Carolina Moore

Heme oxygenase-1 gene transfer inhibits inducible nitric oxide synthase expression and protects genetically fat Zucker rat livers from ischemia-reperfusion injury.

BACKGROUND Ischemia/reperfusion (I/R) injury is a critical factor in the dysfunction of steatotic orthotopic liver transplants. Heme oxygenase-1 (HO-1), a cytoprotective protein, may be important in ameliorating hepatic I/R injury. METHODS We used adenovirus (Ad)-based HO-1 gene transfer to analyze the effects of HO-1 overexpression in a well-established fatty Zucker rat model of I/R followed by orthotopic liver transplantation. RESULTS Ad-HO-1 gene therapy increased recipient survival (80% vs. 40-50% in controls) and significantly diminished hepatocyte injury, as compared with untreated and Ad-beta-galactosidase (Ad-beta-Gal)-treated livers. Orthotopic liver transplants in the Ad-HO-1 group exhibited less macrophage infiltration in the portal areas, as compared with controls. Unlike untreated and Ad-beta-Gal-treated orthotopic liver transplant controls, which showed elevated levels of inducible nitric oxide synthase by infiltrating macrophages, inducible nitric oxide synthase expression in the Ad-HO-1 group was almost absent. In contrast, endothelial nitric oxide synthase was comparable in Ad-HO-1- and Ad-beta-Gal-transduced fatty orthotopic liver transplants. Intragraft expression of antiapoptotic Bcl-2 and Bag-1 was increased in Ad-HO-1-treated orthotopic liver transplants, as compared with Ad-beta-Gal controls. Moreover, increased HO enzymatic activity was accompanied by inhibition of caspase-3 protein expression. CONCLUSIONS HO-1 gene transfer significantly prolongs survival of steatotic orthotopic liver transplants, depresses macrophage infiltration, suppresses local expression of inducible nitric oxide synthase, and modulates pro- and antiapoptotic pathways.

Cyclooxygenase-2 Deficiency Enhances Th2 Immune Responses and Impairs Neutrophil Recruitment in Hepatic Ischemia/Reperfusion Injury

Cyclooxygenase-2 (COX-2) is a prostanoid-synthesizing enzyme that is critically implicated in a variety of pathophysiological processes. Using a COX-2-deficient mouse model, we present data that suggest that COX-2 has an active role in liver ischemia/reperfusion (I/R) injury. We demonstrate that COX-2-deficient mice had a significant reduction in liver damage after I/R insult. The inability of COX-2−/− to elaborate COX-2 products favored a Th2-type response in these mice. COX-2−/− livers after I/R injury showed significantly decreased levels of IL-2, as well as IL-12, a cytokine known to have a central role in Th1 effector cell differentiation. Moreover, such livers expressed enhanced levels of the anti-inflammatory cytokine IL-10, shifting the balance in favor of a Th2 response in COX-2-deficient mice. The lack of COX-2 expression resulted in decreased levels of CXCL2, a neutrophil-activating chemokine, reduced infiltration of MMP-9-positive neutrophils, and impaired late macrophage activation in livers after I/R injury. Additionally, Bcl-2 and Bcl-xL were normally expressed in COX-2−/− livers after injury, whereas respective wild-type controls were almost depleted of these two inhibitors of cell death. In contrast, caspase-3 activation and TUNEL-positive cells were depressed in COX-2−/− livers. Therefore, our data support the concept that COX-2 is involved in the pathogenic events occurring in liver I/R injury. The data also suggest that potential valuable therapeutic approaches in liver I/R injury may result from further studies aimed at identifying specific COX-2-derived prostanoid pathways.

Fibronectin-α4β1 Integrin-Mediated Blockade Protects Genetically Fat Zucker Rat Livers from Ischemia/Reperfusion Injury

We tested a hypothesis that interactions between fibronectin (FN), the major extracellular matrix component, and its integrin α4β1 receptor is important in the development of ischemia/reperfusion injury of steatotic liver transplants. We examined the effect of connecting segment-1 (CS1) peptide-facilitated blockade of FN-α4β1 interaction in a well-established steatotic rat liver model of ex vivo cold ischemia followed by iso-transplantation. In this model, CS1 peptides were administered through the portal vein of steatotic Zucker rat livers before and after cold ischemic storage. Lean Zucker recipients of fatty liver transplants received an additional 3-day course of CS1 peptides after transplant. CS1 peptide therapy significantly inhibited the recruitment of T lymphocytes, neutrophil activation/infiltration, and repressed the expression of proinflammatory tumor necrosis factor-α and interferon-γ. Moreover, it resulted in selective inhibition of inducible nitric oxide synthase expression, peroxynitrite formation, and hepatic necrosis. Importantly, CS1 peptide therapy improved function/histological preservation of steatotic liver grafts, and extended their 14-day survival in lean recipients from 40% in untreated to 100% in CS1-treated OLTs. Thus, CS1 peptide-mediated blockade of FN-α4β1 interaction protects against severe ischemia/reperfusion injury experienced otherwise by steatotic OLTs. These novel findings document the potential of targeting FN-α4β1 in vivo interaction to increase the transplant donor pool through modulation of marginal steatotic livers.

