Birgit Rudolph

Anti-human leukocyte antigen and donor-specific antibodies detected by luminex posttransplant serve as biomarkers for chronic rejection of renal allografts.

Background. Although the incidence of early acute rejection could have been diminished in the past, the long-term renal allograft survival could not benefit from the introduction of more effective immunosuppressive regimens mainly aiming at cellular rejection mechanisms. The cause of chronic rejection is still discussed controversially. Here, we demonstrate to what extent human leukocyte antigen (HLA) antibodies (HLAab) posttransplant contribute to late graft outcome. Methods. A total of 1014 deceased kidney transplant recipients transplanted at the Charité hospital were monitored in a cross-sectional manner for the development of HLAab using Luminex Single Antigen beads. Patients with stable kidney function at a median of 5-years posttransplant were tested once for HLAab and monitored for 5.5 years after testing. Results. Thirty percent of recipients showed HLAab. Donor-specific antibodies (DSA) were found in 31% of antibody positive patients. The presence of DSA was associated with a significantly lower graft survival of 49% vs. 83% in the HLAab negative group (P≤0.0001). Non-DSAs also had an adverse effect on graft survival (70% vs. 83%; P=0.0001). In a prospective analysis of 195 patients with repeatedly no detectable HLAab, the survival probability was 94% as opposed to 79% survival among patients who developed HLAab de novo after the first testing (P=0.05). Conclusions. We confirmed that HLAab produced even late after transplantation are detrimental to graft outcome. DSA were proven to have a strong adverse impact on graft survival. The results indicate that a posttransplant HLAab monitoring routine could be appropriate to improve long-term results.

Donor‐Specific HLA Antibodies in a Cohort Comparing Everolimus With Cyclosporine After Kidney Transplantation

Donor‐specific HLA antibodies (DSA) have a negative impact on kidney graft survival. Therefore, we analyzed the occurrence of DSA and antibody‐mediated rejection (AMR) in patients from two prospective randomized trials in our center. At 3–4.5 months posttransplant 127 patients were randomized to continue cyclosporine or converted to everolimus therapy. The presence of DSA was prospectively assessed using Luminex assays. AMR was defined according to the Banff 2009 classification. Antibody screening was available in 126 patients with a median follow‐up of 1059 days. Seven out of 65 (10.8%) patients on cyclosporine developed DSA after a median of 991 days. In comparison, 14/61 patients (23.0%) randomized to everolimus developed DSA after 551 days (log‐rank: p = 0.048). Eight patients on everolimus compared to two patients on cyclosporine developed AMR (log‐rank: p = 0.036). Four of 10 patients with AMR—all in the everolimus group—lost their graft. A multivariate regression model revealed everolimus, >3 mismatches and living donor as significant risk factors for DSA. Acute rejection within the first year, >3 mismatches, everolimus and living donor were independent risk factors for AMR. This single center analysis demonstrates for the first time that everolimus‐based immunosuppression is associated with an increased risk for the development of DSA and AMR.

Different mRNA and protein expression of matrix metalloproteinases 2 and 9 and tissue inhibitor of metalloproteinases 1 in benign and malignant prostate tissue

OBJECTIVE The aim of this study was to assess the behavior of the matrix metalloproteinases (MMPs) 2 and 9 and the tissue inhibitor of metalloproteinases 1 (TIMP-1) in human prostate cancer. METHODS mRNA and protein expression patterns of MMP-2, MMP-9, and TIMP-1 were studied in cancerous and noncancerous parts of 17 prostates removed by radical prostatectomy. Competitive RT-PCR, gelatin-substrate zymography, and ELISA techniques were used for quantification. RESULTS On the mRNA level, MMP-2 expression was decreased and MMP-9, TIMP-1, the ratios of MMP-2 and MMP-9 to TIMP-1 were unchanged in cancerous tissue compared to the normal counterparts. On the protein level, expression of MMP-9 was significantly higher and TIMP-1 expression was significantly lower, MMP-2 was unchanged and the ratios of MMP-2 and MMP-9 to TIMP-1 were increased in tumor tissue. CONCLUSIONS The higher concentration of MMP-9 as well as the increased ratios of MMP-2 and MMP-9 to TIMP-1 in malignant tissue prove the proteolytic dysbalance in prostate cancer, which does not seem to be associated with the stage and grade of the tumor. Comparison of mRNA and protein expression of MMP-2, MMP-9 and TIMP-1, respectively, did not show any significant relationships illustrating the necessity to study these components at both molecular levels.

