Nataraju Angaswamy

Cooperative signaling for angiogenesis and neovascularization by VEGF and HGF following islet transplantation.

Background. Delayed angiogenesis remains a significant challenge to the survival of transplanted islets. In this study, using a murine model of subcutaneous islet transplantation with matrigel basement membrane matrix, we determined the role of the proangiogenic growth factors in enhancing the islet engraftment. Methods. BALB/c islets were transplanted subcutaneously in growth factor reduced (GFR) or growth factor supplemented (GFS) matrigel into diabetic severe combined immunodeficient mice. GFS matrigel was prepared by supplementing GFR with proangiogenic factors, vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). The functioning grafts were harvested at 15 days and vessel formation was analyzed histopathologically. Results. Our results demonstrate that suboptimal (250) islet equivalents in GFS-VEGF+HGF were able to restore normoglycemia, whereas those transplanted in GFR failed to reverse diabetes. Histopathology of the GFS-VEGF+HGF graft revealed 12±3 blood vessels per field, whereas GFR, GFS-VEGF, and GFS-HGF grafts had only 3±1, 6±2, and 4±1 blood vessels, respectively. Insulin staining demonstrated increased number of islets in matrigel supplemented with VEGF and HGF. Protein and mRNA analysis demonstrated enhanced intercellular adhesion molecule and vascular cell adhesion molecule within the islets when supplemented with both VEGF+HGF suggesting stable blood vessel formation. Transcription factors focal adhesion kinase phosphorylation and extracellular signal-regulated kinase1/2 phosphorylation were also increased (8-fold and 4.6-fold, respectively) when both the growth factors were present. There was weak expression of transcription factors when VEGF or HGF were supplemented alone. Conclusion. We conclude that proangiogenic growth factors, VEGF and HGF, synergistically enhance angiogenesis after islet transplantation leading to stable engraftment.

Interplay between Immune responses to HLA and Non-HLA self-antigens in allograft rejection

Recent studies strongly suggest an increasing role for immune responses against self-antigens (Ags) which are not encoded by the major histocompatibility complex in the immunopathogenesis of allograft rejection. Although, improved surgical techniques coupled with improved methods to detect and avoid sensitization against donor human leukocyte antigen (HLA) have improved the immediate and short term function of transplanted organs. However, acute and chronic rejection still remains a vexing problem for the long term function of the transplanted organ. Immediately following organ transplantation, several factors both immune and non immune mechanisms lead to the development of local inflammatory milieu which sets the stage for allograft rejection. Traditionally, development of antibodies (Abs) against mismatched donor HLA have been implicated in the development of Ab mediated rejection. However, recent studies from our laboratory and others have demonstrated that development of humoral and cellular immune responses against non-HLA self-Ags may contribute in the pathogenesis of allograft rejection. There are reports demonstrating that immune responses to self-Ags especially Abs to the self-Ags as well as cellular immune responses especially through IL17 has significant pro-fibrotic properties leading to chronic allograft failure. This review summarizes recent studies demonstrating the role for immune responses to self-Ags in allograft immunity leading to rejection as well as present recent evidence suggesting there is interplay between allo- and autoimmunity leading to allograft dysfunction.

Regulation of Golgi structure and secretion by receptor-induced G protein βγ complex translocation

We show that receptor induced G protein βγ subunit translocation from the plasma membrane to the Golgi allows a receptor to initiate fragmentation and regulate secretion. A lung epithelial cell line, A549, was shown to contain an endogenous translocating G protein γ subunit and exhibit receptor-induced Golgi fragmentation. Receptor-induced Golgi fragmentation was inhibited by a shRNA specific to the endogenous translocating γ subunit. A kinase defective protein kinase D and a phospholipase C β inhibitor blocked receptor-induced Golgi fragmentation, suggesting a role for this process in secretion. Consistent with βγ translocation dependence, fragmentation induced by receptor activation was inhibited by a dominant negative nontranslocating γ3. Insulin secretion was shown to be induced by muscarinic receptor activation in a pancreatic β cell line, NIT-1. Induction of insulin secretion was also inhibited by the dominant negative γ3 subunit consistent with the Golgi fragmentation induced by βγ complex translocation playing a role in secretion.

