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Posts Tagged ‘immunosuppression’


Newly Found Functions of B Cell

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

The importance of B cells to human health is more than what is already known. Vaccines capable of eradicating disease activate B cells, cancer checkpoint blockade therapies are produced using B cells, and B cell deficiencies have devastating impacts. B cells have been a subject of fascination since at least the 1800s. The notion of a humoral branch to immunity emerged from the work of and contemporaries studying B cells in the early 1900s.

 

Efforts to understand how we could make antibodies from B cells against almost any foreign surface while usually avoiding making them against self, led to Burnet’s clonal selection theory. This was followed by the molecular definition of how a diversity of immunoglobulins can arise by gene rearrangement in developing B cells. Recombination activating gene (RAG)-dependent processes of V-(D)-J rearrangement of immunoglobulin (Ig) gene segments in developing B cells are now known to be able to generate an enormous amount of antibody diversity (theoretically at least 1016 possible variants).

 

With so much already known, B cell biology might be considered ‘‘done’’ with only incremental advances still to be made, but instead, there is great activity in the field today with numerous major challenges that remain. For example, efforts are underway to develop vaccines that induce broadly neutralizing antibody responses, to understand how autoantigen- and allergen-reactive antibodies arise, and to harness B cell-depletion therapies to correct non-autoantibody-mediated diseases, making it evident that there is still an enormous amount we do not know about B cells and much work to be done.

 

Multiple self-tolerance checkpoints exist to remove autoreactive specificities from the B cell repertoire or to limit the ability of such cells to secrete autoantigen-binding antibody. These include receptor editing and deletion in immature B cells, competitive elimination of chronically autoantigen binding B cells in the periphery, and a state of anergy that disfavors PC (plasma cell) differentiation. Autoantibody production can occur due to failures in these checkpoints or in T cell self-tolerance mechanisms. Variants in multiple genes are implicated in increasing the likelihood of checkpoint failure and of autoantibody production occurring.

 

Autoantibodies are pathogenic in a number of human diseases including SLE (Systemic lupus erythematosus), pemphigus vulgaris, Grave’s disease, and myasthenia gravis. B cell depletion therapy using anti-CD20 antibody has been protective in some of these diseases such as pemphigus vulgaris, but not others such as SLE and this appears to reflect the contribution of SLPC (Short lived plasma cells) versus LLPC (Long lived plasma cells) to autoantibody production and the inability of even prolonged anti-CD20 treatment to eliminate the later. These clinical findings have added to the importance of understanding what factors drive SLPC versus LLPC development and what the requirements are to support LLPCs.

 

B cell depletion therapy has also been efficacious in several other autoimmune diseases, including multiple sclerosis (MS), type 1 diabetes, and rheumatoid arthritis (RA). While the potential contributions of autoantibodies to the pathology of these diseases are still being explored, autoantigen presentation has been posited as another mechanism for B cell disease-promoting activity.

 

In addition to autoimmunity, B cells play an important role in allergic diseases. IgE antibodies specific for allergen components sensitize mast cells and basophils for rapid degranulation in response to allergen exposures at various sites, such as in the intestine (food allergy), nose (allergic rhinitis), and lung (allergic asthma). IgE production may thus be favored under conditions that induce weak B cell responses and minimal GC (Germinal center) activity, thereby enabling IgE+ B cells and/or PCs to avoid being outcompeted by IgG+ cells. Aside from IgE antibodies, B cells may also contribute to allergic inflammation through their interactions with T cells.

 

B cells have also emerged as an important source of the immunosuppressive cytokine IL-10. Mouse studies revealed that B cell-derived IL-10 can promote recovery from EAE (Experimental autoimmune encephalomyelitis) and can be protective in models of RA and type 1 diabetes. Moreover, IL-10 production from B cells restrains T cell responses during some viral and bacterial infections. These findings indicate that the influence of B cells on the cytokine milieu will be context dependent.

