Friedrich Thaiss

Differential Activation of NF-κB, AP-1, and C/EBP in Endotoxin-Tolerant Rats: Mechanisms for In Vivo Regulation of Glomerular RANTES/CCL5 Expression

Chemokines play a pivotal role in the regulation of inflammatory cell infiltration in glomerular immune injury. To characterize mechanisms relevant for the regulation of chemokine expression in vivo, the LPS-mediated model of renal inflammation in rats was used in which we have previously demonstrated that the chemokine RANTES/CCL5 is expressed and secreted in glomeruli. Glomerular RANTES/CCL5 expression in this model correlated with an increased glomerular binding activity of the transcription factors AP-1, C/EBP, and NF-κB. To gain further insight into the functional roles of these transcription factors in the regulation of glomerular RANTES/CCL5 expression, we cloned the rat RANTES/CCL5 promoter and established the model of in vivo LPS tolerance. In tolerant rats, LPS-induced glomerular RANTES/CCL5 expression and activation of the transcription factors AP-1 and C/EBP were significantly reduced using both consensus and rat RANTES/CCL5-specific oligonucleotides. Reduced glomerular NF-κB binding activity after LPS injection could be demonstrated in tolerant rats only when using rat RANTES/CCL5-specific oligonucleotides. Reduced binding activity to this RANTES/CCL5-specific NF-κB binding site in the context of broad NF-κB activation might be due to changes in transcription factor interactions or chromatin remodeling processes.

Ubiquitin C-Terminal Hydrolase-L1 Activity Induces Polyubiquitin Accumulation in Podocytes and Increases Proteinuria in Rat Membranous Nephropathy

Ubiquitin C-terminal hydrolase L1 (UCH-L1), a key protease of the ubiquitin-proteasome system (UPS), is associated with neurodegenerative diseases and cancer. Recently, de novo expression of UCH-L1 was described in podocytes in patients with membranous nephropathy (MN), in which UCH-L1 expression correlated with increased ubiquitin content. The objective of the present study was to investigate the role of UCH-L1 in ubiquitin homeostasis and proteasomal degradation in a rat model of MN. After disease induction, UCH-L1 expression increased in podocytes and coincided with decreased glomerular monoubiquitin content. After an initial increase in proteasomal activity, the UPS was impaired. In addition to an increase of ubiquitin in podocytes, aggregates were observed 1 year after disease induction, as in MN in human beings. Inhibition of UCH-L1 hydrolase function in MN reduced UPS impairment and ameliorated proteinuria. In contrast, inhibition of proteasomal activity enhanced UPS impairment, resulting in increased proteinuria. Stable UCH-L1 overexpression in cultured podocytes resulted in accumulation of monoubiquitin and polyubiquitin proteins. In contrast, stable knock-down of UCH-L1 reduced monoubiquitin and polyubiquitin proteins and significantly increased proteasomal activity, indicating that the observed effects in rat MN also occurred in cultured podocytes. These data demonstrate that UCH-L1 activity results in polyubiquitin accumulation, proteasome inhibition, and disease aggravation in experimental models of MN.

Fewer cytomegalovirus complications after kidney transplantation by de novo use of mTOR inhibitors in comparison to mycophenolic acid.

Cytomegalovirus (CMV) is a risk factor for patient and graft survival after kidney transplantation.

Cloning and characterization of an adenoviral vector for highly efficient and doxycycline – suppressible expression of bioactive human single – chain interleukin 12 in colon cancer

