Safety and efficacy of eculizumab in the prevention of antibody-mediated rejection in living-donor kidney transplant recipients requiring desensitization therapy: A randomized trial.

We report results of a phase 2, randomized, multicenter, open-label, two-arm study evaluating the safety and efficacy of eculizumab in preventing acute antibody-mediated rejection (AMR) in sensitized recipients of living-donor kidney transplants requiring pretransplant desensitization (NCT01399593). In total, 102 patients underwent desensitization. Posttransplant, 51 patients received standard of care (SOC) and 51 received eculizumab. The primary end point was week 9 posttransplant treatment failure rate, a composite of: biopsy-proven acute AMR (Banff 2007 grade II or III; assessed by blinded central pathology); graft loss; death; or loss to follow-up. Eculizumab was well tolerated with no new safety concerns. No significant difference in treatment failure rate was observed between eculizumab (9.8%) and SOC (13.7%; P = .760). To determine whether data assessment assumptions affected study outcome, biopsies were reanalyzed by central pathologists using clinical information. The resulting treatment failure rates were 11.8% and 21.6% for the eculizumab and SOC groups, respectively (nominal P = .288). When reassessment included grade I AMR, the treatment failure rates were 11.8% (eculizumab) and 29.4% (SOC; nominal P = .048). This finding suggests a potential benefit for eculizumab compared with SOC in preventing acute AMR in recipients sensitized to their living-donor kidney transplants (EudraCT 2010-019630-28).

Banff survey on antibody-mediated rejection clinical practices in kidney transplantation: Diagnostic misinterpretation has potential therapeutic implications.

The aim of this study was to determine how the Banff antibody-mediated rejection (ABMR) classification for kidney transplantation is interpreted in practice and affects therapy. The Banff Antibody-Mediated Injury Workgroup electronically surveyed clinicians and pathologists worldwide regarding diagnosis and treatment for 6 case-based scenarios. The participants' (95 clinicians and 72 renal pathologists) assigned diagnoses were compared to the Banff intended diagnoses (reference standard). The assigned diagnoses and reference standard differed by 26.1% (SD 28.1%) for pathologists and 34.5% (SD 23.3%) for clinicians. The greatest discordance between the reference standard and clinicians' diagnosis was when histologic features of ABMR were present but donor-specific antibody was undetected (49.4% [43/87]). For pathologists, the greatest discordance was in the case of acute/active ABMR C4d staining negative in a positive crossmatch transplant recipient (33.8% [23/68]). Treatment approaches were heterogeneous but linked to the assigned diagnosis. When acute/active ABMR was diagnosed by the clinician, treatment was recommended 95.3% (SD 18.4%) of the time vs only 77.7% (SD 39.2%) of the time when chronic active ABMR was diagnosed (P < .0001). In conclusion, the Banff ABMR classification is vulnerable to misinterpretation, which potentially has patient management implications. Continued efforts are needed to improve the understanding and standardized application of ABMR classification in the transplant community.

Pharmacodynamic Monitoring of Tacrolimus-Based Immunosuppression in CD14+ Monocytes After Kidney Transplantation.

BACKGROUND: Monocytes significantly contribute to ischemia-reperfusion injury and allograft rejection after kidney transplantation. However, the knowledge about the effects of immunosuppressive drugs on monocyte activation is limited. Conventional pharmacokinetic methods for immunosuppressive drug monitoring are not cell type-specific. In this study, phosphorylation of 3 signaling proteins was measured to determine the pharmacodynamic effects of immunosuppression on monocyte activation in kidney transplant patients. METHODS: Blood samples from 20 kidney transplant recipients were monitored before and during the first year after transplantation. All patients received induction therapy with basiliximab, followed by tacrolimus (TAC), mycophenolate mofetil, and prednisolone maintenance therapy. TAC whole-blood predose concentrations were determined using an antibody-conjugated magnetic immunoassay. Samples were stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin, and phosphorylation of p38MAPK, ERK, and Akt in CD14 monocytes was quantified by phospho-specific flow cytometry. RESULTS: Phosphorylation of p38MAPK and Akt in monocytes of immunosuppressed recipients was lower after 360 days compared with before transplantation in the unstimulated samples [mean reduction in median fluorescence intensity 36%; range -28% to 77% for p-p38MAPK and 20%; range -22% to 53% for p-Akt; P < 0.05]. P-ERK was only decreased at day 4 after transplantation (mean inhibition 23%; range -52% to 73%; P < 0.05). At day 4, when the highest whole-blood predose TAC concentrations were measured, p-p38MAPK and p-Akt, but not p-ERK, correlated inversely with TAC (rs = -0.65; P = 0.01 and rs = -0.58; P = 0.03, respectively). CONCLUSIONS: Immunosuppressive drug combination therapy partially inhibits monocyte activation pathways after kidney transplantation. This inhibition can be determined by phospho-specific flow cytometry, which enables the assessment of the pharmacodynamic effects of immunosuppressive drugs in a cell type-specific manner.

