Eva Zilian

Glomerular mRNA expression of prothrombotic and antithrombotic factors in renal transplants with thrombotic microangiopathy.

Background Thrombotic microangiopathy (TMA) in renal transplants (rTx-TMA) is a serious complication and is usually either recurrent TMA (RecTMA) due to humoral rejection (HR-TMA) or due to calcineurin inhibitor toxicity (CNI-TMA). Although the triggers are known, our knowledge about the thrombogenic transcriptome changes in the microvessels is rudimentary. Methods We examined the expression of several prothrombotic and antithrombotic genes in 25 biopsies with rTx-TMA (6 RecTMA, 9 HR-TMA, and 10 CNI-TMA) and 8 controls. RNA from microdissected glomeruli of paraffin-embedded tissue was isolated and mRNA transcripts were quantified with real-time polymerase chain reaction after preamplification. Results were correlated with clinicopathologic parameters. Results Glomerular mRNA expression of KLF2, KLF4, and tPA was lower and that of PAI-1 was higher in rTx-TMA than in the controls. Glomerular mRNA expression of KLF2 and KLF4 correlated with that of tPA and inversely with that of PAI-1 in rTx-TMA. The mRNA expression of complement regulators CD46 and CD59 were higher in rTx-TMA than in the controls. Only in HR-TMA were glomerular ADAMTS13 and CD55 down-regulated. Conclusions The glomerular capillary bed seems to contribute to all subtypes of rTx-TMA by down-regulation of the endothelial transcription factors KLF2 and KLF4, indicating dedifferentiation with subsequent up-regulation of PAI-1 and down-regulation of tPA, resulting in inhibition of local fibrinolysis. Decreased glomerular expression of ADAMTS13 and CD55 could be an additional pathway toward microthrombosis exclusively in HR-TMA.

Beyond C4d: the ultrastructural appearances of endothelium in ABO-incompatible renal allografts

BACKGROUND ABO incompatibility is no longer a barrier in kidney transplantation. C4d is frequently positive in ABO-incompatible (iABO) biopsies without further signs of microcirculation injury. This phenomenon is assumed to represent graft accommodation. However, ultrastructural examination of glomerular and peritubular capillary endothelium might reveal subtle endothelial damage. METHODS We studied the ultrastructural appearance of the endothelium in 67 biopsies from 21 patients with iABO allografts and compared it with 20 patients (29 biopsies) with ABO-compatible (cABO) grafts with C4d positivity and 25 ABO-compatible control patients (25 biopsies) without serological or histological evidence of humoral rejection (C4d negative). Ten ultrastructural parameters indicative of chronic and acute glomerular and peritubular capillary damage in transmission electron microscopy (TEM) were semi-quantitatively graded and expressed in a sum score. Clinico-pathological data were compared as well as graft function at the time of biopsy and follow-up. RESULTS Ultrastructural parameters did not significantly differ between iABO and controls. In contrast, C4d-positive cABO had the highest TEM sum score (P = 0.001 versus iABO, P = 0.002 versus controls). The sum score did not differ between C4d-positive and C4d-negative iABO but did differ between patients with and without anti-HLA donor-specific antibodies (DSA). Graft function in iABO at the time of biopsy and at follow-up was similar to controls. CONCLUSIONS Our ultrastructural observations support the concept of endothelial accommodation in iABO renal transplants. C4d positivity in the ABO-incompatible situation does not indicate injurious activation of the complement cascade and does not seem to impact on the graft function, in contrast to C4d deposition in cABO with antibody-mediated rejection.

Cell-type-specific downregulation of heme oxygenase-1 by lipopolysaccharide via Bach1 in primary human mononuclear cells

