Martina Seifert

Changing the Antigen Binding Specificity by Single Point Mutations of an Anti-p24 (HIV-1) Antibody

The murine mAb CB4-1 raised against p24 (HIV-1) recognizes a linear epitope of the HIV-1 capsid protein. Additionally, CB4-1 exhibits cross-reactive binding to epitope-homologous peptides and polyspecific reactions to epitope nonhomologous peptides. Crystal structures demonstrate that the epitope peptide (e-pep) and the nonhomologous peptides adopt different conformations within the binding region of CB4-1. Site-directed mutagenesis of the fragment variable (Fv) region was performed using a single-chain (sc)Fv construct of CB4-1 to analyze binding contributions of single amino acid side chains toward the e-pep and toward one epitope nonhomologous peptide. The mutations of Ab amino acid side chains, which are in direct contact with the Ag, show opposite influences on the binding of the two peptides. Whereas the affinity of the e-pep to the CB4-1 scFv mutant heavy chain variable region Tyr32Ala is decreased 250-fold, the binding of the nonhomologous peptide remains unchanged. In contrast, the mutation light chain variable region Phe94Ala reduces the affinity of the nonhomologous peptide 10-fold more than it does for the e-pep. Thus, substantial changes in the specificity can be observed by single amino acid exchanges. Further characterization of the scFv mutants by substitutional analysis of the peptides demonstrates that the effect of a mutation is not restricted to contact residues. This method also reveals an inverse compensatory amino acid exchange for the nonhomologous peptide which increases the affinity to the scFv mutant light chain variable region Phe94Ala up to the level of the e-pep affinity to the wild-type scFv.

Inhibition of keratinocyte apoptosis by IL-15 : a new parameter in the pathogenesis of psoriasis?

Keratinocytes (KC) are important source of and targets for several cytokines. Although KC express IL-15 mRNA, the functional effects of IL-15 on these epithelial cells remain to be dissected. Investigating primary human foreskin KC and HaCaT cells, we show here by semiquantitative RT-PCR and flow cytometric analysis that both translate IL-15 and IL-15R mRNA and express IL-15 and IL-15Rα protein on the cell surface, suggesting that human KC can employ IL-15 for juxtacrine signaling. While IL-15 exerted no significant effect on KC proliferation and IL-6 or IL-8 secretion, IL-15 inhibited both anti-Fas and methylcellulose-induced KC apoptosis in vitro. This is in line with the recognized potent anti-apoptotic effects of IL-15. IL-2, whose receptor shares two components with the IL-15R, failed to inhibit KC apoptosis. Together with the role of IL-15 in sustaining chronic immune reactions, this invited the question of whether a reduction of KC apoptosis by IL-15 may be involved in the pathogenesis of psoriasis, a chronic hyperproliferative inflammatory skin disease characterized by abnormally low KC apoptosis in the epidermis. Remarkably, compared with nonlesional psoriatic skin and skin of healthy volunteers, lesional psoriatic epidermis showed high IL-15 protein expression in the epidermis and enhanced binding activity for IL-15. Therefore, antagonizing the inhibitory effects of IL-15 on KC apoptosis deserves exploration as a novel therapeutic strategy in psoriasis management.

Heme oxygenase-1 inhibits rat and human breast cancer cell proliferation: mutual cross inhibition with indoleamine 2,3-dioxygenase

