refinement, molecular dynamics, and structural bioinformatics. Molecular Medicine laboratory groups utilize many model organisms in their research, including yeast, worms, flies, mice and nonhuman primates. Translational research on human subjects is also vigorously pursued with collaborators in clinical department www.wikipedia.org,

 

Mucosal immunology and vaccine; T Feng and C O Elson:

 

Mucosal immunology is one of the prominent techniques employed all over the world especially in under developed countries.  Many of world scientist and great people like Bill Gates go to places where oral vaccine is required and demonstrate how people can use this technology, his contribution in terms of money to this field is remarkable and it has to be applauded.  Starting from mouth, nose and ears to the end of large intestine is the largest mucosal surface.  It is major surface that encounters all sorts of microorganisms and it has to combat when needed. This mucosal surface and is a major site of multifaceted interactions between the host mucosal immune system and components of the intestinal microbiota.   Host immune responses to the commensal microbiota are tightly controlled and, meanwhile, the microbiota actively shapes intestinal immune responses to itself. Appreciation of these interactions during health and disease may direct therapeutic approaches to a broad range of autoimmune and inflammatory disorders in humans. In this review, we will discuss findings on how the intestinal immune system, especially adaptive immune cells, helps accommodate the large number of resident bacteria, and in turn how the microbiota shapes intestinal immune responses to achieve mutualism. http://www.nature.com

 

One of the key features of the intestinal immune system is its ability to distinguish between pathogenic and symbiotic bacteria, and thus protect against infection while avoiding detrimental and unnecessary inflammatory responses toward the normal microbiota. When these intestinal immune responses are dysregulated, they can result in chronic inflammatory disorders of the gut, including inflammatory bowel disease (IBD), celiac disease, and food allergies.

 

Host immune cells have developed a hierarchy of homeostatic mechanisms to ensure mucosal immune compartmentation and maintain systemic ignorance to commensal bacteria, including a layer of immunoglobulin (Ig) A- and antimicrobial peptide-containing mucus, a physical epithelial barrier, and innate and adaptive immune components. Understanding of the host–microbiota interactions during both steady-state homeostasis and pathological intestinal inflammation may help to direct therapeutic approaches to IBD as well as to a broad range of immune-mediated inflammatory disorders in humans.

 

Intestinal immune system, especially the adaptive immune component, resident microbiota, and in turn how the microbiota shapes intestinal immune responses to achieve mutualism. First, we understand the tight compartmentation of mucosal innate and adaptive immune responses, formed mainly by intestinal IgA reactive to microbial antigens, resulting in immune exclusion of the commensal microbial antigens and systemic immune “ignorance”. This will also highlight recent advances in understanding the influences of regulatory T (Treg) cells and effector T cells in the context of immune homeostasis and dysregulation, and feedback of the microbiota to intestinal T-cell regulation.

 

The human intestine harbors nearly 10x100 billion microorganisms composed of more than 1,000 distinct bacterial species as defined by high-throughput microbial 16S ribosomal RNA gene characterization.

First, the epithelial layer of the intestinal tract is formed by tightly connected intestinal epithelial cells and serves as a physical protective layer, separating luminal contents from the underlying immune compartments, and providing an efficient barrier to block the entry of microflora into the lamina propriety. Specialized intestinal epithelial cells such as mucus-secreting goblet cells and antimicrobial peptide-producing Paneth cells also contribute to the constitution of the mucosal barrier,

 

IgA, secreted by plasma cells and transported by intestinal epithelial cells into the lumen, is more abundant than the sum of all other Ig isotypes combined, and it joins the effort with bactericidal peptides in the mucus layer to form a passive defense line, which sequesters most resident bacteria in the lumen and dramatically reduces the microbial burden of the epithelium. The third layer of intestinal defense is formed by innate and adaptive immunity. Intestinal immune cells are extensively distributed throughout the intestinal mucosa, which is customarily divided into organized inductive and diffusely distributed effector sites.12 Innate and adaptive immune cells accumulate in these mucosal immune compartments and coordinate both to maintain a state of limited mucosal activation and to initiate active immune responses to invading microbes.

 

In contrast to the lack of concomitant systemic immune response, a strong intestinal IgA response to half of the rIBs and to two immunodominant microbiota flagellins, CBir1 and Flax, was identified, indicating a tight intestinal compartmentation of the active immune response to the microbiota, http://www.nature.com/2015-2106;  Xinyang Song, Xiao He, Xiaoxia Li and Youcun Qian have found that the mucosal immune system serves as the front-line defense against pathogens. It also tightly maintains immune tolerance to self-symbiotic bacteria, which are usually called commensals. Sensing both types of microorganisms is modulated by signaling primarily through various pattern-recognition receptors (PRRs) on barrier epithelial cells or immune cells. After sensing, proinflammatory molecules such as cytokines are released by these cells to mediate either defensive or tolerant responses. The interleukin-17 (IL-17) family members belong to a newly characterized cytokine subset that is critical for the maintenance of mucosal homeostasis. In this review, we will summarize recent progress on the diverse functions and signals of this family of cytokines at different mucosal edge.

 

The intestinal mucosa is a particularly dynamic environment in which the host constantly interacts with trillions of commensal microorganisms, known as the microbiota, and periodically interacts with pathogens of diverse nature. In this Review, we discuss how mucosal immunity is controlled in response to enteric bacterial pathogens, with a focus on the species that cause morbidity and mortality in humans. We explain how the microbiota can shape the immune response to pathogenic bacteria, and we detail innate and adaptive immune mechanisms that drive protective immunity against these pathogens. The vast diversity of the microbiota, pathogens and immune responses encountered in the intestines precludes discussion of all of the relevant players in this Review. Instead, we aim to provide a representative overview of how the intestinal immune system responds to pathogenic bacteria.  http://www.nature.com/Araceli Perez-Lopez, Judith Behnsen et al; Nature Reviews Immunology.

 

·       Mucosal immunity reduces the need for elimination of penetrating exogenous antigens by proinflammatory systemic immunity. The adult gut mucosa contains some 80% of the body's activated B cells-differentiated to plasma blasts and plasma cells (PCs). Most mucosal PCs produce dimeric immunoglobulin A (IgA), which, along with pentameric- immunoglobulin M (IgM), can be exported by secretory epithelia expressing the polymeric immunoglobulin receptor. Immune exclusion of antigens is performed mainly by secretory IgA in cooperation with innate defenses, but, in newborns and in IgA deficiency, secretory IgM is important. In the gut, induction and regulation of mucosal immunity occurs primarily in gut-associated lymphoid tissue-particularly the Peyer's patches-and also in mesenteric lymph nodes. Terminal differentiation to PCs is accomplished in the lamina propria to which the activated memory/effector T and B cells home. Lactating mammary glands are part of the secretory immune system, and IgA antibodies in breast milk reflect antigenic stimulation of gut-associated lymphoid tissue and nasopharynx-associated lymphoid tissue such as the tonsils. Breast-milk antibodies are thus highly targeted against infectious agents and other exogenous antigens in the mother's environment, which are those likely to be encountered by the infant. Therefore breast-feeding represents an ingenious immunologic integration of mother and child. https://www.ncbi.nlm.nih.gov.

Mucosal immunology is the study of immune system responses that occur at mucosal membranes of the intestines, the urogenital tract and the respiratory system, i.e., surfaces that are in contact with the external environment.  In healthy states, the mucosal immune system provides protection against pathogens but maintains a tolerance towards non-harmful commensual microbes and benign environmental substances.  It provides three main functions: protecting the mucous membrane against infection; preventing the uptake of antigens, microorganisms, and other foreign materials; and moderating the organism's immune response.

At birth, the neonate's mucosal immune system is relatively undeveloped, but the colonization of intestinal flora promotes its development.  Because of its front-line status within the immune system, the mucosal immune system is being investigated for use in vaccines for various afflictions, including AIDS and allergies, collectively intestinal mucosa covers 400M666^2 area 400m2 (equivalent to one and a half tennis court)  http://www.scq.ubc.ca/

The mucous membranes produce a special type of antibody called secretory IgA or sIgA. In the mucous, this antibody is secreted as a dimer, joined at the non-antigen binding end by a protein known as the J chain as shown in Figure.

mucosalimmunityIgA.gif

 

Figure . Structure of secretory IgA. It consists of at least two IgA molecules covalently linked by a J chain and the secretory component, which is added as the antibody crossed the mucosal epithelial cells into the lumen. http://www.scq.ubc.ca/.

 

This form of the antibody is more stable, less resistant to proteolysis by the digestive enzymes of the gut, and has higher avidity for mucosal surfacesThe mucous membranes are bathed in huge quantities of sIgA, which act as a first line of defense to neutralize invading pathogens.   Experimental evidence shows that the presence of sIgA correlates with resistance to infection by various pathogens, including bacteria, viruses, parasites and fungi.   It has also been shown to neutralize viruses and prevent their adherence to the epithelial cells lining the mucous (thereby preventing infection) as well as mediating excretion of pathogens and preventing the assembly of mature virus particles.

Another important component of mucosal immunity is the T cell-mediated immune response. T cells that specifically recognize pathogens can help antibodies to clear the infection or directly kill the invader themselves. T cells produced in the mucous are capable of traveling throughout the mucosal tissues through special “homing” receptors on their membranes. This means that if an immune response is generated in the gastrointestinal  lining, T cells produced there can travel to other mucosal sites, for example, the lungs or nasal cavity, providing protection over a large surface area;

 Among the newer vaccines designed to induce a protective immune response to HIV is one designed to mimic the mucosal responses seen in these resistant individuals.  In 2001, a US group tested the first mucosal HIV vaccine in rhesus macaques.  While the vaccine showed promising results, but it failed to prevent infection.

·       Organs of the immune system include the thymus, spleen, and lymph nodes. T-lymphocytes develop in the thymus, which is located in the chest directly above the heart. The spleen, which is located in the upper abdomen, makes antibodies and removes old and damaged red blood cells.  The immune system is broadly divided into two major components: innate immunity and adaptive immunity. Innate immunity involves immediate, nonspecific responses to foreign invaders, while adaptive immunity requires more time to develop its complex, specific responses http://lpi.oregonstate.edu/.

Vaccination is one of the most successful applications of immunology and for a long time has depended on parenteral administration protocols. However, recent studies have pointed to the promise of mucosal vaccination because of its ease, economy and efficiency in inducing an immune response not only systemically, but also in the mucosal compartment where many pathogenic infections are initiated. However, successful mucosal vaccination requires the help of an adjuvant for the efficient delivery of vaccine material into the mucosa and the breaking of the tolerogenic environment, especially in oral mucosal immunization. Given that M cells are the main gateway to take up luminal antigens and initiate antigen-specific immune responses, understanding the role and characteristics of M cells is crucial for the development of successful mucosal vaccines. Especially, particular interest has been focused on the regulation of the tolerogenic mucosal microenvironment and the introduction of the luminal antigen into the lymphoid organ by exploiting the molecules of M cells. Here, we review the characteristics of M cells and the immune regulatory factors in mucosa that can be exploited for mucosal vaccine delivery and mucosal immune regulation. Sae-Hae Kim and Yong-Suk Jang; http://www.nature.com/

Figure 1. Immune Cell Phagocytosis. Macrophages are specialized leukocytes that respond to invading pathogens by initiating phagocytosis and the synthesis and release of pro-inflammatory cytokines. Microorganisms like bacteria have pathogen-associated molecular patterns (PAMPs) that are identified by pattern recognition receptors on macrophages. The left side of the figure illustrates the process of phagocytosis, which involves engulfment of the bacterium into an intracellular vesicle called a phagosome, phagosome-lysosome fusion to form a phagolysosome, degradation of the bacterium by enzymes, and cellular release of the degraded material by exocytosis. The right side of the figure illustrates that bacterial binding to surface receptors of the macrophage also signals the transcription of pro-inflammatory cytokines in the cell’s nucleus. Cytokines are then produced in the cytoplasm and these pro-inflammatory proteins are secreted from the cell to affect behavior of nearby cells.

http://lpi.oregonstate.edu/

 

Expression of mucosal IgA immune responses after different routes of vaccination: http://www.nature.com/

The 'common mucosal immune system' is more restricted than previously thought. In humans, immunization studies with cholera toxin B subunit by different mucosal routes have clearly shown that the strongest response takes place at the directly vaccine-exposed mucosa and the second-best responses at adjacent mucosae or at specifically interconnected inductive-expression mucosal systems such as the gut-mammary gland link in lactating women. A notable exception is the fact that nasal mucosal immunization not only stimulates an immune response in the respiratory tract, but also can give rise to a strong genital-vaginal mucosal immune response. Shading indicates strength of response.

Regulatory Mechanisms:  The mucosal immune system has evolved a variety of mechanisms to achieve and maintain tolerance against self-antigens and against the plethora of environmental antigens present in the microflora, in food and among airborne matter. Studies in animal models have identified that mucosal tolerance can be achieved through different mechanisms, including activation-induced cell death, anergy and, most important, the induction of regulatory T cells. Anergy of antigen-specific T cells has been reported after inhalation or ingestion of large quantities of soluble proteins, and deletion of specific T cells only after mucosal administration of massive, non-physiological antigen doses42. Induction of regulatory cells after mucosal delivery of antigens has been reported in animal models for more than 25 years and has received a major attention during the last few years given the potential of such regulatory cells as therapeutic agents in immune-mediated diseases.

In mice, four main types of regulatory T cells have been described: (i) antigen-induced CD4+TH2-like cells that produce IL-4 and IL-10 and antagonize the activity of TH1 effector cells;  (ii) CD4+CD45RBlow Tr1 cells that function through the production of IL-10; (iii) CD4+ or CD8+ T cells producing TGF-beta (TH3 cells);  and apparently most important,  (iv) a population of naturally occurring CD4+CD25+ regulatory T cells (Treg cells) that suppress proliferation through a cell contact−dependent mechanism. Although anergic in vitro,  the latter cells can be expanded in an antigen-specific manner in vivo after immunization.  Notably, these cells may also confer suppressor activity on other CD4+ T cells by inducing the expression of the transcription factor Foxp3 and/or the major histocompatibility complex (MHC) class II−binding molecule LAG-3 in such cells ('infectious tolerance')51, 52. Thereby, they may also provide a direct link between effector T-cell inhibition by Treg, TH3 and Tr1 cells. Thus, natural human CD4+CD25+ Treg expressing the mucosal alpha4beta7 integrin, when co-cultured with conventional CD4+ T cells, induced Tr1-like IL-10−secreting T cells with strong suppressor activity on effector T cells, whereas another, alpha4beta1-positive Treg subset in similar cultures instead induced TH3-like TGF-beta−secreting suppressor T cells51. Recent evidence indicates that all of these different regulatory T cell types and mechanisms can be induced or expanded by mucosal administration of antigens leading to peripheral tolerance (oral tolerance; J.-B. Sun et al., unpublished data).

 

Organs of the immune system include the thymus, spleen, and lymph nodes.  T-lymphocytes develop in the thymus, which is located in the chest directly above the heart. The spleen, which is located in the upper abdomen, makes antibodies and removes old and damaged red blood cells.   The immune system is broadly divided into two major components: innate immunity and adaptive immunity. Innate immunity involves immediate, nonspecific responses to foreign invaders, while adaptive immunity requires more time to develop its complex, specific responses, http://lpi.oregonstate.edu/.

 

Oral Vaccines; Polio vaccine; Trivalent oral poliovirus vaccine (tOPV); The trivalent vaccine was withdrawn in April 2016 and replaced with the bivalent oral poliovirus vaccine (bOPV), which contains only attenuated virus of types 1 and 3. This is because continued use of tOPV threatened to continue seeding new type 2 circulating vaccine-derived polioviruses (cVDPV2), despite the wild type 2 virus being eradicated in 1999.

The first polio vaccine was the inactivated polio vaccine; It was developed by Jonas Salk and came into use in 1955.  The oral polio vaccine was developed by Albert Sabin and came into commercial use in 1961; The wholesale cost in the developing world is about 0.25 USD per dose for the oral form as of 2014.  In the United States it costs between 25 and 50 USD for the inactivated form. OPV also proved to be superior in administration, eliminating the need for sterile syringes and making the vaccine more suitable for mass vaccination campaigns. OPV also provided longer lasting immunity than the Salk vaccine. 

 

13_Polio imm 2

http://polioeradication.org/- live attenuated vaccine-virus in OPV 

 

The oral polio vaccine is simple to administer. A few drops, given multiple times, can protect a child for life. © WHO/Rod CurtisOPV also proved to be superior in administration, eliminating the need for sterile syringes and making the vaccine more suitable for mass vaccination campaigns. OPV also provided longer lasting immunity than the Salk vaccine.

One dose of OPV produces immunity to all three poliovirus serotypes in approximately 50% of recipients. Three doses of live-attenuated OPV produce protective antibodies to all three poliovirus types in more than 95% of recipients. OPV produces excellent immunity in the intestine, the primary site of wild poliovirus entry, which helps prevent infection with wild virus in areas where the virus is endemic. The live virus used in the vaccine is shed in the stool and can be spread to others within a community. The live virus also has stringent requirements for transport and storage, which are a problem in some hot or remote areas. As with other live-virus vaccines, immunity initiated by OPV is probably lifelong.   The trivalent (against wild type 1, 2 and 3) OPV has been used and nearly eradicated polio infection worldwide. Spearheaded by The Global Polio Eradication Initiative, 155 countries switched to use the bivalent (against wild type 1 and 3) between 17 April and 1 May 2016.

The Salk vaccine, or inactivated poliovirus vaccine (IPV), is based on three wild, virulent reference strains, Mahoney (type 1 poliovirus), MEF-1 (type 2 poliovirus), and Saukett (type 3 poliovirus), grown in a type of monkey kidney tissue culture (Vero cell line), which are then inactivated with formalin.  The injected Salk vaccine confers IgG-mediated immunity in the bloodstream, which prevents polio infection from progressing to viremia and protects the motor neurons, thus eliminating the risk of bulbar polio and post-polio syndrome.

In United States, the vaccine was administered along with the diphtheria, tetanus, and acellular pertussis vaccines (DTaP) and a pediatric dose of hepatitis B vaccine.  In the UK, IPV is combined with tetanus, diphtheria, pertussis, and Haemophilus influenzae type b vaccines

Sabin strains were chosen for worldwide distribution.  There are 57 nucleotide substitutions which distinguish the attenuated Sabin 1 strain from its virulent parent (the Mahoney serotype), two nucleotide substitutions attenuate the Sabin 2 strain, and 10 substitutions are involved in attenuating the Sabin 3 strain.  The primary attenuating factor common to all three Sabin vaccines is a mutation located in the virus's internal ribosome entry site (IRES);  In 1961, type 1 and 2 monovalent oral poliovirus vaccine (MOPV) was licensed, and in 1962, type 3 MOPV was licensed. In 1963, trivalent OPV (TOPV) was licensed, and became the vaccine of choice in the United States and most other countries of the world, largely replacing the inactivated polio vaccine.  A second wave of mass immunizations led to a further dramatic decline in the number of polio cases. Between 1962 and 1965 about 100 million Americans (roughly 56% of the population at that time) received the Sabin vaccine. The result was a substantial reduction in the number of poliomyelitis cases, even from the much reduced levels following the introduction of the Salk vaccine.

Indian-The trial also demonstrated the superiority of bOPV compared to the respective Sabin type 1 and 3 strains contained in the trivalent oral poliovirus vaccine (tOPV). Immediately after the trial results were available, the ACPE convened by conference call and reviewed the trial outcomes and current epidemiology of wild poliovirus globally.

Mucosa:

An ulcer (/ˈʌlsər/; from Latin ulcus, "ulcer, sore")  is a break in the skin or mucous membrane with loss of surface tissue and the disintegration and necrosis of epithelial tissue.

Mucosal immunity and vaccines:

There is currently great interest in developing mucosal vaccines against a variety of microbial pathogens. Mucosally induced tolerance also seems to be a promising form of immunomodulation for treating certain autoimmune diseases and allergies. Here we review the properties of the mucosal immune system and discuss advances in the development of mucosal vaccines for protection against infections and for treatment of various inflammatory disorders.www.nature.com

The mucosal immune system has three main functions: (i) to protect the mucous membranes against colonization and invasion by potentially dangerous microbes that may be encountered, (ii) to prevent uptake of not degraded antigens including foreign proteins derived from ingested food, airborne matter and commensal microorganisms, and (iii) to prevent the development of potentially harmful immune responses to these antigens if they do reach the body interior. At variance with the systemic immune apparatus, this functions in a normally sterile milieu and often responds vigorously to invaders, the MALT guard’s organs that are replete with foreign matter. It follows that upon encountering this plethora of antigenic stimuli; the MALT must economically select appropriate effector mechanisms and regulate their intensity to avoid bystander tissue damage and immunological exhaustion. Jan Holmgren & Cecil Czerkinsky

 

GENERIC NAME(S):  Guaifenesin is an expectorant. It works by thinning and loosening mucus in the airways, clearing congestion, and making breathing easier.

 

Mucosal immunity and vaccines: wwwq.nature.com;

The main mucosa-associated lymphoid tissues (MALT) of teleosts are the gut-associated lymphoid tissue (GALT), skin-associated lymphoid tissue (SALT), the gill-associated lymphoid tissue (GIALT) and the recently discovered nasopharynx-associated lymphoid tissue (NALT). Teleost MALT includes diffuse B cells and T cells with specific phenotypes different from their systemic counterparts that have co-evolved to defend the microbe-rich mucosal environment. Both B and T cells respond to mucosal infection or vaccination. Irene Salinas; Both B and T cells respond to mucosal infection or vaccination. Specific antibody responses can be measured in the gills, gut and skin mucosal secretions of teleost fish following mucosal infection or vaccination. Rainbow trout studies have shown that IgT antibodies and IgT+ B cells are the predominant B cell subset in all MALT and respond in a compartmentalized manner to mucosal infection. Our current knowledge on adaptive immunity in teleosts is limited compared to the mammalian literature. New research tools and in vivo models are currently being developed in order to help reveal the great intricacy of teleost mucosal adaptive immunity and help improve mucosal vaccination protocols for use in aquaculture. http://www.mdpi.com/ 

 

Polio vaccine:

The oral polio vaccine is simple to administer. A few drops, given multiple times, can protect a child for life. © WHO/Rod Curtis; http://polioeradication.org/- live attenuated vaccine-virus in OPV .  For example, many vaccines contain  sodium and potassium salt based which are essential for life. People may think of formaldehyde as a man-made chemical, but in small quantities it is also found naturally in the bloodstream.  These are the Active ingredients of the vaccine made from viruses or bacteria (also called ‘antigens’). A few vaccines in the UK schedule are made using recombinant DNA technology; genetically modified organisms (GMOs) is used .  Few vaccines contain  Aluminium (an adjuvant), Thiomersal, also called Thimerosal (a preservative), Gelatine (a stabiliser), emulsifiers, sugar, Lactose, Mannitol , latex, glycerol, glutamate, Egg Proteins (Ovalbumin), acidity regulators are added-Dec 20, 2016.  Oral adjuvents-polio-Type1, type 2 and type 3-40, 8 and 32 units of antigens-expients antibiotics (neomycin, streptomycin and polymyxin are also used.  Some vaccines contain 2-phenoxyethanol as a preservative. Thiomersal cannot be used for IPV. http://www.who.int/vaccine_safety

Trivalent oral poliovirus vaccine (tOPV);

The trivalent vaccine was withdrawn in April 2016 and replaced with the bivalent oral poliovirus vaccine (bOPV), which contains only attenuated virus of types 1 and 3. This is because continued use of tOPV threatened to continue seeding new type 2 circulating vaccine-derived polioviruses (cVDPV2), despite the wild type 2 virus being eradicated in 1999.

 

The first polio vaccine was the inactivated; It was developed by Jonas Salk and came into use in 1955. The oral polio vaccine was developed by Albert Sabin and came into commercial use in 1961; The wholesale cost in the developing world is about 0.25 USD per dose for the oral form as of 2014.  In the United States it costs between 25 and 50 USD for the inactivated form. OPV also proved to be superior in administration, eliminating the need for sterile syringes and making the vaccine more suitable for mass vaccination campaigns. OPV also provided longer lasting immunity than the Salk vaccine.  Attenuated IPV and bPV inactivated Polio and bPVBivalent PV are in use.

