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Intestinal Inflammatory Pharmaceutics, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair
February 10, 2016 | This morning, AbbVie announced a partnership with Synlogic of Cambridge, Mass., to create microbiome-based therapies for the treatment of inflammatory bowel disease (IBD). The two companies have sketched out a suggested three-year timeline for preclinical research and development, after which AbbVie will take over advancing any drug candidates into clinical trials.
Drugs inspired by the microbes that live in the human gut are a hot topic in biotech. Companies like Seres Health and Vedanta Biosciences are pursuing the idea from a variety of angles, from making traditional small molecule drugs that interact with the microbiome, to creating probiotics or microbial cocktails that restore a healthy balance to the gut ecosystem. IBD, including Crohn’s disease and ulcerative colitis, is an especially popular target for these companies, thanks to strong suggestions that bacterial populations can affect the course of the disease. Already, Second Genome and Coronado Biosciences have taken prospective treatments into the clinic (though the latter has been dealt serious setbacks in Phase II trials).
But even among this peculiar batch of startups, Synlogic’s approach to drug design is exquisitely odd. The company calls its products “synthetic biotics”―in fact, they’re genetically engineered bacteria whose DNA contains intricately designed “gene circuits,” built to start producing therapeutic molecules when and only when the patient needs them.
“We are not looking at correcting the dysregulation of microbes in the gut, like other microbiome companies,” CEO José-Carlos Gutiérrez-Ramos tells Bio-IT World. “We have one bacterium, and it’s engineered to do different functions.”
Synlogic was founded in 2013 by two synthetic biologists at MIT, Timothy Lu and Jim Collins. (Bio-IT World has previously spoken with Lu about his academic work on bacterial gene circuits.) Gutiérrez-Ramos joined almost two years later, leaving a position as the head of Pfizer’s BioTherapeutics R&D group, where he had plenty of opportunity to turn emerging biotechnology ideas into drug candidates ready for submission to the FDA.
Still, synthetic biotics are a good deal more unusual than the biologic drugs he worked on at Pfizer.
His new company doesn’t quite spin functions for its microbes out of whole cloth. All the genes the company uses are copied either from the human genome, or from the bacteria living inside us. But by recombining those genes into circuits, Gutiérrez-Ramos believes Synlogic can finely control whether and when genes are expressed, giving its synthetic biotics the same dosage control as a traditional drug. Meanwhile, choosing the right bacterium to engineer―the current favorite is a strain called E. coli Nissle―ensures the biotics do not form stable colonies in the gut, but can be cleared out as soon as a patient stops treatment.
“We’re pharma guys,” he says. “What we want is to have pharmacologically well-defined products.”
The Molecular Circuit Board
Even before the partnership with AbbVie, Synlogic had a pipeline of drug candidates in development, all meant to treat rare genetic disorders caused by single mutations that shut down the activity of a crucial gene. In principle, there seems to be no reason that bacteria carrying the right genes couldn’t pick up the slack. “We know the patient is missing a function that is typically performed by the liver, or the kidney, or the pancreas,” says Gutiérrez-Ramos. “What we do is shift that function from an organ to a stable fraction of the microbiome.”
The approach is in some ways analogous to gene therapy, where a corrected version of a broken gene is inserted into a patient’s own DNA. “We don’t use that word, but the fact is it’s a non-somatic gene therapy,” Gutiérrez-Ramos says. “And if something goes wrong, you can control it just by stopping treatment.” The most advanced synthetic biotic in Synlogic’s pipeline targets urea cycle disorder, exactly the sort of disease that might otherwise be addressed by gene therapy: patients are missing a single enzyme that helps remove nitrogen from the body and prevent it from forming ammonia in the bloodstream. Synlogic will meet with the FDA this March to discuss whether and how this first product can be tested in humans.
The new IBD program with AbbVie, however, adds a whole new level of complexity. Executives from the two companies have been in discussions for around six months, and both agree that no single mechanism will be enough to provide significant relief for patients. Crohn’s and ulcerative colitis are painful autoimmune diseases that involve both a weakening of the epithelial lining in the stomach, and a buildup of inflammatory molecules. The development plan that AbbVie and Synlogic have agreed on includes three separate methods of attack to relieve these symptoms.
“One approach AbbVie is very interested in is for our synthetic biotics to produce substances that could tighten the epithelial barrier,” says Gutiérrez-Ramos. “Another approach is to degrade pro-inflammatory molecules”―the same tack taken by AbbVie’s current leading IBD drug, Humira, which targets the inflammatory protein TNFα. “Finally, we can produce anti-inflammatory molecules.”
Uniquely, synthetic biotics can perform all three functions at once; it’s just a matter of inserting the right genes. But that alone might not be a decisive advantage over some sort of combination therapy. The biggest selling point of Synlogic’s microbes is not the genes they can be engineered to express―what you might call the “output” of their gene circuits―but the input, the DNA elements called “inducible promoters” that decide when those genes should be activated.
The core idea is that patients will have a constant population of synthetic biotics in their bodies, taken daily―but those microbes will only generate their therapeutic payloads when needed. In IBD, Gutiérrez-Ramos explains, “it’s not that the patient is always inflamed, but they have flares. Our vision, and AbbVie’s vision, is that the bacteria that you take every day sense when the flare is coming, and then trigger the genetic output.”
This would be a major improvement over a drug like Humira, which after all is constantly inhibiting a part of the immune system. Patients taking Humira, or one of the many other immunosuppressant drugs for IBD, are at a constantly heightened risk of infection; tuberculosis is a particular specter for these patients. If Synlogic can find a genetic “on-switch” that responds to a reliable indicator of IBD flares, it could potentially create a much more precisely administered treatment, while still giving patients the simple dosing schedule of one pill every day.
The company has leads on two inducible promoters that might do the trick: one that reacts to nitric oxide, and another tied to reactive oxygen species. Of course, there’s no guarantee that either will respond sensitively to IBD flares in a real clinical setting. “This is an early time for the technology,” says Gutiérrez-Ramos. “We have demonstrated this in animals, but we have to demonstrate it in humans.”