CD16+ Monocytes and Skewed Macrophage Polarization toward M2 Type Hallmark Heart Transplant Acute Cellular Rejection.

BACKGROUND: During acute heart transplant rejection, infiltration of lymphocytes and monocytes is followed by endothelial injury and eventually myocardial fibrosis. To date, no information is available on monocyte-macrophage-related cellular shifts and their polarization status during rejection. Here, we aimed to define and correlate monocyte-macrophage endomyocardial tissue profiles obtained at rejection and time points prior to rejection, with corresponding serial blood samples in 25 heart transplant recipients experiencing acute cellular rejection. Additionally, 33 healthy individuals served as control. MATERIALS AND METHODS: Using histology, immunohistochemistry, confocal laser scan microscopy, and digital imaging expression of CD14, CD16, CD56, CD68, CD80, and CD163 were explored to define monocyte and macrophage tissue profiles during rejection. Fibrosis was investigated using Sirius Red stainings of rejection, non-rejection, and 1-year biopsies. Expression of co-stimulatory and migration-related molecules on circulating monocytes, and production potential for pro- and anti-inflammatory cytokines were studied using flow cytometry. RESULTS: At tissue level, striking CD16+ monocyte infiltration was observed during rejection (p < 0.001). Significantly more CD68+CD163+ M2 macrophages were documented during rejection compared to barely present CD68+CD80+ M1 macrophages. Rejection was associated with severe fibrosis in 1-year biopsies (p < 0.001). Irrespective of rejection status, decreased frequencies of circulating CD16+ monocytes were found in patients compared to healthy individuals. Rejection was reflected by significantly increased CD54 and HLA-DR expression on CD16+ monocytes with retained cytokine production potential. CONCLUSION: CD16+ monocytes and M2 macrophages hallmark the correlates of heart transplant acute cellular rejection on tissue level and seem to be associated with fibrosis in the long term.

Targeting the Monocyte-Macrophage Lineage in Solid Organ Transplantation.

There is an unmet clinical need for immunotherapeutic strategies that specifically target the active immune cells participating in the process of rejection after solid organ transplantation. The monocyte-macrophage cell lineage is increasingly recognized as a major player in acute and chronic allograft immunopathology. The dominant presence of cells of this lineage in rejecting allograft tissue is associated with worse graft function and survival. Monocytes and macrophages contribute to alloimmunity via diverse pathways: antigen processing and presentation, costimulation, pro-inflammatory cytokine production, and tissue repair. Cross talk with other recipient immune competent cells and donor endothelial cells leads to amplification of inflammation and a cytolytic response in the graft. Surprisingly, little is known about therapeutic manipulation of the function of cells of the monocyte-macrophage lineage in transplantation by immunosuppressive agents. Although not primarily designed to target monocyte-macrophage lineage cells, multiple categories of currently prescribed immunosuppressive drugs, such as mycophenolate mofetil, mammalian target of rapamycin inhibitors, and calcineurin inhibitors, do have limited inhibitory effects. These effects include diminishing the degree of cytokine production, thereby blocking costimulation and inhibiting the migration of monocytes to the site of rejection. Outside the field of transplantation, some clinical studies have shown that the monoclonal antibodies canakinumab, tocilizumab, and infliximab are effective in inhibiting monocyte functions. Indirect effects have also been shown for simvastatin, a lipid lowering drug, and bromodomain and extra-terminal motif inhibitors that reduce the cytokine production by monocytes-macrophages in patients with diabetes mellitus and rheumatoid arthritis. To date, detailed knowledge concerning the origin, the developmental requirements, and functions of diverse specialized monocyte-macrophage subsets justifies research for therapeutic manipulation. Here, we will discuss the effects of currently prescribed immunosuppressive drugs on monocyte/macrophage features and the future challenges.