The Salk vaccine, or inactivated poliovirus vaccine (IPV), is based on three wild, virulent reference strains, Mahoney (type 1 poliovirus), MEF-1 (type 2 poliovirus), and Saukett (type 3 poliovirus), grown in a type of monkey kidney tissue culture (Vero cell line), which are then inactivated with formalin.  The injected Salk vaccine confers IgG-mediated immunity in the bloodstream, which prevents polio infection from progressing to viremia and protects the motor neurons, thus eliminating the risk of bulbar polio and post-polio syndrome.

In the United States, vaccine is administered along with the diphtheria, tetanus, and acellular pertussis vaccines (DTaP) and a pediatric dose of hepatitis B vaccine.  In the UK, IPV is combined with tetanus, diphtheria, pertussis, and Haemophilus influenzae type b vaccines

Sabin strains were chosen for worldwide distribution. There are 57 nucleotide substitutions which distinguish the attenuated Sabin 1 strain from its virulent parent (the Mahoney serotype), two nucleotide substitutions attenuate the Sabin 2 strain, and 10 substitutions are involved in attenuating the Sabin 3 strain. The primary attenuating factor common to all three Sabin vaccines is a mutation located in the virus's internal ribosome entry site (IRES);   In 1961, type 1 and 2 monovalent oral poliovirus vaccine (MOPV) was licensed, and in 1962, type 3 MOPV was licensed. In 1963, trivalent OPV (TOPV) was licensed, and became the vaccine of choice in the United States and most other countries of the world, largely replacing the inactivated polio vaccine.  A second wave of mass immunizations led to a further dramatic decline in the number of polio cases. Between 1962 and 1965 about 100 million Americans (roughly 56% of the population at that time) received the Sabin vaccine. The result was a substantial reduction in the number of poliomyelitis cases, even from the much reduced levels following the introduction of the Salk vaccine.

Indian made OPV  trial also demonstrated the superiority of bOPV compared to the respective Sabin type 1 and 3 strains contained in the trivalent oral poliovirus vaccine (tOPV). Immediately after the trial results were available, the ACPE convened by conference call and reviewed the trial outcomes and current epidemiology of wild poliovirus globally.

Plant based polio mucosoal vaccine https://www.sciencedaily.com/

Edible vaccine production for veterinary use has received widespread attention because of health initiatives aimed at decreasing antibiotic use in livestock and other animals to avoid the development of antibiotic-resistant strains, especially of epidemic and zoonotic pathogens. These issues have promoted the development of plant-based vaccines, which can easily fulfil these requirements (zoonotic).

Edible vaccines are actually recombinant vaccines in which selected antigens against a particular pathogen are introduced into a plant and grown.  Oral delivery of this plant induces a protective immune response against that particular pathogen in the form of an edible vaccine; Plant cells expressing vaccine antigens or biopharmaceuticals can be lyophilized and stored at ambient temperature for many years maintaining efficacy of expressed protein drugs; Plant-based vaccines have the potential to induce a mucosal immune response and a systemic immune response without the pain and risk associated with needles and injections.

Banana vaccine: Inspired by calf fed on banana- made vaccine in Banana; not succeded, Redig Mandy.  Plant vaccines will be a billion-dollar business, said Professor Arntzen, who leads a team of the world's top plant scientists at Cornell University in New York State.  Successful experiments show that the vaccine worked, he added, and will cost less than one penny a dose to make. A single gene transferred into a tomato or banana plant is reproduced as a protein thousands of times inside the fruit. When eaten it passes into the intestine and then into the blood stream producing antibodies against hepatitis B - working the same way as a traditionally injected but much more expensive vaccine.  A single dried banana chip or tomato paste sandwiched in a wafer contains enough protein to act as a vaccine dose. Traditional vaccines have to be refrigerated and cost £10 for a successful course, making them too expensive for developing countries where hepatitis B kills many people directly and causes the death of many more through subsequent cancer.  Tomatoes and bananas genetically modified to contain hepatitis B vaccine could rid the world of the virus, a leading American scientist said in London yesterday.  Based on this plant based oral vaccine one can develop many such vaccines against a large number of infecting microbes. https://www.theguardian.com;

 

Edible vaccine production for veterinary use has received widespread attention because of health initiatives aimed at decreasing antibiotic use in livestock and other animals to avoid the development of antibiotic-resistant strains, especially of epidemic and zoonotic pathogens. These issues have promoted the development of plant-based vaccines, which can easily fulfil these requirements (zoonotic).

Edible vaccines are actually recombinant vaccines in which selected antigens against a particular pathogen are introduced into a plant. Oral delivery of this plant induces a protective immune response against that particular pathogen in the form of an edible vaccine; Plant cells expressing vaccine antigens or biopharmaceuticals can be lyophilized and stored at ambient temperature for many years maintaining efficacy of expressed protein drugs; Plant-based vaccines have the potential to induce a mucosal immune response and a systemic immune response without the pain and risk associated with needles and injections Banana vaccine: Redig Mandy ; Institution:  Biochemistry-  inspired by calf fed on banana-made vaccine in Banana; Redig Mandy; not come into the market.

 

Plant vaccines will be a billion-dollar business, said Professor Arntzen, who leads a team of the world's top plant scientists at Cornell University in New York state. Successful experiments show that the vaccine worked, he added, and will cost less than one penny a dose to make. A single gene transferred into a tomato or banana plant is reproduced as a protein thousands of times inside the fruit. When eaten it passes into the intestine and then into the blood stream producing antibodies against hepatitis B - working the same way as a traditionally injected but much more expensive vaccine.  A single dried banana chip or tomato paste sandwiched in a wafer contains enough protein to act as a vaccine dose. Traditional vaccines have to be refrigerated and cost £10 for a successful course, making them too expensive for developing countries where hepatitis B kills many people directly and causes the death of many more through subsequent cancer.  Tomatoes and bananas genetically modified to contain hepatitis B vaccine could rid the world of the virus, a leading American scientist said in London. https://www.theguardian.com;  Transgenic potato that expresses the hepatitis B surface antigen which can be used as vaccine.. http://www.news.cornell.edu/ Transgenic potato that expresses the hepatitis B surface antigen which can be used as vaccine. http://www.news.cornell.edu/

Bayer; GMO foods, Genetically modified foods;  sugar, aspartame Papaya, canola, cotton Dairy corn, soybean, sugar beets, potatoes, Tomatoes, squash, oils, golden rice, Zucchini, apple, papaya, squash, corn, sugar beets, Jatropha, BT cotton, wine. Rape seed, vitamins, vegetables, tobacco, peas they are the few of them.

Banana Vaccine-Yasmin Thanavala; BANANA plants and TOMATO plants growing at the Boyce Thompson Institute for Plant Research at Cornell University have been genetically engineered to produce vaccines in their fruit. Bananas are particularly appealing as vaccines because they grow widely in many parts of the developing world, can be eaten raw and are liked by most children.  If such plants are made available to common men, they can grow such plant in their yard and whenever required by anyone can be used. https://www.mcdb.ucla.edu

 

The advantages would be enormous. The plants could be grown locally, and cheaply, using the standard growing methods of a given region. Because many food plants can be regenerated readily, the crops could potentially be produced indefinitely without the growers having to purchase more seeds or plants year after year. Homegrown vaccines would also avoid the logistical and economic problems posed by having to transport traditional preparations over long distances, keeping them cold en route and at their destination. And, being edible, the vaccines would require no syringes— which, aside from costing something, can lead to infections if they become contaminated

JARED SCHNEIDMAN DESIGN; Cut leaf.   Expose leaf to bacteria carrying an antigen gene and an antibiotic- resistance gene. Allow bacteria to deliver the genes into leaf cells.  Expose leaf to an antibiotic to kill cells that lack the new genes.  Wait for surviving (gene-altered) cells to multiply and form a clump (callus).  Then allow callus to sprout shoots and roots. 5 Put in soil. Within three months, the plantlets will grow into plants bearing antigen-laden vaccine potatoes. https://www.mcdb.ucla.edu

 

Tumor necrosis factor (TNF); Treatment for certain tumor cells-Tumor necrosis factor (TNF, tumor necrosis factor alpha, (cachexin, or cachectin)  is a cell signaling protein (cytokine) involved in systemic inflammation and is one of the cytokines that make up the acute phase reaction; it is produced chiefly by activated macrophages, although it can be produced by many other cell types such as CD4+ lymphocytes, NK cells, neutrophils, mast cells, eosinophils, and neurons.   Dysregulation of TNF production has been implicated in a variety of  human diseases including Alzheimer's disease, cancer, major depression, psoriasis and inflammatory bowel disease (IBD).

 

TNF Cell Signaling:

TNF can bind two receptors, TNFR1 (TNF receptor type 1; CD120a; p55/60) and TNFR2 (TNF receptor type 2; CD120b; p75/80). TNFR1 is 55-kDa and TNFR2 is 75-kDa.  TNFR1 is expressed in most tissues, and can be fully activated by both the membrane-bound and soluble trimeric forms of TNF, whereas TNFR2 is found typically in cells of the immune system, and respond to the membrane-bound form of the TNF homo-trimer. As most information regarding TNF signaling is derived from TNFR1, the role of TNFR2 is likely underestimated.

PDB 1du3 EBI.jpg

https://en.wikipedia.orgSignalling Pathway:

TNF can bind two receptors, TNFR1 (TNF receptor type 1; CD120a; p55/60) and TNFR2 (TNF receptor type 2; CD120b; p75/80). TNFR1 is 55-kDa and TNFR2 is 75-kDa.  TNFR1 is expressed in most tissues, and can be fully activated by both the membrane-bound and soluble trimeric forms of TNF, whereas TNFR2 is found typically in cells of the immune system, and respond to the membrane-bound form of the TNF homotrimer. As most information regarding TNF signaling is derived from TNFR1, the role of TNFR2 is likely underestimated. https://en.wikipedia.org;

 TNF – TNF  is a Protein Kinase: 1.  Pathways signaling through PI 3-kinase  and phosphatidylinositol (3,4,5)P3 (PI-3 kinase and protein kinase B/Akt). 2.  Mitogen-activated protein kinases (MAPKinases). NB:  Both group 1and 2 signals also activate protein kinase Cγ and Protein kinase Cζ. 3.  Possible interaction via kinases not coupled to IRS proteins.

It has been suggested that the most dominant is the first group (PI 3-kinase) which converts phosphatidylinositol 3,4 bisphosphate (PIP2) or [PI(3,4)P2] to phosphatidylinositol 3,4,5 triphosphate PIP3 or (PI 3,4,5)P3.  These nucleotides act as anchors, binding down-line protein kinases to the plasma membrane and activating them.  These nucleotides seem to be responsible for the alterations in carbohydrate, protein and lipid metabolism that are initiated by insulin.

It is now clear that a serine-threonine kinase "Akt" (otherwise known as  protein kinase B) is THE central element in the actions of insulin.   Akt is activated by PIP3.   Recent work has shown that PIP3 binding opens Akt to phosphorylation bu phosphoinositide-dependent protein kinase PDK-1 and the mammalian target of rapamycin (mTOR) complex 2 (mTORC2).    Look at the next figure from the work of Professor Peter Shepherd (Acta Physiol Scand 183, 3-12, 2005).  Phosphorylation of the insulin receptor and IRS1 and 2 lead to binding and activation of phosphatidylinositol 3 kinase (PI3K) and formation of PI(3,4,5)P3.  This then binds to the plasma membrane and  associates with phosphoinositol- dependent kinase-1 (PDK-1) and leading to phosphorylation and activation of PKB/Akt. Activated Akt is initiates many of the metabolic actions of insulin. 

This gene encodes a multifunctional proinflammatory cytokine that belongs to the tumor necrosis factor (TNF) superfamily. This cytokine is mainly secreted by macrophages. It can bind to, and thus functions through its receptors TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. This cytokine is involved in the regulation of a wide spectrum of biological processes including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation. This cytokine has been implicated in a variety of diseases, including autoimmune diseases, insulin resistance, and cancer. Knockout studies in mice also suggested the neuroprotective function of this cytokine. [RefSeq, Jul 2008] https://www.ncbi.nlm.nih.gov

 

TNF gene maps to chromosome 6p21.3, spans about 3 kilobases and contains 4 exons. The last exon codes for more than 80% of the secreted protein.[21] The 3' UTR of TNFα contains an AU-rich element (ARE). TNF protein is primarily produced as a 233-amino acid-long type II transmembrane protein arranged in stable homotrimers; heterotrimeric.  From this membrane-integrated form the soluble homotrimeric cytokine (sTNF) is released via proteolytic cleavage by the metalloprotease TNF alpha converting enzyme (TACE, also called ADAM17).  The soluble 51 kDa trimeric sTNF tends to dissociate at concentrations below the nanomolar range, thereby losing its bioactivity. The secreted form of human TNFα takes on a triangular pyramid shape, and weighs around 17-kD. Both the secreted and the membrane bound forms are biologically active, although the specific functions of each is controversial. But, both forms do have overlapping and distinct biology activities

This gene encodes a multifunctional proinflammatory cytokine that belongs to the tumor necrosis factor (TNF) superfamily. This cytokine is mainly secreted by macrophages. It can bind to, and thus functions through its receptors TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. This cytokine is involved in the regulation of a wide spectrum of biological processes including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation. This cytokine has been implicated in a variety of diseases, including autoimmune diseases, insulin resistance, and cancer. Knockout studies in mice also suggested the neuroprotective function of this cytokine. [provided by RefSeq, Jul 2008] https://www.ncbi.nlm.nih.gov

 

 

Interleukin-2 (IL-2).  Cancer treatment, immune deficiency, and HIV infection treatment; Interleukin-2 is the only drug approved in the US for the treatment of metastatic RCC. It is also approved in many other countries. But IL-2 isn't just a drug. IL-2 is a natural part of your immune system, a messenger protein called a cytokine which activates parts of your immune system. IL-2 does not kill tumor cells directly like classical chemotherapy. Instead, IL-2 activates and stimulates the growth of immune cells, most importantly T-Cells, but also Natural Killer Cells (NK Cells), both of which are capable of destroying cancer cells directly.

The term interleukin derives from (inter-) "as a means of communication", and (-leukin) "deriving from the fact that many of these proteins are produced by leukocytes and act on leukocytes".  "as a means of communication".  They are used in Cancer treatment, immune deficiency, and HIV infection; Interleukin-2 is the only drug approved in the US for the treatment of metastatic RCC. It is also approved in many other countries. But IL-2 isn't just a drug. IL-2 is a natural part of your immune system, a messenger protein called a cytokine which activates parts of your immune system. IL-2 does not kill tumor cells directly like classical chemotherapy. Instead, IL-2 activates and stimulates the growth of immune cells, most importantly T-Cells, but also Natural Killer Cells (NK Cells), both of which are capable of destroying cancer cells directly. Some interleukins are classified as lymphokines, lymphocyte-produced cytokines that mediate immune responses.

Human T- and B-cells recognize invaders by the shape of molecules - antigens - on their surfaces. Our immune system can produce a T- and B-cell to fit every possible shape. However, any T- or B-cell that recognized molecules found on your cells were destroyed while you were growing in the womb, to prevent them from attacking your own body. But you were left with millions of others, one for every foreign antigen you might ever encounter.

Having recognized the invader, different types of T-cell then have different jobs to do. Some send chemical instructions (cytokines) to the rest of the immune system. Your body can then produce the most effective weapons against the invaders, which may be bacteria, viruses or parasites. Other types of T-cells recognize and kill virus-infected cells directly. Some help B-cells to make antibodies, which circulate and bind to antigens.

With the help of T-cells, B-cells make special Y-shaped proteins called antibodies. Antibodies stick to antigens on the surface of germs, stopping them in their tracks, creating clumps that alert your body to the presence of intruders. Your body then starts to make toxic substances to fight them. Patrolling defender cells called phagocytes engulf and destroy antibody-covered intruders.

T cells (thymus cells) and B cells (bone marrow- or bursa-derived cells [a]) are the major cellular components of the adaptive immune response. T cells are involved in cell-mediated immunity, whereas B cells are primarily responsible for humoral immunity (relating to antibodies). http://www.sciencemuseum.org.uk/

http://www.rcsb.org/pdb/images/1D9C_bio_r_500.jpg?bioNum=1Image result for A typical Interferon alphahttps://upload.wikimedia.org/wikipedia/commons/thumb/9/99/1AU1_Human_Interferon-Beta.png/200px-1AU1_Human_Interferon-Beta.pnghttp://www.rcsb.org/pdb/images/1RH2_bio_r_500.jpg?bioNum=1

IFB gamma and Interferon Alfa and Interferon Beta and Recombinant IFN alpha 2B- respectively; https://www.dreamstime.com

Image result for A typical Interleukin-protein

Typical Interferon-IL6; https://en.wikipedia.org/

cytokine_fusion_proteins_740

Immuno-Cytokine; Cytokine-a fusion protein- http://www.roche.com

http://www.sciencemuseum.org.uk/~/media/rwscim/whoami/findoutmore/what%20is%20so%20special%20about%20you%20b-cells.jpg?rand=777027712

Immunoglobulin?  http://www.sciencemuseum.org.uk

Antibody-cytokine Fusion Proteins

Antibody-cytokine fusion proteins, which consist of cytokines fused to an antibody, have the properties of all components and acquire advantages compared to proteins alone. Structurally, there are two main types of ACFPs which cytokines can be alternatively fused to either whole immunoglobulin (Ig fusion) or antigen-binding fragments such as Fabs, single-chain variable fragments (scFvs) or divalent derivatives thereof, e.g. diabodies. Moreover, differences in the composition of the fusion proteins are also due to the cytokine itself. Many cytokines are composed of a single monomeric protein domain (e.g. IL-2 and IFN-a), while others are homodimeric (e.g. IL-10 and IFN-γ), homotrimeric molecules (e.g. TNF and TRAIL) or heterodimeric (e.g. IL-12 and IL-27). Each cytokine can be fused at the amino- or carboxy-terminus of the antibody, which depends on the structure of the cytokine and antibody, and in order to conserve the biological activity of both components. Further improvements also include the fusion of different cytokines to an antibody. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumour necrosis factors; Antibody-cytokine fusion proteins; Creative biolabs-http://www.creativebiolabs.net/

What interferon and interleukin are?

Immune function is helped by two kinds of white blood cells. The “B cells” (so-called because they develop in bone marrow) produce antibodies. The “T cells” (so-called because they develop in a small organ called the thymus gland) are responsible for a variety of other immune responses.  Interferon and interleukin are substances made by cells in the body, to communicate with each other.  Interferons (IFNs) are low molecular weight proteins that belong to the class of glycoproteins known as cytokines.  They act as signaling molecules released by host cells in response to pathogens such as viruses, bacteria, parasites and even tumour cells. So tumour cells are foreigners for the body.  IFNs are part of the non-specific immune system and are an important first line of defense against viral infections. They are released by host cells in response to the presence of pathogens such as viruses, bacteria, parasites or tumor cells. IFNs have other functions: they activate immune cells, such as natural killer cells and macrophages; they increase recognition of infection or tumor cells by up-regulating antigen presentation to T lymphocytes; and they increase the ability of uninfected host cells to resist new infection by virus. FNs belong to the large class of proteins known as cytokines, molecules used for communication between cells to trigger the protective defenses of the immune system that help eradicate pathogens.  Interferons are named for their ability to "interfere" with viral replication  by protecting cells from virus infections. IFNs also have various other functions: they activate immune cells, such as natural killer cells and macrophages; they increase host defenses by up-regulating antigen presentation by virtue of increasing the expression of major histocompatibility complex (MHC) antigens. Certain symptoms of infections, such as fever, muscle pain and "flu-like symptoms", are also caused by the production of IFNs and other cytokines. Host symptoms, such as aching muscles and fever, are related to the production of IFNs during infection. They are used to treat diseases such as Multiple Sclerosis (MS) and Hepatitis Interferon and interleukin can boost the immune system, so doctors sometimes use man made versions to treat cancer. Because of the way it works, doctors sometimes call this type of treatment immunotherapy. Some viruses are resistant to Interferons.

Production of interferons occurs mainly in response to microbes, such as viruses and bacteria, and their products. Binding of molecules uniquely found in microbes—viral glycoproteins, viral RNA, bacterial endotoxin (lipopolysaccharide), bacterial flagella, CpG motifs—by pattern recognition receptors, such as membrane bound Toll like receptors or the cytoplasmic receptors RIG-I or MDA5, can trigger release of IFNs. Toll Like Receptor 3 (TLR3) is important for inducing interferons in response to the presence of double-stranded RNA viruses; the ligand for this receptor is double-stranded RNA (dsRNA). After binding dsRNA, this receptor activates the transcription factors IRF3 and NF-κB, which are important for initiating synthesis of many inflammatory proteins. RNA interference technology tools such as siRNA or vector-based reagents can either silence or stimulate interferon pathways.  Release of IFN from cells (specifically IFN-γ in lymphoid cells) is also induced by mitogens. Other cytokines, such as interleukin 1, interleukin 2, interleukin-12, tumor necrosis factor and colony-stimulating factor, can also enhance interferon production. Interferon therapy is used (in combination with chemotherapy and radiation) as a treatment for some cancers.  This treatment can be used in hematological malignancy; leukemia and lymphomas including hairy cell leukemia, chronic myeloid leukemia, nodular lymphoma, and cutaneous T-cell lymphoma.  Patients with recurrent melanomas receive recombinant IFN-α2b.  Both hepatitis B and hepatitis C are treated with IFN-α, often in combination with other antiviral drugs.  Some of those treated with interferon have a sustained virological response and can eliminate hepatitis virus. The most harmful strain—hepatitis C genotype I virus—can be treated with a 60-80% success rate with the current standard-of-care treatment of interferon-α, ribavirin and recently approved protease inhibitors such as Telaprevir (Incivek) May 2011, Boceprevir (Victrelis) May 2011 or the nucleotide analog polymerase inhibitor Sofosbuvir (Sovaldi) December 2013.  Biopsies of patients given the treatment show reductions in liver damage and cirrhosis. Some evidence shows giving interferon immediately following infection can prevent chronic hepatitis C, although diagnosis early in infection is difficult since physical symptoms are sparse in early hepatitis C infection. Control of chronic hepatitis C by IFN is associated with reduced hepatocellular carcinoma.

The term 'interleukin' (IL) has been used to describe a group of cytokines with complex immunomodulatory functions - including cell proliferation, maturation, migration and adhesion. These cytokines also play an important role in immune cell differentiation and activation. Determining the exact function of a particular cytokine is complicated by the influence of the producing cell type, the responding cell type and the phase of the immune response. ILs can also have pro- and anti-inflammatory effects, further complicating their characterization. These molecules are under constant pressure to evolve due to continual competition between the host's immune system and infecting organisms; as such, ILs have undergone significant evolution. This has resulted in little amino acid conservation between orthologous proteins, which further complicates the gene family organization.

Interleukins are a large group of immunomodulatory proteins that elicit a wide variety of responses in cells and tissues. These cytokines comprise a large number of the known immunological 'second messenger' molecules within mammals. Interleukins initiate a response by binding to high-affinity receptors located on the surface of cells; Interleukins function in a paracrine or autocrine fashion, rather than as an endocrine signal, which is more common with steroidal and amino acid-derived hormones. The response of a particular cell to these cytokines depends on the ligands involved, specific receptors expressed on the cell surface and the particular signalling cascades that are activated. ILs modulate growth, differentiation and activation during an immune response. This distinguishes them from chemokines - the main function of which is to direct immune cells to the site of inflammation via chemotaxis - and interferons (IFNs), which predominantly mediate cellular response to viral infection. Despite attempts to separate these three groups based on function, there is a degree of overlap.

 

How ILs work; Interferon and interleukin work in several ways, including; one of several proteins important for lymphocyte proliferation.Interleukin-Interfering with the way cancer cells grow and multiply

·    Stimulating the immune system and encouraging killer T cells and other cells to attack cancer cells

·    Encouraging cancer cells to produce chemicals that attract immune system cells to them

 

Doctors use interferon alpha for several different types of cancer, particularly

·    Kidney cancer (renal cell cancer)

·    Melanoma

·    Multiple myeloma

·    Some types of leukaemia

·    You can have interferon alpha into the bloodstream through a drip (infusion). But you are more likely to have it as an injection just under the skin (subcutaneously). How often you have it depends on which type of cancer you are having treatment for. Most people have interferon 3 times a week but you can have it as a daily injection.

