Search tips
Search criteria

Results 1-13 (13)

Clipboard (0)

Select a Filter Below

Year of Publication
Document Types
1.  Critical Role for IL-17A/F in the Immunopathogenesis of Obliterative Airway Disease Induced by Anti-MHC I Antibodies 
Transplantation  2013;95(2):293-300.
The IL-17 axis is implicated in pathogenesis of chronic rejection following human lung transplantation. Using a murine model of obliterative airway disease (OAD), we recently demonstrated that Abs to MHC class I antigens can induce immune responses to self-antigens that contributes to immunopathogenesis of chronic rejection. Using a murine model of OAD, we determined the role of IL-17 family members in induction of autoimmunity leading to OAD following ligation of MHC class I.
Anti-MHC class I or control antibodies (Abs) were administered intrabronchially to wild-type (WT) and IL-17a knock out (IL-17A−/−) C57BL/6.
By day 30, anti-MHC I administered endobronchially in IL-17A−/− mice demonstrated significant reduction in cellular infiltration, a 36.8% reduction in CD4+ T cells, 62.7% in CD11b+ macrophages, 37.5% in degree of fibrosis, 1.94 fold and 2.17 fold decrease in anti-KAT and anti-Col-V respectively when compared to wild type mice. Analysis of lung infiltrating cells in anti-MHC I WT revealed increase in IL-17A (KAT:92+21,Col-V:103+19spm) and IL-17F (KAT:5.03%,Col-V:2.75%) secreting CD4+ T cells. However, administration of anti-MHC I in IL-17A−/− demonstrated increase only in IL-17F for KAT (13.70%) and Col-V (7.08%). Anti-IL-17(A-F) mAb administration following anti-MHC I abrogated OAD in both WT and IL-17A−/−.
Our findings indicate that IL-17A and IL-17F secreted by CD4+Th17 cells specific to lung self-antigens are critical mediators of autoimmunity leading to the pathogenesis of OAD.
PMCID: PMC3549536  PMID: 23325004
2.  Modulation of immune responses following solid organ transplantation by microRNA 
Organ transplantation, an accepted treatment for end stage organ failure, is often complicated by allograft rejection and disease recurrence. In this review we will discuss the potential role of microRNAs in allograft immunity especially leading to rejection of the transplanted organ. microRNAs (miRNAs), originally identified in C. elegans, are short non-coding 21–24 nucleotide sequences that bind to its complementary sequences in functional messenger RNAs and inhibits post-translational processes through RNA duplex formation resulting in gene silencing (Lau et al., 2001). Gene specific translational silencing by miRNAs regulates pathways for immune responses such as development of innate immunity, inflammation, T-cell and B-cell differentiation and signaling that are implicated in various stages of allograft rejection. miRNAs also play a role in development of post-transplant complicacies like fibrosis, cirrhosis, carcinogenesis often leading to graft loss and poor patient outcome. Recent advancements in the methods for detecting and quantifying miRNA in tissue biopsies, as well as in serum and urine samples, has led to identification of specific miRNA signatures in patients with allograft rejection and have been utilized to predict allograft status and survival. Therefore, miRNAs play a significant role in post-transplant events including allograft rejection, disease recurrence and tumor development impacting patient outcome.
PMCID: PMC3518685  PMID: 23036474
3.  Hepatitis C Virus Induced miR200c Down Modulates FAP-1, a Negative Regulator of Src Signaling and Promotes Hepatic Fibrosis 
PLoS ONE  2013;8(8):e70744.
