Plant-Mediated Zinc Oxide Nanoparticles: Advances in the New Millennium towards Understanding Their Therapeutic Role in Biomedical Applications.

Zinc oxide nanoparticles have become one of the most popular metal oxide nanoparticles and recently emerged as a promising potential candidate in the fields of optical, electrical, food packaging, and biomedical applications due to their biocompatibility, low toxicity, and low cost. They have a role in cell apoptosis, as they trigger excessive reactive oxygen species (ROS) formation and release zinc ions (Zn2+) that induce cell death. The zinc oxide nanoparticles synthesized using the plant extracts appear to be simple, safer, sustainable, and more environmentally friendly compared to the physical and chemical routes. These biosynthesized nanoparticles possess strong biological activities and are in use for various biological applications in several industries. Initially, the present review discusses the synthesis and recent advances of zinc oxide nanoparticles from plant sources (such as leaves, stems, bark, roots, rhizomes, fruits, flowers, and seeds) and their biomedical applications (such as antimicrobial, antioxidant, antidiabetic, anticancer, anti-inflammatory, photocatalytic, wound healing, and drug delivery), followed by their mechanisms of action involved in detail. This review also covers the drug delivery application of plant-mediated zinc oxide nanoparticles, focusing on the drug-loading mechanism, stimuli-responsive controlled release, and therapeutic effect. Finally, the future direction of these synthesized zinc oxide nanoparticles' research and applications are discussed.

Ascorbic acid 6-palmitate: a potent inhibitor of human and soybean lipoxygenase-dependent lipid peroxidation.

OBJECTIVES: Lipoxygenases (LOX) are the key enzymes involved in the biosynthesis of leukotrienes and reactive oxygen species, which are implicated in pathophysiology of inflammatory disorders. This study was conducted to evaluate the inhibitory effect of water-soluble antioxidant ascorbic acid and its lipophilic derivative, ascorbic acid 6-palmitate (Vcpal) on polymorphonuclear lymphocyte 5-LOX and soybean 15-LOX (sLOX) in vitro. METHODS: LOX activity was determined by measuring the end products, 5-hydroperoxy eicosatetraenoic acid (5-HETE) and lipid hydroperoxides, by spectrophotometric and high performance liquid chromatography methods. The substrate-dependent enzyme kinetics and docking studies were carried out to understand the nature of inhibition. KEY FINDINGS: Vcpal potently inhibited 5-LOX when compared with its inhibitory effect on sLOX (IC50; 2.5 and 10.3 µm respectively, P = 0.003). Further, Vcpal inhibited 5-LOX more strongly than the known synthetic drugs: phenidone and nordihydroguaiaretic acid (P = 0.0007). Enzyme kinetic studies demonstrated Vcpal as a non-competitive reversible inhibitor of 5-LOX. In-silico molecular docking revealed high MolDock and Rerank score for Vcpal than ascorbic acid, complementing in-vitro results. CONCLUSION: Both in-vitro and docking studies demonstrated Vcpal but not ascorbic acid as a non-competitive inhibitor of 5-LOX- and sLOX-induced lipid peroxidation, suggesting a key role for lipophilic nature in bringing about inhibition.

Synergism of a natural plant product, oleanolic acid with calcineurin inhibitor in prolonging islet allograft survival.

BACKGROUND: Oleanolic acid (OA) is a natural plant-derived triterpenoid with potent anti-inflammatory properties. Since inflammatory cytokines released following islet transplantation hinders engraftment and long-term function, we determined the synergistic ability of OA to with Cyclosporine-A (CSA), a calcineurin inhibitor in improving islet allograft's function and survival. METHODS: C57BL/6 mice were rendered diabetic using streptozotocin (200mg/kg). BALB/c islets were transplanted under the kidney capsule alone (control) or along with administration of OA alone, CSA alone or a combination of both OA and CSA (OA+CSA). T-cell infiltration was analyzed by immunohistochemistry; cytokine concentration was analyzed by Luminex; T-cell cytokine phenotype was analyzed by ELISpot; and alloimmune response was analyzed by flow cytometry. RESULTS: OA+CSA markedly prolonged islet allograft survival compared to controls (37 ± 5 days vs. 8 ± 3 days). A significant decrease of CD4+ (34 ± 9 vs. 154 ± 42 cells/hpf) and CD8+ T-cellular (46 ± 22 vs. 224 ± 51 cells/hpf, p<0.0001) infiltration into the graft in OA+CSA treated mice compared to controls. A significant decrease in T cell infiltration was demonstrated in the OA+CSA cohort over either compound application individually. An increase in anti-inflammatory molecules, IL-10 (2.4-fold) and vascular endothelial growth factor (1.6-fold), along with decreased pro-inflammatory cytokines IFN-γ, IL-1ß (1.3-2.4-fold) and IL-17 (3.2-fold) was demonstrated. OA+CSA also significantly decreased the frequency of allo-specific T-cell responses. Development of antibodies against donor antigens was also delayed (39 vs 22 days; p<0.05) in the OA+CSA cohort over administration of either agent individually. CONCLUSIONS: OA and CSA exert synergistic effect towards enhancing islet allograft survival and function. This synergistic effect resulted in markedly reduced graft infiltrating cells with attenuation of inflammatory cytokine milieu leading to impairment of both cellular and humoral alloimmune responses. Therefore, novel therapeutic approaches involving combination of OA with calcineurin-inhibitor based immunosuppressant CSA will produce potential beneficial outcomes in clinical islet transplantation.

