The role of immunoglobulin-G subclasses and C1q in de novo HLA-DQ donor-specific antibody kidney transplantation outcomes.

BACKGROUND: Anti-HLA-DQ antibodies are the predominant HLA class II donor-specific antibodies (DSAs) after transplantation. Recently, de novo DQ DSA has been associated with worse allograft outcomes. The aim of this study was to determine the further complement-binding characteristics of the most harmful DQ DSA. METHODS: Single-antigen bead technology was used to screen 284 primary kidney transplant recipients for the presence of posttransplantation DQ DSA. Peak DSA sera of 34 recipients with only de novo DQ DSA and of 20 recipients with de novo DQ plus other DSAs were further analyzed by a modified single-antigen bead assay using immunoglobulin (Ig)-G subclass-specific reporter antibodies and a C1q-binding assay. RESULTS: Compared with recipients who did not have DSA, those with de novo persistent DQ-only DSA and with de novo DQ plus other DSAs had more acute rejection (AR) episodes (22%, P=0.005; and 36%, P=0.0009), increased risk of allograft loss (hazards ratio, 3.7, P=0.03; and hazards ratio, 11.4, P=0.001), and a lower 5-year allograft survival. De novo DQ-only recipients with AR had more IgG1/IgG3 combination and C1q-binding antibodies (51%, P=0.01; and 63%, P=0.001) than patients with no AR. Furthermore, the presence of C1q-binding de novo DQ DSA was associated with a 30% lower 5-year allograft survival (P=0.003). CONCLUSIONS: The presence of de novo persistent, complement-binding DQ DSA negatively impacts kidney allograft outcomes. Therefore, early posttransplantation detection, monitoring, and removal of complement-binding DQ might be crucial for improving long-term kidney transplantation outcomes.

HO-1-STAT3 axis in mouse liver ischemia/reperfusion injury: regulation of TLR4 innate responses through PI3K/PTEN signaling.

BACKGROUND & AIMS: Signal transducer and activator of transcription 3 (STAT3), a key mediator of anti-inflammatory cytokine signaling, is essential for heme oxygenase-1 (HO-1)-induced cytoprotection. The phosphoinositide 3-kinase (PI3K)/phosphatase and tensin homolog delete on chromosome 10 (PTEN) pathways regulate diverse innate immune responses. This study was designed to investigate the role of STAT3 in the regulation of PI3K/PTEN cascade after HO-1 induction in a mouse model of innate immune-dominated liver ischemia/reperfusion injury (IRI). METHODS: Partial warm ischemia was produced in the left and middle hepatic lobes of C57BL/6 mice for 90 min, followed by 6h of reperfusion. RESULTS: Mice subjected to Ad-HO-1 transfer were resistant to liver IRI, and this cytoprotective effect correlated with increased intrahepatic PI3K/Akt and diminished PTEN expression. In contrast, mice undergoing adjunctive Ad-HO-1 treatment and STAT3 knockdown (siRNA) remained susceptible to IR-mediated local inflammatory response and hepatocellular damage. Consistent with decreased cell apoptosis and inhibited TLR4 expression after PI3K/Akt activation, treatment with specific PI3k inhibitor increased local inflammation and recreated liver IRI despite Ad-HO-1 gene transfer. Parallel in vitro studies with bone marrow derived-macrophages have confirmed that HO-1-STAT3 axis-induced PI3K/Akt negatively regulated PTEN expression in TLR4-dependent fashion. CONCLUSIONS: These findings underscore the role of HO-1 induced STAT3 in modulating PI3K/PTEN in liver IRI cascade. Activating PI3K/Akt provides negative feedback mechanism for TLR4-driven inflammation. Identifying molecular pathways of STAT3 modulation in the innate immune system provides the rationale for novel therapeutic approaches for the management of liver inflammation and IRI in transplant patients.

Type I interferon pathway mediates renal ischemia/reperfusion injury.