Some of the side effects that interferon alpha and interleukin 2 can cause include; Fatigue (tiredness and weakness),  Flu like symptoms,  DiarrhoeaLow levels of blood cellsFeeling sick and Loss of appetite; Interleukin 2 can also cause low blood pressure. http://www.cancerresearchuk.org.

There are several types of T-Cells but, without going into detail, certain T-Cells are capable of killing tumor cells if they recognize a specific antigen on the surface of the tumor cell. Antigens are normally proteins. Each T-Cell is specific for only one antigen but you have many different T-Cells. NK Cells have the ability to kill tumor cells without needing to recognize a specific antigen (I'm not sure how!). While this sounds good, NK cells are weaker cancer killers than T-Cells. The so called LAK cells which were used in some of the early immunotherapy experiments are actually NK cells.     There are human 36-37 interleukins in human beeings, produced by different cell types. http://www.cancerguide.org/

 

There are several types of T-Cells but, without going into detail, certain T-Cells are capable of killing tumor cells if they recognize a specific antigen on the surface of the tumor cell. Antigens are normally proteins. Each T-Cell is specific for only one antigen but you have many different T-Cells. NK Cells have the ability to kill tumor cells without needing to recognize a specific antigen (I'm not sure how!). While this sounds good, NK cells are weaker cancer killers than T-Cells. The so called LAK cells which were used in some of the early immunotherapy experiments are actually NK cells. http://www.cancerguide.org/

 

The term 'interleukin' (IL) has been used to describe a group of cytokines with complex immunomodulatory functions - including cell proliferation, maturation, migration and adhesion. These cytokines also play an important role in immune cell differentiation and activation. Determining the exact function of a particular cytokine is complicated by the influence of the producing cell type, the responding cell type and the phase of the immune response. ILs can also have pro- and anti-inflammatory effects, further complicating their characterization. These molecules are under constant pressure to evolve due to continual competition between the host's immune system and infecting organisms; as such, ILs have undergone significant evolution. This has resulted in little amino acid conservation between orthologous proteins, which further complicates the gene family organization.

Interleukins are a large group of immunomodulatory proteins that elicit a wide variety of responses in cells and tissues. These cytokines comprise a large number of the known immunological 'second messenger' molecules within mammals. Interleukins initiate a response by binding to high-affinity receptors located on the surface of cells; Interleukins function in a paracrine or autocrine fashion, rather than as an endocrine signal, which is more common with steroidal and amino acid-derived hormones. The response of a particular cell to these cytokines depends on the ligands involved, specific receptors expressed on the cell surface and the particular signalling cascades that are activated. ILs modulate growth, differentiation and activation during an immune response. This distinguishes them from chemokines - the main function of which is to direct immune cells to the site of inflammation via chemotaxis - and interferons (IFNs), which predominantly mediate cellular response to viral infection. Despite attempts to separate these three groups based on function, there is a degree of overlap.

 

 

The term 'interleukin' (IL) has been used to describe a group of cytokines with complex immunomodulatory functions - including cell proliferation, maturation, migration and adhesion. These cytokines also play an important role in immune cell differentiation and activation. Determining the exact function of a particular cytokine is complicated by the influence of the producing cell type, the responding cell type and the phase of the immune response. ILs can also have pro- and anti-inflammatory effects, further complicating their characterisation. These molecules are under constant pressure to evolve due to continual competition between the host's immune system and infecting organisms; as such, ILs have undergone significant evolution. This has resulted in little amino acid conservation between orthologous proteins, which further complicates the gene family organisation.

Interleukins are a large group of immunomodulatory proteins that elicit a wide variety of responses in cells and tissues. These cytokines comprise a large number of the known immunological 'second messenger' molecules within mammals. Interleukins initiate a response by binding to high-affinity receptors located on the surface of cells; Interleukins function in a paracrine or autocrine fashion, rather than as an endocrine signal, which is more common with steroidal and amino acid-derived hormones. The response of a particular cell to these cytokines depends on the ligands involved, specific receptors expressed on the cell surface and the particular signalling cascades that are activated. ILs modulate growth, differentiation and activation during an immune response. This distinguishes them from chemokines - the main function of which is to direct immune cells to the site of inflammation via chemotaxis - and interferons (IFNs), which predominantly mediate cellular response to viral infection. Despite attempts to separate these three groups based on function, there is a degree of overlap.

 

 

Interferons; (Treatment for cancer and viral infections); Interferons (IFNs) are a group of signaling proteins  made and released by host cells in response to the presence of several pathogens, such as viruses, bacteria, parasites, and also tumor cells.  In a typical scenario, a virus-infected cell will release interferons causing nearby cells to heighten their anti-viral defenses. IFNs belong to the large class of proteins known as cytokines, molecules used for communication between cells to trigger the protective defenses of the immune system that help eradicate pathogens. Interferons are named for their ability to "interfere" with viral replication;  by protecting cells from virus infections. IFNs also have various other functions: they activate immune cells, such as natural killer cells and macrophages; they increase host defenses by up-regulating antigen presentation by virtue of increasing the expression of major histocompatibility complex (MHC) antigens. Certain symptoms of infections, such as fever, muscle pain and "flu-like symptoms", are also caused by the production of IFNs and other cytokines. More than twenty distinct IFN genes and proteins have been identified in animals, including humans. They are typically divided among three classes: Type I IFN, Type II IFN, and Type III IFN. IFNs belonging to all three classes are important for fighting viral infections and for the regulation of the immune system. https://en.wikipedia.org/wiki

The mammalian types are designated IFN-α (alpha), IFN-β (beta), IFN-κ (kappa), IFN-δ (delta), IFN-ε (epsilon), IFN-τ (tau), IFN-ω (omega), and IFN-ζ (zeta, also known as limitin).

 IFNs of different kinds also have various other functions: they activate immune cells, such as natural killer cells and macrophages; they increase host defenses by up-regulating antigen presentation by virtue of increasing the expression of major histocompatibility complex (MHC) antigens. Certain symptoms of infections, such as fever, muscle pain and "flu-like symptoms", are also caused by the production of IFNs and other cytokines.

More than twenty distinct IFN genes and proteins have been identified in animals, including humans. They are typically divided among three classes: Type I IFN, Type II IFN, and Type III IFN. IFNs belonging to all three classes are important for fighting viral infections and for the regulation of the immune system

 

Type 1 interferons (IFN-1) are implicated in the pathogenesis of systemic lupus erythematosus (SLE), but most studies have only reported the effect of IFN-1 on mixed cell populations. We aimed to define modules of IFN-1-associated genes in purified leucocyte populations and use these as a basis for a detailed comparative analysis.

Production of type I interferon (IFN) is an essential component of the innate immune response against invading pathogens. However, its production must be tightly regulated to avoid harmful effects. Compounds that modulate the IFN response are potentially valuable for a variety of applications due to IFN’s beneficial and detrimental roles. We developed and executed a cell-based high-throughput screen (HTS) targeting components that participate in and/or regulate the IRF3 and nuclear factor (NF)–κB branches of the IFN induction pathway. The assay detects activation of the IFN induction pathway via an enhanced green fluorescent protein (eGFP) reporter gene under the control of the IFNβ promoter and was optimized, miniaturized, and demonstrated suitable for HTS as robust Z′ factor scores of >0.6 were consistently achieved. A diversity screening set of 15,667 small molecules was assayed and two novel hit compounds validated that specifically inhibit the IFN induction pathway. We demonstrate that one of these compounds acts at or upstream of IRF3 phosphorylation. A second cell-based assay to detect activation of the IFN signaling (Jak-Stat) pathway via an eGFP reporter gene under the control of an IFN-stimulated response element (ISRE) containing MxA promoter also performed well (robust Z′ factor >0.7) and may therefore be similarly used to identify small molecules that modulate the IFN signaling pathway;

 

Interferon beta-1a and interferon beta-1b:  are used to treat and control multiple sclerosis, an autoimmune disorder. Interferon therapy is used (in combination with chemotherapy and radiation) as a treatment for some cancers.  It is recently demonstrated that type I interferon (IFN-I) signaling was responsible for many of the immune dysfunctions associated with persistent virus infection and in particular the induced expression of the suppressive factors IL-10 and PDL1 by dendritic cells (DCs). Yet, mechanistically how IFN-I signaling specifically generates and programs cells to become immunosuppressive is still unknown. Herein, we define the underlying mechanisms of IFN-I mediated immunosuppression and establish that the induction of factors and the generation of the DCs that express them are separable events integrally reliant on additional inflammatory factors. Further, we demonstrate a similar derivation of the suppressive DCs that emerge in other diseases associated with prolonged inflammation and immunosuppression, specifically in HIV infection, Mycobacterium tuberculosis, and cancer, indicating a conserved origin of immunosuppression and suggesting that targeting the pathways that underlie expression of immunosuppressive cells and factors could be beneficial to treat multiple chronic diseases. https://www.ncbi.nlm.nih.gov/ PLoS Pathog. Zoe O. Gage 2016 Jan; Cameron R. Cunningham,1 Ameya Champhekar et al.

 

Pharmaceutical forms of interferons

Generic name

Trade name

Interferon alpha 2a

Roferon A

Interferon alpha 2b

Intron A/Reliferon/Uniferon

Human leukocyte Interferon-alpha (HuIFN-alpha-Le)

Multiferon

Interferon beta 1a, liquid form

Rebif

Interferon beta 1a, lyophilized

Avonex

Interferon beta 1a, biogeneric (Iran)

Cinnovex

Interferon beta 1b

Betaseron / Betaferon

Interferon gamma 1b

Actimmune

PEGylated interferon alpha 2a

Pegasys

PEGylated interferon alpha 2a (Egypt)

Reiferon Retard

PEGylated interferon alpha 2b

PegIntron

PEGylated interferon alpha 2b plus ribavirin (Canada)

Pegetron

 

 

IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7.’, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17IFNA21, IFNB1, IFNW, IFNE1, IFNK; https://en.wikipedia.org/wiki/Interferon; The above mentioned are some of the Interferons.

 

Interferon beta (IFNβ) is a cytokine that is naturally produced by the immune system in response to biological and chemical stimuli. It signals by binding to the heterodimeric type I IFN receptor composed of the IFNAR1 and IFNAR2 chains, and regulates the expression of a plethora of genes by means of the classical JAK/STAT and other pathways. IFNβ is pleiotropic in that it elicits antiviral, antiproliferative, and immunomodulatory activities on numerous cell types. The biological activities underpin the mechanisms by which the protein is used to treat various diseases such as hepatitis C infection and multiple sclerosis. Despite the success of IFNβ therapy, the drug may evoke the production of antidrug antibodies that may reduce treatment efficiency. Immunogenicity is related to many factors: among them, structural properties, particularly aggregation. and T-cell and B-cell epitopes in the structure of IFNβ, appear to be important. Knowledge of the structural properties of IFNβ and its relation to immunogenicity may help scientists to develop safer and more effective forms. Several methods have been used to predict and reduce the immunogenicity of certain IFNβ drug products. In this chapter, we review the current knowledge on IFNβ from its structure, dynamic conformation, signaling pathway, and mechanism of action to its therapeutic effects. Immunogenicity and its relation to structural properties of IFNβ are also discussed. https://www.researchgate,

 

Interferon (Treatment for cancer and viral infections); Interferons (IFNs) are a group of signaling proteins made and released by host cells in response to the presence of several pathogens, such as viruses, bacteria, parasites, and also tumor cells. https://en.wikipedia.org/wiki In a typical scenario, a virus-infected cell will release interferons causing nearby cells to heighten their anti-viral defenses. IFNs belong to the large class of proteins known as cytokines, molecules used for communication between cells to trigger the protective defenses of the immune system that help eradicate pathogens. Interferons are named for their ability to "interfere" with viral replication,  by protecting cells from virus infections. IFNs also have various other functions: they activate immune cells, such as natural killer cells and macrophages; they increase host defenses by up-regulating antigen presentation by virtue of increasing the expression of major histocompatibility complex (MHC) antigens. Certain symptoms of infections, such as fever, muscle pain and "flu-like symptoms", are also caused by the production of IFNs and other cytokines. More than twenty distinct IFN genes and proteins have been identified in animals, including humans. They are typically divided among three classes: Type I IFN, Type II IFN, and Type III IFN. IFNs belonging to all three classes are important for fighting viral infections and for the regulation of the immune system. https://en.wikipedia.org/wiki/Interferon

IFN-γ is mostly secreted by activated CD4 + , CD8 + T cells and NK cells. This cytokine has immunomodulatory, anti-cancer and anti-microbial effects and is important for prophylaxis, diagnosis and treatment of chronic infections and cancers. Objective: The purpose of this study was to clone the full cDNA of human IFN-γ and express it in CHO cell line. Methods: Lymphocytes from a healthy individual were isolated and activated by phytohaemagglutinin (PHA) in vitro. After 4 hours, total RNA extracted and first cDNA strand was synthesized. cDNA was amplified with primers containing EcoRI and NotI sites. The amplified fragment and the PcDNA3.1 vector were cut by EcoRI and NotI and ligated. The construct (pcDNA3.1-IFN-γ) was transferred into E.coli (DH5α strain) using CaCl2 method and selected by plating on a medium containing ampicillin. The construct sequence was confirmed by PCR and sequence analysis. Construct expression was achieved by performing a calcium phosphate-mediated transfection into CHO cells and followed by selection of stable drug (G418) resistant clones by limiting dilution assay (LDA). The IFN-γ production by transfected CHO cells was measured using ELISA technique. Results and Conclusion: Out of 33 grown transformed bacterial colonies, only 6 had the entire sequences of the inserted fragment and one of them was used for the transfection experiment. Out of 768 wells, 5 clones produced more than 100 ng/ml/10 6 cells of IFN-γ. Among the 5 clones, one with the maximum production of INF-γ (143 ng/ml/10 6 cells) was selected and used for propagation.

IFN-α[edit]

The IFN-α proteins are produced by leukocytes. They are mainly involved in innate immune response against viral infection. The genes responsible for their synthesis come in 13 subtypes that are called IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21. These genes are found together in a cluster on chromosome 9.

IFN-α is also made synthetically as medication in hairy cell leukemia. The International Nonproprietary Name (INN) for the product is interferon alfa. The recombinant type is interferon alfacon-1. The pegylated types are pegylated interferon alfa-2a and pegylated interferon alfa-2b.

IFN-β

The IFN-β proteins are produced in large quantities by fibroblasts. They have antiviral activity that is involved mainly in innate immune response. Two types of IFN-β have been described, IFN-β1 (IFNB1) and IFN-β3 (IFNB3) (a gene designated IFN-β2 is actually IL-6). IFN-β1 is used as a treatment for multiple sclerosis as it reduces the relapse rate.

IFN-β1 is not an appropriate treatment for patients with progressive, non-relapsing forms of multiple sclerosis.[6]

IFN-ε, -κ, -τ, -δ, and -ζ]

IFN-ε, -κ, -τ, and -ζ appear, at this time, to come in a single isoform in humans, IFNK. Only ruminants encode IFN-τ, a variant of IFN-ω. So far, IFN-ζ is only found in mice, while a structural homolog, IFN-δ is found in a diverse array of non-primate and non-rodent placental mammals. Most but not all placental mammals encode functional IFN-ε and IFN-κ genes.

IFN-ω

IFN-ω, although having only one functional form described to date (IFNW1),   has several pseudogenes: IFNWP2, IFNWP4, IFNWP5, IFNWP9, IFNWP15, IFNWP18, and IFNWP19 in humans. Many non-primate placental mammals express multiple IFN-ω subtypes

IFN-ν- [not to be confused with Interferon gamma (IFNγ)]

This subtype of Type I IFN was recently described as a pseudogene in human, but potentially functional in the domestic cat genome. In all other genomes of non-feline placental mammals, IFN-ν is a pseudogene; in some species, the pseudogene is well preserved, while in others, it is badly mutilated or is undetectable. Moreover, in the cat genome, the IFN-ν promoter is deleteriously mutated. It is likely that the IFN-ν gene family was rendered useless prior to mammalian diversification. Its presence on the edge of the Type I IFN locus in mammals may have shielded it from obliteration, allowing its detection.

Sources and functions:

IFN-α and IFN-β are secreted by many cell types including lymphocytes (NK cells, B-cells and T-cells), macrophages, fibroblasts, endothelial cells, osteoblasts and others. They stimulate both macrophages and NK cells to elicit an anti-viral response, and are also active against tumors. Plasmacytoid dendritic cells have been identified as being the most potent producers of type I IFNs in response to antigen, and have thus been coined natural IFN producing cells. Current study findings suggest that by forcing IFN-α expression in tumor-infiltrating macrophages, it is possible to elicit a more effective dendritic cell activation and immune effector cell cytotoxicity.[7]

 

https://upload.wikimedia.org/wikipedia/commons/thumb/f/fb/1AU1_Human_Interferon-Beta01.png/220px-1AU1_Human_Interferon-Beta01.png

https://en.wikipedia.org/

IFN-α acts as a pyrogenic factor by altering the activity of thermosensitive neurons in the hypothalamus thus causing fever. It does this by binding to opioid receptors and eliciting the release of prostaglandin-E2 (PGE2).

A similar mechanism is used by IFN-α to reduce pain; IFN-α interacts with the μ-opioid receptor to act as an analgesic.; IFN-ω is released by leukocytes at the site of viral infection or tumors.

 

In mice, IFN-β inhibits immune cells to produce growth factors, thereby slowing tumor growth, and inhibits other cells from producing vessel producing growth factors, thereby blocking tumor angiogenesis and hindering the tumour from connecting into the blood vessel system;

 

Human Interferon;

Daniela Novick. Author links open the author workspace.Batya Cohen. Author links open the author workspace. Menachem Rubinstein; http://www.sciencedirect.com/ . We describe a universal ligand-binding receptor for human interferons α and interferon β (type I IFNs). A soluble 40 kDa IFN-αβ receptor (p40) that blocks the activity of type I IFNs was purified from urine and sequenced. Antibodies raised against p40 completely block the activity of several type I IFNs and immunoprecipltate both a cellular 102 kDa IFN-αβ receptor and its cross-linked complexes with IFN-α2. The receptor is a disulfide-linked dimer, consisting of 51 kDa subunits. We isolated and expressed a 1.5 kb cDNA, coding for the IFN-αβ receptor. Its 331 amino acid sequence includes a leader and a transmembrane region, while its ectodomain corresponds to p40. IFN-αβ receptor is physically associated with the cytoplasmic Tyr kinase JAK1, hence, in addition to ligand binding, it is directly involved in signal transduction.

 

Interferon α2b gene was amplified by PCR using specific primers containing appropriate restriction enzymes, plant highly expression sequence and Histidine tag sequence. Target sequence was cloned in plant expression vector pCAMBIA1304 and the construct named pCAMINFα. pCAMINFα was transferred to E. coli strain DH5α and plated on LB agar medium containing kanamycin 50 mgl-1. The colonies were confirmed by colony PCR and sequencing. The construct was transferred into Agrobacterium tumefaciens by freeze-thaw method and transformed colonies were confirmed by colony PCR. Tobacco plants (cultivar xanthi) were inoculated with A. tumefaciens strain LBA4404 by leaf disc method. Inoculated explants were cultured on MSII (MS + BAP 1mgl-1 + NAA 0.1 mgl-1) at 28°C and darkness for 48 hours. Then explants were transferred to selection medium containing cephotaxime (250 mgl-1) and hygromycin (15 mgl-1) in a 16/8 (day/night) h photoperiod in growth room with an irradiance of 5000 lux. Transgenic plants were regenerated and transferred to perlite. Genomic DNA was extracted from regenerated plants by Dellaporta method at 5-leaf step and transgenic lines were confirmed by PCR with specific primers. Expression of Interferon α2b gene was confirmed by dot blotting. https://www.ncbi.nlm.nih.gov Shahrzad Ahangarzadeh et al.

 

A cDNA encoding the human interferon-γ receptor was isolated from a λgt11 expression library using a polyclonal antireceptor antiserum. The gene for this receptor was identified in a cosmid library and transfected into mouse cells. The human interferon-γ receptor expressed in mouse cells displayed the same binding properties as in human cells. However, transfected cells were not sensitive to human IFN-γ, suggesting the need for species-specific cofactors in receptor function. As inferred from the cDNA sequence, the human interferon-γ receptor shows no similarities to known proteins and represents a novel transmembrane receptor. It is most likely the product of a single mRNA and a gene located on chromosome 6q. http://www.sciencedirect.com

 

Diabetes:   Sushruta (6th century BCE) identified diabetes and classified it as Madhumeha. He further identified it with obesity and sedentary lifestyle, advising exercises to help "cure" it. The ancient Indians tested for diabetes by observing whether ants were attracted to a person's urine, and called the ailment "sweet urine disease" (Madhumeha).

Genetically Engineered Pharmaceuticals- Insulins:

Insulin for diabetics- by itself N HORMONE; required for the action of other major mammalian hormones that promote growth. Three groups of molecules possibly related to this anabolic function are considered: (1) molecules that relate to the structure of membranes and the initiation of insulin action, including insulin receptors; (2) molecules (such as cyclic nucleotides and cations, e.g. K+ and Mg+ + ) that are potentially concerned with transmission and amplification of information from the plasma membrane to subcellular functional units; (3) molecules that may contribute directly to development of juvenile or maturity-onset diabetes. Two new concepts are proposed: First, the receptor for insulin occupies a locus on the plasma membrane coupled to Mg++-activated (Na+ + K+ )- ATPase; hormone activation stimulates ATPase activity. As a result of the activation of the membrane ATPase enzyme system by insulin, concentrations of K+ and Mg + + increase at critical intracellular loci; these ions then serve as "second messengers." The second new" concept is that the genetic abnormality in human diabetes mellitus lies in a single or multiple enzyme defect that leads to accumulation of a fragment from growth hormone. One such fragment is known to inhibit glucose uptake and fatty acid synthesis.

Insulin and glucagon are two hormones regulating  glucose and fat metabolism in the body. Both are synthesized in the pancreas. Both are proteins, but physiologically they are opposites. 

Insulin is a protein hormone. It contains 51 amino acids. It weighs 5808 Daltons (a unit of weight measurement). It is made up of two protein chains linked together by a disulfide bond.  A gene called INS codes for the precursor of insulin is preproinsulin.  Pancreatic cells called beta cells secrete insulin. These cells are located in clusters called islets of Langerhan.

http://www.medbio.info/horn/time%203-4/homeos2.jpg

http://www.medbio.info/

High blood sugar level promotes the release of insulin from beta cells while stress hormones (adrenalin) inhibit insulin release. In healthy individuals, pancreas secretes insulin in tightly controlled amounts to maintain the blood glucose levels within normal parameters. Insulin is critical in regulating carbohydrates and lipids. It regulates glucose, amino acid, and lipid absorption by cells all over the body. It increases DNA replication and protein synthesis. The action of insulin is widespread but more pronounced in liver, muscle cells, and fat tissue. Liver and skeletal muscle tissues store glucose as glycogen while fat tissue stores it as triglycerides under the influence of insulin. Insulin promotes glycogen synthesis, lipid synthesis, and fat esterification; therefore, glycogen breakdown and fat breakdown occur when insulin levels are low. The body hydrolyses glycogen (a stored form of glucose) to release glucose into the blood stream when blood sugar drops below normal levels. Insulin inhibits the secretion of glucagon which has the opposite action of insulin. It also inhibits the use of lipid as an energy source. The blood level of insulin acts as a signal to alter the direction of biochemical reactions in cells. It also inhibits sodium excretion by kidneys.

 

Glucogon:  Glucagon is a protein hormone. It contains 29 amino acids. It weighs 3485 Daltons.  Genes code for the precursor of glucagon is proglucagon; which is then cleaved into the active form of glucagon in alpha cells of pancreas. But in the intestines proglucagon breaks down to form different products. Low blood sugar level, stress hormones like adrenalin, amino acids like Arginine, Alanine, neurotransmitters like acetylcholine, and hormones like cholecystokinin increase glucagon secretion.  Human growth inhibiting hormones, insulin, and urea inhibit glucagon secretion. Glucagon increases blood sugar level. It promotes glycogenolysis. Even though glucagon promotes glucose synthesis from fatty acids it does not affect fat breakdown.