The Effect of Tacrolimus and Mycophenolic Acid on CD14+ Monocyte Activation and Function.

Monocytes and macrophages play key roles in many disease states, including cellular and humoral rejection after solid organ transplantation (SOT). To suppress alloimmunity after SOT, immunosuppressive drug therapy is necessary. However, little is known about the effects of the immunosuppressive drugs tacrolimus and mycophenolic acid (MPA) on monocyte activation and function. Here, the effect of these immunosuppressants on monocytes was investigated by measuring phosphorylation of three intracellular signaling proteins which all have a major role in monocyte function: p38MAPK, ERK and Akt. In addition, biological functions downstream of these signaling pathways were studied, including cytokine production, phagocytosis and differentiation into macrophages. To this end, blood samples from healthy volunteers were spiked with diverse concentrations of tacrolimus and MPA. Tacrolimus (200 ng/ml) inhibited phosphorylation of p38MAPK by 30% (mean) in CD14+ monocytes which was significantly less than in activated CD3+ T cells (max 60%; p < 0.05). This immunosuppressive agent also partly inhibited p-AKT (14%). MPA, at a therapeutic concentration showed the strongest effect on p-AKT (27% inhibition). p-ERK was inhibited with a maximum of 15% after spiking with either tacrolimus or MPA. The production of IL-1ß and phagocytosis by monocytes were not affected by tacrolimus concentrations, whereas MPA did inhibit IL-1ß production by 50%. Monocyte/macrophage polarization was shifted to an M2-like phenotype in the presence of tacrolimus, while MPA increased the expression of M2 surface markers, including CD163 and CD200R, on M1 macrophages. These results show that tacrolimus and MPA do not strongly affect monocyte function, apart from a change in macrophage polarization, to a clinically relevant degree.

Lipid droplets hypertrophy: a crucial determining factor in insulin regulation by adipocytes.

Lipid droplets (LDs) hypertrophy in adipocytes is the main cause of energy metabolic system dysfunction, obesity and its afflictions such as T2D. However, the role of adipocytes in linking energy metabolic disorders with insulin regulation is unknown in humans. Human adipocytes constitutively synthesize and secrete insulin, which is biologically functional. Insulin concentrations and release are fat mass- and LDs-dependent respectively. Fat reduction mediated by bariatric surgery repairs obesity-associated T2D. The expression of genes, like PCSK1 (proinsulin conversion enzyme), GCG (Glucagon), GPLD1, CD38 and NNAT, involved in insulin regulation/release were differentially expressed in pancreas and adipose tissue (AT). INS (insulin) and GCG expression reduced in human AT-T2D as compared to AT-control, but remained unchanged in pancreas in either state. Insulin levels (mRNA/protein) were higher in AT derived from prediabetes BB rats with destructed pancreatic ß-cells and controls than pancreas derived from the same rats respectively. Insulin expression in 10 human primary cell types including adipocytes and macrophages is an evidence for extrapancreatic insulin-producing cells. The data suggest a crosstalk between AT and pancreas to fine-tune energy metabolic system or may minimize the metabolic damage during diabetes. This study opens new avenues towards T2D therapy with a great impact on public health.

Rotterdam: main port for organ transplantation research in the Netherlands.

This overview describes the full spectrum of current pre-clinical and clinical kidney-, liver-, heart- and lung transplantation research performed in Erasmus MC - University Medical Centre in Rotterdam, The Netherlands. An update is provided on the development of a large living donor kidney transplantation program and on optimization of kidney allocation, including the implementation of a domino kidney-donation program. Our current research efforts to optimize immunosuppressive regimens and find novel targets for immunosuppressive therapy, our recent studies on prevention of ischemia-reperfusion-induced graft injury, our newest findings on stimulation of tissue regeneration, our novel approaches to prevent rejection and viral infection, and our latest insights in the regulation of allograft rejection, are summarized.

