David Bruch

Ginger extract inhibits LPS induced macrophage activation and function.

BackgroundMacrophages play a dual role in host defence. They act as the first line of defence by mounting an inflammatory response to antigen exposure and also act as antigen presenting cells and initiate the adaptive immune response. They are also the primary infiltrating cells at the site of inflammation. Inhibition of macrophage activation is one of the possible approaches towards modulating inflammation. Both conventional and alternative approaches are being studied in this regard. Ginger, an herbal product with broad anti inflammatory actions, is used as an alternative medicine in a number of inflammatory conditions like rheumatic disorders. In the present study we examined the effect of ginger extract on macrophage activation in the presence of LPS stimulation.MethodsMurine peritoneal macrophages were stimulated by LPS in presence or absence of ginger extract and production of proinflammatory cytokines and chemokines were observed. We also studied the effect of ginger extract on the LPS induced expression of MHC II, B7.1, B7.2 and CD40 molecules. We also studied the antigen presenting function of ginger extract treated macrophages by primary mixed lymphocyte reaction.ResultsWe observed that ginger extract inhibited IL-12, TNF-α, IL-1β (pro inflammatory cytokines) and RANTES, MCP-1 (pro inflammatory chemokines) production in LPS stimulated macrophages. Ginger extract also down regulated the expression of B7.1, B7.2 and MHC class II molecules. In addition ginger extract negatively affected the antigen presenting function of macrophages and we observed a significant reduction in T cell proliferation in response to allostimulation, when ginger extract treated macrophages were used as APCs. A significant decrease in IFN-γ and IL-2 production by T cells in response to allostimulation was also observed.ConclusionIn conclusion ginger extract inhibits macrophage activation and APC function and indirectly inhibits T cell activation.

In Vitro Immunomodulatory Effects of Ten Commonly Used Herbs on Murine Lymphocytes

OBJECTIVESnPhysicians are increasingly encountering patients who use herbal products. Some of these products are known to modulate the immune system but their scientific basic is not well established. Because these products can affect the host immune system, they could be beneficial in the treatment of immune-related diseases, or alternatively, they could cause inadvertent side-effects. The purpose of this study was to determine which of these common herbal products modulate lymphocyte proliferation in vitro.nnnMETHODSnLymphocyte proliferation assays using concanavalin A (mitogen stimulation) and mixed lymphocyte culture (alloantigen stimulation) were used as in vitro tests to investigate the immunomodulatory effects of 10 commonly used herbal products.nnnRESULTSnGinger and tea were consistently immunosuppressive while dong quai, milk thistle, and St. Johns wort were consistently immunostimulatory in vitro. Ginseng enhanced lymphocyte proliferation only in the mitogen-stimulation assay. The magnitude of the enhancement or suppression of the individual herbal products was different in the two assays.nnnCONCLUSIONnOur study provides a uniform survey of the immunomodulatory properties of 10 commonly used herbal products and paves the way for testing these effects in vivo and in clinical setting.

Morphological and biochemical effects of immunosuppressive drugs in a capillary tube assay for endothelial dysfunction

Abstract: Immunosuppressive drugs common in clinical transplantation are known to have untoward effects on the vascular system. The effects of some drugs, notably cyclosporinu2003A (CyA), have been studied on the vascular system, while those of others have not. In the vascular system, endothelial cells are the predominant cell type exposed to intravascular concentrations of immunosuppressive drugs. We therefore studied the effects of drugs common in clinical transplantation on endothelial cells in a capillary tube assay. The endothelial cells in the capillary tubes are morphologically more similar to those in the microvasculature than endothelial cells in monolayers. We studied the kinetics and extent of capillary tube formation and prostacyclin (PGI2) and endothelin‐1 (ET‐1) release from the inu2003vitro capillaries to determine the morphological and biochemical effects of five immunosuppressive agents on endothelial function. We found a significant difference in the morphological and biochemical effects of the two common calcineurin inhibitors, CyA and tacrolimus (FK506) on capillary morphology inu2003vitro. The former had a pronounced injurious effect on the morphology of the inu2003vitro capillaries, while the latter did not. CyA also significantly increased ET‐1 release by the capillaries, but FK506 did not. Mycophenolate mofetil (MMF) was the only other agent that had a moderately injurious effect on the morphology of the inu2003vitro capillaries. Sirolimus (rapamycin) and dexamethasone, similar to FK506, had no effect on the capillary morphology. All these agents, except dexamethasone, increased PGI2 release. Our data suggest that CyA adversely affects the morphology of the microvasculature and that this is mediated, at least partly, by an increased ET‐1 release by endothelial cells exposed to CyA. These findings describe a novel effect of CyA and MMF on endothelial cells that could be relevant to understanding the mechanisms of immunosuppressive drug‐mediated endothelial injury in clinical transplantation.