Background Interleukin-12 (IL-12) is well characterized to induce cellular antitumoral immunity by activation of NK-cells and T-lymphocytes. However, systemic administration of recombinant human IL-12 resulted in severe toxicity without perceptible therapeutic benefit. Even though intratumoral expression of IL-12 leads to tumor regression and long-term survival in a variety of animal models, clinical trials have not yet shown a significant therapeutic benefit. One major obstacle in the treatment with IL-12 is to overcome the relatively low expression of the therapeutic gene without compromising the safety of such an approach. Our objective was to generate an adenoviral vector system enabling the regulated expression of very high levels of bioactive, human IL-12. Results High gene expression was obtained utilizing the VP16 herpes simplex transactivator. Strong regulation of gene expression was realized by fusion of the VP16 to a tetracycline repressor with binding of the fusion protein to a flanking tetracycline operator and further enhanced by auto-regulated expression of its fusion gene within a bicistronic promoter construct. Infection of human colon cancer cells (HT29) at a multiplicity of infection (m.o.i.) of 10 resulted in the production of up to 8000 ng/106 cells in 48 h, thus exceeding any published vector system so far. Doxycycline concentrations as low as 30 ng/ml resulted in up to 5000-fold suppression, enabling significant reduction of gene expression in a possible clinical setting. Bioactivity of the human single-chain IL-12 was similar to purified human heterodimeric IL-12. Frozen sections of human colon cancer showed high expression of the coxsackie adenovirus receptor with significant production of human single chain IL-12 in colon cancer biopsies after infection with 3*107 p.f.u. Ad.3r-scIL12. Doxycycline mediated suppression of gene expression was up to 9000-fold in the infected colon cancer tissue. Conclusion VP16 transactivator-mediated and doxycycline-regulated expression of the human interleukin-12 gene enables highly efficient and tightly controlled cytokine expression in human cancer. These data illustrate the potential of the described adenoviral vector system for the safe and superior expression of therapeutic genes in the treatment of colorectal cancer and other malignancies.BackgroundInterleukin-12 (IL-12) is well characterized to induce cellular antitumoral immunity by activation of NK-cells and T-lymphocytes. However, systemic administration of recombinant human IL-12 resulted in severe toxicity without perceptible therapeutic benefit. Even though intratumoral expression of IL-12 leads to tumor regression and long-term survival in a variety of animal models, clinical trials have not yet shown a significant therapeutic benefit. One major obstacle in the treatment with IL-12 is to overcome the relatively low expression of the therapeutic gene without compromising the safety of such an approach. Our objective was to generate an adenoviral vector system enabling the regulated expression of very high levels of bioactive, human IL-12.ResultsHigh gene expression was obtained utilizing the VP16 herpes simplex transactivator. Strong regulation of gene expression was realized by fusion of the VP16 to a tetracycline repressor with binding of the fusion protein to a flanking tetracycline operator and further enhanced by auto-regulated expression of its fusion gene within a bicistronic promoter construct. Infection of human colon cancer cells (HT29) at a multiplicity of infection (m.o.i.) of 10 resulted in the production of up to 8000 ng/106 cells in 48 h, thus exceeding any published vector system so far. Doxycycline concentrations as low as 30 ng/ml resulted in up to 5000-fold suppression, enabling significant reduction of gene expression in a possible clinical setting. Bioactivity of the human single-chain IL-12 was similar to purified human heterodimeric IL-12. Frozen sections of human colon cancer showed high expression of the coxsackie adenovirus receptor with significant production of human single chain IL-12 in colon cancer biopsies after infection with 3*107 p.f.u. Ad.3r-scIL12. Doxycycline mediated suppression of gene expression was up to 9000-fold in the infected colon cancer tissue.ConclusionVP16 transactivator-mediated and doxycycline-regulated expression of the human interleukin-12 gene enables highly efficient and tightly controlled cytokine expression in human cancer. These data illustrate the potential of the described adenoviral vector system for the safe and superior expression of therapeutic genes in the treatment of colorectal cancer and other malignancies.

Incidence of BK virus infection after kidney transplantation is independent of type of immunosuppressive therapy

BK polyomavirus (BKV) infection and BKV nephropathy (BKVN) are risk factors for allograft function and survival.

In vitro and in vivo proof of tolerance after two-step haploidentical bone marrow and kidney transplantation of the same donor.