 

Therapeutic uses of glucagon include relaxation of the lower esophageal sphincter in the esophageal blocks and spasms, severe hypoglycemia, and for treating beta blocker overdose.

 

What is the difference between Insulin and Glucagon?

Both are protein hormones.  High blood sugar level promotes insulin secretion while inhibiting glucagon secretion. • Stress hormones inhibit insulin secretion while promoting glucagon secretion. • The beta cells secrete insulin while the alpha cells secrete glucagon. • Insulin reduces blood sugar while glucagon increases. • Insulin forces substances (glucose, amino acids) into cells while glucagon inhibits it. • Insulin promotes the synthesis of glycogen while glucagon breaks glycogen down. • Insulin promotes lipid synthesis, but glucagon does not break it down. • Insulin inhibits glucagon formation while glucagon does not control insulin secretion.

Main points:  When blood glucose levels  increase over about 5 mmol/l the beta-cells increase their output of insulin and C-peptide.  The glucagon-producing alpha-cells remain quiet, and hold on to their hormone.   A fall in blood glucose under about 4 mmol/l leads to a pronounced decrease in insulin secretion.  The alpha-cells become active and deliver glucagon to the blood.

http://www.medbio.info/images/Time%203-4/homeos18.gif

http://www.medbio.info

http://www.medbio.info/images/time3-4/homeos8.gif

http://medboio.info

Hypothalamic KATP Channels Regulate Hepatic Gluconeogenesis and Postpranial Blood Glucose Levels:

Blood sugar levels are dependent upon glucose uptake after meals and hepatic release of glucose between meals.   The sugar released from the liver comes either from stored glycogen or production of glucose from lactate and amino acids.  This production of glucose is largely responsible for stabilization of postprandial blood sugar levels.  The hyperglycemia noted in type 2 diabetes partially results from lack of control over hepatic glucose formation due to resistance to insulin.  It has recently become clear that part of this insulin effect occurs indirectly through insulin-sensitive receptors in the brain (more precisely, in the hypothalamus).     In a very recent article in Nature, Alessandro Pocai and coauthors presented convincing data that couples  insulin-stimulation of hypothalamic KATP channels with neural control of hepatic gluconeogenesis (Nature 434, 1026-1031, 2005; and an overview by Nature's editors ((click here)). Insulin stimulated opening of hypothalamic KATP channels results in vagal nerve signaling to the liver and inhibition of gluconeogenesis.  This is part of the normal response to meals and following insulin release from the pancreatic ß-cells.  Thus, signaling from the brain is one of the important control mechanisms which establish correct "between-meal" blood sugar levels.  Hypothalamic insulin resistance and therefore loss of control over hepatic gluconeogenesis may well be one of the important factors involved in development of type 2 diabetes.  This model is summarized in this figure from the overview in Nature . (Nature 434, 1026-31, 2005

 

 

 

 

 

 

It stimulates 1. Transport of glucose ions, amino acids, 2. Glycogen formation, 3. Glucose conversion to triglycerides .4. Nucleic acid synthesis and protein synthesis.

 

Image result for How Insulin biochemically acts

http://www.alluvionbiomedical.com/

  Fig 1. Diet-induced physiological changes in the liver that promote improved blood glucose control.

Fig Diet-induced physiological changes in the liver that promote improved blood glucose control. http://www.alluvionbiomedical.com/

 

Insulin for diabetics- It is estimated that 450 million people suffer from this disease.  By itself is an HORMONE; required for the action of other major mammalian hormones that promote growth. It is now genetically engineered protein available in the market all over the world. Three groups of molecules possibly related to this anabolic function are considered: (1) molecules that relate to the structure of membranes and the initiation of insulin action, including insulin receptors; (2) molecules (such as cyclic nucleotides and cations, e.g. K+ and Mg+ + ) that are potentially concerned with transmission and amplification of information from the plasma membrane to subcellular functional units; (3) molecules that may contribute directly to development of juvenile or maturity-onset diabetes. Two new concepts are proposed: First, the receptor for insulin occupies a locus on the plasma membrane coupled to Mg++-activated (Na+ + K+ )- ATPase; hormone activation stimulates ATPase activity. As a result of the activation of the membrane ATPase enzyme system by insulin, concentrations of K+ and Mg + + increase at critical intracellular loci; these ions then serve as "second messengers." The second new" concept is that the genetic abnormality in human diabetes mellitus lies in a single or multiple enzyme defect that leads to accumulation of a fragment from growth hormone. One such fragment is known to inhibit glucose uptake and fatty acid synthesis. DIABETES 21 (Suppl. 2).

 

Diet-induced physiological changes in the liver that promote improved blood glucose control.  Insulin initiates its action by binding to a glycoprotein receptor on the surface of the cell. This receptor consists of an alpha-subunit, which binds the hormone, and a beta-subunit, which is an insulin-stimulated, tyrosine-specific protein kinase. Activation of this kinase is believed to generate a signal that eventually results in insulin's action on glucose, lipid, and protein metabolism. The growth-promoting effects of insulin appear to occur through activation of receptors for the family of related insulin-like growth factors. Both genetic and acquired abnormalities in the number of insulin receptors, the activity of the receptor kinase, and the various post-receptor steps in insulin action occur in disease states leading to tissue resistance to insulin action. It stimulates 1.Transport of glucose ions, Amino acids, 2.Glycogen formation, 3.Glucose conversion to triglycerides, 4.Nucleicacid synthesis and protein synthesis.

 

Diabetes Type 1, It occurs in approximately 10 percent of all cases, is an autoimmune disease in which the immune system, by mistake, attacks its own insulin-producing cells so that insufficient amounts of insulin are produced - or no insulin at all. Type 1 affects predominantly young people and usually makes its debut before the age of 30, and most frequently between the ages of 10 and 14.  Rest 90% are type II;

In the early 1920s, Frederick Banting, John Macleod, George Best and Bertram Collip isolated the hormone insulin and purified it so that it could be administered to humans. This was a major breakthrough in the treatment of diabetes type 1.

Symptoms of type 1 diabetes; Excessive thirst and dehydration, frequent urination, hunger, accompanied by weight loss; blurred vision; weakness, tiredness, or sleepiness; vomiting or nausea, back foot foot breaks ; sudden irritability.

Diabetes Type 1, which occurs in approximately 10 percent of all cases, is an autoimmune disease in which the immune system, by mistake, attacks its own insulin-producing cells so that insufficient amounts of insulin are produced - or no insulin at all. Type 1 affects predominantly young people and usually makes its debut before the age of 30, and most frequently between the ages of 10 and 14. Rest 90% are type II;

In the early 1920s, Frederick Banting, John Macleod, George Best and Bertram Collip isolated the hormone insulin and purified it so that it could be administered to humans. This was a major breakthrough in the treatment of diabetes type 1.

Insulin is produced in the pancreas. To be more specific, it's produced by the beta cells in the islets of Langerhans in the pancreas. . If sugar level becomes too low, it can result in a coma and eventually death. Give orange juice or inject insulin;  If the levels are low, the patient suffers from an overdose of insulin. https://www.nobelprize.org

 

Illustration: Pancreas and neighboring organs - as described in the information

https://www.ncbi.nlm.nih.gov

 

Image result for islets of langerhans diagram

https://www.shutterstock.com

The islets of Langerhans are responsible for the endocrine function of the pancreas. Each islet contains beta, alpha, and delta cells that are responsible for the secretion of  hormones.

Insulin is produced in the pancreas. To be more specific, it's produced by the beta cells in the islets of Langerhans in the pancreas. . If sugar level becomes too low, it can result in a coma and eventually death. Give orange juice or inject insulin;  If the levels are low, the patient suffers from an overdose of insulin; the person can go into coma. https://www.nobelprize.org

 

Type 2:

Type 2 diabetes begins with insulin resistance. This means that the cells don't react to insulin the way they are supposed to do. Diabetes 2 will be 400 million or more in the entire population USA.  https://www.nobelprize.org/

·       fatigue, excessive thirst, frequent urination; blurred vision; mood changes; a high rate of infections; slow healing process.

·       Insulin resistance, compensatory hyperinsulnamia; compensatory hyperinsulinaemia occurs when pancreatic β cell secretion increases to maintain normal blood glucose levels in the setting of peripheral insulin resistance in muscle and adipose tissue; Insulin resistance syndrome, Metabolic syndrome;

Insulin protein consists of two polypeptide chains A and B.  The A chain comprises 21 amino acids and the B chain 30 amino acids. The A chain has an N-terminal helix linked to an anti-parallel C-terminal helix; the B chain has a central helical segment. The two chains are joined by 2 disulphide bonds, which join the N- and C-terminal helices of the A chain to the central helix of the B chain. In pro-insulin, a connecting peptide links the N-terminus of the A chain to the C-terminus of the B chain. If the levels are low, the patient suffers from an overdose of insulin. https://www.nobelprize.org; Insulin resistance, compensatory hyperinsulnamia; compensatory hyperinsulinaemia occurs when pancreatic β cell secretion increases to maintain normal blood glucose levels in the setting of peripheral insulin resistance in muscle and adipose tissue; Insulin resistance syndrome, Metabolic syndrome; Type 2 diabetes begins with insulin resistance. This means that the cells don't react to insulin the way they are supposed to do.  Insulin resistance, compensatory hyperinsulnamia; ompensatory hyperinsulinaemia occurs when pancreatic β cell secretion increases to maintain normal blood glucose levels in the setting of peripheral insulin resistance in muscle and adipose tissue; Insulin resistance syndrome, Metabolic syndrome;

 

The A chain comprises 21 amino acids and the B chain 30 amino acids. The A chain has an N-terminal helix linked to an anti-parallel C-terminal helix; the B chain has a central helical segment. The two chains are joined by 2 disulphide bonds, which join the N- and C-terminal helices of the A chain to the central helix of the B chain. In pro-insulin, a connecting peptide links the N-terminus of the A chain to the C-terminus of the B chain.

Mechanisms of Insulin Secretion;

Increased levels of glucose induce the “first phase” of glucose-mediated insulin secretion by release of insulin from secretory granules in the β cell. Glucose entry into the β cell is sensed by glucokinase, which phosphorylates glucose to glucose-6-phosphate (G6P), generating ATP.12 Closure of K+-ATP-dependent channels results in membrane depolarization and activation of voltage dependent calcium channels leading to an increase in intracellular calcium concentration; this triggers pulsatile insulin secretion.13Augmentation of this response occurs by both a K+-ATP channel-independent Ca2+-dependent pathway and K+-ATP channel-independent Ca2+-independent pathways of glucose action.10 Other mediators of insulin release include activation of phospholipases and protein kinase C (e.g. by acetycholine) and by stimulation of adenylyl cyclase activity and activation of β cell protein kinase A, which potentiates insulin secretion. This latter mechanism may be activated by hormones, such as vasoactive intestinal peptide (VIP), PACAP, GLP-1, and GIP. These factors appear to play a significant role in the second phase of glucose mediated insulin secretion, after refilling of secretory granules translocated from reserve pools.

 

http://jcs.biologists.org/content/joces/127/9/1911/F7.large.jpg?width=800&height=600&carousel=1

Insulin elicits a ROS-activated and an IP3-dependent Ca2+release, which both impinge on GLUT4 translocation http://jcs.biologists.org/conten; Ariel Contreras-Ferrat et al.

Insulin - action on peripheral cells

http://www.medicinehack.com/

 

Insulin binds to receptor on target sites on the surface of hepatocyte cells, muscle cells and brain cells. These sites have an intrinsic tyrosine kinase activity that leads to receptor auto phosphorylation and recruitment of intracellular signaling molecules.  The latter results  is widespread metabolic and mitogenic effects of insulin as shown in the diagram above.

Nutrients in the GI tract stimulate the secretion of hormones known as incretins which amplify glucose-induced insulin release. These account for the greater insulin response to oral, as opposed to intravenous, glucose. GIP and GLP-1 are the two most important incretin hormones. GLP-1 also inhibits glucagon release, delays gastric emptying and reduces appetite. https://www.ncbi.nlm.nih.gov

 

The classical symptoms of diabetes are polyuria (frequent urination), polydipsia (increased thirst) and polyphagia (increased hunger).

 

The receptor for insulin consists of an alpha-subunit, which binds the hormone, and a beta-subunit, which is an insulin-stimulated, tyrosine-specific protein kinase. Activation of this kinase is believed to generate a signal that eventually results in insulin's action on glucose, lipid, and protein metabolism. The growth-promoting effects of insulin appear to occur through activation of receptors for the family of related insulin-like growth factors. Both genetic and acquired abnormalities in the number of insulin receptors, the activity of the receptor kinase, and the various post-receptor steps in insulin action occur in disease states leading to tissue resistance to insulin action.

 

Nutrients in the GI tract stimulate the secretion of hormones known as incretins which amplify glucose-induced insulin release. These account for the greater insulin response to oral, as opposed to intravenous, glucose. GIP and GLP-1 are the two most important incretin hormones. GLP-1 also inhibits glucagon release, delays gastric emptying and reduces appetite. https://www.ncbi.nlm.nih.gov

InsulinHexamer.jpg

Pancreatic beta cells produce insulin, which is a hexamer; https://en.wikipedia.org

 

https://upload.wikimedia.org/wikipedia/commons/thumb/8/8c/Insulin_glucose_metabolism.jpg/400px-Insulin_glucose_metabolism.jpg

 

Effect of insulin on glucose uptake and metabolism: Insulin binds to its receptor (1), which starts many protein activation cascades (2). These include translocation of Glut-4 transporter to the plasma membrane and influx of glucose (3), glycogen synthesis (4), glycolysis (5) and triglyceride synthesis (6).

Increased potassium uptake – forces cells synthesizing glycogen (a very spongy, "wet" substance, that increases the content of intracellular water, and its accompanying K+ions)[56] to absorb potassium from the extracellular fluids; lack of insulin inhibits absorption. Insulin's increase in cellular potassium uptake lowers potassium levels in blood plasma. This possibly occurs via insulin-induced translocation of the Na+/K+-ATPase to the surface of skeletal muscle cells.

Once an insulin molecule has docked onto the receptor and effected its action, it may be released back into the extracellular environment, or it may be degraded by the cell. The two primary sites for insulin clearance are the liver and the kidney. The liver clears most insulin during first-pass transit, whereas the kidney clears most of the insulin in systemic circulation. Degradation normally involves endocytosis of the insulin-receptor complex, followed by the action of insulin-degrading enzyme. An insulin molecule produced endogenously by the pancreatic beta cells is estimated to be degraded within about one hour after its initial release into circulation ((insulin half-life ~ 4–6 minutes).

 

An external file that holds a picture, illustration, etc.
Object name is cbr26_2pg019f2.jpg

NCBi.nlm,nih.gov https://www.ncbi.nlm.nih.gov

 

What causes insulin resistance and Diabetes; Insulin resistance and high levels of insulin and lipids all precede the development of metabolic dysfunction. Which metabolic factor is to blame?

Type 2 diabetes is a metabolic disease categorized, primarily, by reduced insulin sensitivity, β-cell dysfunction, and elevated hepatic glucose production [1]. Insulin resistance is widely accepted as the starting point for the progression from glucose intolerance to overt type 2 diabetes. Therefore understanding the underlining mechanisms of insulin resistance pathophysiology is of great importance to the development of novel and effective treatments.

The notion that 5' AMP-activated protein kinase (AMPK) mediates the anti-hyperglycaemic action of metformin has recently been challenged by genetic loss-of-function studies, thrusting the AMPK-independent effects of the drug into the spotlight for the first time in more than a decade. Key AMPK-independent effects of the drug include the mitochondrial actions that have been known for many years and which are still thought to be the primary site of action of metformin. Coupled with recent evidence of AMPK-independent effects on the counter-regulatory hormone glucagon, new paradigms of AMPK-independent drug action are beginning to take shape. In this review we summarise the recent research developments on the molecular action of metformin.

 

http://www.nature.com/nature/journal/v414/n6865/images/414799a-f2.2.jpg

Alan R. Saltiel & C. Ronald Kahn http://www.nature.com/nature

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http://journal.frontiersin.org/article/

 

 

The insulin receptor is a tyrosine kinase that undergoes auto phosphorylation, and catalyzes the phosphorylation of cellular proteins such as members of the IRS family, Shc and Cbl. Upon tyrosine phosphorylation, these proteins interact with signaling molecules through their SH2 domains, resulting in a diverse series of signaling pathways, including activation of PI(3)K and downstream PtdIns(3,4,5)P3-dependent protein kinases, ras and the MAP kinase cascade, and Cbl/CAP and the activation of TC10. These pathways act in a concerted fashion to coordinate the regulation of vesicle trafficking, protein synthesis, enzyme activation and inactivation, and gene expression, which results in the regulation of glucose, lipid and protein metabolism.

The pathogenesis of insulin resistance, a major component of type 2 diabetes, has been the subject of extensive study, much of which has focused on signaling events or mitochondrial and endoplasmic reticulum dysfunction. A large body of data converges to suggest that insulin resistance also involves transcriptional and epigenomic (i.e., nuclear) events. Several nuclear receptors regulate insulin sensitivity in both positive and negative ways, including peroxisome proliferator-activated receptor γ (PPARγ) and the glucocorticoid receptor (GR). Insulin sensitivity can be affected by many transcription factors, cofactors, and chromatin-modifying enzymes working coordinately in different tissues and cell types.

Diabetic cardiomyopathy (DCM); Diabetic cardiomyopathy (DCM)

Cardiac metab; http://journal.frontiersin.org/article/ Insulin-stimulated glucose uptake in cardiomyocytes is mediated primarily through mobilization of glucose transporter 4 (GLUT4). Basal cardiac glucose uptake is mediated by glucose transporter 1 (GLUT1), however, contraction-mediated activation of GLUT4 translocation may also contribute significantly to myocardial glucose uptake (Abel, 2004). We have reported that GLUT4-mediated glucose uptake in cardiomyocytes can be activated through a G protein, phosphatidylinositol 3-kinase (PI3K)γ and 1,4,5-inositol-triphosphate (IP3) receptor (IP3R) signaling axis, leading to generation of a cytoplasmic Ca+2 signal that mobilizes GLUT4

Insulin resistance in most cases is believed to be manifest at the cellular level via post-receptor defects in insulin signaling;  genetic polymorphisms of tyrosine phosphorylation of the insulin receptor, IRS proteins or PIP-3 kinase, or may involve abnormalities of GLUT 4 function.

Those tissues defined as insulin dependent, based on intracellular glucose transport, are principally adipose tissue and muscle. However, insulin’s actions are pleotropic and widespread, as are the manifestations of insulin resistance and the associated compensatory hyperinsulinemia.

Transport in muscle is via GLUT4, 60% of insulin is taken by muscle cells.  In the fed state insulin promotes glycogen synthesis via activation of glycogen synthase. This enables energy to be released anaerobically via glycolysis, e.g. during intense muscular activity. Adipose tissue; Intracellular glucose transport into adipocytes in the postprandial state is insulin-dependent via GLUT 4;

Since lipoprotein lipase activity is insulin-dependent and impaired by insulin resistance, peripheral uptake of triglycerides from VLDL is also diminished. These mechanisms contribute to the observed hypertriglyceridemia of insulin resistance. Mitogenic effects of insulin (and growth hormone) are mediated via hepatic production of insulin-like growth factors and potentially via suppression of sex-hormone binding globulin (SHBG) production.

Insulin and its actions play an important role in various aspects of endothelial function, e.g. nitric oxide production, while insulin resistance is strongly associated with endothelial dysfunction. While the brain is not insulin-dependent so far as intracellular glucose uptake is concerned, insulin receptors have been located in the brain and are concentrated in the olfactory bulb, hypothalamus, hippocampus,40 retina and vessels of the choroid plexus, as well as in regions of the striatum and cerebral cortex e.g. medial temporal lobes. Insulin is believed to act as a neuropeptide, involved in satiety, appetite regulation, olfaction, memory and cognition. https://www.ncbi.nlm.nih.gov-

 

The molecular mechanism of insulin action.Kahn CR.

 

Insulin initiates its action by binding to a glycoprotein receptor on the surface of the cell. This receptor consists of an alpha-subunit, which binds the hormone, and a beta-subunit, which is an insulin-stimulated, tyrosine-specific protein kinase. Activation of this kinase is believed to generate a signal that eventually results in insulin's action on glucose, lipid, and protein metabolism. Activation of beta subunits lead to GLUT4 to be transported to the cellular PM. This leads to the transport of glucose from external into cells thus creates reduction of sugar in extra cellular fluids. The growth-promoting effects of insulin appear to occur through activation of receptors for the family of related insulin-like growth factors. Both genetic and acquired abnormalities in the number of insulin receptors, the activity of the receptor kinase, and the various post-receptor steps in insulin action occur in disease states leading to tissue resistance to insulin action. https://www.ncbi.nlm.nih.gov, www.slideshare.net

http://arbl.cvmbs.colostate.edu/hbooks/pathphys/endocrine/pancreas/proinsulin.gif

 

 

 

 

 

 

www.slideshare.net?top figure

 

The work is focused on the action of thiazolidinedione (TZDs), oral anti-diabetic drugs which reduce insulin resistance and exert insulin-sensitizing action directly on tissues. According to the professor Carme Caelles, at her laboratory in the Barcelona Science Park, "the action mechanism of TZDs is not well known yet. Their receptor (the PPARγ) has been identified; but we do not know yet how they act at molecular level.

 

http://arbl.cvmbs.colostate.edu/hbooks/pathphys/endocrine/pancreas/insulin.gif

Insulin Polypeptide chain

http://arbl.cvmbs.colostate.edu/hbooks/pathphys/endocrine/pancreas/fatspare.gif

 

 

 

 

 

 

 

 

 

Proinsulin consists of three domains: an amino-terminal B chain, a carboxy-terminal A chain and a connecting peptide in the middle known as the C peptide. Within the endoplasmic reticulum, proinsulin

Comparison of gene expression -Of 6,451 genes surveyed, transcriptional patterns of 85 genes showed alterations in the diabetic patients after withdrawal of treatment, when compared with patterns in the nondiabetic control subject; Insulin treatment reduced the difference in patterns between diabetic and nondiabetic control subjects (improved) in all but 11 gene transcripts, which included genes involved in structural and contractile functions, growth and tissue development, stress response, and energy metabolism. 

 

 These improved transcripts included genes involved in insulin signaling, transcription factors, and mitochondrial maintenance. However, insulin treatment altered the transcription of 29 additional genes involved in signal transduction; structural and contractile functions; growth and tissue development; and protein, fat, and energy metabolism. Type 2 diabetic patients had elevated circulating insulin during the insulin-treated phase, although their blood glucose levels (98.8 ± 6.4 vs. 90.0 ± 2.9 mg/dl for diabetic vs. control) were similar to those of the control subjects.  In contrast,  after withdrawal of treatment, the diabetic patients had reduced SI and elevated blood glucose (224.0 ± 26.2 mg/dl), although their insulin levels were similar to those of the nondiabetic control subjects. This study identified several candidate genes for muscle insulin resistance, complications associated with poor glycemic control, and effects of insulin treatment in people with type 2 diabetes.

 

Insulin inhibits breakdown of fat in adipose tissue by inhibiting the intracellular lipase that hydrolyzes triglycerides to release fatty acids. Insulin also increases the permeability of many cells to potassium, magnesium and phosphate ions. The effect on potassium is clinically important. Insulin activates sodium-potassium ATPases in many cells, causing a flux of potassium into cells. Under certain circumstances, injection of insulin can kill patients because of its ability to acutely suppress plasma potassium concentrations.