BACKGROUND: Ischemia/reperfusion (I/R) injury is a common cause of acute renal failure after kidney transplantation. This study was designed to analyze the role of type I interferon (IFN) signaling downstream of Toll-like receptor 2/Toll-like receptor 4 activation in the mechanism of I/R-triggered kidney damage. METHODS: Local warm ischemia was induced in groups wild-type (WT) and type I IFN receptor (IFNAR)-/- mice (C57BL/6) by clamping both kidney pedicles for 45 min. Mice were killed at 5/24/72 hr after reperfusion for serum and kidney sampling. RESULTS: At 5 hr, serum creatinine and blood urea nitrogen levels were markedly reduced in IFNAR-/- mice as compared with WT. By 24 hr after reperfusion, both serum creatinine/blood urea nitrogen in WT increased further, whereas those in IFNAR-/- mice remained comparable with sham controls. Histological analyses showed significantly higher percentage of tubules in the outer medulla displaying cell necrosis, loss of the brush border, cast formation and tubular dilatation in WT mice, as compared with IFNAR-/-. Immunohistology revealed increased neutrophil and macrophage infiltration in the outer medulla in WT mice. The expression of proinflammatory tumor necrosis factor-α, interleukin-1, interleukin-6, and CXCL-2 was markedly reduced selectively in IFNAR-/- mice. Finally, terminal deoxynucleotide transferase-mediated dUTP nick-end labeling analysis showed significantly decreased frequency of apoptotic tubular epithelial cells in IFNAR-deficient mice, as compared with WT. CONCLUSION: This is the first report, which documents the key role of type I IFN signaling in the mechanism of kidney I/R injury. Type I IFN may thus serve as a novel target for the therapy against renal I/R injury.

Kidney transplantation in the US: an analysis of the OPTN/UNOS registry.

As of January 14, 2011, 112,707 individuals were listed for kidney transplant. In the past 5 years the yearly average of deceased donor and living donor kidney-only transplants was 10,052 and 6,153, respectively. Compared with a previous decade, one-, 3- and 5-year graft survival rates for deceased donor kidney transplants increased 5%, 6%, and 6%, respectively. Long-term graft survival has not really improved over the past 2 decades. During the past decade, the percentage of kidneys from deceased donors over age 60 transplanted in patients of the same age group increased (from 29% to 60%) over the previous decade. The number of kidney transplants through paired-exchange donation was 109 in 2007. In 2010, that number almost quadrupled to 421. The number of unrelated living donors (LD) has also increased dramatically over the past decade, accounting for 28% of all LD transplants in 2010, and being the major source of LD kidney transplants since 2008. The combination of male donor and female recipient yielded the highest short- and long-term graft survival rates. Female donor and male recipient had the lowest short- and long-term graft survival rates. The risk of graft loss with female donor and male recipient was 23% higher than with male donor and female recipient. For re-transplants, risk of graft loss was 48% higher in patients who received no induction than in those who received IL-2RA + steroid. The impact of HLA mismatch on graft survival rates has declined, but it remains significant. There is still a 10% decrease in the 5-year graft survival from zero MM (77%) to 5&6 MM (67%) in the more recent years. National sharing of zero HLA-mismatched kidneys to sensitized adults with PRA > 20% has increased the transplant opportunity for more highly sensitized patients, nevertheless it will also indirectly decreased the benefits in survival that zero HLA-mismatches provides to about 6% of the kidney transplant population each year who were not sensitized. As of June 2011, 198,314 kidney transplant recipients were alive with a functioning graft.

Programmed death-1/B7-H1 negative costimulation protects mouse liver against ischemia and reperfusion injury.

UNLABELLED: Programmed death-1 (PD-1)/B7-H1 costimulation acts as a negative regulator of host alloimmune responses. Although CD4 T cells mediate innate immunity-dominated ischemia and reperfusion injury (IRI) in the liver, the underlying mechanisms remain to be elucidated. This study focused on the role of PD-1/B7-H1 negative signaling in liver IRI. We used an established mouse model of partial liver warm ischemia (90 minutes) followed by reperfusion (6 hours). Although disruption of PD-1 signaling after anti-B7-H1 monoclonal antibody treatment augmented hepatocellular damage, its stimulation following B7-H1 immunoglobulin (B7-H1Ig) fusion protected livers from IRI, as evidenced by low serum alanine aminotransferase levels and well-preserved liver architecture. The therapeutic potential of B7-H1 engagement was evident by diminished intrahepatic T lymphocyte, neutrophil, and macrophage infiltration/activation; reduced cell necrosis/apoptosis but enhanced anti-necrotic/apoptotic Bcl-2/Bcl-xl; and decreased proinflammatory chemokine/cytokine gene expression in parallel with selectively increased interleukin (IL)-10. Neutralization of IL-10 re-created liver IRI and rendered B7-H1Ig-treated hosts susceptible to IRI. These findings were confirmed in T cell-macrophage in vitro coculture in which B7-H1Ig diminished tumor necrosis factor-α/IL-6 levels in an IL-10-dependent manner. Our novel findings document the essential role of the PD-1/B7-H1 pathway in liver IRI. CONCLUSION: This study is the first to demonstrate that stimulating PD-1 signals ameliorated liver IRI by inhibiting T cell activation and Kupffer cell/macrophage function. Harnessing mechanisms of negative costimulation by PD-1 upon T cell-Kupffer cell cross-talk may be instrumental in the maintenance of hepatic homeostasis by minimizing organ damage and promoting IL-10-dependent cytoprotection.

