Quantitative polymerase chain reaction profiling of immunomarkers in rejecting kidney allografts for predicting response to steroid treatment.

BACKGROUND: Steroid-resistant acute rejection is a risk factor for inferior renal allograft outcome. METHODS: From 873 kidney transplant recipients (1995-2005), 108 patients with a first rejection episode were selected for study using strict inclusion criteria and clinical endpoint definition. We aimed to predict response to corticosteroid treatment using gene expression of 65 transcripts. These reflect cytokines, chemokines, and surface and activation markers of various cell types including T cells, macrophages, B cells, and granulocytes. Steroid resistance (40% of the patients) was defined as requirement for antithymocyte globulin treatment within 2 weeks after corticosteroid treatment. RESULTS: None of the clinical and histomorphologic parameters showed a significant association with response to treatment. Univariate logistic regression analysis resulted in 11 messenger RNA markers, including T-cell-related transcripts CD25, lymphocyte activation gene-3, Granzyme B, and interleukin-10, and macrophage-specific transcripts mannose receptor and S100 calcium-binding protein A9, which significantly discriminated steroid resistant from steroid-responsive rejections (P<0.05). In multivariate logistic regression, the combination of T-cell activation markers CD25:CD3e ratio (odds ratio, 8.7; confidence interval, 2.4-31.2) and lymphocyte activation gene-3 (odds ratio, 3.3; confidence interval, 1.4-7.7) represented the best predictive model for steroid response (P<0.0001). Specificity and sensitivity were 78% and 60%, respectively. After internal stratified 10-fold cross-validation, the model remained significant. Inclusion of clinical variables into the model with molecular variables did not enhance prediction. CONCLUSIONS: Differences in intragraft expression profiles reflect variability in the response to antirejection treatment. In acute rejection, molecular markers, particularly those reflecting T-cell activation, offer superior prognostic value compared with conventional parameters.

Setting a research agenda for progressive multiple sclerosis: the International Collaborative on Progressive MS.

Despite significant progress in the development of therapies for relapsing MS, progressive MS remains comparatively disappointing. Our objective, in this paper, is to review the current challenges in developing therapies for progressive MS and identify key priority areas for research. A collaborative was convened by volunteer and staff leaders from several MS societies with the mission to expedite the development of effective disease-modifying and symptom management therapies for progressive forms of multiple sclerosis. Through a series of scientific and strategic planning meetings, the collaborative identified and developed new perspectives on five key priority areas for research: experimental models, identification and validation of targets and repurposing opportunities, proof-of-concept clinical trial strategies, clinical outcome measures, and symptom management and rehabilitation. Our conclusions, tackling the impediments in developing therapies for progressive MS will require an integrated, multi-disciplinary approach to enable effective translation of research into therapies for progressive MS. Engagement of the MS research community through an international effort is needed to address and fund these research priorities with the ultimate goal of expediting the development of disease-modifying and symptom-relief treatments for progressive MS.

Increased influx of myeloid dendritic cells during acute rejection is associated with interstitial fibrosis and tubular atrophy and predicts poor outcome.

Dendritic cells are key players in renal allograft rejection and have been identified as an intrinsic part of the kidney. Here we quantified and phenotyped the dendritic cell populations in well-defined biopsies of 102 patients with acute renal allograft rejection in comparison with 78 available pretransplant biopsies. There was a strong increase in BDCA-1(+) and DC-SIGN(+) myeloid, BDCA-2(+) plasmacytoid, and DC-LAMP(+) mature dendritic cells in rejection biopsies compared with the corresponding pretransplant tissue. Mature dendritic cells were mostly found in clusters of lymphoid infiltrate and showed a strong correlation with the Banff infiltrate score. The presence of both myeloid and plasmacytoid dendritic cell subsets in the kidney during acute rejection correlated with interstitial fibrosis and tubular atrophy. Importantly, the myeloid dendritic cell density at the time of acute rejection was an independent risk factor for loss of renal function after the first year. Thus, acute renal allograft rejection is characterized by an influx of myeloid and plasmacytoid dendritic cells, strongly associated with local damage in the graft. Hence, the density of myeloid dendritic cells during acute rejection could be an important risk factor for the long-term development of chronic changes and loss of graft function.

C4d staining in renal allograft biopsies with early acute rejection and subsequent clinical outcome.