 

In the early 1920s, Frederick Banting, John Macleod, George Best and Bertram Collip isolated the hormone insulin and purified it so that it could be administered to humans. This was a major breakthrough in the treatment of diabetes type 1. Insulin is produced in the pancreas. To be more specific, it's produced by the beta cells in the islets of Langerhans in the pancreas. . If sugar level becomes too low, it can result in a coma and eventually death. Give oranghe juice or inject insulin;  If the levels are low, the patient suffers from an overdose of insulin. https://www.nobelprize.org

Mechanisms of Insulin Secretion

Increased levels of glucose induce the “first phase” of glucose-mediated insulin secretion by release of insulin from secretory granules in the β cell. Glucose entry into the β cell is sensed by glucokinase, which phosphorylates glucose to glucose-6-phosphate (G6P), generating ATP?.  Closure of K+-ATP-dependent channels results in membrane depolarization and activation of voltage dependent calcium channels leading to an increase in intracellular calcium concentration; this triggers pulsatile insulin secretion.13Augmentation of this response occurs by both a K+-ATP channel-independent Ca2+-dependent pathway and K+-ATP channel-independent Ca2+-independent pathways of glucose action.10 Other mediators of insulin release include activation of phospholipases and protein kinase C (e.g. by acetycholine) and by stimulation of adenylyl cyclase activity and activation of β cell protein kinase A, which potentiates insulin secretion. This latter mechanism may be activated by hormones, such as vasoactive intestinal peptide (VIP), PACAP, GLP-1, and GIP. These factors appear to play a significant role in the second phase of glucose mediated insulin secretion, after refilling of secretory granules translocated from reserve pools

Nutrients in the GI tract stimulate the secretion of hormones known as incretins which amplify glucose-induced insulin release. These account for the greater insulin response to oral, as opposed to intravenous, glucose. GIP and GLP-1 are the two most important incretin hormones. GLP-1 also inhibits glucagon release, delays gastric emptying and reduces appetite. https://www.ncbi.nlm.nih.gov.

 

Type 2 diabetes is a metabolic disease categorized, primarily, by reduced insulin sensitivity, β-cell dysfunction, and elevated hepatic glucose production [1]. Insulin resistance is widely accepted as the starting point for the progression from glucose intolerance to overt type 2 diabetes. Therefore understanding the underlining mechanisms of insulin resistance pathophysiology is of great importance to the development of novel and effective treatments.

 

The notion that 5' AMP-activated protein kinase (AMPK) mediates the anti-hyperglycaemic action of metformin has recently been challenged by genetic loss-of-function studies, thrusting the AMPK-independent effects of the drug into the spotlight for the first time in more than a decade. Key AMPK-independent effects of the drug include the mitochondrial actions that have been known for many years and which are still thought to be the primary site of action of metformin. Coupled with recent evidence of AMPK-independent effects on the counter-regulatory hormone glucagon, new paradigms of AMPK-independent drug action are beginning to take shape. In this review we summarise the recent research developments on the molecular action of metformin.

 

The insulin receptor is a tyrosine kinase that undergoes autophosphorylation, and catalyses the phosphorylation of cellular proteins such as members of the IRS family, Shc and Cbl. Upon tyrosine phosphorylation, these proteins interact with signalling molecules through their SH2 domains, resulting in a diverse series of signalling pathways, including activation of PI(3)K and downstream PtdIns(3,4,5)P3-dependent protein kinases, ras and the MAP kinase cascade, and Cbl/CAP and the activation of TC10. These pathways act in a concerted fashion to coordinate the regulation of vesicle trafficking, protein synthesis, enzyme activation and inactivation, and gene expression, which results in the regulation of glucose, lipid and protein metabolism. Alan R. Saltiel & C. Ronald Kahn http://www.nature.com/nature .      The pathogenesis of insulin resistance, a major component of type 2 diabetes, has been the subject of extensive study, much of which has focused on signaling events or mitochondrial and endoplasmic reticulum dysfunction. A large body of data converges to suggest that insulin resistance also involves transcriptional and epigenomic (i.e., nuclear) events. Several nuclear receptors regulate insulin sensitivity in both positive and negative ways, including peroxisome proliferator-activated receptor γ (PPARγ) and the glucocorticoid receptor (GR).  Insulin sensitivity can be affected by many transcription factors, cofactors, and chromatin-modifying enzymes working coordinately in different tissues and cell types. Diabetic cardiomyopathy (DCM); Diabetic cardiomyopathy (DCM).

Cardiac metab; Insulin-stimulated glucose uptake in cardiomyocytes is mediated primarily through mobilization of glucose transporter 4 (GLUT4). Basal cardiac glucose uptake is mediated by glucose transporter 1 (GLUT1), however, contraction-mediated activation of GLUT4 translocation may also contribute significantly to myocardial glucose uptake (Abel, 2004)

We have reported that GLUT4-mediated glucose uptake in cardiomyocytes can be activated through a G protein, phosphatidylinositol 3-kinase (PI3K)γ and 1,4,5-inositol-triphosphate (IP3) receptor (IP3R) signaling axis, leading to generation of a cytoplasmic Ca+2 signal that mobilizes GLUT4

Insulin resistance in most cases is believed to be manifest at the cellular level via post-receptor defects in insulin signaling;  genetic polymorphisms of tyrosine phosphorylation of the insulin receptor, IRS proteins or PIP-3 kinase, or may involve abnormalities of GLUT 4 function.

Those tissues defined as insulin dependent, based on intracellular glucose transport, are principally adipose tissue and muscle. However, insulin’s actions are pleotropic and widespread, as are the manifestations of insulin resistance and the associated compensatory hyperinsulinaemia.

Transport in muscle is via GLUT4, 60% of insulin is Tken by muscle cells.  n the fed state insulin promotes glycogen synthesis via activation of glycogen synthase. This enables energy to be released anaerobically via glycolysis, e.g. during intense muscular activity.

Adipose tissue; Intracellular glucose transport into adipocytes in the postprandial state is insulin-dependent via GLUT 4;

since lipoprotein lipase activity is insulin-dependent and impaired by insulin resistance, peripheral uptake of triglycerides from VLDL is also diminished. These mechanisms contribute to the observed hypertriglyceridaemia of insulin resistance.

Mitogenic effects of insulin (and growth hormone) are mediated via hepatic production of insulin-like growth factors and potentially via suppression of sex-hormone binding globulin (SHBG) production.

Insulin and its actions play an important role in various aspects of endothelial function, e.g. nitric oxide production, while insulin resistance is strongly associated with endothelial dysfunction.

While the brain is not insulin-dependent so far as intracellular glucose uptake is concerned, insulin receptors have been located in the brain and are concentrated in the olfactory bulb, hypothalamus, hippocampus,40 retina and vessels of the choroid plexus, as well as in regions of the striatum and cerebral cortex e.g. medial temporal lobes. Insulin is believed to act as a neuropeptide, involved in satiety, appetite regulation, olfaction, memory and cognition.

https://www.ncbi.nlm.nih.gov-

The molecular mechanism of insulin action. Kahn CR.

Insulin initiates its action by binding to a glycoprotein receptor on the surface of the cell. This receptor consists of an alpha-subunit, which binds the hormone, and a beta-subunit, which is an insulin-stimulated, tyrosine-specific protein kinase. Activation of this kinase is believed to generate a signal that eventually results in insulin's action on glucose, lipid, and protein metabolism. The growth-promoting effects of insulin appear to occur through activation of receptors for the family of related insulin-like growth factors. Both genetic and acquired abnormalities in the number of insulin receptors, the activity of the receptor kinase, and the various post-receptor steps in insulin action occur in disease states leading to tissue resistance to insulin action. https://www.ncbi.nlm.nih.gov. www.slideshare.net.

 

INSULIN AND ITS MECHANISM
OF ACTION

INSULIN AND ITS MECHANISM OF
ACTION

-Ashmita Chaudhuri
B.Pharm, 4th year, 7th semest...

Insulin protein-monomer and hexamer

                                                                                   

http://arbl.cvmbs.colostate.edu/hbooks/pathphys/endocrine/pancreas/insulin_recept.gif

 

 

 

 

 

 

 

 

Insulin receptor- Alpa and Beta subunits bound to cell membrane

 

 

The insulin receptor is a tyrosine kinase. In other words, it functions as an enzyme that transfers phosphate groups from ATP to tyrosine residues on intracellular target proteins. Binding of insulin to the alpha subunits causes the beta subunits to phosphorylate themselves (autophosphorylation), thus activating the catalytic activity of the receptor. The activated receptor then phosphorylates a number of intracellular proteins, which in turn alters their activity, thereby generating a biological response. Several intracellular proteins have been identified as phosphorylation substrates for the insulin receptor, the best-studied of which is insulin receptor substrate 1 or IRS-1. When IRS-1 is activated by phosphorylation, a lot of things happen. Among other things, IRS-1 serves as a type of docking center for recruitment and activation of other enzymes that ultimately mediate insulin's effects. A more detailed look at these processes is presented in the section on insulin transduction.

 Insulin facilitates entry of glucose into muscle, adipose and several other tissue-via GLUT4,  Insulin stimulates the liver to store glucose in the form of glycogen. A well-known effect of insulin is to decrease the concentration of glucose in blood,  Insulin promotes synthesis of fatty acids in the liver.

 

The work is focused on the action of thiazolidinediones (TZDs), oral anti-diabetic drugs which reduce insulin resistance and exert insulin-sensitizing action directly on tissues. According to the professor Carme Caelles, at her laboratory in the Barcelona Science Park, "the action mechanism of TZDs is not well known yet. Their receptor (the PPARγ) has been identified; but we do not know yet how they act at molecular level.

Insulin also increases the permiability of many cells to potassium, magnesium and phosphate ions. The effect on potassium is clinically important. Insulin activates sodium-potassium ATPases in many cells, causing a flux of potassium into cells. Under certain circumstances, injection of insulin can kill patients because of its ability to acutely suppress potassium Plasma concentrations.

Proinsulin consists of three domains: an amino-terminal B chain, a carboxy-terminal A chain and a connecting peptide in the middle known as the C peptide. Within the endoplasmic reticulum, proinsulin

Comparison of gene expression -Of 6,451 genes surveyed, transcriptional patterns of 85 genes showed alterations in the diabetic patients after withdrawal of treatment, when compared with patterns in the nondiabetic control subject; Insulin treatment reduced the difference in patterns between diabetic and nondiabetic control subjects (improved) in all but 11 gene transcripts, which included genes involved in structural and contractile functions, growth and tissue development, stress response, and energy metabolism. 

These improved transcripts included genes involved in insulin signaling, transcription factors, and mitochondrial maintenance. However, insulin treatment altered the transcription of 29 additional genes involved in signal transduction; structural and contractile functions; growth and tissue development; and protein, fat, and energy metabolism. Type 2 diabetic patients had elevated circulating insulin during the insulin-treated phase, although their blood glucose levels (98.8 ± 6.4 vs. 90.0 ± 2.9 mg/dl for diabetic vs. control) were similar to those of the control subjects.   In contrast, after withdrawal of treatment, the diabetic patients had reduced SI and elevated blood glucose (224.0 ± 26.2 mg/dl),  although their insulin levels were similar to those of the nondiabetic control subjects. This study identified several candidate genes for muscle insulin resistance, complications associated with poor glycemic control, and effects of insulin treatment in people with type 2 diabetes.

 

Image result for How Insulin biochemically acts

http://care.diabetesjournals.org/ Harold E. Lebovitz, hlebovitz1@hotmail.com.

 

In type 2 diabetes, the abnormalities in insulin secretion are multiple. One of the initial defects is a loss of the early phase of meal-stimulated insulin secretion. This is followed by an inability of the β-cell to increase insulin secretion sufficient to overcome hepatic and peripheral insulin resistance. Type 2 diabetes is characterized by a progressive decrease in both β-cell mass and secretory function so that, in most individuals, absolute insulin deficiency occurs in the late stages of the disease.

 

In normal physiology, β-cell  insulin  secretion is coupled immediately with changes in the plasma glucose level. The secretory response is rapid (within a minute or two), and because the half-life of insulin is ~5 min, there is little lag time in the glucose regulatory system. Endogenously secreted insulin goes via the portal vein to the liver, where 30–80% of it is either metabolized or used.  The portal vein-to-peripheral arterial insulin ratio is ~2:1. The administration of insulin exogenously eliminates the rapid regulation of plasma glucose, since the insulin must be taken up slowly and without regulation from the subcutaneous injection site. The kinetics are determined by the nature of the injected insulin formulation. Additionally, as illustrated in the figure, it is necessary to create hyperinsulinemia in the periphery to achieve adequate insulin in the liver (portal-to-peripheral insulin levels ~1:2 to appropriately regulate hepatic glucose production and/or glucose uptake.

 

Figure 1

Administration of exogenous insulin provides a different insulin gradient than that occurring after endogenous insulin secretion. Endogenous insulin secretion acts initially on the liver where a major portion of it is taken up and <50% reaches the peripheral tissues. Exogenously administered insulin must circulate through the peripheral tissues before it can reach the liver; therefore, peripheral hyperinsulinemia is necessary to attain adequate insulin to regulate the liver.

 

Image result for How Insulin biochemically acts

http://care.diabetesjournals.org/ Harold E.

 

Lebovitz, hlebovitz1@hotmail.com. In type 2 diabetes, the abnormalities in insulin secretion are multiple. One of the initial defects is a loss of the early phase of meal-stimulated insulin secretion. This is followed by an inability of the β-cell to increase insulin secretion sufficient to overcome hepatic and peripheral insulin resistance. Type 2 diabetes is characterized by a progressive decrease in both β-cell mass and secretory function so that, in most individuals, absolute insulin deficiency occurs in the late stages of the disease.

1.                                In normal physiology, β-cell insulin secretion is coupled immediately with changes in the plasma glucose level (1). The secretory response is rapid (within a minute or two), and because the half-life of insulin is ~5 min, there is little lag time in the glucose regulatory system. Endogenously secreted insulin goes via the portal vein to the liver, where 30–80% of it is either metabolized or used (2). The portal vein-to-peripheral arterial insulin ratio is ~2:1. The administration of insulin exogenously eliminates the rapid regulation of plasma glucose, since the insulin must be taken up slowly and without regulation from the subcutaneous injection site. The kinetics are determined by the nature of the injected insulin formulation. Additionally, as illustrated in Fig. 1, it is necessary to create hyperinsulinemia in the periphery to achieve adequate insulin in the liver (portal-to-peripheral insulin levels ~1:2) to appropriately regulate hepatic glucose production and/or glucose uptake.

 

Administration of exogenous insulin provides a different insulin gradient than that occurring after endogenous insulin secretion. Endogenous insulin secretion acts initially on the liver where a major portion of it is taken up and <50% reaches the peripheral tissues. Exogenously administered insulin must circulate through the peripheral tissues before it can reach the liver; therefore, peripheral hyperinsulinemia is necessary to attain adequate insulin to regulate the liver.

 

Metformin;

Metformin Glucophage is basically a refined herbal medicine derived from a flower called French lilac (Galega officinalis) (goat’s rue or French lilac). “Glucophage” (glucose eater) is for T2D type 2 diabetes. Metformin is derived from Galega officinalis, an herb described to treat several ailments since the seventeenth century. Studies in the early 1900s showed guanidine, a compound isolated from the herb, was able to lower blood sugar in animal models. These models also suggested guanidine by itself was likely too potent and toxic to use clinically in humans.

 

The effectiveness of metformin for treatment of type 2 diabetes is due to its multiple mechanisms of action. Metformin works by decreasing the amount of sugar made by the liver and consequently decreasing the secretion of insulin from the pancreas. Metformin also slows the absorption of sugar in the intestine and increases insulin sensitivity in the muscles and periphery to help the body utilize sugar. Since metformin does not increase the amount of insulin secreted from the pancreas, as seen with other oral anti-diabetic medications such as sulfonylureas, it is does not cause weight gain or excessively reduce blood sugar. - See more at: http://www.rxeconsult.com/ 

 

Evidence from large clinical studies such as the UKPDS (United Kingdom Prospective Diabetes Study) shows early treatment with metformin in type 2-diabetes reduces long-term cardiovascular complications that can lead to death. This clinical finding may be attributed to metformin’s benefits on fat (lipids), oxidative stress, and inflammation. Metformin has shown positive effects on lowering triglycerides, lowering “bad” cholesterol (LDL), and slightly increasing “good” cholesterol (HDL). Additionally, recent mechanistic studies have shown how metformin can have anti-inflammatory effects on the vascular wall and improve lipid metabolism in macrophages (cells that help fight infection in the body). Other in vitro studies are evaluating the mechanism for metformin reducing oxidative stress. Results suggest several possible pathways for reducing reactive oxygen species, including decreases in specific proteins involved in intracellular metabolism. - See more at:

Treatment for prevention and treatment of solid tumor cancers (breast, prostate, colorectal, and endometrial). Numerous in vitro and studies are attempting to explain the mechanism for how metformin may inhibit tumor growth. - See more at:  http://www.rxeconsult.com/healthcare-articles   

 

Metformin: The notion that 5' AMP-activated protein kinase (AMPK) mediates the anti-hyperglycaemic action of metformin has recently been challenged by genetic loss-of-function studies, thrusting the AMPK-independent effects of the drug into the spotlight for the first time in more than a decade. Key AMPK-independent effects of the drug include the mitochondrial actions that have been known for many years and which are still thought to be the primary site of action of metformin. Coupled with recent evidence of AMPK-independent effects on the counter-regulatory hormone glucagon, new paradigms of AMPK-independent drug action are beginning to take shape. In this review we summarise the recent research developments on the molecular action of metformin. Graham Rena; https://www.ncbi.nlm.nih.gov

 

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Object name is 125_2013_2991_Fig1_HTML.jpg

(ab) Schematic diagram of the anti-hyperglycaemic action of metformin on the liver cell. Part (b) shows a simplified version of (a). Metformin is transported into hepatocytes mainly via OCT1, resulting in an inhibition of the mitochondrial respiratory chain (complex I) through a currently unknown mechanism(s). The resulting deficit in energy production is balanced by reducing the consumption of energy in the cell, particularly reduced gluconeogenesis in the liver. This is mediated in two main ways. First, a decrease in ATP and a concomitant increase in AMP concentration occur, which is thought to contribute to the inhibition of gluconeogenesis directly (because of the energy/ATP deficit). Second, increased AMP levels function as a key signalling mediator that has been proposed to (1) allosterically inhibit cAMP–PKA signalling through suppression of adenylate cyclase, (2) allosterically inhibit FBPase, a key gluconeogenic enzyme, and (3) activates AMPK. This leads to inhibition of gluconeogenesis (1 and 2) and lipid/cholesterol synthesis (3), which may contribute to the longer term metabolic and therapeutic responses to the drug. FBPase; fructose-1,6-bisphosphatase

Metformin and Alzheimer’s disease:

Metformin; (other names -Glucophage, Glucophage XR, Glumetza, Riomet);  Nobody could explain how metformin helped, but then Canadian researchers showed that metformin reduces cell mutations and DNA damage.  Spanish scientists published in the journal Cell Cycle that metformin in two ways.  Like a chemotherapy drug, it blocks certain enzymes cancer cells need to reproduce. But the scientists wrote that metformin’s glucose-lowering and insulin-lowering effects may be more important. Metformin mimics the effect of severely restricting calories. The lowered insulin level enables healthy cells to reproduce better, so they don’t become cancerous. As a powerful antioxidant, metformin may actually slow aging. Anti-aging guru Ward Dean, MD, calls metformin “the most effective anti-aging drug there is.  Mostly,  through a mild and transient inhibition of the mitochondrial respiratory-chain complex 1. Metformin also activates the AMP-activated protein kinase (AMPK). Acutely decrease hepatic glucose production, mostly through a mild and transient inhibition of the mitochondrial respiratory chain complex I.

 

Although the molecular target of metformin has been elusive for several years, Zhou et al. reported that the activation of AMPK (AMP-activated protein kinase) was intimately associated with the pleiotropic actions of metformin. AMPK is a phylogenetically conserved serine/threonine protein kinase viewed as a fuel gauge monitoring systemic and cellular energy status, and which plays a crucial role in protecting cellular functions under energy-restricted conditions. AMPK is a heterotrimeric protein consisting of a catalytic α-subunit and two regulatory subunits, β and γ, and each subunits have at least two isoforms. AMPK is activated by an increase in the intracellular AMP/ATP ratio resulting from an imbalance between ATP production and consumption. Activation of AMPK involves AMP binding to regulatory sites on the γ-subunits. This causes conformational changes that allosterically activate the enzyme and inhibit dephosphorylation of Thr within the activation loop of the catalytic α-subunit.  AMPK activation requires phosphorylation of Thr  by upstream kinases, identified as the tumour suppressor STK11 (serine/threonine kinase 11)/LKB1 and CaMKKβ (Ca2+/calmodulin-dependent protein kinase β), which is stimulated further by the allosteric activator AMP.  Moreover, it has been recently shown that ADP, and therefore the ADP/ATP ratio, could also play a regulatory role on AMPK by binding to specific domains in the γ-subunit .  Activated AMPK switches cells from an anabolic to a catabolic state, shutting down the ATP-consuming synthetic pathways and restoring energy balance. This regulation involves phosphorylation by AMPK of key metabolic enzymes and transcription factors/co-activators modulating gene expression. As a result, glucose, lipid and protein synthesis, as well as cell growth, are inhibited, whereas fatty acid oxidation and glucose uptake are stimulated.

 In addition, the resulting decrease in hepatic energy status activates AMPK (AMP-activated protein kinase), a cellular metabolic sensor, providing a generally accepted mechanism for the action of metformin on hepatic gluconeogenesis. 

Owing to its high acid dissociation constant (pKa=12.4), metformin exists in a positively charged protonated form under physiological conditions and, as a result, can only partially cross the plasma membrane by passive- diffusion. Thus its intracellular transport is mediated by different isoforms of OCTs depending on the tissue under consideration (e.g. OCT1 in the liver or OCT2 in the kidney). Once inside the cytosolic compartment, mitochondria then constitute the primary target of metformin. The positive charge of metformin has been proposed to account for its accumulation within the matrix of energized mitochondria, driven by the membrane potential, whereas the apolar hydrocarbon side chain of the drug could also promote binding to hydrophobic structures, especially the phospholipids of mitochondrial membranes.  Although the exact mechanism(s) by which metformin acts at the molecular level remains unknown, it has been shown that the drug inhibits the mitochondrial respiratory chain specifically at the level of complex I without affecting any other steps in the mitochondrial machinery. This unique property of the drug induces a decrease in NADH oxidation, proton pumping across the inner mitochondrial membrane and oxygen consumption rate, leading to lowering of the proton gradient and, ultimately, to a reduction in proton-driven synthesis of ATP from ADP and Pi.

 

The demonstration that the respiratory-chain complex 1, a potent antihyperglycemic agent now recommended as the first line oral therapy for type 2 diabetes (T2D). It is to acutely decrease hepatic glucose production, mostly through a mild and transient inhibition of the mitochondrial respiratory-chain complex 1. In addition, the resulting decrease in hepatic energy status activates the AMP-activated protein kinase (AMPK), a cellular metabolic sensor, providing a generally accepted mechanism for metformin action on hepatic gluconeogenic program. The demonstration that the respiratory-chain complex 1, but not AMPK, is the primary target of metformin was recently strengthened by showing that the metabolic effect of the drug is preserved in liver-specific AMPK-deficient mice.  Metformin restore ovarian function in polycystic ovary syndrome, reduce fatty liver and to lower microvascular and macrovascular complications associated with T2D.  Its use was also recently suggested as an adjuvant treatment for cancer or gestational diabetes, and for the prevention in pre-diabetic populations.

Once treated with metformin, however, CBP was activated to the levels of nondiabetic mice, and their blood glucose levels returned to normal. However, when given to diabetic mice with defective copies of CBP, metformin had no effect on blood glucose levels, a proof that metformin works through CBP. As a cellular energy sensor, AMP-activated protein kinase (AMPK) is activated in response to a variety of conditions that deplete cellular energy levels, such as nutrient starvation (especially glucose), hypoxia and exposure to toxins that inhibit the mitochondrial respiratory chain complex;

 

Metformin and Alzheimer’s disease

Metformin (brand name Glucophage, Glucophage XR, Glumetza, Riomet) ,Nobody could explain how metformin helped, but then Canadian researchers showed that metformin reduces cell mutations and DNA damage.

 

Spanish scientists published in the journal Cell Cycle that metformin seems to block cancer in two ways. Like a chemotherapy drug, it blocks certain enzymes cancer cells need to reproduce. But the scientists wrote that metformin’s glucose-lowering and insulin-lowering effects may be more important. Metformin mimics the effect of severely restricting calories. The lowered insulin level enables healthy cells to reproduce better, so they don’t become cancerous.