Although it’s far too early to say if synthetic biotics will become an ordinary part of the pharma toolkit, AbbVie’s decision to invest in the technology offers the means to test this approach on a large scale. Synlogic expects to raise its own funding for trials of its rare disease products, which the FDA does not expect to enroll huge numbers of patients, but IBD is a problem of a very different order.
“We are very honored to work with truly the leader in treatment of inflammatory bowel disease,” says Gutiérrez-Ramos. With the backing of big pharma, it will be possible to trial microbiome-based therapies for the kinds of common, chronic diseases that are the biggest drain on our healthcare system. What’s more, the AbbVie partnership is an important signal of the industry’s faith in synthetic biology as an approach to treating disease.
In the meantime, we are sharing the encouraging news, that is, that the symptoms of digestive disorders can be alleviated, and often completely eliminated, with the right combination of medication, dietary changes, exercise, weight loss, stress reduction and surgery.
It’s all detailed in an important new research report from Johns Hopkins — rated #1 of America’s best hospitals for 21 consecutive years 1991-2011 by U.S. News & World Report.
The 2013 Johns Hopkins Digestive Disorders White Paper
Your Digestive Expert, H. Franklin Herlong, M.D. Adjunct Professor of MedicineJohns Hopkins University School of Medicine
The expertise you need, in language you can understand and use
In The 2013 Johns Hopkins Digestive Disorders White Paper, you will discover exciting advances and the most useful, current information to help you prevent or treat conditions affecting the digestive tract.
You’ll find a thorough overview of what the medical field knows about upper and lower digestive tract disorders (including everything from gastroesophageal reflux disease [GERD] to peptic ulcers, and irritable bowel syndrome to colorectal polyps) and conditions that affect the liver, gallbladder and pancreas.
You will learn how to prevent these diseases and, when symptoms arise, the best ways for you and your doctor to diagnose and treat them. The Johns Hopkins White Papers redefine the term “informed consumer.” In The 2013 Johns Hopkins Digestive Disorders White Paper, specialists from Johns Hopkins University School of Medicine report in depth on the latest digestive disorders prevention strategies and treatments. Thousands of Americans rely on Johns Hopkins expertise to help them manage their digestive disorders.
In The 2013 Johns Hopkins Digestive Disorders White Paper you’ll get a thorough overview of what the medical field knows about the most common digestive disorders today. You’ll find a wealth of news you can use about:
Celiac disease
Constipation
Crohn’s disease
Diarrhea
Diverticulosis and diverticulitis
Gallstones
Gastritis
GERD
Hiatal hernia
Irritable bowel syndrome
Ulcerative colitis
Ulcers
and more…
Timely Information Backed by Johns Hopkins Resources and Expertise
The symptoms of digestive disorders can be alleviated, and often completely eliminated, with the right combination of medication, dietary changes, exercise, weight loss, stress reduction and, as a last resort, surgery.
Learning as much as possible about the causes, effects and treatments for your digestive disorder is the first step toward living a fuller life with minimal discomfort and physical limitations.
The 2013 Johns Hopkins Digestive Disorders White Paper is designed to help you ensure the best outcome. Use what you learn to help you:
Recognize and respond to symptoms and changes as they occur.
Communicate effectively with your doctor, ask informed questions and understand the answers.
Make the right decisions, based on an understanding of the newest drugs, the latest treatments and the most promising research.
Take control over your condition and act out of knowledge rather than fear.
Tips for optimal digestive health
Maybe It’s Not “Just Heartburn”: Occasional heartburn can be treated with over-the-counter antacids. But if you have any of these symptoms, talk to your doctor to rule out more serious problems.
Should You Try Probiotics? Evidence is mounting that these “friendly bacteria” can help treat many digestive problems, such as IBS and Crohn’s disease. See how they work and are used, and whether they might relieve your gastrointestinal issues.
New Ways to Look Inside: The benefits and drawbacks of patient-friendly imaging tools including the “video pill” and virtual colonoscopy. How do state-of-the-art tools compare with established diagnostic exams?
Making Friends with Fiber: Getting enough dietary fiber is an easy way to prevent or treat a wide variety of digestive complaints. See which foods deliver the most fiber.
How to Avoid a Foodborne Illness: Follow these guidelines to choose, store, prepare and serve food in ways that minimize the health risks that result in 76 million infections and 325,000 hospitalizations annually.
1INSERM, U1043, Centre de Physiopathologie de Toulouse Purpan (CPTP), Toulouse F-31300, France.
2CNRS, U5282, Toulouse F-31300, France.
3CPTP, Université de Toulouse, Université Paul Sabatier (UPS), Toulouse F-31300, France.
4Institut National de la Recherche Agronomique (INRA), UMR 1319 Micalis, Commensal and Probiotics-Host Interactions Laboratory, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France.
5Department of Physiology and Pharmacology, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
6Pôle Digestif, CHU Purpan, Toulouse F-31059, France.
7Division of Gastroenterology, Hepatology and Nutrition, Children’s Hospital, Toulouse F-31059, France.
8Institut Pasteur, Unité de Défense Innée et Inflammation, Paris F-75015, France.
9INSERM U874, Paris F-75724, France.
10Universite Paris Diderot, Sorbonne Paris Cite, Cellule Pasteur F-75013, France.
ABSTRACT
Elafin, a natural protease inhibitor expressed in healthy intestinal mucosa, has pleiotropic anti-inflammatory properties in vitro and in animal models. We found that mucosal expression of Elafin is diminished in patients with inflammatory bowel disease (IBD). This defect is associated with increased elastolytic activity (elastase-like proteolysis) in colon tissue. We engineered two food-grade strains of lactic acid bacteria (LAB) to express and deliver Elafin to the site of inflammation in the colon to assess the potential therapeutic benefits of the Elafin-expressing LAB. In mouse models of acute and chronic colitis, oral administration of Elafin-expressing LAB decreased elastolytic activity and inflammation and restored intestinal homeostasis. Furthermore, when cultures of human intestinal epithelial cells were treated with LAB secreting Elafin, the inflamed epithelium was protected from increased intestinal permeability and from the release of cytokines and chemokines, both of which are characteristic of intestinal dysfunction associated with IBD. Together, these results suggest that oral delivery of LAB secreting Elafin may be useful for treating IBD in humans.