Cross-species comparison of gene expression between human and porcine tissue, using single microarray platform--preliminary results.

Abstract:u2002 Introduction:u2002 Xenotransplantation is a potential solution for inadequate supply of donor organs. Pigs are considered the ideal donor for kidney transplantation to human recipients, therefore it is important to understand the gene regulation in the porcine organs. Oligonucleotide array technology has been utilized largely for human, mouse and rat gene expression studies only. Its use with porcine genes has not been reported. We investigated the possibility of studying gene regulation in porcine kidney with a human GeneChip microarray® platform.

An Interleukin-6-Neutralizing Antibody Prevents Cyclosporine-Induced Nephrotoxicity in Mice

INTRODUCTIONnChronic use of cyclosporine A (CyA) induces nephrotoxicity primarily due to endothelial dysfunction. In our previous studies, potential mechanisms were identified in vitro and implicated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and interleukin-6 (IL-6) as key components in causing endothelial dysfunction. In this study, we tested the hypothesis that NADPH oxidase activity and IL-6 are key components in renal damage in an in vivo model.nnnMETHODSnMale mice C57B/6 mice from Jackson Laboratory (Bar Harbor, ME) at 6-8 wks were subjected to a low-salt diet throughout the trial. After 1 week on a low-salt diet, the mice were injected daily with treatments in 50 muL vehicle composed of 75% cremaphor (Sigma, St. Louis, MO) and ethanol for 5 wks. A vehicle-alone group was also set aside. Mice were weighed and 25 mg/kg/day cyclosporine (Novartis Pharma, St. Louis, MO) was injected daily. Apocynin (Calbiochem, Gibbstown, NJ) 20 mg/kg were injected either alone or concomitantly with CyA. Another group of mice were administered IL-6 antibody (Cat no. MAB406; R&D Systems, Minneapolis, MN) at 2 mug/day along with CyA. The kidneys were removed en bloc immediately and submitted in formalin for paraffin sections. Trichrome stains were performed. Slides were blinded and 10 photographs of cortical areas per treatment group were taken, which covered an estimate of 10% surface area in random fashion. Areas of renal damage, which were determined by tubular necrosis, were identified and quantified by amount of necrosis per photograph. Each photograph was divided into 10 blocks, and the number of blocks that contained necrotic tubules per photo was recorded.nnnRESULTSnThe two control mice (low salt only) had no damage. The four vehicle mice had trace amounts of tubular necrosis. CyA treatment group demonstrated the highest amount of damage (29/70; 41%). CyA with apocynin, a specific NADPH oxidase inhibitor, was found to have 36% (22/60) damage, whereas the CyA with IL-6 antibody only was observed to have 15% (6/40) damage. Comparing imaging analysis, there was no difference between mice treated with CyA alone and with CyA with apocynin. However, the amount of damage in mice treated with CyA and IL-6 antibody was found to be significantly lower than both CyA and CyA with apocynin.nnnCONCLUSIONSnCyA action as a calcineurin inhibitor has allowed prolongation of kidney transplants, but its chronic use has led to devastating consequences such as allograft nephropathy. Previously, we have identified potential mechanisms of CyA-induced endothelial dysfunction in vitro. The current study identifies increased IL-6 expression as a mechanism by which CyA induces renal damage and that the use of an IL-6-neutralizing antibody may be useful in reducing CyA-induced renal damage.