As a powerful antioxidant, metformin may actually slow aging. Anti-aging guru Ward Dean, MD, calls metformin “the most effective anti-aging drug there is mostly through a mild and transient inhibition of the mitochondrial respiratory-chain complex 1, Activates the AMP-activated protein kinase (AMPK), 

 Acutely decrease hepatic glucose production, mostly through a mild and transient inhibition of the mitochondrial respiratory chain complex I; Although the molecular target of metformin has been elusive for several years, Zhou et al.  reported that the activation of AMPK (AMP-activated protein kinase) was intimately associated with the pleiotropic actions of metformin. AMPK is a phylogenetically conserved serine/threonine protein kinase viewed as a fuel gauge monitoring systemic and cellular energy status, and which plays a crucial role in protecting cellular functions under energy-restricted conditions. AMPK is a heterotrimeric protein consisting of a catalytic α-subunit and two regulatory subunits, β and γ, and each subunit has at least two isoforms. AMPK is activated by an increase in the intracellular AMP/ATP ratio resulting from an imbalance between ATP production and consumption. Activation of AMPK involves AMP binding to regulatory sites on the γ-subunits. This causes conformational changes that allosterically activate the enzyme and inhibit dephosphorylation of Thr172 within the activation loop of the catalytic α-subunit. AMPK activation requires phosphorylation on Thr172 by upstream kinases, identified as the tumour suppressor STK11 (serine/threonine kinase 11)/LKB1 and CaMKKβ (Ca2+/calmodulin-dependent protein kinase β), which is stimulated further by the allosteric activator AMP.   Moreover, it has been recently shown that ADP, and therefore the ADP/ATP ratio, could also play a regulatory role on AMPK by binding to specific domains in the γ-subunit [19,20]. Activated AMPK switches cells from an anabolic to a catabolic state, shutting down the ATP-consuming synthetic pathways and restoring energy balance. This regulation involves phosphorylation by AMPK of key metabolic enzymes and transcription factors/co-activators modulating gene expression [18]. As a result, glucose, lipid and protein synthesis, as well as cell growth, are inhibited, whereas fatty acid oxidation and glucose uptake are stimulated.

 In addition, the resulting decrease in hepatic energy status activates AMPK (AMP-activated protein kinase), a cellular metabolic sensor, providing a generally accepted mechanism for the action of metformin on hepatic gluconeogenesis. 

 

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Object name is halms658070f1.jpg

https://www.ncbi.nlm.nih.gov The mitochondrial respiratory chain complex 1 is the primary target of metformin

Owing to its high acid dissociation constant (pKa=12.4), metformin exists in a positively charged protonated form under physiological conditions and, as a result, can only partially cross the plasma membrane by passive diffusion. Thus its intracellular transport is mediated by different isoforms of OCTs depending on the tissue under consideration (e.g. OCT1 in the liver or OCT2 in the kidney). Once inside the cytosolic compartment, mitochondria then constitute the primary target of metformin. The positive charge of metformin has been proposed to account for its accumulation within the matrix of energized mitochondria, driven by the membrane potential, whereas the apolar hydrocarbon side chain of the drug could also promote binding to hydrophobic structures, especially the phospholipids of mitochondrial membranes.  Although the exact mechanism(s) by which metformin acts at the molecular level remains unknown, it has been shown that the drug inhibits the mitochondrial respiratory chain specifically at the level of complex I without affecting any other steps in the mitochondrial machinery. This unique property of the drug induces a decrease in NADH oxidation, proton pumping across the inner mitochondrial membrane and oxygen consumption rate, leading to lowering of the proton gradient and, ultimately, to a reduction in proton-driven synthesis of ATP from ADP and Pi.

 

Restores ovarian function in polycystic ovary syndrome, reduce fatty liver and to lower microvascular and macrovascular complications associated with T2D, Its use was also recently suggested as an adjuvant treatment for cancer or gestational diabetes, and for the prevention in pre-diabetic populations.

 Once treated with metformin, however, CBP was activated to the levels of nondiabetic mice, and their blood glucose levels returned to normal. However, when given to diabetic mice with defective copies of CBP, metformin had no effect on blood glucose levels, a proof that metformin works through CBP. As a cellular energy sensor, AMP-activated protein kinase (AMPK) is activated in response to a variety of conditions that deplete cellular energy levels, such as nutrient starvation (especially glucose), hypoxia and exposure to toxins that inhibit the mitochondrial respiratory chain complex;

 Acutely decreases hepatic glucose production, mostly through a mild and transient inhibition of the mitochondrial respiratory-chain complex 1. In addition, the resulting decrease in hepatic energy status activates the AMP-activated protein kinase (AMPK), a cellular metabolic sensor, providing a generally accepted mechanism for metformin action on hepatic gluconeogenic program. The demonstration that the respiratory-chain complex 1, but not AMPK, is the primary target of metformin was recently strengthened by showing that the metabolic effect of the drug is preserved in liver-specific AMPK-deficient mice.

 

AMPK activation by metformin is not a result of direct activation; instead, metformin inhibits complex I of the mitochondrial respiratory chain, leading to an increased AMP:ATP ratio.  The main effect of this drug from the biguanide family is to acutely decrease hepatic glucose production, mostly through a mild and transient inhibition of the mitochondrial respiratory-chain complex 1. In addition, the resulting decrease in hepatic energy status activates the AMP-activated protein kinase (AMPK), a cellular metabolic sensor, providing a generally accepted mechanism for metformin action on hepatic gluconeogenic program. The demonstration that the respiratory-chain complex 1, but not AMPK, is the primary target of metformin was recently strengthened by showing that the metabolic effect of the drug is preserved in liver-specific AMPK-deficient mice. https://www.ncbi.nlm.nih.gov/

The mitochondrial respiratory-chain complex 1 is the primary target of metformin:

Due to its high acid dissociation constant (pKa=12.4) metformin exists in a positively charged protonated form under physiological conditions and, as a result, can only marginally cross the plasma membrane by passive diffusion. Thus, its intracellular transport is mediated by different isoforms of the organic cation transporters (OCT) depending of the tissue considered (e.g. OCT1 in liver or OCT2 in kidney). Once inside the cytosolic compartment, mitochondria then constitute the primary target of metformin. The positive charge of metformin was proposed to account for its accumulation within the matrix of energized mitochondria, driven by the membrane potential (Δϕ), whereas the apolar hydrocarbon side-chain of the drug could also promote binding to hydrophobic structures, especially the phospholipids of mitochondrial membranes [31]. Although the exact mechanism(s) by which metformin acts at the molecular level remains unknown, it has been shown that the drug inhibits mitochondrial respiratory-chain specifically at the complex 1 level without affecting any other steps of the mitochondrial machinery. This unique property of the drug induces a decrease in NADH oxidation, proton pumping across the inner mitochondrial membrane and oxygen consumption rate, leading to lowering of the proton gradient (Δϕ) and ultimately to a reduction in proton-driven synthesis of ATP from ADP and inorganic phosphate (Pi).

 

Image result for Metformin action on Mitochondrial Complex1

      http://cancerpreventionresearch.aacrjournals.org

Image result for Metformin action on Mitochondrial Complex1

https://bmcbiol.biomedcentral.com/

 

Image result for Metformin action on Mitochondrial Complex1

Rosina Pryor, Filipe Cabreiro;http://www.biochemj.org

 

Potential molecular mechanisms of metformin action on hepatic gluconeogenesis;

After hepatic uptake through OCT1, the mitochondria is the primary target of metformin which exerts specific and AMPK-independent inhibition of respiratory-chain complex 1. The resultant mild decrease in energy status leads to acute and transient inhibition of energy-consuming gluconeogenic pathway. In addition, through AMPK-dependent and -independent regulatory points, metformin can lead to the inhibition of glucose production by disrupting gluconeogenesis gene expression. In parallel, the LKB1-dependent activation of AMPK triggered by ATP depletion could reduce hepatic lipogenesis and exert an indirect effect on hepatic insulin sensitivity to control hepatic glucose output.

 

Glipizide acts by partially blocking potassium channels among beta cells of pancreatic islets of Langerhans. By blocking potassium channels, the cell depolarizes which results in the opening of voltage-gated calcium channels. The resulting calcium influx encourages insulin release from beta cells Wikipedia; Glipizide acts by partially blocking potassium channels among beta cells of pancreatic islets of Langerhans. By blocking potassium channels, the cell depolarizes which results in the opening of voltage-gated calcium channels. The resulting calcium influx encourages insulin release from beta cells.  Acts on Cardiovascular system, Nephrons, Metformin’s action on cancer, anti-age.

 

 

Metformin and vildaglipitin- good for Dibetes-2; Vildaglipitin primarily by enhancing pancreatic (α and β) islet function. Improves Isulin secretion,

 

The main effect of this drug from the biguanide family is to acutely decrease hepatic glucose production, mostly through a mild and transient inhibition of the mitochondrial respiratory-chain complex 1. decrease in hepatic energy status activates the AMP-activated protein kinase (AMPK), a cellular metabolic sensor, providing a generally accepted mechanism for metformin action on hepatic gluconeogenic program.  The main effect of this drug from the biguanide family is to acutely decrease hepatic glucose production, mostly through a mild and transient inhibition of the mitochondrial respiratory-chain complex 1.

Metformin decreases in hepatic energy status, which activates the AMP-activated protein kinase (AMPK), a cellular metabolic sensor, providing a generally accepted mechanism for metformin action on hepatic gluconeogenic program.   In addition, the resulting decrease in hepatic energy status activates the AMP-activated protein kinase (AMPK), a cellular metabolic sensor, providing a generally accepted mechanism for metformin action on hepatic gluconeogenic program. 

may also have therapeutic potential in other conditions including diabetic nephropathy, cardiovascular diseases, polycystic ovary disease and the prevention or treatment of cancer.  Metformin is also frequently described as an insulin sensitizer leading to reduction in insulin resistance and significant reduction of plasma fasting insulin level. The improvement in insulin sensitivity by metformin could be ascribed to its positive effects on insulin receptor expression and tyrosine kinase activity ;

The preferential action of metformin in hepatocytes is due to the predominant expression of the organic cation transporter 1 (OCT1), which has been shown to facilitate cellular uptake of metformin .

Hou et al. reported that the activation of AMP-activated protein kinase (AMPK) was intimately associated with the pleiotropic actions of metformin.  AMPK is a phylogenetically conserved serine/threonine protein kinase viewed as a fuel gauge monitoring systemic and cellular energy status and which plays a crucial role in protecting cellular functions under energy-restricted conditions. AMPK is a heterotrimeric protein consisting of a catalytic α-subunit and two regulatory subunits β and γ and each subunit has at least two isoforms. AMPK is activated by increase in the intracellular AMP-on-ATP ratio resulting from imbalance between ATP production and consumption. Activation of AMPK involves AMP binding to regulatory sites on the γ subunits. This causes conformational changes that allosterically activate the enzyme and inhibit dephosphorylation of Thr172 within the activation loop of the catalytic α subunit. AMPK activation requires phosphorylation on Thr172 by upstream kinases, identified as the tumor suppressor serine/threonine kinase 11 (STK11/LKB1) and CaMKKβ, which is further stimulated by the allosteric activator AMP . Moreover, it has been recently shown that ADP, and therefore the ADP-on-ATP ratio, could also play a regulatory role on AMPK by binding to specific domains on the γ subunit [19, 20]. Activated AMPK switches cells from an anabolic to a catabolic state, shutting down the ATP-consuming synthetic pathways and restoring energy balance. This regulation involves phosphorylation by AMPK of key metabolic enzymes and transcription factors/co-activators modulating gene expression [18]. As a result, glucose, lipid and protein synthesis as well as cell growth are inhibited whereas fatty acid oxidation and glucose uptake are stimulated. https://www.ncbi.nlm.nih.gov

 

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Potential molecular mechanisms of metformin action on hepatic gluconeogenesis;

After hepatic uptake through OCT1, the mitochondria is the primary target of metformin which exerts specific and AMPK-independent inhibition of respiratory-chain complex 1. The resultant mild decrease in energy status leads to acute and transient inhibition of energy-consuming gluconeogenic pathway. In addition, through AMPK-dependent and -independent regulatory points, metformin can lead to the inhibition of glucose production by disrupting gluconeogenesis gene expression. In parallel, the LKB1-dependent activation of AMPK triggered by ATP depletion could reduce hepatic lipogenesis and exert an indirect effect on hepatic insulin sensitivity to control hepatic glucose output. https://www.ncbi.nlm.nih.gov; It also exerts an inhibitory effect on mitochondrial ROS production by selectively blocking the reverse electron flow through the respiratory-chain complex 1.

 

Empagliflozin 10 and 25 mg for 24 weeks as add-on to metformin therapy significantly improved glycemic control, weight, and BP, and were well-tolerated. http://care.diabetesjournals.org/ Metformin mainly acts by reducing hepatic glucose production through inhibition of gluconeogenesis and may increase glucose uptake in peripheral tissue.

The kidney has emerged as a therapeutic target in the treatment of type 2 diabetes. Approximately 90% of glucose filtered by the kidney is reabsorbed by the sodium glucose cotransporter 2 (SGLT2), located in the proximal tubule of the nephron.   SGLT2 inhibitors are novel oral anti-diabetes agents that, by reducing glucose reabsorption, increase urinary glucose excretion and reduce hyperglycemia independent of β-cell function and insulin resistance . This mechanism carries a low risk of hypoglycemia, with additional benefits of weight loss and reductions in blood pressure (BP).

And in the latest analysis, also reported earlier this month at the ADA meeting, empagliflozin was shown to be renoprotective too, significantly reducing the incidence of worsening nephropathy, by 39%. Check for glycated Hemoglobin(A1C),  https://www.ncbi.nlm.nih.gov.   Metformin’s action on cancer, anti-age;  Acts on Cardiovascular system, Nephrons,

Empagliflozin is an orally active, potent, selective inhibitor of SGLT2.  In phase II trials, treatment with empagliflozin as an add-on to metformin therapy was shown to be well-tolerated, with low rates of hypoglycemia, and to lead to significant reductions in HbA1c and body weight that were maintained over 90 weeks; Empagliflozin is an orally active, potent, selective inhibitor of SGLT2.  In phase II trials, treatment with empagliflozin as an add-on to metformin therapy was shown to be well-tolerated, with low rates of hypoglycemia, and to lead to significant reductions in HbA1c and body weight that were maintained over 90 weeks;

And in the latest analysis, also reported earlier this month at the ADA meeting, empagliflozin was shown to be renoprotective too, significantly reducing the incidence of worsening nephropathy, by 39

Metformin and vildaglipitin- good for Dibetes-2; Vildaglipitin primarily by enhancing pancreatic (α and β) islet function. Improves Insulin secretion,

The main effect of this drug from the biguanide family is to acutely decrease hepatic glucose production, mostly through a mild and transient inhibition of the mitochondrial respiratory-chain complex 1. decrease in hepatic energy status activates the AMP-activated protein kinase (AMPK), a cellular metabolic sensor, providing a generally accepted mechanism for metformin action on hepatic gluconeogenic program.  The main effect of this drug from the biguanide family is to acutely decrease hepatic glucose production, mostly through a mild and transient inhibition of the mitochondrial respiratory-chain complex 1.

Metformin decreases in hepatic energy status, which activates the AMP-activated protein kinase (AMPK), a cellular metabolic sensor, providing a generally accepted mechanism for metformin action on hepatic gluconeogenic program.   In addition, the resulting decrease in hepatic energy status activates the AMP-activated protein kinase (AMPK), a cellular metabolic sensor, providing a generally accepted mechanism for metformin action on hepatic gluconeogenic program. 

It may also have therapeutic potential in other conditions including diabetic nephropathy, cardiovascular diseases, polycystic ovary disease and the prevention or treatment of cancer.  Metformin is also frequently described as an insulin sensitizer leading to reduction in insulin resistance and significant reduction of plasma fasting insulin level. The improvement in insulin sensitivity by metformin could be ascribed to its positive effects on insulin receptor expression and tyrosine kinase activity ;

The preferential action of metformin in hepatocytes is due to the predominant expression of the organic cation transporter 1 (OCT1), which has been shown to facilitate cellular uptake of metformin . It is HH   hI

 

reported that the activation of AMP-activated protein kinase (AMPK) was intimately associated with the pleiotropic actions of metformin. AMPK is a phylogenetically conserved serine/threonine protein kinase viewed as a fuel gauge monitoring systemic and cellular energy status and which plays a crucial role in protecting cellular functions under energy-restricted conditions. AMPK is a heterotrimeric protein consisting of a catalytic α-subunit and two regulatory subunits β and γ and each subunit has at least two isoforms. AMPK is activated by increase in the intracellular AMP-on-ATP ratio resulting from imbalance between ATP production and consumption. Activation of AMPK involves AMP binding to regulatory sites on the γ subunits. This causes conformational changes that allosterically activate the enzyme and inhibit dephosphorylation of Thr172 within the activation loop of the catalytic α subunit. AMPK activation requires phosphorylation on Thr172 by upstream kinases, identified as the tumor suppressor serine/threonine kinase 11 (STK11/LKB1) and CaMKKβ, which is further stimulated by the allosteric activator AMP.   Moreover, it has been recently shown that ADP, and therefore the ADP-on-ATP ratio, could also play a regulatory role on AMPK by binding to specific domains on the γ subunit.  Activated AMPK switches cells from an anabolic to a catabolic state, shutting down the ATP-consuming synthetic pathways and restoring energy balance. This regulation involves phosphorylation by AMPK of key metabolic enzymes and transcription factors/co-activators modulating gene expression [18]. As a result, glucose, lipid and protein synthesis as well as cell growth are inhibited whereas fatty acid oxidation and glucose uptake are stimulated. https://www.ncbi.nlm.nih.gov 

 

TB drug- Bedaquiline; http://www.tbfacts.org/

Bedaquiline works by blocking an enzyme inside the Mycobacterium tuberculosis bacteria called ATP synthase. This enzyme is used by the bacteria to generate energy. Without the ability to generate energy, the TB bacteria die and the patient’s condition can start to improve. -  See more at main TB drugs isoniazid (INH) and rifampicin (RMP). This means that the drugs don’t work. - See more at: http://www.tbfacts.org/.  Ethionamide, kanamycin, pyrazinamide, Afloxacin, cycloserine/terizidone are available alternatives.  See more at: costing US$30,000 in the United States; Bedaquiline also called Sirturo.

Cancer:

Despite current successes in hematological cancers, we are only in the beginning of exploring the powerful potential of CAR redirected T cells in the control and elimination of resistant, metastatic, or recurrent non hematological cancers. This review discusses the application of the CAR T cell therapy, its challenges, and strategies for successful clinical and commercial translation.

CARs are genetically engineered receptors that combine the specific binding domains from a tumor targeting antibody with T cell signaling domains to allow specifically targeted antibody redirected T cell activation. 

Using immune system to treat cancer.;

CARs are genetically engineered receptors that combine the specific binding domains from a tumor targeting antibody with T cell signaling domains to allow specifically targeted antibody redirected T cell activation. 

Despite current successes in hematological cancers, we are only in the beginning of exploring the powerful potential of CAR redirected T cells in the control and elimination of resistant, metastatic, or recurrent nonhematological cancers. This review discusses the application of the CAR T cell therapy, its challenges, and strategies for successful clinical and commercial translation. https://www.cancer.gov

Healthy Human T Cell

Cancer Cell; Immune Checkpoint ModulatorsImmune Cell TherapyTherapeutic AntibodiesCancer Treatment Vaccines, Immune System Modulators, Research at NCI, https://www.cancer.gov;

 

The immune system’s natural capacity to detect and destroy abnormal cells may prevent the development of many cancers. However, cancer cells are sometimes able to avoid detection and destruction by the immune system. Cancer cells may:

·       One immunotherapy approach is to block the ability of certain proteins, called immune checkpoint proteins, to limit the strength and duration of immune responses. These proteins normally keep immune responses in check by preventing overly intense responses that might damage normal cells as well as abnormal cells. But, researchers have learned that tumors can commandeer these proteins and use them to suppress immune responses.

·       Blocking the activity of immune checkpoint proteins releases the "brakes" on the immune system, increasing its ability to destroy cancer cells.

·        The first such drug to receive approval by FDA, ipilimumab (Yervoy®), for the treatment of advanced melanoma, blocks the activity of a checkpoint protein known as CTLA4, which is expressed on the surface of activated immune cells called cytotoxic T lymphocytes. CTLA4 acts as a "switch" to inactivate these T cells, thereby reducing the strength of immune responses; ipilimumab binds to CTLA4 and prevents it from sending its inhibitory signal.

 

Immunotherapy; Cancer;

Scanning electron micrograph of a human T lymphocyte (also called a T cell), as shown above, from the immune system of a healthy donor. Source: National Institute of Allergy and Infectious Diseases (NIAID).

·               Therapy; Immune Checkpoint ModulatorsImmune Cell TherapyTherapeutic AntibodiesCancer Treatment Vaccines, Immune System Modulators, Research at NCI,

The immune system’s natural capacity to detect and destroy abnormal cells may prevent the development of many cancers. However, cancer cells are sometimes able to avoid detection and destruction by the immune system. Cancer cells may:

·       One immunotherapy approach is to block the ability of certain proteins, called immune checkpoint proteins, to limit the strength and duration of immune responses. These proteins normally keep immune responses in check by preventing overly intense responses that might damage normal cells as well as abnormal cells. But, researchers have learned that tumors can commandeer these proteins and use them to suppress immune responses.

·       Blocking the activity of immune checkpoint proteins releases the "brakes" on the immune system, increasing its ability to destroy cancer cells.

·        The first such drug to receive approval by FDA, ipilimumab (Yervoy®), for the treatment of advanced melanoma, blocks the activity of a checkpoint protein known as CTLA4, which is expressed on the surface of activated immune cells called cytotoxic T lymphocytes. CTLA4 acts as a "switch" to inactivate these T cells, thereby reducing the strength of immune responses; ipilimumab binds to CTLA4 and prevents it from sending its inhibitory signal.

Immune Cell Therapy

Progress is also being made with an experimental form of immunotherapy called adoptive cell transfer (ACT). In several small clinical trials testing ACT, some patients with very advanced cancer—primarily blood cancers—have had their disease completely eradicated. In some cases, these treatment responses have lasted for years.

 

In one form of ACT, T cells that have infiltrated a patient’s tumor, called tumor-infiltrating lymphocytes(TILs), are collected from samples of the tumor. TILs that show the greatest recognition of the patient's tumor cells in laboratory tests are selected, and large populations of these cells are grown in the laboratory. The cells are then activated by treatment with immune system signaling proteins called cytokines and infused into the patient’s bloodstream.

 

Another form of ACT that is being actively studied is CAR T-cell therapy. In this treatment approach, a patient’s T cells are collected from the blood and genetically modified to express a protein known as a chimeric antigen receptor, or CAR. Next, the modified cells are grown in the laboratory to produce large populations of the cells, which are then infused into the patient.

Therapeutic Antibodies; Therapeutic antibodies are antibodies made in the laboratory that are designed to cause the destruction of cancer cells. One class of therapeutic antibodies, called antibody–drug conjugates (ADCs), has proven to be particularly effective, with several ADCs having been approved by the FDA for the treatment of different cancers.

ADCs are created by chemically linking antibodies, or fragments of antibodies, to a toxic substance. The antibody portion of the ADC allows it to bind to a target molecule that is expressed on the surface of cancer cells. The toxic substance can be a poison, such as a bacterial toxin; a small-molecule drug; or a radioactive compound. Once an ADC binds to a cancer cell, it is taken up by the cell and the toxic substance kills the cell.

The FDA has approved several ADCs for the treatment of patients with cancer, including:

·               ado-trastuzumab emtansine (Kadcyla®) for the treatment of some types of breast cancer

·               brentuximab vedotin (Adcetris®) for Hodgkin lymphoma and a type of non-Hodgkin T-cell lymphoma 

·               ibritumomab tiuxetan (Zevalin®) for a type of non-Hodgkin B-cell lymphoma

 rituximab (Rituxan,  denileukin diftitox (ONTAK®)

Cancer Treatment Using Vaccines:

The use of cancer treatment (or therapeutic) vaccines is another approach to immunotherapy. These vaccines are usually made from a patient’s own tumor cells or from substances produced by tumor cells. They are designed to treat cancers that have already developed by strengthening the body’s natural defenses against the cancer.