Cytokines involved in IBD (Photo credit: Wikipedia)
Metabolism
Front. Physio., 10 October 2012 | doi: 10.3389/fphys.2012.00401
Outlook: membrane junctions enable the metabolic trapping of fatty acids by intracellular acyl-CoA synthetases
Joachim Füllekrug*, Robert Ehehalt and Margarete Poppelreuther
Molecular Cell Biology Laboratory, Internal Medicine IV, University of Heidelberg, Heidelberg, Germany
The mechanism of fatty acid uptake is of high interest for basic research and clinical interventions. Recently, we showed that mammalian long chain fatty acyl-CoA synthetases (ACS) are not only essential enzymes for lipid metabolism but are also involved in cellular fatty acid uptake. Overexpression, RNAi depletion or hormonal stimulation of ACS enzymes lead to corresponding changes of fatty acid uptake. Remarkably, ACS are not localized to the plasma membrane where fatty acids are entering the cell, but are found instead at the endoplasmic reticulum (ER) or other intracellular organelles like mitochondria and lipid droplets. This is in contrast to current models suggesting that ACS enzymes function in complex with transporters at the cell surface. Drawing on recent insights into non-vesicular lipid transport, we suggest a revised model for the cellular fatty acid uptake of mammalian cells which incorporates trafficking of fatty acids across membrane junctions. Intracellular ACS enzymes are then metabolically trapping fatty acids as acyl-CoA derivatives. These local decreases in fatty acid concentration will unbalance the equilibrium of fatty acids across the plasma membrane, and thus provide a driving force for fatty acid uptake.
English: Acyl-CoA from the cytosol to the mitochondrial matrix. Français : Transport de l’Acyl-CoA du Cytosol jusqu’à la matrice mitochondriale. (Photo credit: Wikipedia)
English: The mechanism for Long Chain Fatty Acyl-CoA Synthetase (Photo credit: Wikipedia)
Tofacitinib, an Oral Janus Kinase Inhibitor, in Active Ulcerative Colitis
Reporter: Larry Bernstein, MD
This is an overview of a recently published article about a new treatment for ulcerative colitis. It also reviews the use of a class of drug in inflammatory conditions, and introduces the problem of sepsis.
Ulcerative colitis is a chronic inflammatory disease of the colon that belongs to a group of diseases lumped together as Inflammatory Bowel Disease (IBD). There is a distinction to be made between Crohn’s disease, which may be limited to the small intestine (regional enteritis), the terminal ileum, or a portion of the transverse colon, and ulcerative colitis.
In ulcerative colitis the inflammation is limited to the mucosa and submucosa, but in Crohn’s disease there is a deep penetration of the intestinal wall (fistula) that may extend to the peritoneum causing abscess, scarring, peritonitis and possibly volvulus, obstruction and gangrenous bowel, which necessitate surgical resection. IBD tends to occur in children and young adults, repeats in families, and requires dietary management (fluid intake, Metamucil, restriction of fiber) . It is characterized by abdominal pain, diarrhea, bleeding, weight loss, and episodic fever, but also may be associated with joint pain.
Conservative medical treatment focuses on suppressing the immune response using 5-ASA, azathioprine, 6-mercaptopurine. If severe, biologic therapy is used to treat patients with severe Crohn’s disease that does not respond to any other types of medication, such as a TNF (tumor necrosis factor) inhibitor which can have secondary effects, and they are not universally effective. The importance of immunity can’t be understated, it involves a large portion of immune system and primitive Toll-like receptors (TLRs) that trigger signaling pathways. TLRs represent an important mechanism by which the host detects a variety of microorganisms that colonize in the gut. Endothelial and epithelial cells, and resident macrophages are potent producers of inflammatory cytokines, interleukins, IL-1, IL-6, and TNF-α, which are distinguished from another set that is treated in this study. In addition, there is a balance that has to be achieved between suppression and upregulation in treatment, which is referred to as immunomodulation.
The opposite of immunosuppression is upregulation It is cental to recent advances in chemotherapy of melanolma, small cell carcinoma and NSCCL of lung, and treatment resistant prostate cancer. An example is ipilimumab, whic upregulates cytotoxic T-cells to destroy cancer cells, but it has runaway destructive effects on the GI tract.
This study investigates the use of tofacitinib (CP-690,550), an oral inhibitor of Janus kinases 1, 2, and 3 with in vitro functional specificity for kinases 1 and 3 over kinase 2, which is expected to block signaling involving gamma chain–containing cytokines including interleukins 2, 4, 7, 9, 15, and 21. These cytokines are integral to lymphocyte activation, function, and proliferation.
The mechanism of drug action
Jak 1 and 3 inhibitor, which is targeted at blocking signaling involving gamma chain–containing cytokines including interleukins 2, 4, 7, 9, 15, and 21. The result would be to block signaling involving (gamma chains)–suppressing “lymphokines” 2, 4, 7, 9, 15, and 21. The lymphocyte pool is regional, being the antibody mediated immune system of the Bursa of Fabricius (B-lymphocytes, as opposed to the thymic derived T-cells) that form the largest immune organ extending the length of the intestines and the stomach. The family transmission suggests an epigenetic event.
Gastrointestinal Tract
Oropharynx – Tonsils
Distal small intestine (ilieum) – Peyer’s Patches
Appendix, cecum
However, this classification of the lymphocytes has much greater complexity than I indicate. The so called B-cells have receptors that recognize foreign antigen, but the T-cells have similar receptors and are tied to both the innate and the adaptive immune response. Lymphocytes are the predominant cells of the immune system, but macrophages and plasma cells are present also. Lymphocytes circulate, alternating between the circulatory blood stream and the lymphatic channels. The end result of the immune reaction is the production of specific antibodies and antigen-reactive cells. These cells are called lymphocytes and are found in the blood and in the lymphoid system.