Hepatoprotective action of Orthosiphon diffusus (Benth.) methanol active fraction through antioxidant mechanisms: an in vivo and in vitro evaluation.

ETHNOPHARMACOLOGICAL RELEVANCE: Preparations of Orthosiphon diffusus (Benth.) have been used by folk medicinal practitioners in the Western Ghats of India for treating inflammation, hepatitis and jaundice for many years and their effectiveness is widely acclaimed among the tribal communities. AIM OF THE STUDY: To evaluate the mechanisms behind the antioxidant and hepatoprotective potential of Orthosiphon diffusus methanol active fraction (MAF) using in vivo (rat) and in vitro (cell culture) models. MATERIALS AND METHODS: Neutralization of CCl4-induced hepatotoxicity by MAF was evaluated in rats. Towards this, serum levels of hepatic injury markers (lactate dehydrogenase and alkaline phosphatase), antioxidant enzymes in the liver homogenates, and histological examination were performed. In in vitro studies, mechanisms of neutralization of H2O2-induced toxicity by MAF using MTT, Comet assay and up-regulation of antioxidant enzymes at genetic level (RT-PCR) was performed in HepG2 cells. RESULTS: Rats pre-treated with Orthosiphon diffusus MAF demonstrated significantly reduced levels of serum LDH (1.3-fold, p<0.05) and ALP (1.6-fold, p<0.05). Similarly, multiple dose MAF administration demonstrated significantly enhanced levels (p<0.05) of antioxidant enzymes in the liver homogenates. Histological analysis revealed complete neutralization of CCl4-induced liver injury by the extract. The in vitro studies demonstrated that, pre-treatment of MAF effectively prevented H2O2-induced oxidative stress, genotoxicity and significantly enhanced (~6-fold, p<0.01) expression of genes for antioxidant enzymes. CONCLUSIONS: Orthosiphon diffusus MAF demonstrated significant hepatoprotection against CCl4-induced hepatotoxicity by antioxidant mechanisms comparable to silymarin. H2O2-induced oxidative stress was completely neutralized by MAF through enhanced expression of genes for antioxidant enzymes. Therefore, this study validates the use of Orthosiphon diffusus by folk medicinal practitioners in India. Further, MAF of Orthosiphon diffusus can serve as a strong candidate for the development of herbal hepatoprotective agents.

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.

Role of antibodies to self-antigens in chronic allograft rejection: potential mechanism and therapeutic implications.

Significant progress has been made in preventing acute allograft rejection following solid organ transplantation resulting in improved allograft survival. However, long term function still remains disappointing primarily due to chronic allograft rejection. Alloimmune responses primarily defined by the development of antibodies (Abs) to donor mismatched major histocompatibility antigens during the post-transplantation period have been strongly correlated to the development of chronic rejection. In addition, recent studies have demonstrated an important role for autoimmunity including the development of Abs to organ specific self-antigens in the pathogenesis of chronic allograft rejection. Based on this, a new paradigm has evolved indicating a possible cross-talk between the alloimmune responses and autoimmunity leading to chronic rejection. In this review, we will discuss the emerging concept for the role of cellular and humoral immune responses to self-antigens in the immunopathogenesis of chronic allograft rejection which has the potential to develop new strategies for the prevention and/or treatment of chronic rejection.

LMP-420, a small molecular inhibitor of TNF-α, prolongs islet allograft survival by induction of suppressor of cytokine signaling-1: synergistic effect with cyclosporin-A.