In 2010, the FDA approved the first cancer treatment vaccine, sipuleucel-T (Provenge®), for use in some men with metastatic prostate cancer. Other therapeutic vaccines are being tested in clinical trials to treat a range of cancers, including brain, breast, and lung cancer.

Cancer immunotherapy via dendritic cells: Karolina Palucka1,2 & Jacques Banchereau3  Nature Reviews Cancer 12, 265-277 (April 2012)

Cancer immunotherapy attempts to harness the power and specificity of the immune system to treat tumours. The molecular identification of human cancer-specific antigens has allowed the development of antigen-specific immunotherapy. In one approach, autologous antigen-specific T cells are expanded ex vivo and then re-infused into patients. Another approach is through vaccination; that is, the provision of an antigen together with an adjuvant to elicit therapeutic T cells in vivo. Owing to their properties, dendritic cells (DCs) are often called 'nature's adjuvants' and thus have become the natural agents for antigen delivery. After four decades of research, it is now clear that DCs are at the Centre of the immune system owing to their ability to control both immune tolerance and immunity. Thus, DCs are an essential target in efforts to generate therapeutic immunity against cancer.

Despite current successes in hematological cancers, we are only in the beginning of exploring the powerful potential of CAR redirected T cells in the control and elimination of resistant, metastatic, or recurrent non-hematological cancers. This review discusses the application of the CAR T cell therapy, its challenges, and strategies for successful clinical and commercial translation.

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https://www.ncbi.nlm.nih.gov

Elements involved in TCR and CAR recognition and activation. The TCR is disulfide-linked heterodimer consisting of one α and one β chain expressed in complex with invariant CD3 chains (γ, δ, ζ, and ε). The TCR recognizes intracellular or extracellular proteins presented as peptides by MHC molecules. Costimulation of CD28 through its ligands, CD80/CD86, is required for optimal activation and production of interleukin-2 (IL-2) and other cytokines. While most hematological tumors express costimulatory molecules, solid tumor cells as well as antigen presenting cells in the tumor microenvironment usually lack such molecules. CARs recognize surface antigens in an MHC unrestricted manner. CARs are fusion proteins between single-chain variable fragments (scFv) from a monoclonal antibody and one or more T cell receptor intracellular signaling domains. Various hinges and transmembrane (TM) domains are used to link the recognition and the signaling molecules [5]. While first generation CARs signaled through the CD3ζ chain only, second generation CARs include a signaling domain from a costimulatory molecule, for example, CD28 (illustrated), 4-1BB, OX40, CD27, or ICOS.

 

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Example of manufacturing and delivery pipeline of CAR T cell therapies: Peripheral blood mononuclear cells (PBMCs) are harvested from the patient (or a T cell donor) (a) and transferred to a good manufacturing practice (GMP) facility, where the T cells are isolated and activated in the presence of magnetic beads conjugated with CD3 and CD28 antibodies (b) and subsequently genetically engineered by viral transduction to express the CAR (c). The activated T cells are expanded ex vivo for a period, typically 10–14 days, to reach a therapeutic relevant number (d) before magnetic bead removal (e) and formulation, either for freezing or for adoptive transfer (f). The patient undergoes a conditional chemotherapy prior to infusion of the CAR T cells (g). https://www.ncbi.nlm.nih.gov

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With sequence of a patient, in hand scientists can diagnose genetic disease, identify future disease risk, home in on disease modifiers and predict responses to drugs. sequence of a patient, scientists can diagnose genetic disease, identify future disease risk, home in on disease modifiers and predict responses to drugs. http://www.moleculargenetics.utoronto.ca/

Cell death. 

 

Approved antibodies include alemtuzumab, ipilimumab, nivolumab, ofatumumab and rituximab. https://www.technologyreview.com; Antonio Regalado, https://en.wikipedia.org.

Therapeutic Antibodies; https://www.ncbi.nlm.nih.gov; Masami Suzuki et al; Therapeutic antibodies: their mechanisms of action and the pathological findings they induce in toxicity studies; From the mid-1990’s, a series of therapeutic antibodies were launched that are now being used in clinic. The disease areas that therapeutic antibodies can target have subsequently expanded, and antibodies are currently utilized as pharmaceuticals for cancer, inflammatory disease, organ transplantation, cardiovascular disease, infection, respiratory disease, ophthalmologic disease, and so on.  1) the high level of specificity and affinity to the target molecule or antigen achieves a high level of efficacy and fewer adverse events, 2) their ability to target diverse molecules and the modes of action of the antibodies allow them to be applied to a wide range of therapeutic targets, and 3) modification and refinement by genetic engineering technology and the establishment of recombinant manufacturing technology has made industrial manufacturing possible. The efficacy of therapeutic antibodies stems from various natural functions of antibodies — neutralization, antibody-dependent cell-mediated cytotoxic (ADCC) activity, or complement-dependent cytotoxic (CDC) activity —, or the antibody can be utilized as a drug delivery carrier (missile therapy)

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Mechanisms of action of therapeutic antibodies. https://www.ncbi.nlm.nih.gov

 

America’s biopharmaceutical research companies are using biological processes to develop 907 medicines and vaccines targeting more than 100 diseases. Many biologics are made from a variety of natural sources—human, animal or microorganisms. Like small-molecule drugs, some biologics are intended to treat diseases and medical conditions. Other biologics are used to prevent or diagnose disease. Examples of biological products include but are not limited to.

http://phrma-docs.phrma.org/ monoclonal antibodies • vaccines, including therapeutic vaccines • blood and blood products for transfusion and/or manufacturing into other products • gene therapies • cell therapies

 

The medicines discussed in this report are either in human clinical trials or under review by the U.S. Food and Drug Administration (FDA). These medicines often represent cutting-edge research in which the latest scientific discoveries are translated into novel therapies that provide new treatment options for patients. Increased understanding of the molecular and genetic bases of disease has opened up the development of a range of targeted treatments. For instance, monoclonal antibodies (mAbs) are proteins that help the immune system identify and bind to foreign substances. Thirty years after initial development, these therapies help treat some of the most costly and challenging diseases.

·               Self -antigens that are not normally made by the tissue in which the cancer developed (for example, antigens that are normally made only by embryonic tissue but are expressed in an adult cancer) and, thus, are viewed as foreign by the immune system

·               Newly formed antigens, or neo-antigens, that result from gene mutations in cancer cells and have not been seen previously by the immune system

·               Vaccines are medicines that boost the immune system's natural ability to protect the body against “foreign invaders,” mainly infectious agents,  that may cause disease. Cancer vaccines belong to a class of substances known as biological response modifiers. Biological response modifiers work by stimulating or restoring the immune system’s ability to fight infections and disease. There are two broad types of cancer vaccines: Preventive (or prophylactic) vaccines, which are intended to prevent cancer from developing in healthy people

·               Treatment (or therapeutic) vaccines, which are intended to treat an existing cancer by strengthening the body’s natural immune response against the cancer. Treatment vaccines are a form of immunotherapy.

Two types of cancer preventive vaccines (human papillomavirus vaccines and hepatitis B virus vaccines) are available in the United States, and one treatment vaccine (for metastatic prostate cancer) is available.

 

·               Immune System Modulators

The immune system’s natural capacity to detect and destroy abnormal cells may prevent the development of many cancers. However, cancer cells are sometimes able to avoid detection and destruction by the immune system. Cancer cells may: reduce the expression of tumor antigens on their surface, making it harder for the immune system to detect them; express proteins on their surface that induce immune cell inactivation, induce cells in the surrounding environment (microenvironment) to release substances that suppress immune responses and promote tumor cell proliferation and survival.

Immune check point Modulators:

One immunotherapy approach is to block the ability of certain proteins, called immune checkpoint proteins, to limit the strength and duration of immune responses. These proteins normally keep immune responses in check by preventing overly intense responses that might damage normal cells as well as abnormal cells. But, researchers have learned that tumors can commandeer these proteins and use them to suppress immune responses. Blocking the activity of immune checkpoint proteins releases the "brakes" on the immune system, increasing its ability to destroy cancer cells.

The first such drug to receive approval by FDA,  ipilimumab  (Yervoy®), for the treatment of advanced melanoma, blocks the activity of a checkpoint protein known as CTLA4, which is expressed on the surface of activated immune cells called cytotoxic T lymphocytes. CTLA4 acts as a "switch" to inactivate these T cells, thereby reducing the strength of immune responses; ipilimumab binds to CTLA4 and prevents it from sending its inhibitory signal.

Immune Cell Therapy;

Progress is also being made with an experimental form of immunotherapy called adoptive cell transfer (ACT). In several small clinical trials testing ACT, some patients with very advanced cancer—primarily blood cancers—have had their disease completely eradicated. In some cases, these treatment responses have lasted for years.

In one form of ACT, T cells that have infiltrated a patient’s tumor, called tumor-infiltrating lymphocytes(TILs), are collected from samples of the tumor. TILs that show the greatest recognition of the patient's tumor cells in laboratory tests are selected, and large populations of these cells are grown in the laboratory. The cells are then activated by treatment with immune system signaling proteins called cytokines and infused into the patient’s bloodstream.

Another form of ACT that is being actively studied is CAR T-cell therapy. In this treatment approach, a patient’s T cells are collected from the blood and genetically modified to express a protein known as a chimeric antigen receptor, or CAR. Next, the modified cells are grown in the laboratory to produce large populations of the cells, which are then infused into the patient.

Therapeutic Antibodies; Therapeutic antibodies are antibodies made in the laboratory that are designed to cause the destruction of cancer cells. One class of therapeutic antibodies, called antibody–drug conjugates (ADCs), has proven to be particularly effective, with several ADCs having been approved by the FDA for the treatment of different cancers. ADCs are created by chemically linking antibodies, or fragments of antibodies, to a toxic substance. The antibody portion of the ADC allows it to bind to a target molecule that is expressed on the surface of cancer cells. The toxic substance can be a poison, such as a bacterial toxin; a small-molecule drug; or a radioactive compound. Once an ADC binds to a cancer cell, it is taken up by the cell and the toxic substance kills the cell.

Cancer Treatment Vaccines

The use of cancer treatment (or therapeutic) vaccines is another approach to immunotherapy. These vaccines are usually made from a patient’s own tumor cells or from substances produced by tumor cells. They are designed to treat cancers that have already developed by strengthening the body’s natural defenses against the cancer.

In 2010, the FDA approved the first cancer treatment vaccine, sipuleucel-T (Provenge®), for use in some men with metastatic prostate cancer. Other therapeutic vaccines are being tested in clinical trials to treat a range of cancers, including brain, breast, and lung cancer.

Tumour immunology & immunotherapy;

Cancer immunotherapy via dendritic cells

Karolina Palucka1,2 & Jacques Banchereau3   Nature Reviews Cancer 12, 265-277 (April 2012) 

Cancer immunotherapy attempts to harness the power and specificity of the immune system to treat tumours. The molecular identification of human cancer-specific antigens has allowed the development of antigen-specific immunotherapy. In one approach, autologous antigen-specific T cells are expanded ex vivo and then re-infused into patients. Another approach is through vaccination; that is, the provision of an antigen together with an adjuvant to elicit therapeutic T cells in vivo. Owing to their properties, dendritic cells (DCs) are often called 'nature's adjuvants' and thus have become the natural agents for antigen delivery. After four decades of research, it is now clear that DCs are at the Centre of the immune system owing to their ability to control both immune tolerance and immunity. Thus, DCs are an essential target in efforts to generate therapeutic immunity against cancer.

Despite current successes in hematological cancers, we are only in the beginning of exploring the powerful potential of CAR redirected T cells in the control and elimination of resistant, metastatic, or recurrent nonhematological cancers. This review discusses the application of the CAR T cell therapy, its challenges, and strategies for successful clinical and commercial translation.

 

Gene therapy: The first attempt at modifying human DNA was performed in 1980 by Martin Cline, but the first successful and approved nuclear gene transfer in humans was performed in May 1989; Between 1989 and February 2016, over 2,300 clinical trials had been conducted, more than half of them in phase I, The first attempt, an unsuccessful one, at gene therapy (as well as the first case of medical transfer of foreign genes into humans not counting organ transplantation) was performed by Martin Cline on 10 July 1980-for  treating beta-thalassemia;

 

The first commercial gene therapy, Gendicine, was approved in China in 2003 for the treatment of certain cancers; In 2011 Neovasculgen was registered in Russia as the first-in-class gene-therapy drug for treatment of peripheral artery disease, including critical limb ischemia.  In 2012 Glybera, a treatment for a rare inherited disorder, became the first treatment to be approved for clinical use in either Europe or the United States after its endorsement by the European Commission;

Somatic cell gene therapy - SCGT. Germline gene therapy GGT; A number of viruses have been used for human gene therapy, including retrovirus, adenovirus, lentivirus, herpes simplex, vaccinia and adeno-associated virus.

 

Antisense therapies; Cancer, Cytomegalovirus retinitis, familial hypercholesterol, Hemorrhagic fever viruses; HIV/AIDS, Spinal muscular atrophy, Duchenne muscular dystrophy, Hypertriglyceridemia, Hypertriglyceridemia, color blindness

 

                                    Immune Deficiency

http://learn.genetics.utah.edu/

 

 

 

 

Severe Combined Immune Deficiency (SCID)

 

Hereditary Blindness

http://learn.genetics.utah.edu/c-blindness

Advances in the field of epigenetics have allowed the design of new therapeutic strategies to address complex diseases such as type 1 diabetes (T1D). Clustered regularly interspaced short palindromic repeats (CRISPR)-on is a novel and powerful RNA-guided transcriptional activator system that can turn on specific gene expression; however, it remains unclear whether this system can be widely used or whether its use will be restricted depending on cell types, methylation promoter statuses or the capacity to modulate chromatin state. Our results revealed that the CRISPR-on system fused with transcriptional activators (dCas9-VP160) activated endogenous human INS, which is a silenced gene with a fully methylated promoter. Similarly, we observed a synergistic effect on gene activation when multiple single guide RNAs were used, and the transcriptional activation was maintained until day 21. Regarding the epigenetic profile, the targeted promoter gene did not exhibit alteration in its methylation status but rather exhibited altered levels of H3K9ac following treatment. Importantly, we showed that dCas9-VP160 acts on patients’ cells in vitro, particularly the fibroblasts of patients with T1D. CRISPR-on system for the activation of the endogenous human INS gene; http://www.nature.com/;C A Giménez, M Ielpi, A Mutto, L Grosembacher, P Argibay and F Pereyra-Bonnet

 

 

 

Cancer cells;

Inherited; Genetics specialists estimate that only about 2 or 3 in every 100 cancers diagnosed (2 to 3%) are linked to an inherited gene fault. Pancreatin cancer is prevelant;  Globally, 337,872 new pancreatic cancer cases and 330,391 deaths were estimated in 2012’; BRCA1, BRCA2, and p53 are examples of tumor suppressor genes, DNA repair genes; cell cycle regulatory genes,

 

Cell mediated therapy; Elizabeth J. Akins1 and Purnima Dubey

https://www.ncbi.nlm.nih.gov

 The fairly recent use of molecular imaging techniques to track cell populations has presented researchers and clinicians with a powerful diagnostic tool for determining the efficacy of cell-mediated therapy for the treatment of cancer.  ell mediated therapy /immunotherapy was found to be effective only in few cases. This review highlights the application of whole-body noninvasive radioisotopic, magnetic, and optical imaging methods for monitoring effector cells in vivo. Issues that affect sensitivity of detection, such as methods of cell marking, efficiency of cell labeling, toxicity, and limits of detection of imaging modalities, are discussed.

 

It is thought that the immune system regularly detects and destroys transformed cells, a process termed immune-surveillance ; Immunotherapy seeks to modulate immune function to target the cancerous cells that have evaded immune surveillance. Therefore, harnessing the power of the adaptive cellular immune system to eradicate tumors, while sparing normal tissues, is an attractive potential therapy for the treatment of cancer, particularly metastatic disease.

Major Effector cells used in cancer therapy include CD8+ cytotoxic T cells, CD4+ helper T cells, CD4+ CD25+ regulatory T cells, and natural killer cells CD8-CTLs, CD4 Thl,Th2,  Regulatory T cells, NK1 (Naturla killer cells). Peptide fragments of antigens expressed by tumor cells are presented to the immune system by professional antigen-presenting cells (APCs), such as dendritic cells (DCs). As a result, cytolytic (CD8+) and helper (CD4+) T cells proliferate and release effector cell cytokines. When tumoricidal activity can be detected, cytolytic T cells (CTLs) are the primary effector cells, whereas CD4+ T helper cells provide help in the form of secreted cytokines. The generation of an effective antitumor immune response requires the concerted activity of all of these components of the immune system

 

https://www.ncbi.nlm.nih.gov

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https://www.ncbi.nlm.nih.gov

 

 “Best-case” scenario:  CD8+ T cells are activated in draining lymph node (LN) by DCs expressing tumor antigen peptides on class I MHC (HLA-A, HLA-B, and HLA-C). Once activated, CD8+ T cells home to site of tumor, invading stroma to gain access to tumor cells. Antigen-expressing tumor cells are recognized and lysed by CD8+ T cells. HEV = high endothelial venule.

Therapeutic monoclonal antibodies target specific antigens found on the cell surface, such as transmembrane receptors or extracellular growth factors. In some cases, monoclonal antibodies are conjugated to radio-isotopes or toxins to allow specific delivery of these cytotoxic agents to the intended cancer cell target.

Small molecules can penetrate the cell membrane to interact with targets inside a cell. Small molecules are usually designed to interfere with the enzymatic activity of the target protein. For example, the small molecule STI-571 became known as imatinib (generic name) and is marketed by Novartis under the brand name Gleevec.

https://molcelltherapies.biomedcentral.com- read, it looks  GOOOOD

Genetically Engineered Pharmaceuticals;

Factor VIII for males suffering from hemophilia A; Hemophilia A (HA, OMIM) is an X-linked bleeding disorder caused by heterogeneous mutations in the factor VIII gene (F8). The FVIII protein is required for propagation of the intrinsic coagulation pathway [1]. Hemophilia A, or congenital factor VIII deficiency, is the most common of the inherited bleeding disorders, its incidence is estimated to be between 1:5,000 and 1:10,000 in men http://www.sciencedirect.com/ Factor VIII (F8) is the only gene known to be associated with hemophilia A. F8 maps to the distal end of the long arm of the X-chromosome (Xq28) and spans 186 kb of genomic DNA. It consists of 26 exons that encode a 2351 amino acid precursor polypeptide. The mature FVIII protein consists of three homologous A domains, two homologous C domains and the unique B domain, which are arranged in the order A1-A2-B-A3-C1-C2 from the amino terminus to the carboxyl-terminal end. The different domains play an important role in the function of FVIII as each domain contains specific binding sites for different components of the clotting cascade  and  Genetic defects can affect these interaction sites and cause HA.

Factor IX; FactorX for hemophilia B- Hemophilia B is a rare genetic bleeding disorder in which affected individuals have insufficient levels of a blood protein called factor IX. Factor IX is a clotting factor. Clotting factors are specialized proteins needed for blood clotting, the process by which blood seals a wound to stop bleeding and promote healing. Individuals with hemophilia B do not bleed faster than unaffected individuals, they bleed longer. This is because they are missing a protein involved in blood clotting and are unable to effectively stop the flow of blood from a wound, injury or bleeding site. This is sometimes referred to as prolonged bleeding or a bleeding episode. The FDA approved the drug Idelvion, coagulation Factor IX (recombinant), albumin fusion protein in 2016. The drug is available for children and adults affected by Hemophilia B. Idelvion are manufactured by CSL Behring. https://rarediseases.org;

Ixinity, a recombinant factor IX product, was approved in 2015 for the control and prevention of bleeding episodes and for perioperative management in adults and children 12 years of age or older with hemophilia B. This medication is manufactured by Emergent BioSolutions, Inc.

Recombinant Factor IX: Recombinant factor IX products are manufactured in a laboratory. These genetically engineered products do not contain animal or human protein and are not derived from human blood; they are theoretically considered to be free of the risk of transmitting viruses. Recombinant factor IX therapy is the recommended treatment for individuals with hemophilia B. In the U.S., the currently available recombinant factor IX products are BeneFIX®, Rixubis®, and Alprolix®.  https://rarediseases.org

Manufacturingdrugs; Genetically modified organism, Gene therapy https://en.wikipedia.org

Organisms are genetically engineered to discover the functions of certain genes. This could be the effect on the phenotype of the organism, where the gene is expressed or what other genes it interacts with. These experiments generally involve loss of function, gain of function, tracking and expression.

·                Loss of function experiments, such as in a gene knockout experiment, in which an organism is engineered to lack the activity of one or more genes. A knockout experiment involves the creation and manipulation of a DNA construct in vitro, which, in a simple knockout, consists of a copy of the desired gene, which has been altered such that it is non-functional. Embryonic stem cells incorporate the altered gene, which replaces the already present functional copy. These stem cells are injected into blastocysts, which are implanted into surrogate mothers. This allows the experimenter to analyze the defects caused by this mutation and thereby determine the role of particular genes. It is used especially frequently in developmental biology. Another method, useful in organisms such as Drosophila (fruit fly), is to induce mutations in a large population and then screen the progeny for the desired mutation. A similar process can be used in both plants and prokaryotes. Loss of function tells whether or not a protein is required for a function, but it does not always mean it's sufficient, especially if a function requires multiple proteins and lose the said function if one protein is missing.

·                Gain of function experiments, the logical counterpart of knockouts. These are sometimes performed in conjunction with knockout experiments to more finely establish the function of the desired gene. The process is much the same as that in knockout engineering, except that the construct is designed to increase the function of the gene, usually by providing extra copies of the gene or inducing synthesis of the protein more frequently. Gain of function is used to tell whether or not a protein is sufficient for a function, but it does not always mean it's required. Especially when dealing with genetic/functional redundancy.

·                Tracking experiments, which seek to gain information about the localization and interaction of the desired protein. One way to do this is to replace the wild-type gene with a 'fusion' gene, which is a juxtaposition of the wild-type gene with a reporting element such as green fluorescent protein (GFP) that will allow easy visualization of the products of the genetic modification. While this is a useful technique, the manipulation can destroy the function of the gene, creating secondary effects and possibly calling into question the results of the experiment. More sophisticated techniques are now in development that can track protein products without mitigating their function, such as the addition of small sequences that will serve as binding motifs to monoclonal antibodies.

·                Expression studies aim to discover where and when specific proteins are produced. In these experiments, the DNA sequence before the DNA that codes for a protein, known as a gene's promoter, is reintroduced into an organism with the protein coding region replaced by a reporter gene such as GFP or an enzyme that catalyzes the production of a dye. Thus the time and place where a particular protein is produced can be observed. Expression studies can be taken a step further by altering the promoter to find which pieces are crucial for the proper expression of the gene and are actually bound by transcription factor proteins; this process is known as promoter bashing.

 

One can manufacture mass quantities of the protein by growing the transformed organism in bioreactor equipment using techniques of industrial fermentation, and then purifying the protein.[94] Some genes do not work well in bacteria, so yeast, insect cells, or mammalians cells, each a eukaryote, can also be used. For example, in Alzheimer's disease, brain cells, or neurons, start to die off because of defective DNA. If doctors could grow new neurons from the patient's stem cells, they could replace the dying cells in the brain with cells engineered to have normal DNA, curing the disease. Currently, there is no cure for Alzheimer's and people's neurons slowly deteriorate over time, eventually making them unable to do even the most basic self-care. http://study.com/academy,

 

Some of the vast range of human substances being genetically made;

o   Human epidermal growth factor (940);  This gene encodes a member of the epidermal growth factor superfamily. The gene is located in chromosome 4.  The encoded preproprotein is proteolytically processed to generate the 53-amino acid epidermal growth factor peptide. This protein acts a potent mitogenic factor that plays an important role in the growth, proliferation and differentiation of numerous cell types. This protein acts by binding with high affinity to the cell surface receptor, epidermal growth factor receptor. Defects in this gene are the cause of hypomagnesemia type 4. Dysregulation of this gene has been associated with the growth and progression of certain cancers. Alternative splicing results in multiple transcript variants, at least one of which encodes a preproprotein that is proteolytically processed. [provided by RefSeq, Jan 2016] https://www.ncbi.nlm.nih.gov

 

Epidermal growth factor (EGF) is a growth factor that stimulates cell growth, proliferation, and differentiation by binding to its receptor EGFR-6045 Da protein-with 53a.a.