 

The presence of B cells in a variety of solid tumor types, including breast cancer, ovarian cancer, and melanoma, has been associated in some studies with a positive prognosis. The mechanism involved is unclear but could include antigen presentation to CD4 and CD8 T cells, antibody production and subsequent enhancement of presentation, or by promoting tertiary lymphoid tissue formation and local T cell accumulation. It is also noteworthy that B cells frequently make antibody responses to cancer antigens and this has led to efforts to use antibodies from cancer patients as biomarkers of disease and to identify immunotherapy targets.

 

Malignancies of B cells themselves are a common form of hematopoietic cancer. This predilection arises because the gene modifications that B cells undergo during development and in immune responses are not perfect in their fidelity, and antibody responses require extensive B cell proliferation. The study of B cell lymphomas and their associated genetic derangements continues to be illuminating about requirements for normal B cell differentiation and signaling while also leading to the development of targeted therapies.

 

Overall this study attempted to capture some of the advances in the understanding of B cell biology that have occurred since the turn of the century. These include important steps forward in understanding how B cells encounter antigens, the co-stimulatory and cytokine requirements for their proliferation and differentiation, and how properties of the B cell receptor, the antigen, and helper T cells influence B cell responses. Many advances continue to transform the field including the impact of deep sequencing technologies on understanding B cell repertoires, the IgA-inducing microbiome, and the genetic defects in humans that compromise or exaggerate B cell responses or give rise to B cell malignancies.

 

Other advances that are providing insight include:

  • single-cell approaches to define B cell heterogeneity,
  • glycomic approaches to study effector sugars on antibodies,
  • new methods to study human B cell responses including CRISPR-based manipulation, and
  • the use of systems biology to study changes at the whole organism level.

With the recognition that B cells and antibodies are involved in most types of immune response and the realization that inflammatory processes contribute to a wider range of diseases than previously believed, including, for example, metabolic syndrome and neurodegeneration, it is expected that further

  • basic research-driven discovery about B cell biology will lead to more and improved approaches to maintain health and fight disease in the future.

 

References:

 

https://www.cell.com/cell/fulltext/S0092-8674(19)30278-8

 

https://onlinelibrary.wiley.com/doi/full/10.1002/hon.2405

 

https://www.pnas.org/content/115/18/4743

 

https://onlinelibrary.wiley.com/doi/full/10.1111/all.12911

 

https://cshperspectives.cshlp.org/content/10/5/a028795

 

https://www.sciencedirect.com/science/article/abs/pii/S0049017218304955

 

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Immunopathogenesis Advances in Diabetes and Lymphomas

Larry H Bernstein, MD, FCAP, Curator

LPBI

 

 

 Science team says they’ve taken another step toward a potential cure for diabetes

Wednesday, January 27, 2016 | By John Carroll
Building on years of work on developing new insulin-producing cells that could one day control glucose levels and cure diabetes, a group of investigators led by scientists at MIT and Boston Children’s Hospital say they’ve developed a promising new gel capsule that protected the cells from an immune system assault.

Dr. Jose Oberholzer, a professor of bioengineering at the University of Illinois at Chicago, tested a variety of chemically modified alginate hydrogel spheres to see which ones would be best at protecting the islet cells created from human stem cells.

The team concluded that 1.5-millimeter spheres of triazole-thiomorphine dioxide (TMTD) alginate were best at protecting the cells and allowing insulin to seep out without spurring an errant immune system attack or the development of scar tissue–two key threats to making this work in humans.

They maintained healthy glucose levels in the rodents for 174 days, the equivalent to decades for humans.

“While this is a very promising step towards an eventual cure for diabetes, a lot more testing is needed to ensure that the islet cells don’t de-differentiate back toward their stem-cell states or become cancerous,” said Oberholzer.