See Appendix
Trial features: double-blind, placebo-controlled, phase 2 trial; Patients were randomly assigned to receive tofacitinib at a dose of 0.5 mg, 3 mg, 10 mg, or 15 mg or placebo twice daily for 8 weeks. Study goal: evaluated the efficacy of tofacitinib in 194 adults with moderately to severely active ulcerative colitis.
Primary outcome: a clinical response at 8 weeks, defined as an absolute decrease from baseline in the score on the Mayo scoring system for assessment of ulcerative colitis activity (possible score, 0 to 12, with higher scores indicating more severe disease) of 3 or more and a relative decrease from baseline of 30% or more with an accompanying decrease in the rectal bleeding subscore of 1 point or more or an absolute rectal bleeding subscore of 0 or 1. Results and conclusion: The primary outcome, clinical response at 8 weeks, occurred in 32%, 48%, 61%, and 78% of patients receiving tofacitinib at a dose of 0.5 mg (P=0.39), 3 mg (P=0.55), 10 mg (P=0.10), and 15 mg (P<0.001), respectively, as compared with 42% of patients receiving placebo.
Clinical remission (defined as a Mayo score ≤2, with no subscore >1) at 8 weeks occurred in 13%, 33%, 48%, and 41% of patients receiving tofacitinib at a dose of 0.5 mg (P=0.76), 3 mg (P=0.01), 10 mg (P<0.001), and 15 mg (P<0.001), respectively, as compared with 10% of patients receiving placebo. Three patients treated with tofacitinib had an absolute neutrophil count of less than 1500.
Patients with moderately to severely active ulcerative colitis treated with tofacitinib were more likely to have clinical response and remission than those receiving placebo. (Funded by Pfizer; ClinicalTrials.gov number, NCT00787202.) Commentary: The study is only phase 2, and it is also limited to disease of the descending colon. The next phase will be necessary to determine the effect on a larger population at the selected dose, and will be necessary to determine both the size of the effect and identify unexpected adverse effects. We also have to keep in mind that the success of the study would limit the treatment to a subset of patients with IBD.
Efficacy of Proposed Treatment:
it is effective at about 40% remission for 8 weeks compared to 10% for placebo, or an adjusted actual 30% for 8 weeks.
A much larger study needs to be done to see how well the dose holds up, as well as the dosing interval. There are two factors that will affect the t1/2 of the drug so that 1/2 dose could be replaced at the end of t1/2.
The dose of 15 mg was no better for clinical response.
I would think that the next trial might give a loading dose of 15 mg, and then 7 mg (better that 3 mg) would be replaced every t1/2. But this is more complicated than usual.
I identified two steps, not one direct effect.
The inhibitor has to balance the production rate versus the removal rate of the T-cell population. The drug itself is not measured, only the effect. I know that albumin, the liver produced protein, has a half-life of removal of 21 days. Platelets are short shelf-life as well as rapid turnaround in plasma.
I don’t know what is the local production and removal rate of lymphocytes in the gut. That would be the key determinant for dosing.
The following may shed some light on what has been discussed:
Common characteristics of the lymphoid system.
The lymphoid system involves organs and tissues where lymphocytic cells originate as lymphocyte precursors that mature and differentiate, and either lodge in the lymphoid organs or move throughout the body.
Precursor cells originate in the yolk sac, liver, spleen, or bursa of Fabricius (or its mammalian equivalent, the bone marrow) in an embryo or fetus.
Stem cells from bone marrow or embryonic tissues are deposited and mature into lymphocytes in the central or primary lymphoid organs, which include the thymus and the bursa or bone marrow. Upon maturation, the lymphocytes undergo further maturation toward immunocompetence and production of immunoglobulins or sensitized lymphocytes.
Adaptive immunity has 2 main classes:
Antibody-mediated – B Lymphocyte
Cell-mediated – T Lymphocyte
Lymph follicles are our point of reference:
Organized concentrations of Lymphocytes
No capsule, covered by epithelia
Nodules are unit structure seen in a node
Oval concentrations in meshwork of reticular cells
If pathogens initially evade constitutive defenses, they may yet be attacked by more specific inducible defenses. The inducible defenses are so-called because they are induced upon primary exposure to a pathogen or one of its products. The inducible defenses must be triggered in a host, take time to develop, and are a function of the immune response. The type of resistance thus developed in the host is called acquired immunity.
Three important features of the immunological system relevant to host defense and/or “immunity are:
1. Specificity. An antibody or reactive T cell will react specifically with the antigen that induced its formation; it will not react with other antigens. Generally, this specificity is of the same order as that of enzyme-substrate specificity or receptor-ligand specificity.
The specificity of the immune response is explained on the basis of the clonal selection hypothesis: during the primary immune response, a specific antigen selects a pre-existing clone of specific lymphocytes and stimulates exclusively its activation, proliferation and differentiation.
2. Memory. The immunological system has a “memory”.
Once the immunological response has reacted to produce a specific type of antibody or reactive T cell, it is capable of producing more of the antibody or activated T cell more rapidly and in larger amounts.
3. Tolerance. An animal generally does not undergo an immunological response to its own (potentially-antigenic) components.
The animal is said to be tolerant, or unable to react to its own potentially-antigenic components.
Gene expression – CD28 signal transduction , λδ T repertoire and antigen reactivity
Efficient lymphokine gene expression appears to require both T-cell antigen receptor (TCR) signal transduction and an uncharacterized second or costimulatory signal. CD28 is a T-cell differentiation antigen that can generate intracellular signals that synergize with those of the TCR to increase T-cell activation and interleukin-2 (IL-2) gene expression.
These investigators examined the effect of CD28 signal transduction on granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), and gamma interferon (IFN-gamma) promoter activity.
Stimulation of CD28 in the presence of TCR-like signals increases the activity of the GM-CSF, IL-3, and IFN-gamma promoters by three- to sixfold.
