79 research outputs found
Anti-inflammatory effects of withaferin A in islet transplantation and pancreatitis.
No full textIslet transplantation is a promising treatment for patients severely affected by type 1 diabetes and chronic pancreatitis. Islets are transplanted into the portal vein of the liver, and direct contact of islets with blood initiates a strong innate immune response called instant blood-mediated inflammation response (IBMIR). Approximately 50% to 60% of islets may be lost during the peritransplant period due to IBMIR. A transcription factor known as nuclear factor kappa B (NFκB) primarily mediates the proinflammatory response in islets. Preliminary studies using withaferin A (WA), a plant-derived inhibitor of NFκB, showed protection of islets from proinflammatory cytokine-mediated damage. An in vitro tube model for IBMIR was developed to determine the inhibitory effect of WA. After transplantation, islet damage was evaluated using C-peptide and proinsulin levels in serum, which are influenced by metabolic stimuli. Identification of a biomarker specific for islet damage uninfluenced by physiological changes is a critical need. MicroRNA 375 is highly expressed in β cells of islets, and damage of islets results in release of this miRNA into the bloodstream. Analysis of serum samples during and after islet infusion revealed high levels of microRNA 375, indicating damage by IBMIR, and the estimated islet loss was comparable to that in previous reports. Serum microRNA 375 was also analyzed to validate the inhibitory effect of WA on IBMIR in vitro. Chronic pancreatitis is an inflammatory disease affecting the exocrine and subsequently endocrine function of the pancreas. Currently, there is no therapeutic approach to delay progression of the disease. The underlying mechanism of pathogenesis of chronic pancreatitis is not clearly understood. The activation of NFκB in acinar cells has been reported to enhance the severity of chronic pancreatitis. NFκB inhibition by WA was tested in a cerulein-induced pancreatitis mouse model. The sustained inflammation that leads to chronic pancreatitis is potentiated by endoplasmic reticulum stress and inflammasome activation within the pancreas. Treatment with WA prevented activation of these mechanisms, as evidenced by molecular analysis and histological assessment. In summary, inhibition of NFκB by WA could be a promising strategy to treat chronic pancreatic diseases
Targeting toll-like receptor 4 related inflammation using small molecule, TAK-242, to attenuate inflammatory damage in pancreatitis and islet transplantation.
No full textInflammation is usually a carefully controlled process within the body consisting of the pro-inflammatory phase where damaged or infected tissue is removed followed by an anti-inflammatory stage when tissue repair and regeneration takes place. If the pro- or anti-inflammatory stages are not appropriately controlled, the pro-inflammatory process can lead to medical complications caused by excessive damage and the anti-inflammatory process can lead to complications relating to excessive fibrosis. Pancreatitis is a condition where excessive inflammation can result in pancreas and remote organ damage. Excessive inflammation also results in detrimental outcomes in pancreatic islet transplantation which can be utilized to replace beta cell mass lost during type 1 or type 3c diabetes. Toll-like receptor 4, a pattern recognition receptor found on various immune, endothelial, and epithelial cells, can respond to both endogenous and exogenous ligands resulting in enhanced cytokine and chemokine production as well as immune infiltration and activation. In this study, I have used TAK-242, a small molecule inhibitor of TLR4 to analyze the beneficial effects of inhibition of TLR4 in pancreatitis and islet transplantation. In my fist aim I demonstrate the therapeutic potential of inhibition of TLR4 signaling in the context of sterile inflammation using a mouse model of cerulein-induced pancreatitis. In this study I show that administration of TAK-242 prior to cerulein injections results in a less inflammatory environment within the pancreas of mice according to molecular, structural, and flow cytometric analysis. In my next aim I show that targeting TLR4 with TAK-242 can result in enhanced islet transplant outcomes by attenuating innate inflammatory responses immediately after transplantation. In this study, I validate miR-375 and miR-200c as reliable biomarkers for measuring islet graft damage following total pancreatectomy with islet autologous transplantation. I then demonstrate a reduction in these biomarkers and decreases in immune cell activation through inhibition of TLR4. Finally, I show not only does inhibition of TLR4 have effects by directly targeting TLR4 on immune cells, but it also modulates exosome production and cargo of islets in an inflammatory state. These exosomes by themselves have the ability to stimulate macrophage activation which can be subdued by inhibition of TLR4 signaling using TAK-242
Anti-inflammatory effects of withaferin A in islet transplantation and pancreatitis.