Millions of diabetics have effectively controlled the chronic disease with existing therapies, but there’s still a huge unmet medical need to consider. While diabetes companies like Novo ($NVO) like to cite the fact that a third of diabetics have the disease under control, a third are on meds but don’t control it well and a third haven’t been diagnosed. An actual cure for the disease, which has been growing by leaps and bounds all over the world, would be revolutionary.

Their study was published in Nature Medicine.

– here’s the release
– get the journal abstract

 

Long-term glycemic control using polymer-encapsulated human stem cell–derived beta cells in immune-competent mice

Arturo J Vegas, Omid Veiseh, Mads Gürtler,…, Robert Langer & Daniel G Anderson

Nature Medicine (2016)   http://dx.doi.org:/10.1038/nm.4030

The transplantation of glucose-responsive, insulin-producing cells offers the potential for restoring glycemic control in individuals with diabetes1. Pancreas transplantation and the infusion of cadaveric islets are currently implemented clinically2, but these approaches are limited by the adverse effects of immunosuppressive therapy over the lifetime of the recipient and the limited supply of donor tissue3. The latter concern may be addressed by recently described glucose-responsive mature beta cells that are derived from human embryonic stem cells (referred to as SC-β cells), which may represent an unlimited source of human cells for pancreas replacement therapy4. Strategies to address the immunosuppression concerns include immunoisolation of insulin-producing cells with porous biomaterials that function as an immune barrier56. However, clinical implementation has been challenging because of host immune responses to the implant materials7. Here we report the first long-term glycemic correction of a diabetic, immunocompetent animal model using human SC-β cells. SC-β cells were encapsulated with alginate derivatives capable of mitigating foreign-body responses in vivo and implanted into the intraperitoneal space of C57BL/6J mice treated with streptozotocin, which is an animal model for chemically induced type 1 diabetes. These implants induced glycemic correction without any immunosuppression until their removal at 174 d after implantation. Human C-peptide concentrations and in vivo glucose responsiveness demonstrated therapeutically relevant glycemic control. Implants retrieved after 174 d contained viable insulin-producing cells.

Subject terms: Regenerative medicine  Type 1 diabetes

Figure 1: SC-β cells encapsulated with TMTD alginate sustain normoglycemia in STZ-treated immune-competent C57BL/6J mice.close

(a) Top, schematic representation of the last three stages of differentiation of human embryonic stem cells to SC-β cells. Stage 4 cells (pancreatic progenitors 2) co-express pancreatic and duodenal homeobox 1 (PDX-1) and NK6 homeobox 1…

 

Potential Cure for Diabetes Discovered  
http://www.rdmag.com/news/2016/01/potential-cure-diabetes-discovered   01/27/2016

Two new scientific papers published on Monday demonstrated tools that could result in potential therapies for patients diagnosed with type 1 diabetes, a condition in which the immune system limits the production of insulin, typically in adolescents.  See —

Bubble Technique Could Create Type 1 Diabetes Therapy

http://www.dddmag.com/news/2016/01/bubble-technique-could-create-type-1-diabetes-therapy

Two new scientific papers published on Monday demonstrated tools that could result in potential therapies for patients diagnosed with type 1 diabetes, a condition in which the immune system limits the production of insulin, typically in adolescents.

Previous treatments for this disease have involved injecting beta cells from dead donors into patients to help their pancreas generate healthy-insulin cells, writes STAT. However, this method has resulted in the immune system targeting these new cells as “foreign” so transplant recipients have had to take immune-suppressing medications for the rest of their lives.

The first paper published in the journal Nature Biotechnology explained how scientists analyzed a seaweed extract called alginate to gauge its effectiveness in supporting the flow of sugar and insulin between cells and the body. An estimated 774 variations were tested in mice and monkeys in which results indicated only a handful could reduce the body’s response to foreign invaders, explains STAT.

The other paper in the journal Nature Medicine detailed a process where scientists developed small capsules infused with alginate and embryonic stem cells. A six-month observation period revealed this “protective bubble” technique “began to produce insulin in response to blood glucose levels” after transplantation in mice subjects with a condition similar to type 1 diabetes, reports Gizmodo.