As previously demonstrated for the IL-2 promoter, the IL-3 and GM-CSF promoters contain distinct elements of similar sequence which specifically bind a CD28-induced nuclear complex.
Mutation of the CD28 response elements in the IL-3 and GM-CSF promoters abrogates the CD28-induced activity without affecting phorbol ester- and calcium ionophore-induced activity.
These studies indicate that the TCR and CD28-regulated signal transduction pathways, coordinately regulate the transcription of several lymphokines, and the influence of CD28 signals on transcription is mediated by a common complex.
These investigators looked at the relevance λδ T repertoire and the antigen reactivity of clones isolated from CSF in multiple sclerosis (MS).
they found an increased percentage of V delta 1+ cells as compared to peripheral blood of the same donors.
Phenotypic analysis of cells from MS CSF with V gamma- and V delta-specific monoclonal antibodies (mAb) showed that the V delta 1 chain is most frequently associated with gamma chains belonging to the V gamma 1 family.
Sequence analysis of TCR genes revealed heterogeneity of junctional regions in both delta and gamma genes indicating polyclonal expansion. gamma delta clones were established and some recognized glioblastoma, astrocytoma or monocytic cell lines.
Stimulation with these targets induced serine esterase release and lymphokine expression characteristic of the TH0-like phenotype.
Remarkably, these tumor-reactive gamma delta cells were not detected in the peripheral blood using PCR oligotyping, but were found in other CSF lines independently established from the same MS patient.
in the CSF there is a skewed TCR gamma delta repertoire and suggest that gamma delta cells reacting against brain-derived antigens might have been locally expanded.
users.rcn.com/jkimball.ma.ultranet/…/B/B_and_Tcells.htmlShareAIDS; Building the T-cell Repertoire; Gamma/Delta T Cells … T cells specific for this structure (i.e., with complementary TCRs) bind the B cell and; secrete lymphokines that: … Each chain has a variable (V) region and a constant (C) region.
Although mature lymphocytes all look pretty much alike, they are extraordinarily diverse in their functions. The most abundant lymphocytes are:
B lymphocytes (often simply called B cells) and
T lymphocytes (likewise called T cells).
B cells are produced in the bone marrow.
The precursors of T cells are also produced in the bone marrow but leave the bone marrow and mature in the thymus (which accounts for their designation).
Each B cell and T cell is specific for a particular antigen. What this means is that each is able to bind to a particular molecular structure.
The specificity of binding resides in a receptor for antigen:
the B cell receptor (BCR) for antigen and
the T cell receptor (TCR) respectively.
Both BCRs and TCRs share these properties:
They are integral membrane proteins.
They are present in thousands of identical copies exposed at the cell surface.
They are made before the cell ever encounters an antigen.
They are encoded by genes assembled by the recombination of segments of DNA.
How antigen receptor diversity is generated.
They have a unique binding site.
This site binds to a portion of the antigen called an antigenic determinant or epitope.
The binding, like that between an enzyme and its substrate depends on complementarity of the surface of the receptor and the surface of the epitope.
The binding occurs by non-covalent forces (again, like an enzyme binding to its substrate).
Successful binding of the antigen receptor to the epitope, if accompanied by additional signals, results in:
stimulation of the cell to leave G0 and enter the cell cycle.
Repeated mitosis leads to the development of a clone of cells bearing the same antigen receptor; that is, a clone of cells of the identical specificity.
BCRs and TCRs differ in:
their structure;
the genes that encode them;
the type of epitope to which they bind.
heavy (H) plus kappa (κ) or lambda (λ) chains for BCRs;
alpha (α) and beta (β) or gamma (γ) and delta (δ) chains for TCRs)
……is encoded by several different gene segments.
The genome contains a pool of gene segments for each type of chain. Random assortment of these segments makes the largest contribution to receptor diversity.
There are two types of T cells that differ in their TCR:
alpha/beta (αβ) T cells. Their TCR is a heterodimer of an alpha chain with a beta chain. Each chain has a variable (V) region and a constant (C) region. The V regions each contain 3 hypervariable regions that make up the antigen-binding site. [Link]
gamma/delta (γδ) T cells. Their TCR is also a heterodimer of a gamma chain paired with a delta chain.
The discussion that follows now concerns alpha/beta T cells. Gamma/delta T cells, which are less well understood, are discussed at the end [Link].
The TCR (of alpha/beta T cells) binds a bimolecular complex displayed at the surface of some other cell called an antigen-presenting cell (APC).
Most of the T cells in the body belong to one of two subsets. These are distinguished by the presence on their surface of one or the other of two glycoproteins designated:
CD8+ T cells bind epitopes that are part of class I histocompatibility molecules. Almost all the cells of the body express class I molecules.
CD4+ T cells bind epitopes that are part of class II histocompatibility molecules. Only specialized antigen-presenting cells express class II molecules.
These include:
dendritic cells
phagocytic cells like macrophages and
B cells!
Building the T-cell Repertoire
T cells have receptors (TCRs) that bind to antigen fragments nestled in MHC molecules. But,
all cells express class I MHC molecules containing fragments derived from self proteins;
many cells express class II MHC molecules that also contain self peptides.
This presents a risk of the T cells recognizing these self-peptide/self-MHC complexes and mounting an autoimmune attack against them. Fortunately, this is usually avoided by a process of selection that goes on in the thymus (where all T cells develop).
Appendix
FDA approves Abbott Humira as Ulcerative Colitis therapy
PBR Staff Writer Published 01 October 2012
The USFDA has approved Abbott’s Humira (adalimumab) for the treatment of adult patients with moderate to severe Ulcerative Colitis (UC) when certain other medicines have not worked well enough.
Humira, which works by inhibiting tumour necrosis factor-alpha (TNF-alpha), was previously approved for the treatment of moderate to severe Crohn’s disease.
Abbott Global Pharmaceutical Research and Development senior vice president John Leonard said, “Since the first FDA approval of HUMIRA in late 2002, Abbott has continued to investigate the medication in multiple conditions with the goal of bringing this treatment option to more patients who may benefit from it.”
