Our results, in line with the extended experience of other groups, demonstrate favourable short-term allograft survival and function after ABOi renal transplantation after desensitization with antigen-specific IA, IVIG and rituximab. However, the intensified desensitization was associated with an increased risk of infectious complications. This observation prompted us to briefly escalate the desensitization protocol in ABOi kidney recipients in our centre.

Hydrogels have become one of the most popular platforms for three-dimensional (3D) cultivation of mammalian cells. The enormous versatility of hydrogel materials makes it possible to design scaffolds with predefined mechanical properties, as well as with desired biofunctionality. 3D hydrogel constructs have been used for a variety of applications, including tissue engineering of microorgan systems, drug delivery, cytotoxicity testing, and drug screening. Moreover, 3D culture is applied for investigating cellular physiology, stem cell differentiation, and tumor models and for studying interaction mechanisms between the extracellular matrix and cells. In this paper, we review current examples of performance-based hydrogel design for 3D cell culture applications. A major emphasis is placed on a description of how standard analytical protocols and imaging techniques are being adapted to analysis of 3D cell culture in hydrogel systems.

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In the context of kidney transplantation, little is known about the involvement of natural killer (NK) cells in the immune reaction leading to either rejection or immunological tolerance under immunosuppression. Therefore, the peripheral NK cell repertoire of patients after kidney transplantation was investigated in order to identify NK cell subsets that may be associated with the individual immune status at the time of their protocol biopsies for histopathological evaluation of the graft. Alterations in the peripheral NK cell repertoire could be correlated to the type of immunosuppression, i.e., calcineurin-inhibitors like Cyclosporin A vs. Tacrolimus with or without addition of mTOR inhibitors. Here, we could demonstrate that the NK cell repertoire in peripheral blood of kidney transplant patients differs significantly from healthy individuals. The presence of donor-specific antibodies was associated with reduced numbers of CD56dim NK cells. Moreover, in patients, down-modulation of CD16 and CD6 on CD56dim NK cells was observed with significant differences between Cyclosporin A- and Tac-treated patients. Tac-treatment was associated with decreased CD69, HLA-DR, and increased CD94/NKG2A expression in CD56dim NK cells indicating that the quality of the immunosuppressive treatment impinges on the peripheral NK cell repertoire. In vitro studies with peripheral blood mononuclear cells of healthy donors showed that this modulation of CD16, CD6, CD69, and HLA-DR could also be induced experimentally. The presence of calcineurin or mTOR inhibitors had also functional consequences regarding degranulation and interferon-γ-production against K562 target cells, respectively. In summary, we postulate that the NK cell composition in peripheral blood of kidney transplanted patients represents an important hallmark of the efficacy of immunosuppression and may be even informative for the immune status after transplantation in terms of rejection vs. drug-induced allograft tolerance. Thus, NK cells can serve as sensors for immunosuppression and may be utilized for future strategies of an individualized adjustment of immunosuppression.

Background: Infections are the most common cause of late complications in cardiopulmonary bypass (CPB) surgery patients, and are difficult to predict. Here we studied the diagnostic value of a standardized immune monitoring program based on recent advances in flow cytometry (exact quantification of surfacemarker expression) and cytokine determination (semiautomatic systems).Methods: CPB patients (56) at risk for complications (age >70 years and/or preoperative left-ventricular ejection fraction < 25 %) were classified into three groups: without (33), with suspected (14), and with confirmed (9) infection. Applying the Quantibrite™-system, we daily quantified the expression of CD11b, CD64, CD71, CD86, and HLA-DR on monocytes/granulocytes. Furthermore, the ex vivo secretion of tumor necrosis factor (TNF)-␣ as well as the plasma interleukin (IL)-10 levels were determined by a semiautomatic system. Ex vivo elastase release was measured by enzyme-linked immunosorbent assay (ELISA).Results: All patients showed signs of granulocyte activation and monocyte deactivation. Monocytic HLA-DR and plasma IL-10 were the best markers to discriminate patients with infection from those without as early as day 1. Using a cutoff of 5792 HLA-DR molecules per cell, both sensitivity and negative predictive value for patients who developed microbiologically confirmed infection was 1.0, and the area under the curve (AUC) was 0.85.Conclusions: Our data suggest that a standardized immune monitoring at day 1 might be useful for early discrimination of patients at elevated risk for infections. Cytometry Part B (Clin. Cytometry) 53B:54 -62, 2003.

To explore phenotype and function of NK cells in kidney transplant recipients, we investigated the peripheral NK cell repertoire, capacity to respond to various stimuli and impact of immunosuppressive drugs on NK cell activity in kidney transplant recipients. CD56dim NK cells of kidney transplanted patients displayed an activated phenotype characterized by significantly decreased surface expression of CD16 (p=0.0003), CD226 (p<0.0001), CD161 (p=0.0139) and simultaneously increased expression of activation markers like HLA-DR (p=0.0011) and CD25 (p=0.0015). Upon in vitro stimulation via Ca++-dependent signals, down-modulation of CD16 was associated with induction of interferon (IFN)-γ expression. CD16 modulation and secretion of NFAT-dependent cytokines such as IFN-γ, TNF-α, IL-10 and IL-31 were significantly suppressed by treatment of isolated NK cells with calcineurin inhibitors but not with mTOR inhibitors. In kidney transplant recipients, IFN-γ production was retained in response to HLA class I-negative target cells and to non-specific stimuli, respectively. However, secretion of other cytokines like IL-13, IL-17, IL-22 and IL-31 was significantly reduced compared to healthy donors. In contrast to suppression of cytokine expression at the transcriptional level, cytotoxin release, i.e. perforin, granzyme A/B, was not affected by immunosuppression in vitro and in vivo in patients as well as in healthy donors. Thus, immunosuppressive treatment affects NK cell function at the level of NFAT-dependent gene expression whereby calcineurin inhibitors primarily impair cytokine secretion while mTOR inhibitors have only marginal effects. Taken together, NK cells may serve as indicators for immunosuppression and may facilitate a personalized adjustment of immunosuppressive medication in kidney transplant recipients.

Under the influence of flosulide, a highly COX-2-selective inhibitor, we observed an antiproteinuric drug effect. The inflammation in PHN induced COX-2 protein expression that was not affected by low-dose flosulide. COX-1 and COX-2 protein expression was affected by high-dose flosulide, which therefore might lose its selectivity. High-dose flosulide induced a decrease in glomerular prostanoid production possibly because of COX-1 inhibition. Our results suggest that the therapeutic use of flosulide in proteinuria seems advantageous and deserves further studies because the basal prostaglandin levels remain unchanged in the low-dose-treated group, indicating that the compensatory capacity of prostaglandin production, which is essential for the regulation of renal hemodynamics, is maintained.

A treatment regimen consisting of de novo immunosuppression with basiliximab, CsA, and prednisolone, followed by treatment with everolimus and low-dose CsA combined with steroid withdrawal may be a promising therapy after pediatric KTX.