BACKGROUND AND OBJECTIVES: Diffuse C4d staining in peritubular capillaries (PTCs) during an acute rejection episode (ARE) is the footprint of antibody-mediated rejection. In current clinical practice, diffuse C4d+ staining during acute rejection is regarded as an inferior prognostic sign. This case-control study investigated the prognostic role of mere C4d staining for graft outcome during an ARE in a well defined cohort of similarly ARE-treated patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: All kidney transplant recipients in the authors' center from January 1, 1995 to December 31, 2005 were reviewed. From these patients, 151 had a clinical ARE. Paraffin and/or frozen material was available for 128 patients showing a histologically proven ARE within the first 6 months after transplantation. All ARE patients were treated similarly with high-dose pulse steroids and in the case of steroid unresponsiveness with anti-thymocyte globulin. Biopsies were scored according to Banff criteria. Frozen and paraffin sections were stained by immunofluorescence (IF) and immunohistochemistry (IHC) for C4d, respectively, and scored for PTC positivity. RESULTS: Diffuse C4d+ staining in PTCs was found in 12.5% and 4.2% sections stained by IF or by IHC, respectively. Four patients showed diffuse positive staining with both methods but showed no different risk profile from other patients. No relation between C4d staining and clinical parameters at baseline was found. C4d staining was not associated with steroid responsiveness, graft, or patient survival. CONCLUSIONS: This study shows that C4d staining is not related to clinical outcome in this cohort of histologically proven early AREs.

Complement activation by tubular cells is mediated by properdin binding.

Activation of filtered complement products on the brush border of the tubular epithelium is thought to be a key factor underlying proteinuria-induced tubulointerstitial injury. However, the mechanism of tubular complement activation is still unclear. Recent studies on mechanisms of complement activation indicate a key role for properdin in the initiation of an alternative pathway. We hypothesized that properdin serves as a focal point for complement activation on the tubulus. We observed a strong staining for properdin on the luminal surface of the tubules in kidney biopsies from patients with proteinuric renal disease. In vitro experiments revealed dose-dependent binding of properdin to proximal tubular epithelial cells (PTEC), whereas no significant binding to endothelial cells was detected. Exposure of PTEC with normal human serum as a source of complement resulted in complement activation with deposition of C3 and generation of C5b-9. These effects were virtually absent with properdin-deficient serum. Preincubation of PTEC with properdin before addition of properdin-depleted serum fully restored complement activation on the cells, strongly suggesting a key role for properdin in the activation of complement at the tubular surface. In proteinuric renal disease, filtered properdin may bind to PTEC and act as a focal point for alternative pathway activation. We propose that this contribution of properdin is pivotal in tubular complement activation and subsequent damage. Interference with properdin binding to tubular cells may provide an option for the treatment of proteinuric renal disease.

VEGF release by MMP-9 mediated heparan sulphate cleavage induces colorectal cancer angiogenesis.

Angiogenesis is crucial for the progression of colorectal carcinomas in which the bioavailability of Vascular Endothelial Growth Factor (VEGF) plays a major role. VEGF bioavailability is regulated by proteolytic release or cleavage. In colorectal cancer patients, we observed a significant correlation between circulating VEGF and tumour tissue Matrix Metalloproteinase-9 (MMP-9) levels but not with MMP-2. Therefore, we evaluated the role of MMP-9 in regulating VEGF bioavailability and subsequent angiogenesis in 3-dimensional human cell culture models. MMP-9 treatment released VEGF dose-dependently from HT29 colon carcinoma spheroids, comparable to heparitinase, a known mediator of VEGF release. Conditioned medium from human neutrophils, containing high amounts of active MMP-9, released VEGF comparable to recombinant MMP-9, in contrast to myofibroblast medium. MMP-9 treated spheroids showed decreased extracellular levels of heparan sulphates, required for VEGF binding to the matrix, whereas the levels in the medium were increased. Western blot analysis revealed that VEGF(165) is the major isoform released by MMP-9 treatment. In vitro experiments indicated that MMP-9 is not capable to cleave VEGF(165) into smaller isoforms, like plasmin does. These data suggested that MMP-9 mediates release rather than the cleavage of larger VEGF isoforms. Medium from MMP-9 treated HT29 spheroids induced endothelial cell sprouting in an angiogenesis assay, comparable to the effect of recombinant VEGF(165). Anti-VEGF antibody treatment resulted in a strongly reduced number of sprouts. In conclusion, we have shown that neutrophil-derived MMP-9 is able to release biologically active VEGF(165) from the ECM of colon cancer cells by the cleavage of heparan sulphates.

Endothelium specific matrilysin (MMP-7) expression in human cancers.

Over-expression of matrilysin (MMP-7) is predominantly associated with epithelial (pre)malignant cells. In the present study MMP-7 expression is also found in endothelial cells in various human cancer types. Endothelial MMP-7 was associated with CD34 and/or CD105 expression. These immunohistochemical data were confirmed by RT-PCR on VEGF-stimulated endothelial cells. In addition, MMP-7 was also identified in sprouting endothelial cells in vitro. The potential clinical relevance of endothelial MMP-7 was assessed for cervical cancer patients by evaluating the association with overall survival. In contrast to MMP-7 in malignant epithelial cells, MMP-7 expression in endothelial cells showed a significant association with poor survival (LR 5.12, P=0.02, n=30). Our data suggest that MMP-7 is involved in tumor angiogenesis, thereby contributing to malignant growth and hence associated with decreased survival.