CXCR3+CD4+ T cells are enriched in inflamed kidneys and urine and provide a new biomarker for acute nephritis flares in systemic lupus erythematosus patients.

OBJECTIVE The high frequency of CD4+ T cells in interstitial infiltrates of patients with lupus nephritis suggests a contribution of these cells to local pathogenesis. The aim of this study was to examine the role of CXCR3 and the chemokine CXCL10 in recruiting these cells into the kidney and to determine whether the infiltrating T cells could be monitored in the urine to provide a reliable biomarker for acute lupus nephritis. METHODS The frequencies of CD3+ T cells, CXCR3+ cells, and CXCL10+ cells were determined by immunohistochemical and immunofluorescence analyses of kidney sections from 18 patients with lupus nephritis. The frequency of CXCR3+CD4+ T cells was determined by flow cytometry of peripheral blood and urine from 38 patients with systemic lupus erythematosus (SLE), and the values were compared with disease activity as determined by the Systemic Lupus Erythematosus Disease Activity Index. RESULTS In renal biopsy tissues from patients with lupus nephritis, a mean of 63% of the infiltrating cells expressed CXCR3, approximately 60% of them were T cells, and the CXCR3+ cells colocalized with CXCL10-producing cells. In biopsy tissues from SLE patients with acute nephritis, approximately 50% of the urinary CD4+ T cells were CXCR3+, as compared with 22% in the peripheral blood, and the frequency of urinary CXCR3+CD4+ T cells correlated with disease activity. Moreover, the number of urinary CD4+ T cells reflected nephritis activity, and elevation above 800 CD4+ T cells per 100 ml of urine sharply delineated active from inactive nephritis. CONCLUSION CXCR3+ T cells are recruited into the inflamed kidneys, are enriched in the urine, and are a valuable marker of nephritis activity in SLE. They also present a potential target for future therapies.

Comparison between bortezomib and rituximab in the treatment of antibody-mediated renal allograft rejection

BACKGROUND Antibody-mediated rejection (ABMR) following kidney transplantation is associated with poor allograft survival. Conventional treatment based on plasmapheresis (PPH) and the administration of intravenous immunoglobulins (IVIG) is not satisfactory. Two compounds, more specifically targeting B cells and plasma cells, may help to improve the prognosis: rituximab, a B-cell-depleting monoclonal antibody, and bortezomib, a proteasome inhibitor causing apoptosis of plasma cells. METHODS Starting in February 2009, we treated 10 consecutive patients with ABMR according to current Banff criteria with one cycle of bortezomib [1.3 mg/m(2) intravenously (i.v.), Day 1, 4, 8, 11]. This group was compared to a historical control group of patients (n = 9) treated with a fixed single dose of rituximab (500 mg i.v.). All patients received PPH (6×) and IVIG (30 g). Patients with acute ABMR additionally received methylprednisolone (3 × 500 mg i.v.). RESULTS Patient survival in both groups was 100%. At 18 months after treatment, graft survival was 6/10 in the bortezomib group as compared to 1/9 functioning grafts in the rituximab group (P = 0.071). Renal function was superior in patients treated with bortezomib as compared to rituximab-treated patients (serum creatinine at 9 months: 2.5 ± 0.6 versus 5.1 ± 2.1 mg/dL, P = 0.008). Proteinuria was not different between both groups (9 months: 1.3 ± 1.0 versus 1.6 ± 1.6 g/day, P = n.s.). CONCLUSIONS Treatment of ABMR with bortezomib in addition to standard therapy was partially effective, whereas treatment with a fixed dose of rituximab in addition to standard therapy with PPH and IVIG did not result in sufficient long-term graft survival. In the future, new strategies including the combination of both substances and the application of higher doses must be discussed.

Extended liver resection for intrahepatic cholangiocarcinoma: A comparison of the prognostic accuracy of the fifth and sixth editions of the TNM classification.