Essentially, this cured the mice of their diabetes, and the beta cells worked as well as the body’s own cells, according to the researchers. Human trials could still be a few years away, but this experiment could yield a safer alternative to insulin injections.

 

Combinatorial hydrogel library enables identification of materials that mitigate the foreign body response in primates

Arturo J Vegas, Omid Veiseh, Joshua C Doloff, et al.

Nature Biotechnology (2016)    http://dx.doi.org:/10.1038/nbt.3462

The foreign body response is an immune-mediated reaction that can lead to the failure of implanted medical devices and discomfort for the recipient1, 2, 3, 4, 5, 6. There is a critical need for biomaterials that overcome this key challenge in the development of medical devices. Here we use a combinatorial approach for covalent chemical modification to generate a large library of variants of one of the most widely used hydrogel biomaterials, alginate. We evaluated the materials in vivo and identified three triazole-containing analogs that substantially reduce foreign body reactions in both rodents and, for at least 6 months, in non-human primates. The distribution of the triazole modification creates a unique hydrogel surface that inhibits recognition by macrophages and fibrous deposition. In addition to the utility of the compounds reported here, our approach may enable the discovery of other materials that mitigate the foreign body response.

 

Video 1: Intravital imaging of 300 μm SLG20 microcapsules.

Video 2: Intravital imaging of 300 μm Z2-Y12 microcapsules.

Video 3: NHP Laparoscopic procedure for the retrieval of Z2-Y12 spheres.

 

Clinical Focus on Follicular Lymphoma: CAR T-Cells Active in Relapsed Blood Cancers

MedPage Today

CAR T-Cells Active in Relapsed Blood Cancers

Complete responses in half of patients

by Charles Bankhead

Patients with relapsed and refractory B-cell malignancies have responded to treatment with modified T-cells added to conventional chemotherapy, data from an ongoing Swedish study showed.

Six of the first 11 evaluable patients achieved complete responses with increasing doses of chimeric antigen receptor (CAR)-modified T-cells that target the CD19 antigen, although two subsequently relapsed.

Five of the six responding patients received preconditioning chemotherapy the day before CAR T-cell infusion, in addition to chemotherapy administered up to 90 days before T-cell infusion to reduce tumor-cell burden. The remaining five patients received only the earlier chemotherapy, according to a presentation at the inaugural International Cancer Immunotherapy Conference in New York City.

“The complete responses in lymphoma patients despite the fact that they received only low doses of preconditioning compared with other published data surprised us,” Angelica Loskog, PhD, of Uppsala University in Sweden, said in a statement. “The strategy of both providing tumor-reductive chemotherapy for weeks prior to CAR T-cell infusion combined with preconditioning just before CAR T-cell infusion seems to offer promise.

CAR T-cells have demonstrated activity in a variety of studies involving patients with B-cell malignancies. Much of the work has focused on patients with leukemia, including trials in the U.S. B-cell lymphomas have proven more difficult to treat with CAR T-cells because the diseases are associated with higher concentration of immunosuppressive cells that can inhibit CAR T-cell activity, said Loskog. Moreover, blood-vessel abnormalities and accumulation of fibrotic tissue can hinder tumor penetration by therapeutic T-cells.

Each laboratory has its own process for modifying T-cells. Loskog and colleagues in Sweden and at Baylor College of Medicine in Houston have developed third-generation CAR T-cells that contain signaling domains for CD28 and 4-1BB, which act as co-stimulatory molecules. In preclinical models, third-generation CAR T-cells have demonstrated increased activation and proliferation in response to antigen challenge. Additionally, they have chosen to experiment with tumor burden-reducing chemotherapy, a preconditioning chemotherapy to counter the higher immunosuppressive cell count in lymphoma patients.