 

1a3p egf.png

https://en.wikipedia.org

EGF Family of proteins; Heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor-α (TGF-α), Amphiregulin (AR), Epiregulin (EPR), EpigenBetacellulin (BTC), neuregulin-1 (NRG1) neuregulin-2 (NRG2) neuregulin-3 (NRG3).neuregulin-4 (NRG4). Recombinant human epidermal growth factor, sold under the brand name Heberprot-P, is used to treat diabetic foot ulcers. This epidermal growth factor I cloned in plant soybean

 

 

5. Tissue plasminogen activator (980); Tissue plasminogen activator (abbreviated tPA or PLAT) is a protein involved in the breakdown of blood clots. It is a serine protease (EC 3.4.21.68) found on endothelial cells, the cells that line the blood vessels.  As an enzyme, it catalyzes the conversion of plasminogen to plasmin, the major enzyme responsible for clot breakdown. Because, it works on the clotting system, tPA (such as alteplase, reteplase, and tenecteplase) is used in clinical medicine to treat embolic or thrombotic stroke. Use is contraindicated in hemorrhagic stroke and head trauma. The antidote for tPA in case of toxicity is aminocaproic acid. tPA may be manufactured using recombinant biotechnology techniques. tPA created this way may be referred to as recombinant tissue plasminogen activator (rtPA). It is sold as alteplase.

tPA is used in some cases of diseases that feature blood clots, such as pulmonary embolism, myocardial infarction, and stroke, in a medical treatment called thrombolysis. The most common use is for ischemic stroke. It can either be administered systemically, in the case of acute myocardial infarction, acute ischemic stroke, and most cases of acute massive pulmonary embolism, or administered through an arterial catheter directly to the site of occlusion in the case of peripheral arterial thrombi and thrombi in the proximal deep veins of the leg.[3] It has been suggested that if tPA is effective in ischemic stroke, it must be administered as early as possible after the onset of stroke symptoms.

 

https://upload.wikimedia.org/wikipedia/commons/thumb/0/0e/Fibrinolysis.png/250px-Fibrinolysis.png

A simplified illustration demonstrates clot breakdown (fibrinolysis), with blue arrows denoting stimulation, and red arrows inhibition. https://en.wikipedia.org/ https://en.wikipedia.org/

 

6. Human adenosine deaminase (990); Gene therapy for adenosine deaminase; Fabio Candotti,1 Kit L. Shaw,2 Linda Muul, 1 G. Jayashree Jagadeesh1 et al.

 

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https://www.ncbi.nlm.nih.gov

 

Ex vivo gene transfer to bone marrow-derived CD34 hematopoietic stem cells (HSCs) of a patient with inherited severe immune deficiency. Autologous HSCs are cultured and infected with a recombinant retroviral or lentiviral vector carrying a functional copy of the defective gene (for example, ADA, gamma-c, or ABCD1). The gene-corrected cells are then injected back into the patient. For some protocols, the patients may receive mild myeloablation prior to infusion of the HSCs.

 

 Adenosine deaminase (ADA) is an enzyme involved in purine metabolism and is essential for lymphocyte development, survival, and function. ADA deficiency is an autosomal-recessive inherited disorder that can result in severe combined immunodeficiency (SCID). Affected infants with ADA-deficient SCID usually are diagnosed by 6 months of age, after presenting with failure to thrive and recurrent opportunistic infections, because of profound pan-lymphopenia and the virtual absence of humoral and cellular immunity. ADA-deficient SCID is almost always fatal by 2 years of age if immunity is not restored.

We conducted a gene therapy trial in 10 patients with adenosine deaminase (ADA)–deficient severe combined immunodeficiency using 2 slightly different retroviral vectors for the transduction of patients' bone marrow CD34+ cells.  Four subjects were treated without pretransplantation cytoreduction and remained on ADA enzyme-replacement therapy (ERT) throughout the procedure. Only transient (months), low-level (< 0.01%) gene marking was observed in PBMCs of 2 older subjects (15 and 20 years of age), whereas some gene marking of PBMC has persisted for the past 9 years in 2 younger subjects (4 and 6 years). Six additional subjects were treated using the same gene transfer protocol, but after withdrawal of ERT and administration of low-dose busulfan (65-90 mg/m2). Three of these remain well, off ERT (5, 4, and 3 years postprocedure), with gene marking in PBMC of 1%-10%, and ADA enzyme expression in PBMC near or in the normal range. Two subjects were restarted on ERT because of poor gene marking and immune recovery, and one had a subsequent allogeneic hematopoietic stem cell transplantation. These studies directly demonstrate the importance of providing nonmyeloablative pretransplantation conditioning to achieve therapeutic benefits with gene therapy for ADA-deficient severe combined immunodeficiency.

 

Human Factor VIII (1040)-  Factor VIII (FVIII), an essential blood coagulation protein, is a key component of the fluid phase blood coagulation system. Human factor VIII is a single chain of about 300 kDa consisting of domains described as A1-A2-B-A3-C1- C2. The protein undergoes processing prior to secretion into blood resulting in a heavy chain of 200 kDa (A1-A2-B) and a light chain of 80 kDa (A3-C1-C2) linked by metal ions. The role of factor VIII is to increase the catalytic efficiency of factor IXa in the activation of factor X. Variants of these factors lead frequently also to severe bleeding disorders. http://www.actabp.pl Anna Mazurkiewicz-Pisarek et al

 

Hepatitis B vaccine SK & F 1986 Hepatitis B vaccine; Hepatitis B vaccine produced cheaply and efficiently using GMO corn.  SkepticalRapto According to the World Health Organization, nearly 800,000 individuals die each year worldwide as a result of hepatitis B, an infectious virus that afflicts the liver. A safe and effective vaccine has been available since 1982 and is typically administered via three intramuscular injections over a period of about six months. The vaccine is recommended for babies, children and adults. https://www.geneticliteracyproject.org/

 

http://images.dailykos.com/images/117268/story_image/gmo_corn_vaccines.jpg?1416783405

http://www.dailykos.com/story

 

According to the World Health Organization, nearly 800,000 individuals die each year worldwide as a result of hepatitis B, an infectious virus that afflicts the liver. A safe and effective vaccine has been made available since 1982 and is typically administered via three intramuscular injections over a period of about 6 months. The vaccine is recommended for babies, children and adults.

·            Engerix B; Hapatitis B vaccination; Recombivax HB: 1 mL (10 mcg) IM at 0, 1, and 6 months. Diabetics who 60 years or older at the discretion of the treating clinician based on increased need for assisted blood glucose monitoring in long-term care facilities, likelihood of acquiring hepatitis B infection, its complications or chronic sequelae, and likelihood of immune response to vaccination.

 

Digoxin monoclonal: 

Your heart is an amazing powerhouse that pumps and circulates 5 or 6 gallons of blood each minute through your entire body.

http://www.medicinenet.com/digoxin/article.htm

 

Well come 1986 Digoxin antidote; brand name Lanoxin, Lanoxin pediatricDigoxin is indicated for the treatment of mild to moderate heart failure in adults. Digoxin increases left ventricular ejection fraction and improves heart failure symptoms as evidenced by improved exercise capacity and decreased heart failure-related hospitalizations and emergency care, while having no effect on mortality. Where possible, digoxin should be used in combination with a diuretic and an angiotensin-converting enzyme (ACE) inhibitor.

 

Digoxin is used to treat heart failure, usually along with other medications. It is also used to treat a certain type of irregular heartbeat (chronic atrial fibrillation); Cardiac glycosides include digoxin, digitoxin, digitalis and ouabain. Of these, only digoxin is in regular use in the UK. Prescribing digoxin is not difficult, providing certain principles are followed. Digoxin acts by inhibiting cell membrane sodium/potassium ATPase which leads to reversal of the usual sodium/calcium exchange. An increased intracellular calcium level results which, in myocardial muscle, has the effect of enhancing the strength of contraction (positive ionotropism). It also affects the electrical physiology of the heart, blocking atrioventricular (AV) conduction and reducing the heart rate by enhancing vagal nerve activity (negative chronotropy). The principal indication is permanent/persistent Atrial fibrillation AF with a fast ventricular rate - although it it is not the preferred first-line medication (especially as digoxin prevents the normal rise in heart rate associated with exertion). A loading dose should be given of 15 micrograms/kg of lean body weight. For a woman with a lean body weight of 50 kg this would work out at a total dose of 15 x 50 = 750 micrograms. Lean body weight is defined as total body mass minus fat mass. There are a number of methods for determining this but where it is clinically significant the simplest method in primary care is the use of skin calipers; Digoxin, sold under the brand name Lanoxin among others, is a medication used to treat various heart conditions. Most frequently it is used for atrial fibrillation, atrial flutter, and heart failure. Digoxin is taken by mouth or by injection into a vein. Digoxin was first isolated in 1930 from the foxglove plant, Digitalis lanata. The most common indications for digoxin are atrial fibrillation and atrial flutter with rapid ventricular response.

 

Antibody (Digibind)

Orthoclonal OKT3 Cilag 1986 Rejection prophylaxis; Muromonab-CD3 (trade name Orthoclone OKT3, marketed by Janssen-Cilag) is an immunosuppressant drug given to reduce acute rejection in patients with organ transplants.  It is a monoclonal antibody targeted at the CD3 receptor,[3] a membrane protein on the surface of T cells. It was the first monoclonal antibody to be approved for clinical use in humans; T cells recognize antigens primarily via the T cell receptor. This receptor needs various co-receptors to function, one of which is CD3. The T cell receptor-CD3 complex transduces the signal for the T cell to proliferate and attack the antigen.

Muromonab-CD3 is a murine (mouse) monoclonal IgG2a antibody which was created using hybridoma technology. It binds to the T cell receptor-CD3-complex (specifically the CD3 epsilon chain) on the surface of circulating T cells, initially leading to activation, but subsequently inducing blockage and apoptosis of the T cells. This protects the transplant against the T cells.   After application of muromonab-CD3, normal T cell function is said to be restored within a week. When administered for transplant induction, the drug is administered daily thereafter for up to 7 days.

DIGIBIND, Digoxin Immune Fab (Ovine), is a sterile lyophilized powder of antigen binding fragments (Fab) derived from specific antidigoxin antibodies raised in sheep. Production of antibodies specific for digoxin involves conjugation of digoxin as a hapten to human albumin. Sheep are immunized with this material to produce antibodies specific for the antigenic determinants of the digoxin molecule. The antibody is then papain-digested and digoxin-specific Fab fragments of the antibody are isolated and purified by affinity chromatography. These antibody fragments have a molecular weight of approximately 46,200.

 

Each vial, which will bind approximately 0.5 mg of digoxin (or digitoxin), contains 38 mg of digoxin-specific Fab fragments derived from sheep plus 75 mg of sorbitol as a stabilizer and 28 mg of sodium chloride. The vial contains no preservatives. DIGIBIND (digoxin immune fab) is administered by intravenous injection after reconstitution with Sterile Water for Injection (4 mL per vial). http://www.rxlist.com.

Somatotropin ( Eli Lilly 1987 ) Growth hormone (Huma trope) deficiency in children; Growth hormone (GH) is a 191 amino-acid single chain polypeptide, which is secreted by the somatotrophs in the anterior pituitary. With the recognition of its multiple and complex effects in the early 1960s, the physiology and regulation of GH has become a major area of research interest in the field of endocrinology. https://www.ncbi.nlm.nih.gov/ Its secretion is regulated by several factors including GHRH, somatostatin, ghrelin and IGF-1. Apart from its primary function of stimulation of GH secretion, GHRH plays an essential role in pituitary somatotroph development and proliferation. Somatostatin on the other hand is the main inhibitor of GH secretion. Along with its receptors, somatostatin has been extensively studied over the last decade as a treatment for acromegaly. Several somatostatin receptors (SSTR) have been identified, of which SSTR2 and SSTR5 exhibit greater inhibition on the secretion of GH by the somatotrophs. Somatostatin receptor ligands to SSTR2 and 5, such as octreotide, lanreotide and pasireotide, are approved treatment modalities for acromegaly in some countries. GH acts both directly through its own receptors and indirectly through the induced production of Insulin-like Growth Factor I (IGF-I). The “IGF-1 axis” holds a significant place in the field of endocrinology, with numerous research been done on its pharmacokinetics and pharmacodynamics, affecting different organ systems in humans. Its physiological effects have been demonstrated not only in tissue growth, but also in glucose / lipid metabolism, coronary disease, diabetes mellitus and vascular aging. The use of recombinant IGF-1 in IGF-1 deficiency and insulin insensitivity and the use of IGF-1 receptor inhibitors in the promotion of cellular apoptosis, especially in the management of malignancies, are two other main areas of research in prospect.

Next Gen, sequencing: http://www.ebi.ac.uk/

 

ASPIRIN: William L.smith et al;

Around 400 BC, Hippocrates, the "father of medicine" and the namesake of the Hippocratic oath taken by all physicians, administered willow-leaf tea ; Friedrich Bayer developed aspirin (acetylsalicylic acid) in 1897. Aspirin blocks COX which produces PG. Now more than 80 million tablets of it are being consumed every day in the United States alone, and for many more purposes.   Aspirin (Bayer) ,is called as Ecotrin, Ibuprofen (Advil, Motrin), and Naproxen (Aleve). Aspirin is that it irritates the stomach and in some cases cause ulcer and internal bleeding Chemically it is made up of acetylsalicylic acid, a derivative of salicylic acid; notably white willow and meadowsweet (Spirea ulmaria).

 

Although aspirin was introduced as a pain reliever nearly 100 years ago, how it actually works has remained a mystery until now. It is a non-steroidal anti-inflammatory drugs (NSAIDs). Aspirin like ibuprofen and indomethacin, aspirin emerged in 1899;  inhibits an enzyme that produces prostaglandins’  hormone-like messenger molecules that trigger processes in the body, including inflammation.

Prostaglandin H2 synthase, or PGHS. Using X-rays to probe the positions of atoms in tiny crystals of the enzyme, they showed that PGHS has a tunnel running into the middle of it. The raw material must pass through this tunnel to reach the core of the enzyme, where it will be converted into prostaglandin. Aspirin permanently attaches a portion of itself inside the tunnel, where it acts as a gate, blocking prostaglandin's precursor from reaching the "active site" of the enzyme.  There are two PGs-PGHS1 and PGHS2. It reduces swelling and is used to treat gout, rheumatoid arthritis, and inflammatory ailments.  Many people take low dosages (below 100 milligrams) daily for preventing recurrent stroke or heart attack.  Recent studies found it effective in reducing risks for colon and breast cancers.  Evidence is accumulating for similar effects in Alzheimer and other diseases.

Aspirin for primary prevention of cardiovascular and all-cause mortality events in diabetes: updated meta-analysis of randomized controlled trials. Nearly 2400 years ago Willow tree extracts were used for headache remedy. It contains Acetylsalicylic acid; 

 

                                                            http://higheredbcs.wiley.com/legacy/college/boyer/0471661791/cutting_edge/aspirin/aspirin.gif

http://annals.org/

 

 It suppresses the action of the enzyme COX, stops the production of prostaglandin, thus disrupting the pathways to pain, inflammation, elevated temperature, and stomach protection. At the molecular level, aspirin inhibits COX activity by forming a covalent bond with the enzyme. This effectively barricades the COX active site and blocks the synthesis of prostaglandin. COX-2, the therapeutic target of aspirin, is  made up of two identical subunits that form a dimer.

 

Aspirin's acetyl group attaches to serine 530, an amino acid that extends into a narrow, hydrophobic channel within the COX-2 enzyme that leads to its active site. When this amino acid is acetylated, the channel is blocked and arachidonic acid, the precursor to prostaglandin, can no longer reach the COX-2 catalytic site.

Aspirin effectively treats headaches, back and muscle pain, and other general aches and pains. Acts on arthritis; rheumatologic diseases, including osteoarthritis, rheumatoid arthritis, the spondyloarthropathies (inflammatory disorders that involve arthritis with other inflammatory and autoimmune responses), and the arthritis and pleurisy that can accompany lupus.  Heart Attack, stroke, fever Aspirin (1930) is derivative of salicylic acid. http://higheredbcs.wiley.com/l ;

How does aspirin accomplish all these effects? The enzyme that metabolizes arachidonic acid, the precursor to the prostaglandin product, is called cyclooxygenase. There are two forms of cyclooxygenase—cyclooxygenase 1 (COX-1), which produces prostaglandins in a normal physiological state; and cyclooxygenase 2 (COX-2), which mediates pain and inflammation in response to tissue damage. Aspirin inhibits both COX-1 and COX-2 irreversibly. While COX-2 is the therapeutic target of aspirin, it is aspirin's interaction with COX-1 in the gastrointestinal tract that causes the drug's unwanted side effects. COX-1 is needed to maintain a thick stomach lining. Because aspirin disables the COX-1 enzyme, regular use of this drug can lead to a thinning of the mucus that protects the stomach from gastric juices. Ulcers, stomach bleeding, and, in some cases, perforation of the stomach can occur. Thus, aspirin has both good and bad effects. It is very effective in alleviating pain through inhibition of the COX-2 pathway. However, when used for long periods and in high doses, aspirin can cause substantial medical problems by stopping the action of COX-1. It Acts on Cancer?

 

Ecosprin;  Ecosprin Tablet is a medicine that is used for the treatment of Blood thinning in heart disease and stroke, Heart attack, Nerve pain, Headache, Migraine, Toothache and other conditions. Ecosprin Tablet contains Aspirin as an active ingredient. Ecosprin Tablet works by reducing prostaglandin thus helping blood thinning and prevents clotting. Detailed information related to Ecosprin Tablet's uses, composition, dosage, side effects and reviews is listed below. Ecosprin Tablet is a medicine that is used for the treatment of Blood thinning in heart disease and stroke, Heart attack, Nerve pain, Headache, Migraine, Toothache and other conditions.  It is often called Aspirin delayed release tablets called Ecosprin-75.

 

Structure Of  Acetylsalicylic Acid

 

Ecosprin tablet contains Aspirin as an active ingredient(Acetyl salicyclic acid). Ecosprin Tablet works by reducing prostaglandin thus helping blood thinning and prevents clotting. Detailed information related to Ecosprin Tablet's uses, composition, dosage, side effects and reviews is listed below. Ecosprin Tablet is a medicine that is used for the treatment of Blood thinning in heart disease and stroke, Heart attack, Nerve pain, Headache, Migraine, Toothache, Sore throat, Joint pain and other conditionsInactivation of the cyclooxygenase (COX; officially known as prostaglandin-endoperoxide synthase, PTGS).  Greek word glukus (γλυκύς), meaning "sweet". The suffix "-ose" denotes a sugar. The name "dextrose" and the 'D-' prefix come from Latin dexter ("right"), referring to the handedness of the molecules. Aspirin (75 Mg)     .  The treatment of High cholesterol, Heart complications, Blood clots formation in arteries, Stroke, Headache, Migraine and other conditions. Lipikind Plus Capsule contains Aspirin, Atorvastatin, and Clopidogrel as active ingredients. Lipikind Plus Capsule works by blocking the enzyme required for the production of cholesterol in the body; slowing the platelets from sticking to blood vessels; reducing prostaglandin thus helping blood thinning and prevents clotting; http://www.medschat.com/

 

Saridon:  Propyphenazone, a pyrazolone derivative with anti-inflammatory, analgesic and antipyretic activity, was introduced in 1951 for the treatment of rheumatic disorders. Dyscrias;  replacement drug for aminophenazoneSaridon was assessed as more efficacious than ibuprofen, paracetamol, aspirin, and placebo. The currently global base formulation contains 135 mg of propyphenazone, 260 mg of paracetamol and 55 mg of caffeine- fast acting within 15 min. It contains caffeine.  The currently global base formulation contains 135 mg of propyphenazone, 260 mg of paracetamol and 55 mg of caffeine- fast acting within 15 min. 

Two skeletal formulas: left – caffeine, right – adenosine.

 

Manufacturers of caffeine tablets claim that using caffeine of pharmaceutical quality improves mental alertness- students see at the time of examination (Propyphenazone- non selective COX inhhibitor). The effects of caffeine on learning, memory, performance and coordination are rather related to the methylxanthine action on arousal, vigilance and fatigue. Caffeine exerts obvious effects on anxiety and sleep which vary according to individual sensitivity to the methylxanthine.

 

Lipikind Plus:

2-Lipikind Plus Capsule is composed of the following active ingredients (salts), Lipikind  tablet- Atorvastatin (10 Mg), Fenofibrate , Clopidogrel (75 Mg); Aspirin (75 Mg).

http://www.sigmaaldrich.com/content/dam/sigma-aldrich/structure2/082/mfcd00078159.eps/_jcr_content/renditions/mfcd00078159-large.png

 

Lipikind Plus Capsule is composed of the following 2 methoxy-1-naphthadehyde; active ingredients (salts); Lipikind F Tablet works by blocking the enzyme required for the production of cholesterol in the body; increasing the action of enzyme lipoprotein lipase to decrease the level of fats in the blood; active ingredients (salts), Atorvastatin (10 Mg), Clopidogrel (75 Mg), Aspirin (75 Mg). The treatment of High cholesterol, Heart complications, Blood clots formation in arteries, Stroke, Headache, Migraine and other conditions. Lipikind Plus Capsule contains Aspirin, Atorvastatin, and Clopidogrel as active ingredients. Lipikind Plus Capsule works by blocking the enzyme required for the production of cholesterol in the body; slowing the platelets from sticking to blood vessels; reducing prostaglandin thus helping blood thinning and prevents clotting; http://www.medschat.com/  http://www.tabletwise.com/

 

Pentakind: Pentakind; COMPANY NAME IS MANKIND. This medication contains the active ingredient Pantoprazole, it is a proton pump inhibitor that's used to help reduce the amount of acid produced by the stomach to help prevent or treat conditions such as heartburn, GERD, ulcers and gastritis. Side effects may include nausea, dizziness, flatulence and stomach pain.  Pantoprazole, it is a proton pump inhibitor  that's used to help reduce the amount of acid produced by the stomach to help prevent or treat conditions such as heartburn, This medication is a proton-pump inhibitor, Nexium-pentakind like;  http://www.medschat.com/.

 

Abilify: Hydrochlorothiazide; Azithromycin; Generic Prilosec ), Generic Norvasc (amlodipine besylate) (omeprazole), Seroquel- antipsychotic, Crestor, a cholesterol-lowering statin drug; Actos, a diabetes drug.

 

Rank

Drug (Brand Name)

1

Abilify

2

Nexium

3

Humira

4

Crestor

5

Advair Diskus

6

Enbrel

7

Remicade

8

Cymbalta

9

Copaxone

10

Neulasta

11

Lantus Solostar

12

Rituxan

13

Spiriva Handihaler

14

Januvia

15

Atripla

16

Lantus

17

Avastin

18

Lyrica

19

Oxycontin

20

Epogen

21

Celebrex

22

Truvada

23

Diovan

24

Gleevec

25

Herceptin

26

Lucentis

27

Namenda

28

Vyvanse

29

Zetia

30

Levemir

31

Symbicort

32

Sovaldi

33

Novolog Flexpen

34

Novolog

35

Tecfidera

36

Suboxone

37

Humalog

38

Xarelto

39

Seroquel XR

40

Viagra

41

Alimta

42

Victoza 3-Pak

43

Avonex

44

Nasonex

45

Cialis

46

Gilenya

47

Stelara

48

Flovent HFA

49

Prezista

50

Procrit

51

Isentress

52

Janumet

53

Renvela

54

Humalog Kwikpen

55

Orencia

56

Dexilant

57

Vesicare

58

Neupogen

59

Reyataz

60

Lunesta

61

Synthroid

62

Pradaxa

63

Zytiga

64

Benicar

65

Xolair

66

Avonex Pen

67

Vytorin

68

Invega Sustenna

69

Sensipar

70

Xgeva

71

Prevnar 13

72

Evista

73

Aranesp

74

Betaseron

75

Stribild

76

Combivent Respimat

77

Xeloda

78

Afinitor

79

Ventolin HFA

80

Synagis

81

Zyvox

82

Bystolic

83

Sandostatin LAR

84

Complera

85

Benicar HCT

86

Treanda

87

Gardasil

88

Zostavax

89

Pristiq

90