No full textIslet transplantation is a promising treatment for patients severely affected by type 1 diabetes and chronic pancreatitis. Islets are transplanted into the portal vein of the liver, and direct contact of islets with blood initiates a strong innate immune response called instant blood-mediated inflammation response (IBMIR). Approximately 50% to 60% of islets may be lost during the peritransplant period due to IBMIR. A transcription factor known as nuclear factor kappa B (NFκB) primarily mediates the proinflammatory response in islets. Preliminary studies using withaferin A (WA), a plant-derived inhibitor of NFκB, showed protection of islets from proinflammatory cytokine-mediated damage. An in vitro tube model for IBMIR was developed to determine the inhibitory effect of WA. After transplantation, islet damage was evaluated using C-peptide and proinsulin levels in serum, which are influenced by metabolic stimuli. Identification of a biomarker specific for islet damage uninfluenced by physiological changes is a critical need. MicroRNA 375 is highly expressed in β cells of islets, and damage of islets results in release of this miRNA into the bloodstream. Analysis of serum samples during and after islet infusion revealed high levels of microRNA 375, indicating damage by IBMIR, and the estimated islet loss was comparable to that in previous reports. Serum microRNA 375 was also analyzed to validate the inhibitory effect of WA on IBMIR in vitro. Chronic pancreatitis is an inflammatory disease affecting the exocrine and subsequently endocrine function of the pancreas. Currently, there is no therapeutic approach to delay progression of the disease. The underlying mechanism of pathogenesis of chronic pancreatitis is not clearly understood. The activation of NFκB in acinar cells has been reported to enhance the severity of chronic pancreatitis. NFκB inhibition by WA was tested in a cerulein-induced pancreatitis mouse model. The sustained inflammation that leads to chronic pancreatitis is potentiated by endoplasmic reticulum stress and inflammasome activation within the pancreas. Treatment with WA prevented activation of these mechanisms, as evidenced by molecular analysis and histological assessment. In summary, inhibition of NFκB by WA could be a promising strategy to treat chronic pancreatic diseases
Inhibition of TLR4 minimizes islet damage due to sterile inflammation and improves islet transplant outcomes.
No full textIslet transplantation has emerged as an important treatment option for brittle type 1 diabetes and as an adjunct procedure after total pancreatectomy to prevent brittle diabetes. The efficacy and long-term function of islet transplantation have significantly improved over the last two decades. However, transplant outcomes are still largely compromised due to inflammation mediated prior to and after transplantation which results in the loss of as much as 50% of the islet graft. Toll-like receptor 4 (TLR4) has been identified as a major pro-inflammatory mediator of sterile inflammation by sensing damage-associated molecular patterns (DAMPs) and compromising graft function, making it a putative therapeutic target. Here, we study the effects of TLR4 blockade during the peri-transplant period on islet transplant outcomes using TAK-242, a small molecule inhibitor of TLR4, and a combination of basic biological assays as well as in vivo transplant models in mice. The results of early TLR4 blockade during islet isolation demonstrate a markedly reduced inflammatory profile in islets post-isolation which translated to reduced islet damage post-transplant and overall improved transplant outcomes with a cure rate of 75% for treated islets and 29% for untreated islets. Next, we developed a TLR4-antagonist prodrug and a chemical conjugation method to link the prodrug to the surface of islets which is slowly released, creating drug-eluting islets. Transplantation of a marginal dose of 100 modified islets into the kidney subcapsular space resulted in a cure rate of 100% compared to 0% for unmodified islets. In conclusion, we demonstrate that TLR4 is a major mediator of islet graft loss during the peri-transplant period. Therapies directed to inhibit this receptor, before and after transplant, are a promising avenue for improving islet transplant outcomes. The addition of TAK-242 to media during the isolation process is a rapidly translatable approach to clinical use, while the surface modification technique opens a broad range of possible transplant applications
Inhibition of TLR4 minimizes islet damage due to sterile inflammation and improves islet transplant outcomes.
No full textIslet transplantation has emerged as an important treatment option for brittle type 1 diabetes and as an adjunct procedure after total pancreatectomy to prevent brittle diabetes. The efficacy and long-term function of islet transplantation have significantly improved over the last two decades. However, transplant outcomes are still largely compromised due to inflammation mediated prior to and after transplantation which results in the loss of as much as 50% of the islet graft. Toll-like receptor 4 (TLR4) has been identified as a major pro-inflammatory mediator of sterile inflammation by sensing damage-associated molecular patterns (DAMPs) and compromising graft function, making it a putative therapeutic target. Here, we study the effects of TLR4 blockade during the peri-transplant period on islet transplant outcomes using TAK-242, a small molecule inhibitor of TLR4, and a combination of basic biological assays as well as in vivo transplant models in mice. The results of early TLR4 blockade during islet isolation demonstrate a markedly reduced inflammatory profile in islets post-isolation which translated to reduced islet damage post-transplant and overall improved transplant outcomes with a cure rate of 75% for treated islets and 29% for untreated islets. Next, we developed a TLR4-antagonist prodrug and a chemical conjugation method to link the prodrug to the surface of islets which is slowly released, creating drug-eluting islets. Transplantation of a marginal dose of 100 modified islets into the kidney subcapsular space resulted in a cure rate of 100% compared to 0% for unmodified islets. In conclusion, we demonstrate that TLR4 is a major mediator of islet graft loss during the peri-transplant period. Therapies directed to inhibit this receptor, before and after transplant, are a promising avenue for improving islet transplant outcomes. The addition of TAK-242 to media during the isolation process is a rapidly translatable approach to clinical use, while the surface modification technique opens a broad range of possible transplant applications
Analysis of inflammatory changes in human pancreatic islet cells.