Inflammatory insults following islet transplantation (ITx) hinders engraftment and long-term function of the transplanted (Tx) islets. Using a murine model of ITx, we determined the role of LMP-420, a novel TNF-α inhibitor, both individually and in combination with the immunosuppressant cyclosporine A (CSA) in islet engraftment and survival. Diabetic C57BL/6 mice were Tx with 500 BALB/c islets under the kidney capsule. Four cohorts were used: LMP-420 only, CSA only, combination of LMP-420 and CSA (LMP+CSA), and control (n = 12 per cohort). Serial monitoring of blood glucose levels revealed that LMP+CSA (35 ± 5 days) prolonged stable blood insulin levels compared to control (6 ± 4 days). Immunohistology demonstrated that coadministration (LMP+CSA) results in a significant decrease in CD8(+) T-cell infiltration (LMP+CSA: 31 ± 18 vs. control: 224 ± 51 cells, p < 0.001). Serum cytokine analysis revealed that LMP-420 administration resulted in an increase in the anti-inflammatory cytokine IL-10 (2.5-fold), and a decrease in TNF-α (threefold) with no change in IL-2. However, coadministration resulted in a marked decrease in both IL-2 and TNF-α (threefold) along with increase in IL-10 (threefold). Coadministration also demonstrated increase of antiapoptotic SOCS-1 and Mn-SOD expression and significant reduction of donor-specific antibodies (p < 0.005). In conclusion, LMP-420 administration with CSA results in the upregulation of anti-inflammatory and antiapoptotic mechanisms which facilitate islet allograft engraftment and survival.

Mizoribine--an inosine monophosphate dehydrogenase inhibitor--acts synergistically with cyclosporine A in prolonging survival of murine islet cell and heart transplants across major histocompatibility barrier.

INTRODUCTION: Mizoribine (MZR) is an inosine monophosphate dehydrogenase inhibitor. It has been widely used in Japan in the treatment of autoimmune diseases and is known to inhibit T and B cell proliferation. The aim of this study was to evaluate the efficacy of MZR as an immunosuppressive agent and determine its ability to synergize with a commonly used calcineurin inhibitor Cyclosporine A (CsA) in prolonging survival of murine islet cells and heart transplanted across major histocompatibility barrier. METHODS: Murine allogeneic islet cell transplantation between Balb/c donor mice and C57BL/6 recipient mice and heterotopic heart transplantation was done between C3H/He donor mice and Balb/c recipient mice. Recipients were divided into groups based on immunosuppression: Group 1-No immunosuppression, Group 2-MZR alone (20 mg/kg/day), Group 3-CsA alone (20 mg/kg/day), Group 4-MZR+CsA (20 mg/kg/day). Donor specific IFN-γ, IL-10, IL-2, IL-4 secreting cells were enumerated by ELISpot. Serum cytokine and chemokine concentration was measured by Luminex. RESULTS: Islet cell allograft recipients treated with CsA and MZR had prolonged islet function compared to other groups [normoglycemia (blood glucose <200 mg/dL) up to 32±4 days, p<0.05]. Similarly, heart allograft survival was significantly improved in mice treated with CsA and MZR compared to other groups (50% 30-day survival, p=0.04). Donor specific IFN-γ, IL-4, IL-2 secreting cells were significantly decreased in recipients treated with CsA and MZR with marked increase in IL-10 secreting cells (p<0.05). There was also an increase in serum IL-10 with decrease in IFN-γ, IL-4, IL-2, MCP-1, and IL-6 in mice treated with CsA and MZR CONCLUSION: MZR and CsA when used in combination are potent immunosuppressive agents in murine islet cell and heart transplantation models. These agents lead to a decrease in donor specific IFN-γ with increase in IL-10 secreting cells leading to improved allograft survival and function.

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-γ) 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-γ (P<0.05) secreting cells when compared with group 1. This was associated with increase in serum IL-17, IL-10, IL-1ß, IL-6, IL-5, and decreased IFN-γ. 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.

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.

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.

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.

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 ß-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 ß-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 ß-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 ß-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 ß-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.

Lipid raft facilitated ligation of K-alpha1-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.

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

We show that receptor induced G protein betagamma 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 gamma subunit and exhibit receptor-induced Golgi fragmentation. Receptor-induced Golgi fragmentation was inhibited by a shRNA specific to the endogenous translocating gamma subunit. A kinase defective protein kinase D and a phospholipase C beta inhibitor blocked receptor-induced Golgi fragmentation, suggesting a role for this process in secretion. Consistent with betagamma translocation dependence, fragmentation induced by receptor activation was inhibited by a dominant negative nontranslocating gamma3. Insulin secretion was shown to be induced by muscarinic receptor activation in a pancreatic beta cell line, NIT-1. Induction of insulin secretion was also inhibited by the dominant negative gamma3 subunit consistent with the Golgi fragmentation induced by betagamma complex translocation playing a role in secretion.

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.