Despite continuous improvement of immunosuppressive drugs and appropriate clinical protocols in transplantation medicine, chronic graft rejection and long-term side effects of immunosuppression (IS) still represent major hurdles after kidney transplantation (KTx) (1, 2). Recent research projects in this field therefore focus on induction of tolerance after solid organ transplantation (SOT). Different approaches for establishment of tolerance have been pursued in the past (3). Clinical trials applying nonmyeloablative conditioning combined with donorcell infusion and simultaneous KTx have reported promising results (4, 5). Nevertheless, most comprehensive clinical data concerning tolerance still exist from patients with two-step bone marrow transplantation (BMT) and SOT (6, 7). However, the number of reported cases of SOT after haploidentical BMT from the same donor is limited (8, 9), but an IS-free long-term graft survival could be demonstrated (10 –12). So far, BMT from the same donor before SOT is the only option to reliably provide tolerance, but BMT itself is associated with possible lifethreatening side effects (13). Thus, as long as efficient protocols for induction of tolerance are missing, there is the need to identify patients suitable for reduction or withdrawal of IS without risk of graft rejection. Here, we report on a case of KTx in a 23-year-old woman more than 20 years after haploidentical BMT from the same living-related donor (her mother). With help of this unusual case, we were able to prove a difference between tolerance after haploidentical BMT and hyporesponsiveness to human leukocyte antigen (HLA)-matched donor cells with and without ongoing IS. For this, recipient’s peripheral blood mononuclear cells (PBMCs) obtained 6 weeks before and after IS withdrawal were used for mixed lymphocyte culture and ELISpot applying third-party controls (n 11) with diverse numbers of HLA mismatches (0 – 6) as previously described (14). Concerning the medical history of this patient, she had severe combined immunodeficiency syndrome and received allogeneic haploidentical BMT from her mother within the first year of life due to GvHD following maternofetal transfusion syndrome. She developed renal insufficiency at the age of 10. Following 2 years of dialysis, she was transplanted with a fully matched (HLA-A, -B, and -DR alleles) kidney from a deceased donor. This organ was lost due to recurrent infections 8 years after KTx. Subsequently, the patient’s mother (the bone marrow donor 22 years ago) provided her organ to the daughter in August 2010. KTx was performed at the University Hospital Hamburg-Eppendorf with initial graft function and IS consisting of low-dose cyclosporine A (CsA) and steroids. IS was stopped 10 weeks after KTx without any episodes of acute rejection (Fig. 1A). The patient has stable kidney function for more than 1 year. Before secondary KTx, Band Tcell crossmatchings, screening for cytotoxic antibodies, and HLA antibody measurement (Luminex) were all negative. HLA typing of donor and recipient showed a complete chimerism in the peripheral blood while the tissue of mother and daughter is haploidentical. In vitro testing showed that no differences in T-cell proliferation were observed when comparing the autologous control to donor PBMCs (5406 1408 vs. 5414 1344 cpm, respectively). Interestingly, even stimulation with PBMCs of a HLA-matched third-party control (full match of HLA-A, -B, and -DR alleles) resulted in a significantly increased proliferative response (factor 3.4) when compared with stimulation with donor antigen (18,41

High B-cell activating factor is not associated with worse 3-year graft outcome in blood group-incompatible kidney transplantation with rituximab induction.

B cells and their regulation by B‐cell activating factor BAFF are of growing interest in kidney transplantation (KTx). There is evidence that high serum (s) BAFF leads to increased allosensitization and impaired long‐term graft function. We prospectively investigated sBAFF, peripheral blood lymphocytes (PBL), and donor‐specific HLA antibodies (DSA) in patients after ABOi with B‐cell depleting rituximab induction treatment and compared them to a group of blood group‐compatible (ABOc) living donor kidney recipients. Twelve patients after ABOi and 18 after ABOc were included. After rituximab treatment prior to ABOi, B cells remained significantly lower 1 year after KTx (1.2% (0.0–17.8) compared to ABOc of 8.6% (2.8–35.0), p = 0.0004, and also BAFF‐R expression was significantly lower in ABOi (p < 0.006). sBAFF remained elevated 1 year post‐Tx compared to ABOc (3615 ± 1800 vs. 1394 ± 493 pg/mL, p < 0.004). Kidney function was not significantly different between both groups after 1, 2, and 3 years. The use of rituximab in ABOi together with maintenance immunosuppression leads to significant elevation of sBAFF and lowering of B‐cell numbers for more than 1 year, and this does not correlate with worse 3‐year graft outcome.