Heme oxygenase‐1 (HO‐1) is the rate limiting enzyme of heme catabolism whereas indoleamine 2,3 dioxygenase (IDO) catabolizes tryptophan through the kynurenine pathway. We analyzed the expression and biological effects of these enzymes in rat and human breast cancer cell lines. We show that rat (NMU and 13762) but not human cells (MCF‐7 and T47D) express HO‐1. When overexpressed, we found this enzyme to have anti‐proliferative and proapoptotic effects by antioxidant mechanisms in these four cell lines. We show that IDO is expressed by rat and human breast cancer cells. IDO inhibition with 1‐MT and siRNA leads to diminished proliferation in rat cells. In contrast, HO‐1 negative human cell lines increase proliferation upon IDO inhibition. Since we also demonstrate that IDO inhibits the anti‐proliferative HO‐1, we propose that IDO has opposite effects on proliferation depending on the coexpression or not of HO‐1. We also describe that HO‐1 inhibits IDO at the post‐translational level through heme starvation. In vivo, we show that rat normal breast expresses HO‐1 and IDO. In contrast, N‐nitrosomethylurea‐induced breast adenocarcinomas only express IDO. In conclusion, we show that HO‐1/IDO cross‐regulation modulates apoptosis and proliferation in rat and human breast cancer cells.—Hill, M., Pereira, V., Chauveau, C., Zagani, R., Remy, S., Tesson, L., Mazal, D., Ubillos, L., Brion, R., Ashgar, K., Mashreghi, M. F., Kotsch, K., Moffett, J., Doebis, C., Seifert, M., Boczkowski, J., Osinaga, E., Anegon, I. Heme oxygenase‐1 inhibits rat and human breast cancer cells proliferation: mutual cross inhibition with indoleamine 2,3‐dioxygenase. FASEB J. 19, 1957–1968 (2005)

Human immune responses to porcine xenogeneic matrices and their extracellular matrix constituents in vitro.

Several tissue engineering approaches for the treatment of cardiovascular diseases are based on a xenogeneic extracellular matrix. However, the application of engineered heart valves has failed in some patients, causing severe signs of inflammation by so far undetermined processes. Therefore we investigated the immune-mediated responses to porcine valve matrices (native, decellularized and glutaraldehyde-fixed) and to purified xenogeneic extracellular matrix proteins (ECMp). The induction of human immune responses in vitro was evaluated by analyzing the co-stimulatory effects of matrices and ECMp collagen and elastin on the proliferation of immune cell sub-populations via CFSE-based proliferation assays. The pattern of cytokine release was also determined. In porcine matrix punches we demonstrated strong immune responses with the native as well as the decellularized type, in contrast to attenuated effects with glutaraldehyde-fixed matrices. Furthermore, our results indicate that collagen type I (porcine and human) and human elastin were able to elicit proliferation in co-stimulation with anti-CD3 antibody, accompanied by a strong release of Th1 cytokines (IFN-gamma, TNF-alpha). In contrast, porcine elastin did not elicit any response at all. This low immunogenic potential of porcine elastin suggests its suitability for the creation of new tissue engineering heart valve scaffolds in the future.

Advancement of mesenchymal stem cell therapy in solid organ transplantation (MISOT).

There is evolving interest in the use of mesenchymal stem cells (MSC) in solid organ transplantation. Pre-clinical transplantation models show efficacy of MSC in prolonging graft survival and a number of clinical studies are planned or underway. At a recent meeting of the MISOT consortium (MSC In Solid Organ Transplantation) the advances of these studies were evaluated and mechanisms underlying the potential effects of MSC discussed. Continued discussion is required for definition of safety and eventually efficacy endpoints for MSC therapy in solid organ transplantation.

Toward MSC in Solid Organ Transplantation: 2008 Position Paper of the MISOT Study Group

The following position paper summarizes the recommendations for early clinical trials and ongoing basic research in the field of mesenchymal stem cell-induced solid organ graft acceptance—agreed upon on the first meeting of the Mesenchymal Stem Cells In Solid Organ Transplantation (MISOT) study group in late 2008.

Improved long-term graft survival after HO-1 induction in brain-dead donors

Brain death (BD) of the donor, a risk factor uniquely relevant for organs derived from cadaver donors, influences organ quality by induction of various inflammatory events. Consequently ischemia/reperfusion injury is deteriorated and acute and chronic rejections accelerated. Donor treatment might be an approach to improve the quality of the graft. The induction of heme oxygenase 1 (HO‐1) has been shown to exert beneficial effects in living‐donor transplantation models. Therefore, we examined the impact of donor treatment with the selective inducer of HO‐1, cobalt protoporphyrin (CoPP), on organ quality and transplant outcome in a standardized BD model in a F344→LEW kidney transplant rat model. Immediately after BD induction, donor animals were administered a single dose of CoPP (5 mg/kg) and in control groups, HO‐1 activity was blocked with zinc protoporphyrin (ZnPP, 20 mg/kg). Recipients of organs from brain‐dead donors treated with CoPP survived significantly better than those from untreated brain‐dead donors (p < 0.05) and intra‐graft analysis showed improved histology (p < 0.05). Blockade of HO‐1 with ZnPP decreased the survival rates (p < 0.05) comparable to untreated brain‐dead donors. Our results demonstrate that HO‐1 induction by one single treatment of CoPP in brain‐dead donors leads to enhanced allograft survival.