Hepatitis C virus (HCV) induced liver disease is the leading indication for liver transplantation (LTx). Reinfection and accelerated development of fibrosis is a universal phenomenon following LTx. The molecular events that lead to fibrosis following HCV infection still remains poorly defined. In this study, we determined microRNA (miRNA) and mRNA expression profiles in livers from chronic HCV patients and normals using microarrays. Using Genego software and pathway finder we performed an interactive analysis to identify target genes that are modulated by miRNAs. 22 miRNAs were up regulated (>2 fold) and 35 miRNAs were down regulated (>2fold) compared to controls. Liver from HCV patients demonstrated increased expression of 306 genes (>3 fold) and reduced expression of 133 genes (>3 fold). Combinatorial analysis of the networks modulated by the miRNAs identified regulation of the phospholipase C pathway (miR200c, miR20b, and miR31through cellular proto-oncogene tyrosine-protein kinase Src (cSrc)), response to growth factors and hormones (miR141, miR107 and miR200c through peroxisome proliferator-activated receptor alpha and extracellular-signal-regulated kinases, and regulation of cellular proliferation (miR20b, miR10b, and miR141 through cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1 p21). Real time PCR (RT-PCR) validation of the miRNA in HCV infected livers demonstrated a 3.3 ±0.9 fold increase in miR200c. In vitro transfection of fibroblasts with miR200c resulted in a 2.2 fold reduction in expression of tyrosine-protein phosphatase non-receptor type 13 or FAS associated phosphatase 1 (FAP-1) and 2.3 fold increase in expression of cSrc. miR200c transfection resulted in significant increases in expression of collagen and fibroblast growth factor (2.8 and 3.4 fold, p<0.05). Therefore, we propose that HCV induced increased expression of miR200c can down modulate the expression of FAP1, a critical regulator of Src and MAP kinase pathway that play an important role in the production of fibrogenic growth factors and development of fibrosis.
PMCID: PMC3741284  PMID: 23950995
Transplant immunology  2012;26(4):201-206.
Steatotic liver grafts tolerate ischemia-reperfusion (I/R) injury poorly, contributing to increased primary graft nonfunction following transplantation. Activation of nuclear factor kappa-B (NFκB) following I/R injury plays a crucial role in activation of pro-inflammatory responses leading to injury.
We evaluated the role of NFκB in steatotic liver injury by using an orthotopic liver transplant (OLT) model in Zucker rats (lean to lean or obese to lean) to define the mechanisms of steatotic liver injury. Obese donors were treated with bortezomib to assess the role of NF-κB in steatotic liver I/R injury. Hepatic levels of NF-κB and pro-inflammatory cytokines were analyzed by ELISA. Serum transaminase levels and histopathological analysis were performed to assess associated graft injury.
I/R injury in steatotic liver results in significant increases in activation of NF-κB (40%, p<0.003), specifically the p65 subunit following transplantation. Steatotic donor pretreatment with proteasome inhibitor bortezomib (0.1 mg/kg) resulted in significant reduction in levels of activated NF-κB (0.58±0.18 vs. 1.37±0.06 O.D./min/10μg protein, p<0.003). Bortezomib treatment also reduced expression of pro-inflammatory cytokines MIP-2 compared with control treated steatotic and lean liver transplants respectively (106±17.5 vs. 443.3±49.9 vs. 176±10.6 pg/mL, p=0.02), TNF-α (223.8±29.9 vs. 518.5±66.5 vs 264.5±30.1 pg/2μg protein, p=0.003) and IL-1β (6.0±0.91 vs. 19.8±5.2 vs 5±1.7 pg/10μg protein, p= 0.02) along with a significant reduction in ALT levels (715±71 vs 3712.5±437.5 vs 606±286 U/L, p=0.01).
These results suggest that I/R injury in steatotic liver transplantation are associated with exaggerated activation of NFκB subunit p65, leading to an inflammatory mechanism of reperfusion injury and necrosis. Proteasome inhibition in steatotic liver donor reduces NFκB p65 activation and inflammatory I/R injury, improving transplant outcomes of steatotic grafts in a rat model.
PMCID: PMC3675789  PMID: 22286145
hepatic steatosis; I/R injury; liver transplantation; NFκB; PS-341; bortezomib; obese; marginal graft
5.  Donor Graft Steatosis Influences Immunity to Hepatitis C Virus and Allograft Outcome After Liver Transplantation 
Transplantation  2011;92(11):1259-1268.
Hepatitis C (HCV) recurrence following orthotopic liver transplantation (OLT) is universal, often with accelerated allograft fibrosis. Donor liver steatosis is frequently encountered and often associated with poor early post-operative outcome. The study’s aim was to test the hypothesis that allograft steatosis alters immune responses to HCV and self-antigens promoting allograft fibrosis.