Loskog reported details of an ongoing phase I/IIa clinical trial involving patients with relapsed or refractory CD19-positive B-cell malignancies. Altogether, investigators have treated 12 patients with increasing doses (2 x 107 to 2 x 108 cells/m2) of CAR T-cells. One patient (with mixed follicular/Burkitt lymphoma) has yet to be evaluated for response. The remaining 11 included three patients with diffuse large B-cell lymphoma (DLBCL), one with follicular lymphoma transformed to DLBCL, two with chronic lymphocytic leukemia, two with mantle cell lymphoma, and three with acute lymphoblastic leukemia.

All of the patients with lymphoma received standard tumor cell-reducing chemotherapy, beginning 3 to 90 days before administration of CAR T-cells. Beginning with the sixth patient in the cohort, patients also received preconditioning chemotherapy (cyclophosphamide/fludarabine) 1 to 2 days before T-cell infusion to reduce the number and activity of immunosuppressive cells.

Cytokine release syndrome is a common effect of CAR T-cell therapy and occurred in several patients treated. In general, the syndrome has been manageable and has not interfered with treatment or response to the modified T-cells.

On the basis of the data produced thus far, the investigators have proceeded with patient evaluation and enrollment. They have already begun cell production for the next patient that will be treated with autologous CAR T-cells.

Although laboratories have their own cell production techniques, the treatment strategy has broad applicability to the treatment of B-cell malignancies, said Loskog.

“The results using different CARs and different techniques for manufacturing them is very similar in the clinic, in terms of initial complete response,” she told MedPage Today. “By using 4-1BB as a co-stimulator in the CAR intracellular region, it seems possible to achieve long-term complete responses in some patients. However, preconditioning of the patients with chemotherapy to reduce the regulatory immune cells seems crucial for effect.”

In an effort to manage the effect of patients’ immunosuppressive cells, the investigators have begun studying each the immune profile before and after treatment. Preliminary results suggest that the population of immunosuppressive cells increases over time, which has the potential to interfere with CAR T-cell responses.

“Especially for lymphoma, it may be crucial to deplete such cells prior to CAR infusion,” said Loskog. “It may even be necessary with supportive treatment for some time after CAR T-cell infusion. A supportive treatment needs to specifically regulate the suppressive cells while sparing the effect of CARs.”

The immunotherapy conference is jointly sponsored by the American Association for Cancer Research, the Cancer Research Institute, the Association for Cancer Immunotherapy, and the European Academy of Tumor Immunology.

 

PET-CT Best for FL Response Assessment

PET-CT associated with better progression-free and overall survival rates in follicular lymphoma.

Kay Jackson

PET-CT (PET) rather than contrast-enhanced CT scanning should be considered the new gold standard for response assessment after first-line rituximab therapy for high-tumor burden follicular lymphoma (FL), a pooled analysis of a central review in three multicenter studies indicated.

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After Cardiac Transplantation: Sirolimus acts as immunosuppressant Attenuates Allograft Vasculopathy

Writer and Curator: Larry H Bernstein, MD, FCAP

and

Curator: Aviva Lev-Ari, PhD, RN 

 

Sirolimus as primary immunosuppression attenuates allograft vasculopathy with improved late survival and decreased cardiac events after cardiac transplantation

Topilsky Y, Hasin T, Raichlin E, Boilson BA, Schirger JA, et al.
Circulation. 2012 Feb 7;125(5):708-20.    http://dx.doi.org/10.1161/CIRCULATIONAHA.111.040360. Epub 2011 Dec 29

BACKGROUND: We retrospectively analyzed the potential of sirolimus as a primary immunosuppressant

  1. in the long-term attenuation of cardiac allograft vasculopathy progression and
  2. the effects on cardiac-related morbidity and mortality.
METHODS:  Forty-five cardiac transplant recipients were converted to sirolimus 1.2 years (0.2, 4.0) after transplantation with complete calcineurin inhibitor withdrawal. Fifty-eight control subjects 2.0 years (0.2, 6.5 years) from transplantation were maintained on calcineurin inhibitors.
  • Age,
  • sex,
  • ejection fraction, and
  • time from transplantation to baseline intravascular ultrasound study were not different (P>0.2 for all) between the groups;
  • neither were secondary immunosuppressants and
  • use of steroids.