T-cell immunoglobulin mucin-3 determines severity of liver ischemia/reperfusion injury in mice in a TLR4-dependent manner.

BACKGROUND & AIMS: T-cell immunoglobulin mucin (TIM) genes are expressed by T cells and regulate host immunity and tolerance. CD4(+) T cells mediate innate immunity-dominated liver ischemia-reperfusion injury (IRI) by unknown mechanisms. TIM-1 is involved in liver IRI, which is activated in part by the Toll-like receptor (TLR)4; we investigated the role of TIM-3 and TLR4 in IRI. METHODS: Using an antibody against TIM-3 (anti-TIM-3), we studied TIM-3 signaling in mice following partial warm liver ischemia and reperfusion. RESULTS: Mice given anti-TIM-3 had more liver damage than controls. Histological studies revealed that anti-TIM-3 increased hepatocellular damage and local neutrophil infiltration, facilitated local accumulation of T cells and macrophages, and promoted liver cell apoptosis. Intrahepatic neutrophil activity; induction of proinflammatory cytokines and chemokines; and expression of cleaved caspase-3, nuclear factor-κB, and TLR4 all increased in mice given anti-TIM-3. Administration of anti-TIM-3 followed by anti-galectin-9 (Gal-9 is a TIM-3 ligand) increased production of interferon-γ by concanavalin A (ConA)-stimulated spleen T cells and expression of tumor necrosis factor-α and interleukin-6 in ConA-stimulated macrophages co-cultured with T cells. Anti-TIM-3 did not affect liver IRI in TLR4-deficient mice. CONCLUSION: TIM-3 blockade exacerbated local inflammation and liver damage, indicating the importance of TIM-3-Gal-9 signaling in maintaining hepatic homeostasis. TIM-3-TLR4 cross-regulation determined the severity of liver IRI in TLR4-dependent manner; these findings provide important information about the modulation of innate vs adaptive responses in patients that received liver transplants. Negative co-stimulation signaling by hepatic T-cells might be developed to minimize innate immunity-mediated liver tissue damage.

Blockade of Janus kinase-2 signaling ameliorates mouse liver damage due to ischemia and reperfusion.

Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling is one of the major pathways for cytokine signal transduction. However, the role of the JAK/STAT pathway in liver ischemia/reperfusion is not clear. This study focuses on Janus kinase-2 (JAK2), which functions upstream of signal transducer and activator of transcription 1 (STAT1) in JAK/STAT, and its role in the mechanism of liver ischemia/reperfusion injury (IRI). Partial warm ischemia was produced in the hepatic lobes of C57BL/6 mice for 90 minutes, and this was followed by 6 hours of reperfusion. Mice were treated with a JAK2 inhibitor (tyrphostin AG490; 40 mg/kg intraperitoneally) or vehicle 60 minutes prior to ischemic insult. JAK2 blockade resulted in a significant reduction of hepatocyte apoptosis and liver injury. Macrophage and neutrophil infiltration, as assessed by immunohistochemistry, was markedly decreased in AG490-treated livers in comparison with controls. The expression of pro-inflammatory cytokines [tumor necrosis factor alpha, interleukin 6 (IL-6), and IL-1beta] and chemokines [chemokine (C-X-C motif) ligand 10 (CXCL-10) and CXCL-2] was also significantly reduced in the AG490-treated group in comparison with controls. AG490-treated livers showed fewer cells positive for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and reduced cleaved caspase-3 protein expression in parallel with increased B-cell lymphoma extra large expression. We employed AG490 (75 mM) in primary bone marrow-derived macrophage (BMM) and hepatoma cell (CRL1830) cultures, which were both stimulated with lipopolysaccharide (LPS; 10 ng/mL). In BMM cultures, AG490 depressed otherwise LPS-induced pro-inflammatory gene expression programs (IL-6, IL-12p40, IL-1beta, CXCL-10, and inducible nitric oxide synthase). In hepatoma cells, AG490 reduced cleaved caspase-3 expression. Moreover, JAK2 blockade inhibited STAT1 and STAT3 phosphorylation. This is the first report documenting that JAK2 signaling is essential in the pathophysiology of liver IRI, as its selective blockage ameliorated the disease process and protected livers from inflammation and apoptosis.