Pretransplant IgG Reactivity to Apoptotic Cells Correlates With Late kidney Allograft Loss

Preexisting serum antibodies have long been associated with graft loss in transplant recipients. While most studies have focused on HLA‐specific antibodies, the contribution of non‐HLA‐reactive antibodies has been largely overlooked. We have recently characterized mAbs secreted by B cell clones derived from kidney allograft recipients with rejection that bind to apoptotic cells. Here, we assessed the presence of such antibodies in pretransplant serum from 300 kidney transplant recipients and examined their contribution to the graft outcomes. Kaplan–Meier survival analysis revealed that patients with high pretransplant IgG reactivity to apoptotic cells had a significantly increased rate of late graft loss. The effect was only apparent after approximately 1 year posttransplant. Moreover, the association between pretransplant IgG reactivity to apoptotic cells and graft loss was still significant after excluding patients with high reactivity to HLA. This reactivity was almost exclusively mediated by IgG1 and IgG3 with complement fixing and activating properties. Overall, our findings support the view that IgG reactive to apoptotic cells contribute to presensitization. Taking these antibodies into consideration alongside anti‐HLA antibodies during candidate evaluation would likely improve the transplant risk assessment.

Evidence to Support a Contribution of Polyreactive Antibodies to HLA Serum Reactivity.

Background Assessing the serum reactivity to HLA is essential for the evaluation of transplant candidates and the follow-up of allograft recipients. In this study, we look for evidence at the clonal level that polyreactive antibodies cross-reactive to apoptotic cells and multiple autoantigens can also react to HLA and contribute to the overall serum reactivity. Methods We immortalized B cell clones from the blood of 2 kidney transplant recipients and characterized their reactivity to self-antigens, apoptotic cells as well as native, denatured, and cryptic HLA determinants using enzyme-linked immunosorbent assay (ELISA), immunofluorescence, flow cytometry and Luminex assays. We also assessed the reactivity of 300 pretransplant serum specimens to HLA and apoptotic cells. Results We report here 4 distinct B cell clones cross-reactive to self and HLA class I. All 4 clones reacted to numerous HLA class I alleles but did not appear to target canonical “shared” epitopes. In parallel experiments, we observed a strong correlation between IgG reactivity to HLA and apoptotic cells in pretransplant serum samples collected from 300 kidney transplant recipients. Further analysis revealed that samples with higher reactivity to apoptotic cells displayed significantly higher class I percent panel-reactive antibodies compared to samples with low reactivity to apoptotic cells. Conclusions We provide here (1) proof of principle at the clonal level that human polyreactive antibodies can cross-react to HLA, multiple self-antigens and apoptotic cells and (2) supportive evidence that polyreactive antibodies contribute to overall HLA reactivity in the serum of patients awaiting kidney transplant.

The human thymus perivascular space is a functional niche for viral-specific plasma cells

The human thymus perivascular space houses viral-specific plasma cells and memory B cells producing protective antibodies. Not in the thymic house The human thymus is the seat of T cell production, and thymic infection can alter both thymopoiesis and tolerance. Circulating antibodies from bone marrow–resident plasma cells have been shown to help protect the thymus from infection; however, the contribution of thymic-resident B cells has remained unclear. Now, Nuñez et al. report that plasma cells reside in the human thymus and that these plasma cells produce antibodies reactive to common viral proteins. These cells inhabit the thymic perivascular space, located between the thymic epithelial areas and the circulation, and therefore may fortify the thymus against pathogen invasion. These cells are maintained in aging individuals, suggesting that thymic plasma cells play a key role in thymic protection throughout the human life span. The human thymus is susceptible to viral infections that can severely alter thymopoiesis and compromise the mechanisms of acquired tolerance to self-antigens. In humans, plasma cells (PCs) residing primarily in the bone marrow confer long-lasting protection to common viruses by secreting antigen-specific antibodies. Because the thymus also houses B cells, we examined the phenotypic complexity of these thymic resident cells and their possible protective role against viral infections. Using tissue specimens collected from patients ranging in age from 5 days to 71 years, we found that, starting during the first year of life, CD138+ PCs begin accumulating in the thymic perivascular space (PVS), where they constitutively produce immunoglobulin G (IgG) without the need for additional stimulation. These thymic PCs almost exclusively secrete IgG1 and IgG3, the two main complement-fixing effector IgG subclasses. Moreover, using antigen-specific enzyme-linked immunospot assays, we demonstrated that thymic PCs include a high frequency of cells reactive to common viral proteins. Our study reveals an unrecognized role of the PVS as a functional niche for viral-specific PCs. The PVS is located between the thymic epithelial areas and the circulation. PCs located in this compartment may therefore provide internal protection against pathogen infections and preserve the integrity and function of the organ.