Donor-specific antibodies to human leukocyte antigens are associated with and precede antibodies to major histocompatibility complex class I–related chain A in antibody-mediated rejection and cardiac allograft vasculopathy after human cardiac transplantation

Humoral immune responses to mismatched donor human leukocyte antigen (HLA) and major histocompatibility complex (MHC) class I-related chain A (MICA) have been reported to contribute to immunopathogenesis of antibody-mediated rejection (AMR) in the early period and cardiac allograft vasculopathy (CAV) in the late period after cardiac transplantation (HTx). The goal of this study is to define the roles of donor-specific antibodies (DSA) and anti-MICA in AMR and CAV. A total of 95 post-HTx recipients were enrolled; 43 patients in the early period (≤ 12 months post-HTx) and 52 patients in the late period (>12 months post-HTx). Development of DSA and anti-MICA were serially monitored using Luminex. Development of DSA (AMR+: n = 6/8.75%, AMR-: n = 4/35.11%, p = 0.009) and anti-MICA (AMR+: n = 5/8.63%, AMR-: n = 4/35.11%, p = 0.002) was significantly associated with AMR. AMR+DSA+ patients demonstrated increased anti-MICA levels compared with AMR+DSA- patients (p=0.01). Serial monitoring revealed DSA (2.7 ± 1.4 months) preceded development of anti-MICA (6.5 ± 2.1 months) in recipients diagnosed with AMR at 8.3 ± 2.5 months post-HTx. Development of DSA (CAV+: n = 8/12.67%, CAV-: n = 5/40.13%, p = 0.004) and anti-MICA (CAV+: n = 9/12.75%, CAV-: n = 5/40.13%, p = 0.001) was significantly associated with CAV. CAV+DSA+ patients demonstrated increased anti-MICA levels compared with CAV+DSA- patients (p = 0.01). Antibodies to HLA are associated with and precede development of anti-MICA in AMR and CAV. Therefore, DSA and anti-MICA can be used as noninvasive markers for monitoring AMR and CAV.

Development of antibodies to human leukocyte antigen precedes development of antibodies to major histocompatibility class I-related chain A and are significantly associated with development of chronic rejection after human lung transplantation

The development of antibodies (Abs) to major histocompatibility (MHC) class I-related chain A (MICA) and human leukocyte antigen (HLA) and their role in the immunopathogenesis of chronic rejection (bronchiolitis obliterans syndrome [BOS]) after human lung transplantation (LTx) was analyzed. Sera from 80 LTx recipients were analyzed for anti-MICA and anti-HLA Abs using Luminex and flow PRA (panel reactive assay). Development of Abs either to MICA alone or MICA and HLA together significantly correlated (p < 0.01) with development of BOS. Kinetic analysis in the post-LTx period revealed that development of anti-HLA Abs (7.6 +/- 4.7 months) preceded the development of anti-MICA Abs (10.0 +/- 3.5 months). Abs to MICA alleles (*001 and *009) developed approximately 6 months after LTx and peak titers were present at the time of clinical diagnosis of BOS (16.3 +/- 2.7 months). The development of Abs to both MICA and HLA was strongly associated with the development of BOS thereby suggesting a synergistic effect. Furthermore, immune response to mismatched HLA can lead to development of Abs to other MHC related antigens expressed on the airway epithelial cells. Cumulatively, these immune responses contribute to the pathogenesis of chronic rejection following human LTx.

Donor graft steatosis influences immunity to hepatitis C virus and allograft outcome after liver transplantation.