Cyclosporine Inhibition of Angiogenesis Involves the Transcription Factor HESR1

PURPOSEnAngiogenesis is critical in normal development and in tumor growth. Experimentally, cyclosporine A (CyA) inhibits angiogenesis in an in vivo mouse model and an in vitro capillary tube model. The mechanisms behind its antiangiogenic effects are not well characterized. To determine which nuclear factor, if any, may be involved in the antiangiogenic effects of CyA, we performed a microarray analysis of human aortic endothelial cells (HAEC) subjected to CyA and another calcineurin inhibitor, FK 506.nnnMETHODSnHAEC were divided into four groups: (1) HAEC incubated with CyA 2 microg/mL; (2) HAEC incubated with CyA 10 microg/mL; (3) HAEC incubated with FK 506 1 microg/mLl for 24 h; and (4) HAEC as control. We used Affymetrix GeneChip U133-A for gene expression analysis and validated our results with quantitative reverse transcription-polymerase chain reaction.nnnRESULTSnAt a 2 microg/mL dose, CyA treated HAEC revealed a 44-fold increase in the expression of hairy enhancer of split-related protein 1 (HESR1) and 1.73-fold down-regulation of transcripts encoding for the vascular endothelial growth factor (VEGF) receptor (VEGFR2). At 10 microg/mL, the expression of the HESR1 transcript was 57-fold higher than control, and VEGFR2 exhibited a 1.93-fold down-regulation. Quantitative reverse transcription-polymerase chain reaction confirmed a significant (P < 0.0001) increase in expression of HESR1 in CyA treated cells. In contrast, the expression level of HESR1 was not affected by the FK 506 treatment.nnnCONCLUSIONnCyA demonstrate antiangiogenic activities linked to an overexpression of HESR1 transcription factor, and down-regulation of VEGFR2. Thus, use of high-dose CyA may provide a novel treatment in angiogenesis dependent disease.

Green Tea Extract Prolongs Allograft Survival as an Adjunctive Therapy Along With Low Dose Cyclosporine A

The current immunosuppressive drugs are successful in prolonging allograft survival but fail to achieve transplantation tolerance or prevent chronic rejection. Consequently, there is ongoing research to develop novel combinatorial therapies that are more efficacious in prolonging allograft survival as well as induce tolerance toward the transplanted organ. The present study aims to study the efficacy of green tea extract (GTE) in combination with low dose cyclosporine A (CyA) in prolonging allograft survival in mice. Numerous studies have reported the anti-inflammatory and immunomodulatory properties of GTE and its various catechin components. GTE is also known to attenuate CyA induced nephrotoxicity. Therefore, we hypothesized that GTE alone or in combination with CyA will prolong graft survival. Our study demonstrates that GTE in combination with low dose CyA significantly prolongs graft survival as well as increase the production of immunosuppressive cytokine, IL-10. GTE also decreases CyA induced high TGF-beta production, which is incriminated in CyA induced nephrotoxicity. We also observed that GTE inhibits both nonspecific and antigen-specific proliferation of T cells in vitro. These results indicate the potential of GTE as an adjunctive therapy in combination with CyA to prolong allograft survival and to reduce CyA induced nephrotoxicity.

A novel extracorporeal kidney perfusion system: a concept model.

The number of patients awaiting kidney transplantation has more than doubled in the past decade while the number of available donor organs has seen only a modest increase, leading to a critical shortage of organs. In response to this extreme shortage, the criteria for accepting organs have been modified to include marginal donors such as non-heart beating donors (NHBD). In these kidneys, determining viability is important for success of transplantation. Therefore, a study was undertaken to develop a system that would allow the extra-corporeal assessment of function and compatibility of the donor organ before the patient is exposed to the risks associated with surgery. Following bilateral nephrectomy, the kidneys of 10 pigs (~30 kg) were connected to a commercially available hypothermic pulsatile kidney perfusion apparatus. This system was modified to allow for normothermic pulsatile renal perfusion using the potential recipient’s blood, via vascular access. These kidneys were perfused with the animal’s blood for a minimum of two hours while various parameters were monitored. Perfusion pressures were kept between 60 and 90 mmHg, which correlated to flows between 70 and 150 mL/min. A decrease in perfusion pressure with a concomitant rise in flow over the two-hour period served as a good predictor of a viable and compatible graft. The modified kidney preservation system allows the normothermic, pulsatile extracorporeal perfusion of donor kidneys with the ability to monitor resistance to flow and urine production. This model also allows observation of the kidney for signs of hyperacute rejection. Further research needs to be conducted in order to determine if the system represents a methodology to increase the pool of available donor organs.