No full textIncludes bibliographical references (p. 126-143).Pancreatic islet cell transplantation is a promising investigational research treatment for labile type 1 diabetes mellitus. However, several obstacles still exist to the implementation of clinical islet cell transplantation as a standard therapy. These obstacles include a shortage of donor pancreata, an imperfect islet isolation procedure, significant loss of islets during the peri-transplant period, and islet toxicity of immunosuppressive drugs. The present study is focused on addressing two of these major hurdles; namely, identification of factors affecting the islet isolation, and understanding the mechanism involved in the peri-transplant loss of islets. Initial analysis was centered on the effect of cold ischemia time (CIT) on the islet isolation outcome. Comparison of varying CIT and several factors that determine the quantity and quality of islets obtained from 52 isolations was performed. This analysis showed that CIT of less than four hours significantly improved islet isolation results. This finding could help improve the current strategy used in clinical islet transplantation. We hypothesized that following transplantation, the inflammatory environment will specifically alter gene expression in transplanted islets, and also will induce surface expression of HLA-class II molecules. This, in turn, could cause anti-donor response resulting in islet destruction. To test the above hypotheses, two studies were performed. Islet cells were treated with control or type 1 diabetic serum. Gene expression was then analyzed using micro array and confirmed by real-time PCR. Islets treated with diabetic serum demonstrated specific induction of multiple genes reported to have secondary roles in angiogenesis while inhibiting transcription of genes with protective attributes against environmental stresses. Islet cells were treated with IFNγ and TNFα and analyzed for HLA class II induction by real-time PCR analysis, flow cytometry, and immunofluorescent imaging.Cytokine treated islets demonstrated significantly upregulated HLA class II gene transcription and surface expression. Importantly, islet transplant recipient serum showed increased binding and cytotoxicity specifically directed against cytokine treated islets. Together these data suggest that in the context of hepatic portal vein transplantation, islet cells contribute to the innate and adaptive immune response during the peri-transplant period resulting in islet destruction.by Andrew M. Jackson.Ph.D
A comparison of the inflammatory response following autologous compared with allogenic islet cell transplantation.
No full textBackground: The initial response to islet transplantation and the subsequent acute inflammation is responsible for significant attrition of islets following both autologous and allogenic procedures. This multicentre study compares this inflammatory response using cytokine profiles and complement activation. Methods: Inflammatory cytokine and complement pathway activity were examined in two cohorts of patients undergoing total pancreatectomy followed either by autologous (n=11) or allogenic (n=6) islet transplantation. Two patients who underwent total pancreatectomy alone (n=2) served as controls. Results: The peak of cytokine production occurred immediately following induction of anaesthesia and during surgery. There was found to be a greater elevation of the following cytokines: TNF-alpha (P<0.01), MCP-1 (P=0.0013), MIP-1α (P=0.001), MIP-1β (P=0.00020), IP-10 (P=0.001), IL-8 (P=0.004), IL-1α (P=0.001), IL-1ra (0.0018), IL-10 (P=0.001), GM-CSF (P=0.001), G-CSF (P=0.0198), and Eotaxin (P=0.01) in the allogenic group compared to autografts and controls. Complement activation and consumption was observed in all three pathways, and there were no significant differences in between the groups although following allogenic transplantation ΔIL-10 and ΔVEGF levels were significantly elevated those patients who became insulin-independent compared with those who were insulin-dependent. Conclusions: The cytokine profiles following islet transplantation suggests a significantly greater acute inflammatory response following allogenic islet transplantation compared with auto-transplantation although a significant, non-specific inflammatory response occurs following both forms of islet transplantation.Wen Yuan Chung, Cristina A. Pollard, Rohan Kumar, Christopher J. Drogemuller, Bashoo Naziruddin, Cordula Stover, Eyad Issa, John Isherwood, Jill Cooke, Marlon F. Levy, P. Toby H. Coates, Giuseppe Garcea, Ashley R. Denniso
Gadolinium chloride inhibits Kupffer cell nitric oxide synthase (iNOS) induction
No full textAbstract
Kupffer cells (KC) are the phagocytic macrophages of the liver. The rare earth metal, gadolinium (GdCl3), is a lanthanide, which, after phagocytosis by the KC, has been found to alter various aspects of KC physiology. In this study, we describe for the first time that the in vivo administration of GdCl3 to rate decreases the release of NO by isolated rat KC in response to lipopolysaccharide. Western blot analysis shows decreased expression of both imlucible nitric oxide synthase as well as total cellular calmodulin after GdCl3 treatment. Possible mechanisms for this phenomenon are suggested.</jats:p
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