Limited efficacy of immunosuppressive drugs on CD8+ T cell-mediated and natural killer cell-mediated lysis of human renal tubular epithelial cells.

BACKGROUND: Although CD8+ T cell-mediated and natural killer (NK) cell-mediated cytotoxicity against renal tubular epithelial cells (TECs) plays a crucial role during rejection, the degree of inhibition of these lytic immune responses by immunosuppressive drugs is unknown. We investigated the CD8 T-cell and NK cell responses induced by TECs in vitro and questioned how these processes are affected by immunosuppressive drugs. METHODS: Donor-derived TECs were co-cultured with recipient peripheral blood monocyte cells. Proliferation of CD8+ T cells and NK cell subsets was assessed using PKH dilution assay. CD107a degranulation and europium release assay were performed to explore CD8+-mediated and NK cell-mediated TEC lysis. Experiments were conducted in the absence or presence of tacrolimus (10 ng/mL), everolimus (10 ng/mL), and prednisolone (200 ng/mL). RESULTS: Tubular epithelial cells induce significant CD8+ T-cell and NK cell proliferation. All immunosuppressive drugs significantly inhibited TEC-induced CD8+ T-cell proliferation. Interestingly, prednisolone was the most powerful inhibitor of NK cell proliferation. CD8-mediated and NK cell-mediated early lytic responses were marked by strong degranulation after an encounter of unstimulated TECs, represented by a high cell surface expression of CD107a. However, with the use of interferon-γ-activated and tumor necrosis factor-α-activated TECs, the NK degranulation response was significantly reduced and CD8 degranulation response was even more enhanced (P<0.05). Tubular epithelial cell-induced CD8 degranulation and CD8-mediated TEC lysis were preferentially inhibited by tacrolimus and prednisolone, and not by everolimus. Although tacrolimus showed the most inhibitory effect on the degranulation of NK cells, NK cell-mediated TEC lysis was efficiently inhibited by prednisolone (P<0.05). CONCLUSION: Overall, our data point to a limited efficacy of immunosuppressive drugs on CD8+ T cell-mediated and NK cell-mediated lysis of human renal TECs.

Human monocytes produce interferon-gamma upon stimulation with LPS.

Representing a crucial T-helper 1 cytokine, IFN-γ acts as an important bridge between innate and adaptive immunity and is involved in many acute and chronic pathologic states, such as autoimmune diseases and solid organ transplant rejection. At present, debate still prevails about the ability of human monocytes to produce IFN-γ. We aimed to investigate whether human monocytes possess the capacity to produce IFN-γ at mRNA and protein level. Using real time PCR, flow cytometric analysis and ELISA, we investigated the capacity of freshly isolated CD14+ monocytes of healthy individuals and kidney transplant recipients to produce IFN-γ after stimulation with IFN-γ and LPS or LPS alone. We observed increased IFN-γ mRNA levels in CD14+ monocytes after stimulation as compared to the unstimulated controls in both populations. In addition, stimulation with IFN-γ and LPS or LPS alone led to a significant increase in the percentage of CD14+ monocytes producing TNF-α and IFN-γ at protein level (p<0.05). A trend towards increased secreted IFN-γ production in supernatants was also observed after LPS stimulation using ELISA. We conclude that human monocytes from healthy individuals and kidney transplant recipients possess the capacity to produce IFN-γ.

Substantial proliferation of human renal tubular epithelial cell-reactive CD4+CD28null memory T cells, which is resistant to tacrolimus and everolimus.