Heme oxygenase (HO)-1 is the inducible isoform of the heme-degrading enzyme HO, which is upregulated by multiple stress stimuli. HO-1 has major immunomodulatory and anti-inflammatory effects via its cell-type-specific functions in mononuclear cells. Contradictory findings have been reported on HO-1 regulation by the Toll-like receptor (TLR) 4 ligand lipopolysaccharide (LPS) in these cells. Therefore, we reinvestigated the effects of LPS on HO-1 gene expression in human and murine mononuclear cells in vitro and in vivo. Remarkably, LPS downregulated HO-1 in primary human peripheral blood mononuclear cells (PBMCs), CD14(+) monocytes, macrophages, dendritic cells, and granulocytes, but upregulated this enzyme in primary murine macrophages and human monocytic leukemia cell lines. Furthermore, experiments with human CD14(+) monocytes revealed that activation of other TLRs including TLR1, -2, -5, -6, -8, and -9 decreased HO-1 mRNA expression. LPS-dependent downregulation of HO-1 was specific, because expression of cyclooxygenase-2, NADP(H)-quinone oxidoreductase-1, and peroxiredoxin-1 was increased under the same experimental conditions. Notably, LPS upregulated expression of Bach1, a critical transcriptional repressor of HO-1. Moreover, knockdown of this nuclear factor enhanced basal and LPS-dependent HO-1 expression in mononuclear cells. Finally, downregulation of HO-1 in response to LPS was confirmed in PBMCs from human individuals subjected to experimental endotoxemia. In conclusion, LPS downregulates HO-1 expression in primary human mononuclear cells via a Bach1-mediated pathway. As LPS-dependent HO-1 regulation is cell-type- and species-specific, experimental findings in cell lines and animal models need careful interpretation.

Indicators of treatment responsiveness to rituximab and plasmapheresis in antibody-mediated rejection after kidney transplantation.

Background Treatment of patients with antibody-mediated rejection (AMR) after kidney transplantation by rituximab and plasmapheresis is ambiguous. Because of its unknown efficiency and serious side effects, biomarkers, which are predictive for responsiveness to this treatment in AMR patients, are required. Methods Twenty renal transplant patients were included in this retrospective study. Selection was based on Renal Index Biopsies, classified according to Banff within 3 months before treatment. Patients were categorized into responders (R) and nonresponders (NR) depending on whether they returned to dialysis within 6 months after initiation of rituximab treatment. Clinical, histopathologic (Banff classification) and serologic parameters were compared between both groups by t test, Mann-Whitney U test, or likelihood ratio chi-square test. Results In comparisons between the groups, the R group showed a 1.5-fold higher level of estimated glomerular filtration rate and a fourfold lower level of proteinuria. By contrast, there were no differences in the histologic scores for chronic transplant lesions between the groups. The t and i scores were higher in NRs, whereas Banff-C4d scores of peritubular capillaries were increased in the Rs. Transplant biopsies in the Rs exhibited more CD138+ cell infiltrates. Serologic determination of human leukocyte antigen antibodies showed higher positivity for human leukocyte antigen class II donor-specific antibodies in the R group. No significant differences in other clinical criteria were found. Conclusion Increased proteinuria, decreased graft function, and a higher grade of tubulitis and inflammation in AMR are negative predictors for responsiveness to rituximab therapy. Rituximab therapy therefore should be initiated in an early phase of AMR.

PECAM-1-dependent heme oxygenase-1 regulation via an Nrf2-mediated pathway in endothelial cells

The antioxidant enzyme heme oxygenase (HO)-1, which catalyses the first and rate-limiting step of heme degradation, has major anti-inflammatory and immunomodulatory effects via its cell-type-specific functions in the endothelium. In the current study, we investigated whether the key endothelial adhesion and signalling receptor PECAM-1 (CD31) might be involved in the regulation of HO-1 gene expression in human endothelial cells (ECs). To this end PECAM-1 expression was down-regulated in human umbilical vein ECs (HUVECs) by an adenoviral vector-based knockdown approach. PECAM-1 knockdown markedly induced HO-1, but not the constitutive HO isoform HO-2. Nuclear translocation of the transcription factor NF-E2-related factor-2 (Nrf2), which is a master regulator of the inducible antioxidant cell response, and intracellular levels of reactive oxygen species (ROS) were increased in PECAM-1-deficient HUVECs, respectively. PECAM-1-dependent HO-1 regulation was also examined in PECAM-1 over-expressing Chinese hamster ovary and murine L-cells. Endogenous HO-1 gene expression and reporter gene activity of transiently transfected luciferase HO-1 promoter constructs with Nrf2 target sequences were decreased in PECAM-1 over-expressing cells. Moreover, a regulatory role of ROS for HO-1 regulation in these cells is demonstrated by studies with the antioxidant N-acetylcysteine and exogenous hydrogenperoxide. Finally, direct interaction of PECAM-1 with a native complex of its binding partner NB1 (CD177) and serine proteinase 3 (PR3) from human neutrophils, markedly induced HO-1 expression in HUVECs. Taken together, we demonstrate a functional link between HO-1 gene expression and PECAM-1 in human ECs, which might play a critical role in the regulation of inflammation.