Signal Transduction Pathway in Endothelial Dysfunction

BACKGROUNDnEndothelial dysfunction is an important feature of sepsis, acute respiratory distress syndrome (ARDS), and other infectious conditions. Previously, we reported an in vitro model to study endothelial dysfunction, in which endothelial cells are induced to form capillary tube networks by culturing on a basement membrane matrix (Matrigel). In this study, we defined the signal transduction pathways that lead to endothelial cell function and capillary disruption characteristic of sepsis and other infectious conditions.nnnMETHODSnHuman aortic endothelial cells (HAEC) were cultured on a laminin-rich matrix to form capillary-like networks. The HAECs were treated with a protein tyrosine phosphatase inhibitor (sodium orthovanadate), a phosphoinositon-3-phosphate inhibitor (wortmannin), or a protein kinase C inhibitor (bisindolylmaleimide) before capillary tubes had formed or after the capillary tubes had matured. The degree of capillary tube formation was quantified by counting the intersection of capillary networks in triplicate wells. Statistical significance was determined by analysis of variance.nnnRESULTSnEndothelial dysfunction occurred after inhibition of protein tyrosine phosphatase or protein kinase C. Whereas inhibition of phosphoinositon-3-phosphate did not cause endothelial dysfunction, sodium orthovanadate (2-20 microM) and bisindolylmaleimide (2-10 microM) significantly reduced capillary networks. The mean +/- SD of the number of capillary tubes in the control, sodium orthovanadate-treated, and bisindolylmaleimide-treated groups were 251.0 +/- 7.0, 65.6 +/- 9.9 (p < 0.001), and 181.7 +/- 0.1 (p < 0.001), respectively. Sodium orthovanadate (20-200 microM) and bisindolylmaleimide (10-100 microM) inhibited capillary tube formation. At higher concentrations, sodium orthovanadate (> 200 microM) and bisindolylmaleimide (>100 microM) disrupted mature capillary tubes.nnnCONCLUSIONSnOur results suggest that PKC and protein tyrosine phosphatase play a role in endothelial dysfunction by interfering with the phosphorylation signals within endothelial cells. These mechanisms may be important in the endothelial dysfunction in sepsis and other infectious conditions.

Initial angiogenic response in reduced renal mass after transplantation

INTRODUCTIONnShortage of organs is a major problem in kidney transplantation and requires novel strategies to increase the number of kidney transplants. To reduce the shortage of kidneys, we have proposed transplantation of two halves of one kidney into two recipients (hemirenal transplantation, HRT) and have shown its feasibility in pig and human kidneys. However, reduced renal mass can lead to progressive renal failure in rodents and can reduce the longevity of kidney transplants in humans. Recent studies suggest that derangement of angiogenesis plays a role in the progressive renal failure after reduction in renal mass in rodents. However, since the renal physiology of rats is different from that of large animals, we studied angiogenesis in reduced renal mass transplants in pigs and determined if the reduction in renal mass has the same effect in large animals as that in rodents.nnnMATERIALS AND METHODSnKidney autotransplantation was performed in domestic outbred swine. Heminephrectomy of the autotransplanted kidney and nephrectomy of the contralateral kidney were performed 1 week after transplantation to reduce the renal mass. Four weeks after transplantation, the pigs were sacrificed and the hemirenal and control nephrectomy specimens were processed for morphometric analysis of glomerular capillary density and immunohistochemical analysis of VEGF expression. Soluble extracts from the kidneys were tested in an in vitro angiogenesis assay to determine their activity to influence angiogenesis. Statistical analysis with ANOVA was performed on the glomerular capillary density in kidney specimens.nnnRESULTSnAll these parameters of angiogenesis were increased in the reduced renal mass autotransplants as compared to normal kidneys or whole kidney autotransplants. Glomerular capillary density was increased significantly after reduction in renal mass. VEGF expression also was increased progressively by the third week after reduction in renal mass. Soluble extract from the reduced renal mass transplants significantly increased the in vitro angiogenesis.nnnCONCLUSIONnThis is the first study to demonstrate that angiogenesis is increased in the initial stages of reduction in renal mass after transplantation in a large animal model. Increased angiogenesis was found in this model earlier than reported in small animal models (2 weeks in pigs versus 6 weeks in rats). Taken together with other studies, our data suggest that derangement in angiogenesis could play an important role in long-term graft function after hemirenal transplantation.