Background. Hepatitis C virus (HCV) recurrence after orthotopic liver transplantation (OLT) is universal, often with accelerated allograft fibrosis. Donor liver steatosis is frequently encountered and often associated with poor early postoperative outcome. The aim of this study was to test the hypothesis that allograft steatosis alters immune responses to HCV and self-antigens promoting allograft fibrosis. Methods. Forty-eight HCV OLT recipients (OLTr) were enrolled and classified based on amount of allograft macrovesicular steatosis at time of OLT. Group 1: no steatosis (0%–5% steatosis, n=21), group 2: mild (5%–35%, n=16), and group 3: moderate (>35%, n=11). Cells secreting interleukin (IL)-17, IL-10, and interferon gamma (IFN-&ggr;) in response to HCV antigens were enumerated by Enzyme Linked Immunospot Assay. Serum cytokines were measured by Luminex, antibodies to Collagen I, II, III, IV, and V by ELISA. Results. OLTr of moderate steatotic grafts had the highest incidence of advanced fibrosis in protocol 1 year post-OLT biopsy (10.8% vs. 15.8% vs. 36.6%, r=0.157, P<0.05). OLTr from groups 2 and 3 had increased HCV-specific IL-17 (P<0.05) and IL-10 (P<0.05) with reduced IFN-&ggr; (P<0.05) secreting cells when compared with group 1. This was associated with increase in serum IL-17, IL-10, IL-1&bgr;, IL-6, IL-5, and decreased IFN-&ggr;. In addition, there was development of antibodies to Collagen I, II, III and V in OLTr with increased steatosis (P<0.05). Conclusion. The results demonstrate that allograft steatosis influences post-OLT HCV-specific immune responses leading to an IL-17 T-helper response and activation of humoral immune responses to liver-associated self-antigens that may contribute to allograft fibrosis and poor outcome.

Immune Responses to Collagen‐IV and Fibronectin in Renal Transplant Recipients With Transplant Glomerulopathy

Antibodies (Abs) to donor HLA (donor‐specific antibodies [DSA]) have been associated with transplant glomerulopathy (TG) following kidney transplantation (KTx). Immune responses to tissue‐restricted self‐antigens (self‐Ags) have been proposed to play a role in chronic rejection. We determined whether KTx with TG have immune responses to self‐Ags, Collagen‐IV (Col‐IV) and fibronectin (FN). DSA were determined by solid phase assay, Abs against Col‐IV and FN by enzyme‐linked immunosorbent assay and CD4+ T cells secreting interferon gamma (IFN‐γ), IL‐17 or IL‐10 by ELISPOT. Development of Abs to self‐Ags following KTx increased the risk for TG with an odds ratio of 22 (p‐value = 0.001). Abs to self‐Ags were IgG and IgM isotypes. Pretransplant Abs to self‐Ags increased the risk of TG (22% vs. 10%, p < 0.05). Abs to self‐Ags were identified frequently in KTx with DSA. TG patients demonstrated increased Col‐IV and FN specific CD4+ T cells secreting IFN‐γ and IL‐17 with reduction in IL‐10. We conclude that development of Abs to self‐Ags is a risk factor and having both DSA and Abs to self‐Ags increases the risk for TG. The increased frequency of self‐Ag‐specific IFN‐γ and IL‐17 cells with reduction in IL‐10 demonstrate tolerance breakdown to self‐Ags which we propose play a role in the pathogenesis of TG.

Lipid raft facilitated ligation of K-α1-Tubulin by specific antibodies on epithelial cells: Role in pathogenesis of chronic rejection following human lung transplantation

Long term function of human lung allografts is hindered by development of chronic rejection manifested as Bronchiolitis Obliterans Syndrome (BOS). We have previously identified the development of antibodies (Abs) following lung transplantation to K-alpha1-tubulin (KAT), an epithelial surface gap junction cytoskeletal protein, in patients who develop BOS. However, the biochemical and molecular basis of the interactions and signaling cascades mediated by KAT Abs are yet to be defined. In this report, we investigated the biophysical basis of the epithelial cell membrane surface interaction between KAT and its specific Abs. Towards this, we analyzed the role of the lipid raft-domains in the membrane interactions which lead to cell signaling and ultimately increased growth factor expression. Normal human bronchial epithelial (NHBE) cells, upon specific ligation with Abs to KAT obtained either from the serum of BOS(+) patients or monoclonal KAT Abs, resulted in upregulation of growth factors VEGF, PDGF, and bFGF (6.4+/-1.1-, 3.2+/-0.9-, and 3.4+/-1.1-fold increase, respectively) all of which are important in the pathogenesis of BOS. To define the role for lipid raft in augmenting surface interactions, we analyzed the changes in the growth factor expression pattern upon depletion and enrichment with lipid raft following the ligation of the epithelial cell membranes with Abs specific for KAT. NHBE cells cultured in the presence of beta-methyl cyclodextran (betaMCD) had significantly reduced growth factor expression (1.3+/-0.3, vs betaMCD untreated being 6.4+/-1.1-fold increase) upon stimulation with KAT Abs. Depletion of cholesterol on NHBE cells upon treatment with betaMCD also resulted in decreased partitioning of caveolin in the membrane fraction indicating a decrease in raft-domains. In conclusion, our results demonstrate an important role for lipid raft-mediated ligation of Abs to KAT on the epithelial cell membrane, which results in the upregulation of growth factor cascades involved in the pathogenesis of BOS following human lung transplantation.