The approval was based on the data from two phase 3 studies, ULTRA 1 and ULTRA 2, both of which enrolled adult patients who had moderately to severely active UC despite concurrent or prior treatment with immunosuppressants. This should have special significance in view of the past history, which may be explainable, but also keep in mind the serious risks of complications.
It is worthy of comment that anti-TNF treatment was previously rejected in trials for use in sepsis leading to Multiple Organ Dysfunction Syndrome and cardiovascular collapse (shock). More recently an anti-Factor Xa drug, Xygris, to prevent hypercoagulability only in severe sepsis was withdrawn.
Anti TNF for sepsis
1. In a group of patients with elevated interleukin-6 levels, the mortality rate was 243 of 510 (47.6%) in the placebo group and 213 of 488 (43.6%) in the afelimomab group. Using a logistic regression analysis, treatment with afelimomab was associated with an adjusted reduction in the risk of death of 5.8% (p = .041) and a corresponding reduction of relative risk of death of 11.9%. Mortality rates for the placebo and afelimomab groups in the interleukin-6 test negative population were 234 of 819 (28.6%) and 208 of 817 (25.5%), respectively. In the overall population of interleukin-6 test positive and negative patients, the placebo and afelimomab mortality rates were 477 of 1,329 (35.9%)and 421 of 1,305 (32.2%), respectively.
2. No survival benefit was found for the total study population, but patients with increased circulating TNF concentrations at study entry appeared to benefit by the high dose anti-TNF antibody treatment. Increased interleukin (IL)-6 levels predicted a fatal outcome (p =.003), but TNF levels were not found to be a prognostic indicator. TNFlevels were higher (206.7 +/- 60.7 vs. 85.9 +/- 26.1 pg/mL; p <.001) and outcome was poor (41% vs. 71% survival; p =.007) in patients who were in shock at study entry when compared with septic patients not in shock.
Fisher CJ Jr, Opal SM, Dhainaut JF, Stephens S, et al. Influence of an anti-tumor necrosis factor monoclonal antibody on cytokine levels in patients with sepsis. The CB0006 Sepsis Syndrome Study Group. Critical Care Medicine [1993, 21(3):318-327] (PMID:8440099)
3. Large clinical trials involving anti-TNF-alpha MAb have proven to be less conclusive and less successful than clinicians had hoped. The International Sepsis Trial (INTERSEPT), reported by Cohen and Carlet,[14] was designed to assess the safety and efficacy of Bay x 1351, a murine MAb to recombinant human TNF-alpha in patients with sepsis. The INTERSEPT trial was an international, multicenter trial involving 564 patients, 420 of whom were in septic shock. The main study end point — 28-day survival — showed no significant benefit for the treatment group vs controls. Prospectively, the researchers identified 2 secondary variables: shock reversal and frequency of organ failure. Post-28-day survival, treatment groups showed a more rapid reversal of shock compared with placebo, as well as a significant delay in time to first organ failure. The researchers concluded that the anti-TNF-alpha antibody may have a role as adjunctive therapy, but that such a putative role requires more in the way of clinical trial confirmation.
In the TNF-alpha MAb Sepsis Study Group trial, also called the North American Sepsis Trial I (NORASEPT I), Abraham and associates[15] evaluated the efficacy and safety of an anti-TNF-alpha MAb in the treatment of patients with sepsis syndrome. A total of 994 patients in 31 hospitals were enrolled in a randomized, prospective, multicenter, double-blind, placebo-controlled clinical trial. Patients were stratified into shock/nonshock subgroups, then randomized to receive a single infusion of 15 mg/kg of anti-TNF-alpha MAb, 7.5 mg/kg of anti-TNF-alpha MAb, or placebo. The researchers found that among all infused patients, there was no difference in mortality among those receiving therapy and those on placebo. In septic shock patients (n = 478), however, there was a trend toward a reduction in all-cause mortality, which was most evident 3 days after infusion. At day 3, 25 of 162 patients treated with the 15 mg/kg dose died; 22 of 156 treated with 7.5 mg/kg died, but 44 of 160 placebo-treated patients died (15 mg/kg: 44% mortality reduction vs placebo, P = .01; 7.5 mg/kg: 48% reduction vs placebo, P = .004). However, at day 28, the reduction in mortality of shock patients was not significant for either dose of the anti-TNF-alpha MAb relative to placebo.
All studies of MAb against TNF in septic patients and found an absolute risk reduction of 3.5%. The most recently published clinical trial found an absolute reduction in mortality of 3.7%.
Of note, therapy with MAb against TNF has been proven efficacious for treatment of rheumatoid arthritis and is approved by the US Food and Drug Administration for this purpose.
This would be sufficient for another discussion. That can be left for another day.
Sepsis
Sepsis syndrome, or sepsis, is an adverse systemic response to infection that includes fever, rapid heartbeat and respiration, low blood pressure and organ dysfunction associated with compromised circulation.
LPS is a major constituent of Gram-negative bacterial cell walls (see section 3-0) and is essential for membrane integrity. The portion of LPS that causes shock is the innermost and most highly conserved phosphoglycolipid, lipid A. Lipid A is a phosphoglycolipid consisting of a core hexosamine disaccharide with ester- and amide-linked acylated fatty acid tails arranged in either asymmetric or symmetric arrays that anchor the structure in the membrane. It acts by potently inducing inflammatory responses that are life-threatening when systemic, and is known as bacterial endotoxin. Mice deficient in any of the LPS receptor components are more
susceptible to Gram-negative bacterial infection but, at the same time, are less susceptible to the sepsis syndrome.
TLRs have a lethal function in the septic shock syndrome. The physiological function of signaling through phagocyte TLRs is to induce the release of the cytokines TNF, IL-1, IL-6, IL-8 and IL-12 and trigger the inflammatory response, which is critical to containing bacterial infection in the tissues. However, if infection disseminates in the blood, the widespread activation of phagocytes in the bloodstream is catastrophic. Increase in the numbers of circulating neutrophils, or neutrophilia, is driven by effects of colony stimulating factors, such as G-CSF.