BACKGROUND: In spite of maintenance treatment with immunosuppressive drugs, tubulitis still occurs and can lead to structural kidney graft damage. We hypothesize that human renal tubular epithelial cells (TECs) trigger selective proliferation of recipient T-cell subsets with variable sensitivity to immunosuppressive drugs. METHODS: Recipient peripheral blood mononuclear cells were cocultured with donor-derived TECs for 7 days. The proliferation of the total CD4 T-cell pool was assessed. Next, we analyzed which CD4 T-cell subset proliferated and how this response was affected by tacrolimus, everolimus, prednisolone, and mycophenolic acid (MPA) in clinically relevant concentrations. RESULTS: CD4 T-cell proliferation upon TEC encounter was mainly executed by memory T cells. Interestingly, 38%±7% of the proliferating CD4 T-cell pool showed a CD28 phenotype. These proliferating CD4CD28 memory T cells produced high levels of interferon-γ, tumor necrosis factor-α, and the cytolitic protease granzyme B. TEC-reactive CD4 T-cell proliferation was significantly suppressed by tacrolimus, everolimus, prednisolone, and MPA (P<0.05). Surprisingly and in contrast to prednisolone and MPA, neither tacrolimus nor everolimus could inhibit the CD4CD28 T-cell proliferative response. CONCLUSION: Our data show substantial proliferation of TEC-reactive CD4CD28 memory T cells, which are resistant to tacrolimus and everolimus. This phenomenon might play an important mechanistic role during cellular rejection under full immunosuppression.

A shift towards pro-inflammatory CD16+ monocyte subsets with preserved cytokine production potential after kidney transplantation.

BACKGROUND: The presence of monocyte-macrophage lineage cells in rejecting kidney transplants is associated with worse graft outcome. At present, it is still unclear how the monocyte-macrophage related responses develop after transplantation. Here, we studied the dynamics, phenotypic and functional characteristics of circulating monocytes during the first 6 months after transplantation and aimed to establish the differences between kidney transplant recipients and healthy individuals. METHODS: Phenotype, activation status and cytokine production capacity of classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++), monocytes were determined by flow cytometry in a cohort of 33 healthy individuals, 30 renal transplant recipients at transplantation, 19 recipients at 3 months and 16 recipients at 6 months after transplantation using a cross-sectional approach. RESULTS: The percentage of both CD16+ monocyte subsets was significantly increased in transplant recipients compared to healthy individuals, indicative of triggered innate immunity (p≤0.039). Enhanced production capacity of tumor necrosis factor-α, interferon-γ and interleukin-1ß was observed by monocytes at transplantation compared to healthy individuals. Remarkably, three months post-transplant, in presence of potent immunosuppressive drugs and despite improved kidney function, interferon-γ, tumor necrosis factor-α and interleukin-10 production capacity still remained significantly increased. CONCLUSION: Our data demonstrate a skewed balance towards pro-inflammatory CD16+ monocytes that is present at the time of transplantation and retained for at least 6 months after transplantation. This shift could be one of the important drivers of early post-transplant cellular immunity.

Differential effects of activated human renal epithelial cells on T-cell migration.

BACKGROUND: Renal tubular epithelial cells (TECs) are one of the main targets of inflammatory insults during interstitial nephritis and kidney transplant rejection. While Th1 cells are know to be essential in the pathogenesis of rejection, the role of Th17 is still under debate. We hypothesize that TECs modulate the outcome of rejection process by production of distinct chemokines and cytokines that determine the attraction of different T-cell subsets. Therefore, we studied differential effects of activated human renal epithelial cells on T-cell migration. METHODS: Human primary TECs were stimulated by IFN-γ and TNF-α in vitro. Chemokines and cytokines produced by activated TECs were measured using Luminex or ELISA. Chemotaxis assay was performed using activated peripheral blood mononuclear cells composed of CD4+CXCR3+ and CD4+CCR6+ T cells migrating towards stimulated and unstimulated TECs. RESULTS: While activated TECs secreted abundant amounts of the pro-inflammatory cytokines IL-6 and IL-8, the T helper cell differentiation cytokines IL-1ß, IL-12p70, IL-23 or TGF-ß1 were not produced. The production of Th1 chemokines CXCL9, CXCL10 and CCL5 were significantly upregulated after TEC stimulation. In contrast, Th17 chemokine CCL20 could not be detected. Finally, activated TECs attracted significantly higher numbers of CD4+CXCR3+ T cells as compared to unstimulated TECs. No migration of CD4+CCR6+ T cells could be observed. CONCLUSION: Activated primary renal tubular epithelial cells do not attract Th17 cells nor produce cytokines promoting Th17 cell differentiation in our experimental system mimicking the proinflammatory microenvironment of rejection.