Immuno-histological analysis of dendritic cells in nasal biopsies of IgA nephropathy patients.

BACKGROUND: IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide. Intranasal vaccination of patients with IgAN has shown mucosal and systemic IgA hyporesponsiveness. Here, we investigated whether this IgA hyporesponse in IgAN patients can be explained by reduced numbers or altered subset distribution of dendritic cells (DCs) in nasal mucosa. METHODS: Eighteen IgAN patients and 18 healthy volunteers were recruited for this study. Nasal biopsies were taken, after local anaesthesia, from the lower edge of the inferior turbinate. Staining for different subsets of DCs was performed using specific monoclonal antibodies. To detect myeloid DCs, we used CD1a, DC-SIGN and blood dendritic cell antigen-1 (BDCA-1) as a marker and for plasmacytoid DCs we used BDCA-2. DC-cell numbers in the epithelium and in lamina propria were counted separately and expressed as positively stained cells per mm(2). RESULTS: Both myeloid and plasmacytoid DC could be demonstrated in nasal biopsies. Quantification showed that IgAN patients contained significantly more DC-SIGN-positive cells in the lamina propria compared to controls. In addition, in IgAN patients, we observed more CD1a-positive cells in the epithelium. No differences in BDCA-1 and BDCA-2-positive cells were found between patients and controls. The number of positively stained cells in the epithelial layer correlated strongly with the number of positively stained cells in the lamina propria. CONCLUSIONS: Patients with IgAN have higher numbers of CD1a-positive cells in the epithelial layer and more DC-SIGN-positive cells in the lamina propria. Therefore, the earlier observed IgA hyporesponsiveness in IgAN patients after mucosal vaccination cannot be explained by lower numbers of nasal DCs.

Clinical evidence for a protective role of lipocalin-2 against MMP-9 autodegradation and the impact for gastric cancer.

Recently, complexes of matrix metalloproteinase matrix metalloproteinase-9 (MMP-9) with lipocalin-2 (neutrophil gelatinase-associated lipocalin) were found in the urine obtained from breast cancer patients, while these were completely absent in that obtained from healthy controls. In vitro data suggested a possible role for lipocalin-2 in the protection of MMP-9 against autolysis. To establish this effect in vivo, we determined the presence of MMP-9, lipocalin-2 and their complex in tumour tissue from 81 gastric cancer patients. The effect of the presence of the individual parameters, the complexes, and the inhibitors TIMP-1 and TIMP-2 on MMP-9 activity was evaluated with a bioactivity assay. Immuno-histochemical (double) staining identified epithelial cells as the most likely cellular source. Finally, evaluation of all these parameters with clinico-pathological scores revealed that tumour MMP-9/lipocalin-2 complexes were significantly related with the classifications of Laurén and WHO, and highly associated with worse survival in Cox's univariate (HR 2.087, P=0.006) and multivariate analyses (HR 2.095, P=0.025).

EMMPRIN-induced MMP-2 activation cascade in human cervical squamous cell carcinoma.

Tumor progression and recurrence of cervical cancer is associated with upregulation of matrix metalloproteinase 2 (MMP-2). We evaluated the location, origin and activity of MMP-2 in cervical squamous cell carcinomas in comparison with MT1-MMP (MMP-14), TIMP-2 and extracellular matrix metalloproteinase inducer (EMMPRIN). Positive immunostaining for MMP-2 in malignant cells was detected in 83% of the patients. Two patterns of tumor cell MMP-2 staining were observed: either homogenous in all tumor cells or confined to the cells neighboring the stroma (tumor-border staining pattern, TBS). Fluorescence in situ zymography showed active MMP-2 mainly around tumor nodules displaying TBS. The MMP-2 staining of TBS tumors correlated significantly with the presence of TIMP-2 and MT1-MMP, proteins involved in docking MMP-2 to the cell surface and essential for MMP-2 activation. In situ mRNA hybridization in TBS tumors demonstrated more abundant presence of MMP-2 mRNA in neighboring myofibroblasts than in the adjacent tumor cells. Moreover, the TBS MMP-2 pattern correlated with the presence of EMMPRIN (p = 0.023), suggesting that tumor cells induce MMP-2 production in nearby stromal cells. This pro-MMP-2 could subsequently be activated on tumor cells via the presence of MT1-MMP and TIMP-2. The biological relevance of this locally activated MMP-2 was underscored by the observation that only the TBS pattern of MMP-2 significantly correlated with decreased survival. In conclusion, the colocalization of EMMPRIN, MT1-MMP and TIMP-2 in human cervical carcinomas seems to be involved in a specific distribution pattern of tumor cell bound MMP-2, which is related with local proteolytic activity and therefore might be associated with worse prognosis of the patients.