Time course of sepsis. The clinical manifestations of sepsis are manifested by successive waves of the serum cytokine cascade. In humans injected with purified LPS, TNF rises almost immediately and peaks at 1.5 h; the sharp decline of TNF may be due to modulation by its soluble receptor sTNFR. A second wave of cytokines that peaks at 3 h activates the acute-phase response
in the liver, the systemic pituitary response (via IL-6 and IL-1), and the activation and chemotaxis of neutrophils (via IL-6, IL-8 and G-CSF). Neutrophil activation results in the release of lactoferrin from neutrophil secondary granules; the activation of endothelial procoagulants with the rise of tissue plasminogen activator (t-PA). Pituitary-derived adrenocorticotropic hormone (ACTH) and migration inhibition factor (MIF) peak at 5 h and coincide with peak levels of the regulatory cytokines IL-Ra and IL-10 that counteract the release or activity of inflammatory cytokines. Diffuse endothelial activation is shown by the appearance of soluble E-selectin that peaks at about 8 h and remains elevated for several days.
Susceptibility to LPS Toxicity in Gene Knockout Mice
Defect:
High LPS; Low LPS/D-Gal
Proteins
LPS recognition
CD14
LBP
TLR4
MD-2
MyD88
SR-A
phagocyte function
Hck/Fgr
CAM-1
L-selectin
GM-CSF
TNFR1
inflammation
TNFR2
IL-1Ra
IL-1β
IFN-γR
caspase 1
The proteins encoded by the deleted genes are listed. SR-A is scavenger receptor A; Hck and Fgr are Src-family kinases with an essential role in integrin-mediated migration of neutrophils out of the bloodstream.
An experimental immunology drug from Eli Lilly has scored positive data in a mid-stage study of Crohn’s disease patients.
The study enrolled 180 participants, who received either placebo or one of three doses of Lilly’s mirikizumab. After 12 weeks of treatment, all three doses of mirikizumab significantly outperformed placebo on the study’s primary endpoint, which was a 50% or greater reduction in the severity of each patient’s Crohn’s disease.
Specifically, 26% of the 200 mg group, 38% of the 600 mg group, and 44% of the 1,000 mg group achieved a response, versus 11% of the placebo arm. Five of the patients receiving mirikizumab had at least one serious adverse event, while 81 — or 64% — had a treatment emergent adverse event during the trial’s induction phase. Lilly intends to push mirikizumab into late-stage testing for Crohn’s disease.
Crohn’s disease driven by inflammation – not genetics, reports study
Inflammation — not genetic susceptibility — drives the growth of intestinal bacteria and invasive E. coli linked to Crohn’s disease (CD), reports a new Cornell study.
Scientists have long wondered about the role of bacteria in CD. Recent studies have shown marked changes in the composition of the intestinal bacteria in people with CD, leading researchers to ask: Are microbial abnormalities a direct consequence of genetic abnormalities linked to Crohn’s and precede and initiate inflammation, or does intestinal inflammation bring on the bugs?
Inflammation, in fact, drives microbial imbalances (dysbiosis) and the proliferation of a specific type of E. coli that is adherent, invasive and found in the ileum, reported Cornell researchers July 31 in PLoS (7[7]). And genetics, they said, do play a role in determining the threshold and magnitude of dysbiosis in response to acute inflammation induced by environmental triggers.
This study also reports that a common therapy directed against intestinal inflammation decreases dysbiosis. In addition, the study found that the lack of a receptor that helps recruit T cells, which are needed for cell-mediated immunity, to the gut also decreases inflammation and dysbiosis, offering a new option for therapeutic intervention.
“Today, remission is our mission,” said Kenneth Simpson, professor of small animal medicine at Cornell’s College of Veterinary Medicine and principal investigator. “Crohn’s disease is a highly complex condition that finds its strength in the combination of negatives: environmental factors, genetic mutations and immune system malfunctions. Ultimately, there may be a cure. Until then, we need to find ways to relieve suffering.”
CD is a chronic debilitating inflammatory bowel disease that involves a complex interaction of host genes, the immune system, the intestinal microbiome and the environment. Afflicting more than half a million people in North America, CD can trigger mild to severe diarrhea, fever, fatigue, anemia, reduced appetite and weight loss.
To mirror the complex nature of the disease, Simpson’s team designed a study that incorporated inflammatory triggers related to relapse of CD and ileal inflammation. Unlike previous studies that have focused on colonic or fecal dysbiosis, the team focused on ileal dysbiosis, which is prevalent in 70 percent of CD cases. Also novel to this study, the team used a variety of contemporary techniques to generate a comprehensive picture of the composition and spatial distribution of the ileal microbiome. Particular attention was paid to pinpointing the number, pathotype and location of E. coli associated with intestinal inflammation in people, dogs and mice.
“Our findings clearly demonstrate that inflammation drives ileal dysbiosis and proliferation of CD-associated adherent invasive E. coli. Further, in the context of a patient with Crohn’s, we found that the host genotype and therapeutically blocking inflammation both impact the onset and extent of ileal dysbiosis. These novel findings are of high relevance to Crohn’s disease.”
The investigation leveraged the knowledge and resources of researchers in the labs of Erik Denker, Dwight Bowman and Sean McDonough labs. Building on findings in patients with Crohn’s disease evaluated by Dr. Ellen Scherl’s group at Weill Cornell Medical College, this collaboration shed new light on this debilitating disease.
“It appears that we harbor our own powder keg,” said Simpson. “The bacteria are already seeded. It’s what controls the relative balance between the different species of bacteria and their numbers, relative proportions, our ability to deal with them, and the cross-talk between the bacteria and host that is important.”
Zebrafish, popular as aquarium fish, now have an important place in research labs as a model organism for studying human diseases.
At the 2012 International Zebrafish Development Conference, held June 20-24 in Madison, Wisconsin, numerous presentations highlighted the utility of the zebrafish for examining the basic biological mechanisms underlying human disorders and identifying potential treatment approaches for an impressive array of organ and systemic diseases.