Expansion and somatic hypermutation of B-cell clones in rejected human kidney grafts.

Background B-cell infiltrates are common in rejected kidney allografts, yet their composition is still unclear. The aim of our study was to characterize the clonal composition of B-cell infiltrates of rejected human kidney grafts. Methods We used a molecular approach to characterize the partial B-cell repertoires of 5 failed human kidney grafts with detectable B-cell infiltrates. A comparison between the intragraft and blood repertoire was also conducted for 1 case. Results Redundant sequences were observed in both blood and graft, although the level of clonal amplification was significantly higher for the graft. Somatic hypermutations (SHMs) were also more frequent in sequences found in the graft compared to the blood. The rate of nonsilent mutations was significantly higher in complementarity determining regions (CDRs) compared to framework regions in blood sequences as well as in graft sequences found at low frequency. In contrast, this preferential distribution was lost in sequences found at high frequency in the graft, suggesting a lack of affinity maturation in situ. Lastly, follicular dendritic cells were undetectable in CD20+ infiltrates in all samples examined. Conclusions We provide here evidence that B-cell clones expand and undergo SHMs in situ. However, the even distribution of nonsilent SHM in high-frequency graft sequences together with the absence of follicular dendritic cells do not support the view that infiltrating B cells are part of functional germinal centers.

Dynamics of B Cell Recovery In Kidney/Bone Marrow Transplant Recipients

Background Previous studies identified B cell gene signatures and predominance of specific B cell subsets as a marker of operational tolerance after kidney transplantation. These findings suggested a role for B cells in the establishment or maintenance of tolerance. Here we analyzed B cell recovery in 4 subjects, 3 of whom achieved tolerance after combined kidney/bone marrow transplantation. Methods Peripheral B cell subsets were examined longitudinally by flow cytometry. Immunoglobulin heavy chain repertoire analysis was performed using next-generation sequencing. Lastly, the patients’ serum reactivity to HLA was assessed by Luminex. Results B cell counts recovered approximately 1 year posttransplant except for 1 subject who experienced delayed reconstitution. This subject resumed immunosuppression for acute rejection at 10 months posttransplant and underwent preemptive retransplantation at 3 years for chronic rejection. B cell recovery was accompanied by a high frequency of CD20 + CD24highCD38high transitional B cells and a diversified clonal repertoire. However, all 4 subjects showed prevalence of CD20 + CD27+ memory B cells around 6 months posttransplant when B cell counts were still low and the clonal B cell repertoire very limited. The predominance of memory B cells was also associated with high levels of somatically mutated immunoglobulin heavy chain variable sequences and transient serum reactivity to HLA. Conclusions Our observations reveal the presence of memory B cells early posttransplant that likely escaped the preparative regimen at a time consistent with the establishment of tolerance. Further studies are warranted to characterize the functional properties of these persisting memory cells and evaluate their potential contribution to tolerance induction.

Polyreactive Antibodies Crossreactive to HLA and Self-Antigens Contribute to Overall Serum Reactivity.: Abstract# 1437