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), Group 3 – moderate (>35%, n=11). Cells secreting IL-17, IL-10, IFN-γ in response to HCV antigens were enumerated by ELISpot. Serum cytokines were measured by Luminex, antibodies (Abs) to Collagen (Col) I, II, III, IV, V by ELISA.
OLTr of moderate steatotic grafts had the highest incidence of advanced fibrosis in protocol one-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 to group 1. This was associated with increase in serum IL-17, IL-10, IL-1β, IL-6, IL-5 and decreased IFN-γ. In addition, there was development of Abs to Col I, II, III and V in OLTr with increased steatosis (p<0.05).
The results demonstrate that allograft steatosis influences post-OLT HCV specific immune responses leading to a IL-17 T-helper response and activation of humoral immune responses to liver associated self antigens which may contribute to allograft fibrosis and poor outcome.
PMCID: PMC3223266  PMID: 22011763
Allograft Steatosis; Hepatitis C; Recurrence; Fibrosis; Liver Transplantation
6.  The role of molecular chaperonins in warm ischemia and reperfusion injury in the steatotic liver: A proteomic study 
BMC Biochemistry  2012;13:17.
The molecular basis of the increased susceptibility of steatotic livers to warm ischemia/reperfusion (I/R) injury during transplantation remains undefined. Animal model for warm I/R injury was induced in obese Zucker rats. Lean Zucker rats provided controls. Two dimensional differential gel electrophoresis was performed with liver protein extracts. Protein features with significant abundance ratios (p < 0.01) between the two cohorts were selected and analyzed with HPLC/MS. Proteins were identified by Uniprot database. Interactive protein networks were generated using Ingenuity Pathway Analysis and GRANITE software.
The relative abundance of 105 proteins was observed in warm I/R injury. Functional grouping revealed four categories of importance: molecular chaperones/endoplasmic reticulum (ER) stress, oxidative stress, metabolism, and cell structure. Hypoxia up-regulated 1, calcium binding protein 1, calreticulin, heat shock protein (HSP) 60, HSP-90, and protein disulfide isomerase 3 were chaperonins significantly (p < 0.01) down-regulated and only one chaperonin, HSP-1was significantly upregulated in steatotic liver following I/R.
Down-regulation of the chaperones identified in this analysis may contribute to the increased ER stress and, consequently, apoptosis and necrosis. This study provides an initial platform for future investigation of the role of chaperones and therapeutic targets for increasing the viability of steatotic liver allografts.
PMCID: PMC3445822  PMID: 22962947
Ischemia repurfusion injury; Two dimensional gel electrophoresis; Mass spectrometry; Liver transplantation; Chaperonins; Endoplasmic reticulum (ER) stress
Bronchiolitis obliterans syndrome (BOS) is a major cause of morbidity and mortality post lung transplantation (LTx). We sought to determine the relationship between alloimmune responses and autoimmunity, and subsequently how autoimmunity leads to chronic rejection.
We analyzed the development of donor specific antibodies (Abs) in LTx by flow PRA and the development of Abs to K-α1 tubulin (K-α1T) and collagen V (ColV) by ELISA. The frequency of K-α1T and ColV specific T cells that secrete IFN-γ, IL-17 and IL-10 in LTx recipients was measured by ELSIPOT.
In a retrospective analysis of 42 LTx recipients, we demonstrated a strong correlation between development of donor specific anti-HLA Abs, Abs to self-antigens, and BOS (p<0.05). To test the hypothesis that alloimmunity is related to an immune response to self-antigens, we analyzed 103 LTx patients prospectively for the development of donor specific Abs (DSA) and Abs to self-antigens. 42.7% of recipients developed DSA and 30.1% developed Abs to K-α1T and ColV. Development of DSA preceded development of Abs to self-antigens. BOS+ patients had higher frequency of T-cells secreting IL-17 (p<0.01) and IFNγ (p<0.05) with decreased IL-10 (p<0.05) compared to BOS- patients.
Based on these results we propose that alloimmune responses to donor HLA can induce autoimmune responses to airway epithelial self-antigens, characterized by activation of the IL-17 pathway. These immune responses to self-antigens along with alloimmunity contribute to the pathogenesis of BOS. Strategies to prevent development of autoimmunity may be important in preventing the development of chronic rejection.