Objective:The present study was conducted to analyze the outcome after liver resection for intrahepatic cholangiocarcinoma (IHC) and to compare the prognostic accuracy of the fifth and sixth editions of the TNM classification of malignant tumors. Summary Background Data:A comparison of the prognostic accuracy of the fifth and sixth editions of the TNM classification of malignant tumors is missing for IHC as yet. The present report is, to our knowledge, the largest series on surgical resection of IHC in the world literature and the first comparison of long-term outcome according to the fifth and sixth edition of the TNM classification of malignant tumors. Methods:From 1988 to 2007, 195 liver resections for IHC were performed in our institution. Staging was performed according to the liver chapters of the fifth and sixth edition of the TNM classification of malignant tumors. Results:In a multivariate analysis of prognostic variables, R0-resection, UICC-stage I/II according to the sixth edition, highly or moderately differentiated IHC, and lymph node negative IHC were identified as favorable prognostic variables. UICC-stage IIIc of the sixth edition, which was almost identical to the group of lymph node positive IHC was identified as unfavorable predictor of postoperative prognosis. Formally, curative resections (R0-resections) were achieved in 138 patients (71%). One- and 5-year survival rates after R0-resections were 72.4% and 30.4%, respectively. Conclusions:Extended resections for IHC resulted in a favorable rate of R0-resection, which is the most important prognostic variable. Staging of IHC according to sixth edition of the TNM classification is superior in comparison with the fifth edition as indicated by the results of the multivariate analysis.

Description of B lymphocytes and plasma cells, complement, and chemokines/receptors in acute liver allograft rejection.

Background. Although antibody mechanisms play a pathogenetic role in liver allograft rejection, no data exist on B lymphocytes, plasma cells, complement, and chemokines in rejected liver tissue. Methods. Liver biopsy specimens from 25 patients with acute allograft rejection (AR) (rejection activity index, RAI score: 1–9) were analyzed by immunohistochemistry (IH) and reverse transcriptase-polymerase chain reaction (RT-PCR) and compared with biopsy specimens taken prior to implantation (PI). The number of CD20+ and CD138+ cells was evaluated, and the presence and abundance of the chemokines macrophage inflammatory protein (MIP)-3&agr;, CXCL9, CXCL10, CXCL11, CXCL12, and their receptors CCR-6, CXCR3, and CXCR4 were examined. Complement depositions were visualized by C4d IH. Results. The numbers of B lymphocytes (P=0.002) and plasma cells (P=0.022) were significantly higher in AR biopsy specimens compared with PI biopsy specimens. MIP-3&agr;+ and CCR-6+ cells were detected in the portal fields of all AR biopsy specimens. IH double staining revealed a colocalization of MIP-3&agr;+/CD20+ cells; C4d deposits could be demonstrated along the portal capillaries. All examined chemokines and receptors could be detected in normal liver tissue and in AR biopsy specimens by RT-PCR and semiquantitative RT-PCR, demonstrating an overexpression of CXCL10 and -11. Conclusions. The significant increase of B lymphocytes and plasma cells during acute rejection, together with the lack of a significant increase of proliferating cells, indicates that the migration of B lymphocytes and plasma cells—promoted by the expression of B-cell activating chemokines/receptors—plays a key role in acute liver rejection. The C4d deposits along the portal capillaries indicate a humorally mediated alloresponse caused by the accumulated B and plasma cells.

Systemic activation of the immune system induces aberrant BAFF and APRIL expression in B cells in patients with systemic lupus erythematosus

OBJECTIVE Elevated levels of BAFF and APRIL are characteristic of patients with systemic lupus erythematosus (SLE). The reasons for enhanced cytokine production are not well understood. This study was undertaken to identify the cells responsible for the overproduction of these cytokines. METHODS BAFF expression was analyzed on peripheral blood mononuclear cells by multiparameter flow cytometry and in tissue samples by immunofluorescence staining. The levels of BAFF and APRIL mRNA were quantified in sorted B cells. In vitro cultures were used to analyze whether B cell survival and differentiation was supported by autocrine BAFF and/or APRIL. RESULTS Aberrant activation of B cells in patients with SLE was associated with a significant up-regulation of BAFF expression in naive, memory, and plasma cells. Furthermore, strong expression of BAFF and APRIL was found in plasma cells from the lymph node, bone marrow, and kidney. The levels of BAFF and APRIL mRNA in CD19+ B cells correlated both with the titer of anti-double stranded DNA antibodies and with the SLE Disease Activity Index. In vitro experiments demonstrated that B cells released functional BAFF/APRIL upon activation. CONCLUSION Our data show that B cells contribute to the enhanced levels of circulating BAFF and APRIL. The aberrant up-regulation of these cytokines may initiate a vicious circle in which enhanced levels of BAFF and APRIL act in an autocrine manner to reinforce the systemic activation of the humoral immune system.