Ischemia-reperfusion injury: beneficial effects of mesenchymal stromal cells.

Purpose of reviewOrgan transplantation and other major surgeries are impacted by ischemia–reperfusion injury (IRI). Mesenchymal stromal cells (MSCs) recently became an attractive alternative therapeutic tool to combat IRI. The present review highlights the effects of MSCs in the preclinical animal models of IRI and clinical trials, and explains their potential modes of action based on the pathophysiological IRI cascade. Recent findingsThe application of MSCs in animal models of IRI show anti-inflammatory and anti-apoptotic effects, particularly for damage to the kidneys, heart and lungs. The mechanism of MSC action remains unclear, but may involve paracrine factors which could include the transfer of microvesicles, RNA or mitochondria. Although few clinical trials have reached completion, adverse effects appear minimal. SummaryMSCs show promise in protecting against IRI-induced damage. They appear to help recovery mainly by affecting the levels of inflammation and apoptosis during the organ repair process. In addition, they may mediate immunomodulatory effects on the innate and adaptive immune processes triggered during reperfusion and reduce fibrosis. Success in preclinical animal models has led to the initiation of ongoing clinical trials.

Immunomodulative Efficacy of Bone Marrow-Derived Mesenchymal Stem Cells Cultured in Human Platelet Lysate

Human mesenchymal stem cells (hMSCs) are considered to be a promising tool for novel cell-based therapies. Clinical applications in solid organ transplantation were hampered by the dependence on animal serum for hMSCs clinical scale expansion until substitution with human platelet lysate (HPL) became a promising alternative. Therefore we focused on a direct comparison of immunomodulatory properties of hMSCs cultured in HPL or fetal calf serum (FCS). Phenotypic characterization, detection of cytokine secretion and effects on alloantigen- and mitogen-induced lymphocyte proliferation as well as degranulation of cytomegalovirus-specific cytotoxic T cells were applied in potency assays. We demonstrated that HPL-cultured MSCs have comparable immunomodulatory capacities to their FCS-cultured counterparts. The observed immunomodulatory properties include a beneficial inhibitory effect on immune cell proliferation and an unaffected viral T cell immunity. Thus, culturing hMSCs in HPL generates an efficient and safe expansion combined with intriguing immunomodulatory properties making these cells an attractive cell therapeutic tool.

In vivo effect of bone marrow-derived mesenchymal stem cells in a rat kidney transplantation model with prolonged cold ischemia

Brain death and prolonged cold ischemia are major contributors to the poorer long‐term outcome of transplants from deceased donor kidney transplants, with an even higher impact if expanded criteria donors (‘marginal organs’) are used. Targeting ischemia‐reperfusion injury‐related intragraft inflammation is an attractive concept to improve the outcome of those grafts. As mesenchymal stem cells (MSCs) express both immunomodulatory and tissue repair properties, we evaluated their therapeutic efficacy in a rat kidney transplant model of prolonged cold ischemia. The in vitro immunomodulatory capacity of bone marrow‐derived rat MSCs was tested in co‐cultures with rat lymph node cells. For in vivo studies, Dark Agouti rat kidneys were cold preserved and transplanted into Lewis rats. Syngeneic Lewis MSCs were administered intravenously. Transplants were harvested on day 3, and inflammation was examined by quantitative RT‐PCR and histology. Similarly to MSCs from other species, rat MSCs in vitro also showed a dose‐dependent immunomodulatory capacity. Most importantly, in vivo administration of MSCs reduced the intragraft gene expression of different pro‐inflammatory cytokines, chemokines, and intercellular adhesion molecule‐1. In addition, fewer antigen‐presenting cells were recruited into the renal allograft. In conclusion, rat MSCs ameliorate inflammation induced by prolonged cold ischemia in kidney transplantation.