FoxP3 T cells and the pathophysiologic effects of brain death and warm ischemia in donor kidneys.

BACKGROUND AND OBJECTIVES: Forkhead box P3 regulatory T cells control inflammatory responses, but it remains unclear whether they inhibit brain death-initiated inflammation and tissue injury in deceased kidney donors. DESIGN, SETTING, PARTICIPANTS, MEASUREMENT: To study the actions of regulatory T cells at various stages of the donation and transplantation procedure, forkhead box P3, regulatory and inflammatory cytokine expression, and tissue injury markers were determined in time 0 kidney biopsies from deceased and living donors. Additionally, the interaction between forkhead box P3+ T cells and kidney injury molecule-1 by activated primary tubular epithelial cells was studied. RESULTS: After cold storage, the deceased donor kidneys expressed the higher mRNA levels of kidney injury molecule-1 and CD3ε. In these samples, the inflammatory cytokines IL-8 and IFN-γ and markers associated with regulation (forkhead box P3, TGF-ß, and IL-10) were highly expressed compared with living donor kidneys. Correlations were found between mRNA expression levels of forkhead box P3 and kidney injury molecule-1 and forkhead box P3 and IFN-γ. Immunohistochemical analysis confirmed the presence of forkhead box P3+ T cells in donor kidneys. Renal function (analyzed by serum creatinine levels) at the first week posttransplantation correlated with kidney injury molecule-1 and forkhead box P3 mRNA levels. In vitro studies showed that kidney injury molecule-1 expression by primary tubular epithelial cells was 63% (mean) lower when cocultured with regulatory T cells compared with control T cells. CONCLUSIONS: These results show that donor forkhead box P3+ T cells infiltrate the deceased donor kidney, where they may control inflammatory and injury responses.

Viral double-stranded RNA sensors induce antiviral, pro-inflammatory, and pro-apoptotic responses in human renal tubular epithelial cells.

Viral infection in the kidney is characterized by tubular injury induced directly by the virus and/or by cytotoxic lymphocytes. Previously, we found that human tubular epithelial cells express Toll-like receptor 3 (TLR3), melanoma differentiation-associated gene 5 (MDA5), and retinoic acid-inducible gene-I (RIG-I), all sensors of double-stranded RNA (dsRNA) and potent inducers of antiviral activity. Here, we demonstrate increased expression of these three dsRNA sensors in kidney transplant biopsies during cytomegalovirus or BK virus infection. In primary tubular epithelial cells, dsRNA sensor activation induced the production of pro-inflammatory TNF-α and antiviral IFN-ß. Notably, dsRNA also enhanced the expression of pro-apoptotic proteins; however, dsRNA alone did not cause cell death due to the expression of anti-apoptotic proteins. The dsRNA sensitized tubular epithelial cells to apoptosis induced by an agonistic antibody against the Fas receptor (CD95), an apoptotic pathway that eliminates infected cells. These findings indicate that tubular epithelial cells require at least two signals to undergo apoptosis, which can help preserve tubular integrity even under inflammatory conditions. Thus, sensors of viral dsRNA promote antiviral, pro-inflammatory, and pro-apoptotic responses in tubular epithelial cells, which may orchestrate the control of viral infection in the kidney.

SerpinB9 expression in human renal tubular epithelial cells is induced by triggering of the viral dsRNA sensors TLR3, MDA5 and RIG-I.