Cathepsins B, H, L and cysteine protease inhibitors in malignant prostate cell lines, primary cultured prostatic cells and prostatic tissue

Elevated activities of cysteine proteinases, the cathepsins B, H, L (CB, CH, CL) and diminished cysteine protease inhibitors (CPI) have been demonstrated in a variety of tumours and have been suggested to contribute to invasion and metastasis. The situation for prostate cancer is still unknown. In this study, using fluorimetric assays, the catalytic activities of CB, CH, CL were measured in prostatic tissue samples after radical prostatectomy, adenomectomy, transurethral resection of the prostate, in cell cultures grown from cancerous and non-cancerous parts of human prostate after prostatectomy and in the cell lines LNCaP, DU 145 and PC 3. CPIs were determined using heat activation before testing their inhibitory activity against purified CB. Comparing matched pairs of normal and cancerous tissue samples from the prostate, significantly decreased levels of CB, CL in malignant parts of the prostate were found. In contrast, primary cell cultures from cancerous samples showed elevated levels of CB, CH, CL and increased ratios of cathepsins to CPI compared with cell cultures from normal prostate. Established cell lines showed a similar distribution pattern of each cathepsin, DU 145 containing the highest levels, followed by LNCaP and PC 3. Our results suggest that elevated cathepsin levels and consequently increased ratios of cathepsins to CPI in primary cell cultures from cancerous versus non-cancerous parts of the prostate may be indicative of a cellular proteolytic imbalance in prostatic cancer cells. In this respect, primary cell culture experiments should be preferred to determinations in tissue samples.

Evaluation of normal prostate tissue, chronic prostatitis, and prostate cancer by quantitative perfusion analysis using a dynamic contrast-enhanced inversion-prepared dual-contrast gradient echo sequence.

Objective:To quantify independent pharmacokinetic parameters for differentiation of prostate pathology. Material and Methods:Twenty-seven patients with biopsy-proven prostate cancer (PSA: 1.4–16.1 ng/mL) underwent magnetic resonance imaging with a new dynamic contrast-enhanced, inversion-prepared dual-contrast gradient echo sequence (T1/T2*-weighted, 1.65 seconds temporal resolution) using a combined endorectal/body phased-array coil at 1.5 Tesla. Perfusion, blood volume, mean transit time, delay, and dispersion were calculated using a sequential 3-compartment model. Twenty-three patients underwent prostatectomy. For histologic correlation a pathologist mapped areas of normal prostate tissue, chronic prostatitis, and prostate cancer (total of 63 areas) on histologic sections corresponding to the magnetic resonance imaging planes. Results:Compared with normal prostate tissue, low-grade cancer (Gleason score ≤6) only showed higher perfusion (1.01 mL/cm3/min vs. 0.26 mL/cm3/min, P = 0.050), whereas high-grade cancer showed higher perfusion (1.21 mL/cm3/min vs. 0.26 mL/cm3/min, P ≤ 0.001), higher blood volume (1.44% vs. 0.95%, P = 0.005), shorter mean transit time (3.55 seconds vs. 4.40 seconds, P = 0.019), shorter delay (10.15 seconds vs. 13.36 seconds, P = 0.015), and smaller dispersion (8.56 seconds vs. 12.11 seconds, P = 0.020). High-grade cancer showed higher perfusion than chronic prostatitis (1.21 mL/cm3/min vs. 0.90 mL/cm3/min, P = 0.041). Chronic prostatitis showed higher perfusion (0.90 mL/cm3/min vs. 0.26 mL/cm3/min, P = 0.006), higher blood volume (1.53% vs. 0.95%, P = 0.046), shorter delay (11.42 seconds vs. 13.36 seconds, P = 0.015), and smaller dispersion (10.49 seconds vs. 12.11 seconds, P = 0.020) than normal prostate tissue. There were no statistically significant differences between low-grade and high-grade cancer or between low-grade cancer and chronic prostatitis. Conclusion:The pharmacokinetic parameters investigated, especially perfusion, allow statistically significant in situ differentiation of normal prostate tissue from cancer and chronic prostatitis and of high-grade cancer from chronic prostatitis.