PMCID: PMC3091959  PMID: 21414808
8.  Endoplasmic Reticulum Stress is a Mediator of Post-Transplant Injury in Severely Steatotic Liver Allografts 
Hepatic steatosis continues to present a major challenge in liver transplantation. These organs have been shown to have an increased susceptibility to cold ischemia and reperfusion (CIR) injury compared to otherwise comparable lean livers; the mechanisms governing this increased susceptibility to CIR injury are not fully understood. Endoplasmic reticulum (ER) stress is an important link between hepatic steatosis, insulin resistance and the metabolic syndrome. In this study, we investigated ER stress signaling and blockade in the mediation of CIR injury in severely steatotic rodent allografts. Steatotic allografts from genetically leptin-resistant rodents had increased ER stress responses and increased markers of hepatocellular injury following liver transplantation into strain-matched lean recipients. ER stress response components were decreased by the chemical chaperone, TUDCA, resulting in improvement of the allograft injury. TUDCA treatment decreased NF-κB activation, and the pro-inflammatory cytokines IL-6 and IL-1β. However, the predominant response was decreased expression of the ER stress cell death mediator, CHOP. Further, activation of the inflammation-associated caspase 11 was decreased linking ER Stress/CHOP to pro-inflammatory cytokine production following steatotic liver transplantation. These data confirm ER stress in steatotic allografts, and implicate this as a mediating mechanism of inflammation and hepatocyte death in the steatotic liver allograft.
PMCID: PMC3056557  PMID: 21280192
Liver Transplantation; Endoplasmic Reticulum Stress; Hepatic Steatosis; Ischemia-Reperfusion Injury
This study aims to determine the role of antibodies (Abs) to donor mismatched HLA developed during the post-transplant period in inducing defensins and their synergistic role in the pathogenesis of chronic rejection, Bronchiolitis Obliterans Syndrome (BOS), following human lung transplantation (LTx).
Bronchoalveolar Lavage (BAL) and serum from twenty-one BOS+ LTx patients were assayed for β-defensins HNP1-3 (ELISA) and Anti-HLA Abs (Luminex). Human Airway Epithelial Cells (AEC) were treated with anti-HLA Abs, HNP-1/2 or both and the levels of β-defensin was measured by ELISA. Using a mouse model of obliterative airway disease induced by anti-MHC class-I Abs, we quantitatively and qualitatively determined neutrophil infiltration (Myeloperoxidase (MPO) staining) and activity (MPO assay) and defensin levels in the BAL.
In human LTx patients, higher defensin levels correlated with presence of circulating anti-HLA Abs (p<0.05). AEC treated with anti-HLA Abs or HNP-1/2, produced β-defensin with synergistic effects in combination (612±06 vs. 520±23 anti-HLA Ab or 590±10 pg/ml for HNP treatment, p<0.05).Neutrophil numbers (6 fold) and activity (5.5 folds) was higher in the lungs of mice treated with anti-MHC Abs compared to control. Two-fold increase in α-defensin and β-defensin levels was also present in BAL on day 5 following anti-MHC administrations.
Anti-HLA Abs developed during the post-transplant period and α-defensins stimulate β-defensin production by epithelial cells leading to increased cellular infiltration and inflammation. Chronic stimulation of epithelium by Abs to MHC and resulting increased levels of defensins induce growth factor production and epithelial proliferation contributing towards development of chronic rejection following LTx.
PMCID: PMC2991612  PMID: 20691611
Human Neutrophil Peptide; Bronchiolitis Obliterans Syndrome; Bronchoalveolar Lavage; Human Beta Defensin 2; Airway Epithelial Cells
10.  Development of Antibodies to HLA Precedes Development of Antibodies to MICA and Are Significantly Associated With Development of Chronic Rejection Following Human Lung Transplantation 
Human immunology  2010;71(6):560-565.
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)) following 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 following 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.