Inflammatory bowel disease (IBD), while rarely fatal, can have a substantial negative impact on an individual’s quality of life due to abdominal pain, diarrhea, vomiting, bleeding, and severe cramps. The causes of this chronic inflammatory disorder are largely unknown and existing treatments, usually anti-inflammatory drugs, are often not effective. In addition, IBD is often associated with increased risk of developing intestinal cancer.
Researchers from the University of Pittsburgh are using zebrafish to study the biological mechanisms that lead to intestinal inflammation, as often seen in IBD, providing additional understanding that may allow development of better therapies. Prakash Thakur, a research associate working with Nathan Bahary, M.D., Ph.D., described a mutant zebrafish strain that shows many pathological characteristics similar to IBD, including inflammation, abnormal villous architecture, disorganized epithelial cells, increased bacterial growth and high numbers of dying cells in the intestine. “Most of the hallmark features of the disease are seen in this mutant. We are utilizing this fish as a tool to unravel fundamental mechanisms of intestinal pathologies that may contribute to intestinal inflammatory disorders, ” Mr. Thakur said.
The fish have a genetic mutation that disrupts de novo synthesis of an important signaling molecule called phosphatidylinositol (PI). The lack of de novo PI synthesis, Mr. Thakur and his colleagues found, leads to chronic levels of cellular stress, particularly the endoplasmic reticum stress and, ultimately, inflammation. Drugs or other interventions targeting the cellular stress response pathway, rather than just inflammation, helped restore a healthy intestinal structure and increase cell survival in the fish intestine, suggesting this mechanism as a potential therapeutic target for patients with inflammatory disorders, including IBD.
Doxorubicin-Induced Heart Failure
Doxorubicin is a potent chemotherapy drug used to treat many types of cancer, including leukemia, lymphoma, carcinoma, soft tissue sarcoma, and bladder, breast, lung, stomach and ovarian cancers. Unfortunately, drug-induced cardiomyopathy is a common side effect and can lead to heart failure in cancer patients, not only during treatment, but months or years later.
“We hope to identify some drug which only blocks the side effect of doxorubicin but preserves the therapeutic effect,” said Yan Liu, Ph.D., a postdoctoral researcher working in Dr. Randall Peterson’s lab at the Massachusetts General Hospital.
Dr. Liu developed a zebrafish model of doxorubicin-induced cardiomyopathy. The fish experience heart failure within two days of treatment with symptoms similar to those seen in humans, including fewer heart muscle cells, ventricular collapse, and ineffective heartbeats.
The researchers used the model to screen through thousands of potential drug compounds and identified two — visnagin and diphenylurea — that both improved cardiac function and reduced doxorubicin-induced cell death in the heart. Importantly, both compounds specifically protected heart tissue, but not tumor cells, from the toxic effects of doxorubicin. Both seem to act through the suppression of a particular signaling pathway, the c-Jun N-terminal kinase pathway, in the heart cells but not tumor cells.
Dr. Liu also reported promising preliminary results with mice showing reduced cell death and improved cardiac function, indicating that these compounds may also be active in mammals and giving hope for therapies that specifically treat doxorubicin’s side effects without negating its anti-tumor activity.
Spinal muscular atrophy (SMA) is a group of progressive neurodegenerative diseases that affect the nerves in the spinal cord that control muscles, leading to weakness, movement difficulties, poor posture, and trouble breathing and eating.
SMA is linked to mutations in a specific motor neuron survival gene, SMN1. Though mouse studies have reported immature and ineffective synaptic connections between motor neurons and muscles, little is known about the molecular mechanisms leading to those problems or how they might be fixed.
Graduate student Kelvin See, working with Associate Professor Christoph Winkler, Ph.D., at the National University of Singapore used zebrafish with activity-sensitive fluorescence to provide a visual readout of motor neuron activation. They confirmed that low SMN1 levels are associated with low neuronal influx of calcium ions, which play a critical role in triggering neurotransmitter release and thus stimulating the muscles. With their zebrafish model, Mr. See and Dr. Winkler also identified another gene with a similar effect, neurexin, which is important in synaptic structure but had never been implicated in SMA.
In a surprise discovery, the researchers found they could use the same sensor to see activation of a neighboring cell type called Schwann cells. “This gives us the unique opportunity to look at the role of SMN1 not just in motor neurons but also in the surrounding tissue,” said Mr. See.
They saw reduced excitability in Schwann cells also, suggesting that a full understanding of SMA will require a broader view of the affected cell populations. Their results provide several new insights into the fundamental processes disrupted in SMA.
Acute T-cell Lymphoblastic Leukemia and Lymphoma (T-ALL/T-LBL)
Human acute T-cell lymphoblastic leukemias (ALL) and lymphomas (LBL) have high relapse rates in pediatric patients and high mortality rates in adults. Hui Feng, M.D., Ph.D., currently at the Pharmacology Department and Center for Cancer Research at Boston University School of Medicine, is using a zebrafish model of leukemia to search for promising targets for new molecular treatments for these diseases.
To date, studies have identified several biological pathways involved in ALL and LBL, all with a known oncogene in common called c-Myc. However, Myc is so common, involved in regulating more than 15 percent of all genes, that it is very hard to study.
“Because this is a huge list of downstream targets, it is very challenging to predict which genes in the pathway to target to treat Myc-related cancers,” said Dr. Feng.
In work performed in collaboration with Thomas Look, M.D., at the Dana-Farber Cancer Institute, Dr. Feng is combining the power of zebrafish genetics with human clinical studies to hone in on potential genes of interest.
Using a fish strain that reliably develops T-cell lymphoma by two months of age, they identified a novel gene called DLST that is involved in metabolism and energy production in cells. Evidence from human cancer cell lines and patients indicate that abnormally high levels of the protein may be involved in the human disease as well.
Reducing DLST activity in the fish significantly delayed tumor progression and growth, suggesting it is a promising target for developing new therapies for ALL and LBL.
2012pharmaceutical
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