Modulation of heme oxygenase-1 by metalloporphyrins increases anti-viral T cell responses

Heme oxygenase (HO)‐1, the inducible isoform of HO, has immunomodulatory functions and is considered a target for therapeutic interventions. In the present study, we investigated whether modulation of HO‐1 might have regulatory effects on in‐vitro T cell activation. The study examined whether: (i) HO‐1 induction by cobalt‐protoporphyrin (CoPP) or inhibition by tin‐mesoporphyrin (SnMP) can affect expansion and function of virus‐specific T cells, (ii) HO‐1 modulation might have a functional effect on other cell populations mediating effects on proliferating T cells [e.g. dendritic cells (DCs), regulatory T cells (Tregs) and natural killer cells] and (iii) HO‐1‐modulated anti‐viral T cells might be suitable for adoptive immunotherapy. Inhibition of HO‐1 via SnMP in cytomegalovirus (CMV)pp65‐peptide‐pulsed peripheral blood mononuclear cells (PBMCs) led to increased anti‐viral T cell activation and the generation of a higher proportion of effector memory T cells (CD45RA− CD62L−) with increased capability to secrete interferon (IFN)‐γ and granzyme B. Treg depletion and SnMP exposure increased the number of anti‐viral T cells 15‐fold. To test the possibility that HO‐1 modulation might be clinically applicable in conformity with good manufacturing practice (GMP), SnMP was tested in isolated anti‐viral T cells using the cytokine secretion assay. Compared to control, SnMP treatment resulted in higher cell counts and purity without negative impact on quality and effector function [CD107a, IFN‐γ and tumour necrosis factor (TNF)‐α levels were stable]. These results suggest an important role of HO‐1 in the modulation of adaptive immune responses. HO‐1 inhibition resulted in markedly more effective generation of functionally active T cells suitable for adoptive T cell therapy.

The new HLA-C variant HLA-C*05:26 is likely to be structurally identical to the C*05:01P alleles.

The novel allele HLA-C*05:26 differs from HLA-C*05:01 by the non-synonymous amino acid exchange Gly16Ser.

Heme Oxygenase-1 Inhibits HLA Class I Antibody-Dependent Endothelial Cell Activation.

Antibody-mediated rejection (AMR) is a key limiting factor for long-term graft survival in solid organ transplantation. Human leukocyte antigen (HLA) class I (HLA I) antibodies (Abs) play a major role in the pathogenesis of AMR via their interactions with HLA molecules on vascular endothelial cells (ECs). The antioxidant enzyme heme oxygenase (HO)-1 has anti-inflammatory functions in the endothelium. As complement-independent effects of HLA I Abs can activate ECs, it was the goal of the current study to investigate the role of HO-1 on activation of human ECs by HLA I Abs. In cell cultures of various primary human macro- and microvascular ECs treatment with monoclonal pan- and allele-specific HLA I Abs up-regulated the expression of inducible proinflammatory adhesion molecules and chemokines (vascular cell adhesion molecule-1 [VCAM-1], intercellular cell adhesion molecule-1 [ICAM-1], interleukin-8 [IL-8] and monocyte chemotactic protein 1 [MCP-1]). Pharmacological induction of HO-1 with cobalt-protoporphyrin IX reduced, whereas inhibition of HO-1 with either zinc-protoporphyrin IX or siRNA-mediated knockdown increased HLA I Ab-dependent up-regulation of VCAM-1. Treatment with two carbon monoxide (CO)-releasing molecules, which liberate the gaseous HO product CO, blocked HLA I Ab-dependent EC activation. Finally, in an in vitro adhesion assay exposure of ECs to HLA I Abs led to increased monocyte binding, which was counteracted by up-regulation of HO-1. In conclusion, HLA I Ab-dependent EC activation is modulated by endothelial HO-1 and targeted induction of this enzyme may be a novel therapeutic approach for the treatment of AMR in solid organ transplantation.