PMCID: PMC2874120  PMID: 20211214
MICA; HLA; antibodies; lung transplantation; BOS; Chronic rejection
11.  Antibodies to MHC Class I induces autoimmunity: Role in the pathogenesis of chronic rejection¶ 
Alloimmunity to mismatched donor HLA-antigens and autoimmunity to self-antigens have been hypothesized to play an important role in immunopathogenesis of chronic rejection of transplanted organs. However, it is not known what role, if any, alloimmune response plays in inducing autoimmunity. To test whether antibody developed post-transplantation to mismatched donor-MHC induces autoimmunity and chronic rejection, we developed a murine model wherein anti-MHC class I antibodies or control (C1.18.4/anti-keratin) were administered intra-bronchially into native lungs. Animals receiving anti-MHC class I, but not control antibodies, developed marked cellular infiltration around vessels and bronchiole of lung by day 15 followed by epithelial hyperplasia, fibrosis and occlusion of the distal airways similar to chronic rejection following human lung transplantation. Lungs of mice receiving anti-MHC class I showed increased expression of chemokines, their receptors and growth factors and induced IL-17 as well as de novo antibodies to self-antigens, K-α1 tubulin and collagenV. IL-17 neutralization by anti-IL-17 resulted in reduction of autoantibody and lesions induced by anti-MHC class I antibodies. Thus, our results indicate that antibodies to donor-MHC can induce autoimmunity, mediated by IL-17, which plays a pivotal role in chronic rejection post-lung transplantation. Therefore, approaches to prevent autoimmunity should be considered for the treatment of chronic rejection post-lung transplantation.
PMCID: PMC2802821  PMID: 19109162
chronic rejection; autoimmunity; antibodies; MHC; pathogenesis
12.  De Novo Production of K-α1 Tubulin Specific Antibodies: Role in Chronic Lung Allograft Rejection 
Lung transplantation is the treatment option for a variety of end stage pulmonary diseases. Post transplant development of antibodies (Abs) against donor HLA and non-HLA antigens have been associated with acute and chronic rejection of transplanted organs. Development of bronchiolitis obliterans syndrome (BOS) following lung transplantation has been correlated with de novo production of anti-donor-HLA Abs. However, only a portion of the patients with BOS demonstrate detectable anti-donor-HLA Abs. Airway epithelium is considered as a major target for lung allograft rejection. In this study we demonstrate that many BOS+ patients (12 of 36) develop Abs reactive to epithelial cell antigen that is distinct from HLA. Further, de novo production of anti-epithelial cell antibody precedes clinical onset of BOS. N-terminal sequencing and BLASTX analysis as well as blocking with K-alpha1 tubulin specific antibody identified the epithelial antigen as K-α1 tubulin. Binding of the de novo produced anti-K-α1 tubulin antibodies to the airway epithelial cells resulted in the increased expression of transcription factors (TCF5 and c-Myc), leading to increased expression of fibrogenic growth factors, activation of cell cycle signaling and fibro-proliferation, the central events in immunopathogenesis of BOS following human lung transplantation.
PMCID: PMC2796833  PMID: 18354170
human; lung; transplantation; antibodies
13.  Soluble CD30 levels as a diagnostic marker for bronchiolitis obliterans syndrome following human lung transplantation 
Transplant immunology  2007;18(3):260-263.
The long term survival of human lung allograft is hampered by the occurrence of chronic rejection, Bronchiolitis Obliterans Syndrome (BOS). This end-stage disease is normally diagnosed clinically by using the pulmonary function tests. This results in delay of BOS diagnosis and consequently prevents early intervention. It is generally accepted that alloimmunity plays an important role in chronic rejection of the allograft. In this study we analyzed serial serum samples from BOS+ and BOS− patients for sCD30 levels to determine the role of sCD30 to predict the onset of BOS. In contrast to BOS negative patients and normal subjects, 6 out of 9 BOS+ patients (P<0.05) studied had an increase in the sCD30 levels. Significantly, the rise was noted 7.57 ±2.63 months before the clinical diagnosis was evident. Therefore, we propose that the rise in serum sCD30 levels can be used as a marker for the detection of patients who are at risk of development of BOS.
PMCID: PMC2170881  PMID: 18047935
lung transplantation; sCD30; chronic rejection; Bronchiolitis obliterans syndrome (BOS); FEV1

Results 1-13 (13)