The protective function of neutrophil elastase inhibitor in liver ischemia/reperfusion injury.

BACKGROUND.: A neutrophil elastase (NE) inhibitor, Sivelestat, has been approved for the treatment of acute lung injury associated with systemic inflammation in humans. Some reports have also shown its protective effects in liver inflammatory states. We have recently documented the importance of NE in the pathophysiology of liver ischemia/reperfusion injury, a local Ag-independent inflammation response. This study was designed to explore putative cytoprotective functions of clinically available Sivelestat in liver ischemia/reperfusion injury. METHODS.: Partial warm ischemia was produced in the left and middle hepatic lobes of C57BL/6 mice for 90 min, followed by 6 or 24 hr of reperfusion. The mice were given Sivelestat (100 mg/kg, subcutaneous) at 10 min before ischemia, 10 min before reperfusion, and at 1 and 3 hr of reperfusion thereafter. RESULTS.: Sivelestat treatment significantly reduced serum alanine aminotransferase levels and NE activity, when compared with controls. Histological liver examination has revealed that unlike in controls, Sivelestat ameliorated the hepatocellular damage and decreased local neutrophil activity and infiltration. The expression of proinflammatory cytokines (tumor necrosis factor-alpha and interleukin-6), chemokines (CXCL-1, CXCL-2, and CXCL-10), and toll-like receptor 4 was significantly reduced in the treatment group, along with diminished apoptosis through caspase-3 pathway. Moreover, in vitro studies confirmed downregulation of proinflammatory cytokine and chemokine programs in mouse macrophage cell cultures, along with depression of innate toll-like receptor 4 signaling. CONCLUSION.: Sivelestat-mediated NE inhibition may represent an effective therapeutic option in liver transplantation and other inflammation disease states.

The emerging role of T cell immunoglobulin mucin-1 in the mechanism of liver ischemia and reperfusion injury in the mouse.

The T cell immunoglobulin and mucin domain-containing molecules (TIM) protein family, which is expressed by T cells, plays a crucial role in regulating host adaptive immunity and tolerance. However, its role in local inflammation, such as innate immunity-dominated organ ischemia-reperfusion injury (IRI), remains unknown. Liver IRI occurs frequently after major hepatic resection or liver transplantation. Using an antagonistic anti-TIM-1 antibody (Ab), we studied the role of TIM-1 signaling in the model of partial warm liver ischemia followed by reperfusion. Anti-TIM-1 Ab monotherapy ameliorated the hepatocellular damage and improved liver function due to IR, as compared with controls. Histological examination has revealed that anti-TIM-1 Ab treatment decreased local neutrophil infiltration, inhibited sequestration of T lymphocytes, macrophages, TIM-1 ligand-expressing TIM-4(+) cells, and reduced liver cell apoptosis. Intrahepatic neutrophil activity and induction of proinflammatory cytokines/chemokines were also reduced in the treatment group. In parallel in vitro studies, anti-TIM-1 Ab suppressed interferon-gamma (IFN-gamma) production in concanavalin A (conA)-stimulated spleen T cells, and diminished tumor necrosis factor alpha (TNF-alpha)/interleukin (IL)-6 expression in a macrophage/spleen T cell coculture system. This is the first study to provide evidence for the novel role of TIM-1 signaling in the mechanism of liver IRI. TIM-1 regulates not only T for the role of cell activation but may also affect macrophage function in the local inflammation response. These results provide compelling data for further investigation of TIM-1 pathway in the mechanism of IRI, to improve liver function, expand the organ donor pool, and improve the overall success of liver transplantation.

Trend in lung transplantation in the U.S.: an analysis of the UNOS registry.

The number of lung transplants continues to increase in the U.S. The most significant change over the last decade occurred after the 2005 implementation of LAS. When the percentage of patients being transplanted increased even further, while time-to-transplant and the number of patients dying on the waiting list significantly declined. As a result of implementation of LAS in 2005, IPF recipients became the largest group to receive a lung transplant. And the number of transplants for patients age 60 and over has increased significantly. The number of DL transplants performed yearly increased while the number of SL transplants has remained relatively consistent throughout the last decade. Though the gender distribution of recipients has fluctuated each year, the proportion of females receiving lung transplant has decreased. Of the deceased-donor DL and SL transplant recipients, 69% had a cold ischemia time between 3-6 hrs. And 79% of primary DL and SL transplant recipients had a 0% PRA. 6. A higher number of HLA mismatches impacts unfavorably on graft survival rates; yet, surprisingly, zero HLA A-B-DR MM also have an unfavorable impact; Recipients with less than two hours of cold ischemia-time (n = 815, 4.3%) have the worst five-year graft survival; PRA levels greater than 25% have an unfavorable impact on graft survival.