1438 The 2K to 1K Transition. A Podocyte Perspective. Y. Yang,1 S. Wang,1 J. Hodgin,2 F. Afshinnia,1 L. Wickman,3 M. Chowdhury,1 M. Samaniego,1 R. Wiggins.1 1Internal Medicine, University of Michigan, Ann Arbor, MI; 2Pathology, University of Michigan, Ann Arbor, MI; 3Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, MI. Although short term outcome following nephrectomy is good, accumulating evidence suggests that increased prevalence of end stage kidney disease (ESKD) occurs in kidney donors. Furthermore allograft half-life, even in apparently “stable” recipients, has not improved in spite of better immunosuppression. This raises the question of whether the one kidney (1K) state per se carries risk for progression to ESKD. Podocytes are post-mitotic cells with limited capacity for replacement which must cover the fi ltration surface to maintain the fi ltration barrier. In model systems depletion of >30% podocytes causes glomerular destabilization associated with detachment of podocytes that can be monitored in the urine. Minor pathologic changes occur after 30% podocyte depletion while glomerulosclerosis occur after >50% podocyte depletion. To evaluate whether the 2K to 1K transition would cause podocyte stress we performed paired analysis of kidney biopsies obtained at the time of implantation and three months later (n=18). All biopsies were pathologically “normal”. Podocyte nuclear density by TLE4+ immunofl uorescence and podocyte area by peroxidase staining for Glepp1 were measured in the same section and quantitated using imaging software. The correlation between these two independent methods was R2=0.70. There was an increase in glomerular volume (GV) (+21%, P=0.02) and average individual podocyte volume (+19%, P=0.02), but no signifi cant change in podocyte number per tuft (-4%, P=0.5). Therefore transitioning from the 2K to the 1K state resulted in a glomerular volume increase which was compensated mainly by podocyte hypertrophy with a resulting 20% decrease in podocyte nuclear density (P<0.001). To determine whether this degree of podocyte stress would be associated with increased rate of podocyte detachment we measured podocin mRNA:creatinine ratio (UPodCR) in the urine cellular pellet of allograft recipients. Stable allograft recipients excreted an increased level of UPodCR (4-fold above control, P<0.001). Since the 1K recipient has only half the number of podocytes of the 2K control, this represents an approximately 8-fold increase in UPodCR, and by extension an 8-fold increase in relative podocyte detachment rate. This level of podocyte detachment would be expected to impact allograft half-life. Abstract# 1439 Analysis of Early BK Viremia and Development of Antibodies to Self-Antigens (Fibronectin and Collagen IV) Following Kidney Transplantation: Effect On Long-Term Outcomes. M. Seifert,1,2 D. Brennan,2 T. Mohanakumar.2 1Southern Illinois University, Springfi eld; 2Washington University, St. Louis. Background: Immune responses to tissue-restricted self-antigens (SAgs) are emerging as important risk factors for graft failure. We previously demonstrated that antibodies (Abs) to SAgs fi bronectin (FN) and collagen type IV (ColIV) are associated with BK viremia and transplant (txp) glomerulopathy. The effect of early BK viremia and FN/ColIV Abs on long-term txp outcomes is unknown. We hypothesized that early BK viremia and FN/ColIV Abs portend worse long-term txp outcomes than either/no phenotype. Methods: We measured FN/ColIV Abs in plasmas from kidney txp recipients in a BK virus monitoring study (n=20). Subjects were monitored for BK viruria and/or viremia during year-1 post-txp with at least 5 years of follow-up. Non-txp patients with stage 3 chronic kidney disease (CKD, n=12) and kidney txp donors (Donors, n=12) served as controls. Using ELISA, we measured FN/ColIV Abs preand 1, 4, and 8 months post-txp, and in each control group. We compared trends in FN/ColIV Abs status between the BK viremic (n=10) and the BK negative group (n=10) and examined the association between BK status, FN/Col4 Abs status, and a composite outcome of acute rejection, graft failure, or death with a functioning graft. Results: Overall, 5/20 (25%) pre-txp, 5/20 (25%) post-txp, 3/12 (25%) CKD, and 0/12 Donors developed FN/ColIV Abs. By 8 months post-txp, 4/10 (40%) and 5/10 (50%) of BK viremic subjects developed FN and ColIV Abs, respectively. No BK negative subjects developed FN or ColIV Abs (p=0.03). The composite outcome was reached in 6/20 (30%) txp recipients; those who were double-positive for FN/ColIV Abs had higher risk for reaching the composite outcome than doublenegative recipients, independent of BK status (Kaplan-Meier and Cox regression analysis, Figure). Conclusions: Abs to SAgs FN/ColIV are present in non-txp stage 3 CKD, preand post-kidney txp, but not healthy donors. Early immune responses to SAgs FN and ColIV are an independent predictor of long-term txp outcomes and may serve as important diagnostic and therapeutic targets in the early post-txp period. Biomarkers and Immune Monitoring II Wednesday, July 30, 2014 4:00 PM 5:30 PM Room 3020 Abstract# 2200 Tolerance and Rejection Associated Transcripts in Low-Risk Kidney Transplant Recipients: Prospective Study. P. Hruba, I. Brabcova, E. Krepsova, J. Slatinska, I. Striz, A. Sekerkova, O. Viklicky. Institute for Clinical and Experimental Medicine, Prague, Czech Republic. 2200 Tolerance and Rejection Associated Transcripts in Low-Risk Kidney Transplant Recipients: Prospective Study. P. Hruba, I. Brabcova, E. Krepsova, J. Slatinska, I. Striz, A. Sekerkova, O. Viklicky. Institute for Clinical and Experimental Medicine, Prague, Czech Republic. Numerous B and T cells associated gene transcripts were identifi ed in operationally tolerant and in transplant recipients suffering from rejection but little is known about their long-term expression in low risk kidney recipients under immunosuppression. Methods: In this prospective single center study, 46 consecutive low-risk (PRA<20%), fi rst kidney recipients treated with TAC/MMF based immunosuppression were enrolled. The expressions of 28 genes, previously found to be associated with