Three-dimensional intravascular ultrasound studies were performed at baseline and 3.1 years (1.3, 4.6 years) later.

RESULTS:  Plaque index progression (plaque volume/vessel volume) was attenuated in the sirolimus group (0.7±10.5% versus 9.3±10.8%; P=0.0003) owing to
  1. reduced plaque volume in patients converted to sirolimus early (<2 years) after transplantation (P=0.05) and
  2. improved positive vascular remodeling (P=0.01) in patients analyzed late (>2 years) after transplantation.
Outcome analysis in 160 consecutive patients maintained on 1 therapy was performed regardless of performance of intravascular ultrasound examinations.
  1. Five-year survival was improved with sirolimus (97.4±1.8% versus 81.8±4.9%; P=0.006),
  2. There was freedom from cardiac-related events (93.6±3.2% versus 76.9±5.5%; P=0.002).
CONCLUSIONS:  Substituting calcineurin inhibitor with sirolimus as primary immunosuppressant
  1. attenuates long-term cardiac allograft vasculopathy progression and
  2. may improve long-term allograft survival owing to favorable coronary remodeling.
Because of the lack of randomization and retrospective nature of our analysis, the differences in outcome should be interpreted cautiously, and prospective clinical trials are required.

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Svelte Drug-Eluting Stent Utilizing New Class of Bioabsorbable Drug Coating Attains 0% Clinically-Driven Events Through 12-Months in First-In-Man Study
Aviva Lev-Ari, PhD, RN
Biomaterials Technology: Models of Tissue Engineering for Reperfusion and Implantable Devices for Revascularization
Larry h Benstein, MD, FCAP
Vascular Repair: Stents and Biologically Active Implants
Larry h Benstein, MD, FCAP
New Drug-Eluting Stent Works Well in STEMI
Aviva Lev-Ari, PhD, RN
Coronary Artery DiseaseMedical Devices Solutions: From First-In-Man Stent Implantation, via Medical Ethical Dilemmas to Drug Eluting Stents
Aviva Lev-Ari, PhD, RN
Table 1 Illustration

Table 1 Illustration (Photo credit: Libertas Academica)

Photograph of the Taxus drug-eluting stent, fr...

Photograph of the Taxus drug-eluting stent, from the web site of the U.S. Food and Drug Administration. (Photo credit: Wikipedia)

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Reporter: Aviva Lev-Ari, PhD, RN

 

The healing element is also the enemy – an enigma probed by Hebrew University Lautenberg Center researchers

April 3, 2013

Jerusalem – The same factor in our immune system that is instrumental in enabling us to fight off severe and dangerous inflammatory ailments is also a player in doing the opposite at a later stage, causing the suppression of our immune response.