Synergistic effect of antibodies to human leukocyte antigens and defensins in pathogenesis of bronchiolitis obliterans syndrome after human lung transplantation.

BACKGROUND This study aims to determine the role of antibodies to donor-mismatched human leukocyte antigen (HLA) developed during the post-transplant period in inducing defensins and their synergistic role in the pathogenesis of chronic rejection, bronchiolitis obliterans syndrome (BOS), after human lung transplantation (LTx). METHODS Bronchoalveolar lavage (BAL) and serum from 21 BOS+ LTx patients were assayed for β-defensins human neutrophil peptides (HNP) 1-3 (enzyme-linked immunosorbent assay [ELISA]) and anti-HLA antibodies (Luminex, Luminex Corp, Austin, TX). Human airway epithelial cells (AEC) were treated with anti-HLA antibodies, HNP-1/2, or both, and the levels of β-defensin were measured by ELISA. Using a mouse model of obliterative airway disease induced by anti-major histocompatibility (MHC) class-I antibodies, we quantitatively and qualitatively determined neutrophil infiltration by myeloperoxidase (MPO) staining and activity by MPO assay, and defensin levels in the BAL. RESULTS In human LTx patients, higher defensin levels correlated with presence of circulating anti-HLA antibodies (p < 0.05). AEC treated with anti-HLA antibodies or HNP-1/2, produced β-defensin with synergistic effects in combination (612 ± 06 vs 520 ± 23 pg/ml anti-HLA antibody, or 590 ± 10 pg/ml for HNP treatment; p < 0.05). Neutrophil numbers (6-fold) and activity (5.5-fold) were higher in the lungs of mice treated with anti-MHC antibodies vs control. A 2-fold increase in α-defensin and β-defensin levels was also present in BAL on Day 5 after anti-MHC administrations. CONCLUSIONS Anti-HLA antibodies developed during the post-transplant period and α-defensins stimulated β-defensin production by epithelial cells, leading to increased cellular infiltration and inflammation. Chronic stimulation of epithelium by antibodies to MHC and resulting increased levels of defensins induce growth factor production and epithelial proliferation contributing to the development of chronic rejection after LTx.

Phytochemical screening and evaluation of in vitro angiotensin-converting enzyme inhibitory activity of Artocarpus altilis leaf

This study investigates the effect of Artocarpus altilis leaf extracts on angiotensin-converting enzyme (ACE) activity. Among the extracts tested, hot ethanol extract exhibited a potent ACE-inhibitory activity with an IC50 value of 54.08 ± 0.29 µg mL−1 followed by cold ethyl acetate extract (IC50 of 85.44 ± 0.85 µg mL−1). In contrast, the hot aqueous extracts showed minimum inhibition with the IC50 value of 765.52 ± 11.97 µg mL−1 at the maximum concentration tested. Further, the phytochemical analysis indicated the varied distribution of tannins, phenolics, glycosides, saponins, steroids, terpenoids and anthraquinones in cold and hot leaf extracts. The correlation between the phytochemical analysis and ACE-inhibitory activity suggests that the high content of glycosidic and phenolic compounds could be involved in exerting ACE-inhibitory activity. In conclusion, this study supports the utilisation of A. altilis leaf in the folk medicine for the better treatment of hypertension. Further studies on isolation and characterisation of specific ACE-inhibitory molecule(s) from ethyl acetate, ethanol and methanol extracts of A. altilis leaf would be highly interesting.