The inhibition of neutrophil elastase ameliorates mouse liver damage due to ischemia and reperfusion.

Neutrophils are considered crucial effector cells in the pathophysiology of organ ischemia/reperfusion injury (IRI). Although neutrophil elastase (NE) accounts for a substantial portion of the neutrophil activity, the function of NE in liver IRI remains unclear. This study focuses on the role of NE in the mechanism of liver IRI. Partial warm ischemia was produced in the left and middle hepatic lobes of C57BL/6 mice for 90 minutes, and this was followed by 6 to 24 hours of reperfusion. Mice were treated with neutrophil elastase inhibitor (NEI; 2 mg/kg per os) at 60 minutes prior to the ischemia insult. NEI treatment significantly reduced serum alanine aminotransferase levels in comparison with controls. Histological examination of liver sections revealed that unlike in controls, NEI treatment ameliorated hepatocellular damage and decreased local neutrophil infiltration, as assessed by myeloperoxidase assay, naphthol AS-D chloroacetate esterase stains, and immunohistochemistry (anti-Ly-6G). The expression of pro-inflammatory cytokines (tumor necrosis factor alpha and interleukin 6) and chemokines [chemokine (C-X-C motif) ligand 1 (CXCL-1), CXCL-2, and CXCL-10] was significantly reduced in the NEI treatment group, along with diminished apoptosis, according to terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and caspase-3 activity. In addition, toll-like receptor 4 (TLR4) expression was diminished in NEI-pretreated livers, and this implies a putative role of NE in the TLR4 signal transduction pathway. Thus, targeting NE represents a useful approach for preventing liver IRI and hence expanding the organ donor pool and improving the overall success of liver transplantation. Liver Transpl 15:939-947, 2009. (c) 2009 AASLD.

Prevalence of vancomycin-resistant Enterococcus fecal colonization among kidney transplant patients.

BACKGROUND: End stage renal disease patients are at risk of Vancomycin-Resistant Enterococcus (VRE) infections. The first reports of VRE isolation were from hemodialysis patients. However, to date, VRE fecal colonization rates as well as associated risk factors in kidney transplant patients have not yet been established in prospective studies. METHODS: We collected one or two stool samples from 280 kidney transplant patients and analysed the prevalence of VRE and its associated risk factors. Patients were evaluated according to the post-transplant period: group 1, less than 30 days after transplantation (102 patients), group 2, one to 6 months after transplantation (73 patients) and group 3, more than 6 months after transplantation (105 patients). RESULTS: The overall prevalence rate of fecal VRE colonization was 13.6% (38/280), respectively 13.7% for Group 1, 15.1% for group 2 and 12.4% for group 3. E. faecium and E. faecalis comprised 50% of all VRE isolates. No immunologic variables were clearly correlated with VRE colonization and no infections related to VRE colonization were reported. CONCLUSION: Fecal VRE colonization rates in kidney transplant patients were as high as those reported for other high-risk groups, such as critical care and hemodialysis patients. This high rate of VRE colonization observed in kidney transplant recipients may have clinical relevance in infectious complications.

Effect of temporary catheter and late referral on hospitalization and mortality during the first year of hemodialysis treatment.

Late referral (LR) to dialysis therapy has been associated with poor outcomes in people with end-stage renal disease. This had been ascribed to the frequent use of temporary vascular catheters (TVCs) in LR patients. The effects of LR and TVC on the outcomes of an incident hemodialysis population (n = 101) were investigated. There was a higher incidence of vascular access infection, longer period of hospitalization, and lower survival in TVC and LR groups, compared with arteriovenous fistula and early referral (ER) groups, respectively. Late referral patients had higher number of hospitalizations than ER patients. In univariate analysis, LR (hazard ratio [HR] 10.8, P = 0.02) and albumin (HR 0.23, P < 0.0001) were associated with mortality. Late referral and body mass index were associated with the increased risk of hospitalization in univariate analysis. In multivariate analysis, LR was the only risk factor associated with hospitalization (HR 3.51, P = 0.002). In conclusion, LR was associated with increased risk of mortality and increased risk of hospitalization independently of the presence of a TVC.