Why and how this happens and what can be done to mediate this process for the benefit of mankind is the subject of an article published online in the journal Immunity by Ph.D. student Moshe Sade-Feldman and Professor Michal Baniyash of the Lautenberg Center for General and Tumor Immunology at The Hebrew University Faculty of Medicine.
Chronic inflammation poses a major global health problem and is common to different pathologies — such as autoimmune diseases (diabetes, rheumatoid arthritis, lupus and Crohn’s), chronic inflammatory disorders, chronic infections (HIV, leprosy, leishmaniasis) and cancer. Cumulative data indicate that at a certain stage of each of these diseases, the immune system becomes suppressed and results in disease progression.
In their previous work, The Hebrew University researchers had shown that in the course of chronic inflammation, unique immune system cells with suppressive features termed myeloid derived suppressor cells (MDSCs) are generated in the bone marrow and migrate into the body’s organs and blood, imposing a general immune suppression.
A complex network of inflammatory compounds persistently secreted by the body’s normal or cancerous cells support MDSC accumulation, activation and suppressive functions. One of these compounds is tumor necrosis factor-a (TNF-a), which under acute immune responses (short episodes), displays beneficial effects in the initiation of immune responses directed against invading pathogens and tumor cells.
However, TNF-a also displays harmful features under chronic responses, as described in pathologies such as rheumatoid arthritis, psoriasis, type II diabetes, Crohn’s disease and cancer, leading to complications and disease progression. Therefore, today several FDA- approved TNF-a blocking reagents are used in the clinic for the treatment of such pathologies.
What has remained unclear until now, however, is just how TNF-a plays its deleterious role in manipulating the host’s immune system towards the generation of a suppressive environment.
In their work, The Hebrew University researchers discovered the mechanisms underlying the TNF-a  function, a key to controlling this factor and manipulating it, perhaps, for the benefit of humans.  Using experimental mouse models, they showed unequivocally how TNF-a is critical in the induction of immune suppression generated during chronic inflammation. The TNF-a was seen to directly affect the accumulation and suppressive function of MDSCs, leading to an impaired host’s immune responses as reflected by the inability to respond against invading pathogens or against developing tumors.
Further, the direct role of how TNF-a works in humans was mimicked by injecting the FDA-approved anti-TNF-a drug, etanercept, into mice at the exacerbated stage of an inflammatory response, when MDSC accumulation was observed in the blood. The etanercept treatment changed the features of MDSCs and abolished their suppressive activity, leading to the restoration of the host’s immune function.
Taken together, the results show clearly how the TNF-a-mediated inflammatory response, whether acute or chronic, will dictate its beneficial or harmful consequence on the immune system. While during acute inflammation TNF-a is vital for immediate immune defense against pathogens and clearance of tumor cells, during chronic inflammation — under conditions where the host is unable to clear the pathogen or the tumor cells — TNF-a is harmful due to the induction of immune suppression.
These results, providing new insight into the relationship between TNF-a and the development of an immune suppression during chronic inflammation, may aid in the generation of better therapeutic strategies against various pathologies when elevated TNF-a and MDSC levels are detected, as seen, for example, in tumor growths.

 

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Ulcerative colitis

Ulcerative colitis (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.

Tofacitinib, an Oral Janus Kinase Inhibitor, in Active Ulcerative Colitis.
WJ Sandborn, S Ghosh, J Panes, I Vranic, C Su, for the Study A3921063 Investigators
N Engl J Med 2012; 367:616-624 August 16, 2012
http://www.nejm.org/doi/full/10.1056/NEJMoa1112168?query=TOC

 

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.

Fraser JD, Weiss A.  Regulation of T-cell lymphokine gene transcription by the accessory molecule CD28. Mol Cell Biol. 1992 Oct;12(10):4357-63.

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.

Nick S, Pileri P, Tongiani S, Uematsu Y, Kappos L, De Libero G. T cell receptor gamma delta repertoire is skewed in cerebrospinal fluid of multiple sclerosis patients: molecular and functional analyses of antigen-reactive gamma delta clones. Eur J Immunol. 1995 Feb;25(2):355-63. PMID: 1328852 [PubMed – indexed for MEDLINE] PMCID: PMC360359 Free PMC Article

B Cells and T Cells:  Addendum

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.

Panacek EAMarshall JCAlbertson TEJohnson DH, at al.  Efficacy and safety of the monoclonal anti-tumor necrosis factor antibody F(ab’)2 fragment afelimomab in patients with severe sepsis and elevated interleukin-6 levelsCrit Care Med. 2004 Nov;32(11):2173-82.

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 JrOpal SMDhainaut JFStephens 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.

New directions in research on severe sepsis. Human trials with TNF alpha.  Medscape.

4. Why the poor results with sepsis?

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.

The Immune Response to Bacterial InfectionSepsis Syndrome: Bacterial Endotoxin
Chapter 9-3.  2007. p 232-233. New Science Press Ltd

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