BACKGROUND: Serine protease inhibitor B9 (serpinB9) protects against granzyme B-mediated apoptosis and could help to reduce tubular damage under inflammatory conditions like interstitial nephritis. Previously, we found that tubular serpinB9 expression was increased during subclinical rejection. Here, we studied the regulation of serpinB9 expression in tubular epithelial cells (TECs) under inflammatory conditions. METHODS: SerpinB9 expression was analysed on messenger RNA (mRNA), and protein levels in primary human TECs were stimulated with various cytokines and pattern recognition receptor ligands and in kidney transplant biopsies obtained during different types of viral infection. RESULTS: Of the inflammatory stimuli tested, only the double-stranded RNA (dsRNA) analogue poly(I:C) promoted serpinB9 mRNA and protein expression. We found that TECs express the viral dsRNA receptors Toll-like receptor 3 (TLR3), melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene-I (RIG-I). dsRNA receptor ligands enhanced serpinB9 expression, which involved nuclear factor-kappaB (NF-κB) activation, did not require Type I interferon production and was a direct result of dsRNA receptor-induced gene transcription. In kidney transplants, serpinB9 transcription was increased during infection with cytomegalovirus, Epstein-Barr virus or BK virus compared to stable grafts. Immunohistochemistry showed that tubuli and lymphocytes expressed the inhibitor. CONCLUSION: SerpinB9 expression in human TECs is induced by triggering of the viral dsRNA sensors TLR3, MDA5 and RIG-I. Viral dsRNA may increase the threshold for granzyme B-mediated apoptosis in TECs via serpinB9 upregulation and thus help to protect the kidney against cytotoxic insults during viral infection.

Urinary granzyme A mRNA is a biomarker to diagnose subclinical and acute cellular rejection in kidney transplant recipients.

The distinction between T-cell-mediated rejection (TCMR) and other causes of kidney transplant dysfunction such as tubular necrosis requires biopsy. Subclinical rejection (SCR), an established risk factor for chronic allograft dysfunction, can only be diagnosed by protocol biopsy. A specific non-invasive biomarker to monitor immunological graft status would facilitate diagnosis and treatment of common transplantation-related complications. To identify possible markers, we measured urinary mRNA levels of several cytolytic proteins by quantitative PCR. Our cohort of 70 renal transplant recipients had biopsy proven type I and type II TCMR, acute tubular necrosis, SCR, calcineurin inhibitor-toxicity, cytomegalovirus infection, and stable graft function with normal histology. Granzyme A (GzmA) mRNA was significantly higher in subclinical and acute cellular rejection compared to patients with stable grafts or those with tubular necrosis with 80% sensitivity and up to 100% specificity. Granzyme B and perforin mRNA levels could significantly discriminate acute rejection from stable or tubular necrosis, but were not significantly elevated during SCR. Importantly, only GzmA mRNA remained below detection limits from grafts that were stable and most with tubular necrosis. Hence, the presented data indicate that urinary GzmA mRNA levels may entail a diagnostic non-invasive biomarker to distinguish patients with subclinical and acute cellular rejection from those with tubular necrosis or stable grafts.

Advancement of mesenchymal stem cell therapy in solid organ transplantation (MISOT).

There is evolving interest in the use of mesenchymal stem cells (MSC) in solid organ transplantation. Pre-clinical transplantation models show efficacy of MSC in prolonging graft survival and a number of clinical studies are planned or underway. At a recent meeting of the MISOT consortium (MSC In Solid Organ Transplantation) the advances of these studies were evaluated and mechanisms underlying the potential effects of MSC discussed. Continued discussion is required for definition of safety and eventually efficacy endpoints for MSC therapy in solid organ transplantation.

Serine proteases of the human immune system in health and disease.

Serine proteases form a large family of protein-cleaving enzymes that play an essential role in processes like blood coagulation, apoptosis and inflammation. Immune cells express a wide variety of serine proteases such as granzymes in cytotoxic lymphocytes, neutrophil elastase, cathepsin G and proteinase 3 in neutrophils and chymase and tryptase in mast cells. Regulation of proteolysis induced by these serine proteases is essential to prevent self-induced damage. Hence, there are specialized serine protease inhibitors, serpins, which are broadly distributed. Here, we discuss the function of human serine proteases in inflammation, apoptosis and tissue remodeling. Furthermore, we address their impact on development and progression of immune mediated-diseases. Understanding the mode of action of serine proteases will help to unravel molecular processes involved in immunological disorders and will facilitate the identification of new therapeutic targets.