SUMMARYThe generation of myelinating cells in the central nervous system requires the initiation of specific gene expression programs in oligodendrocytes. We reasoned that microRNAs (miRNAs) could play an important role in this process by regulating crucial developmental genes. Microarray profiling of cultured oligodendrocytes identified the miR-17-92 miRNA cluster as highly enriched in oligodendrocytes. We specifically deleted the miR-17-92 cluster in oligodendrocytes using 2Ј,3Ј-cyclic nucleotide 3Ј phosphodiesterase (Cnp)-Cre mice. Absence of miR-17-92 leads to a reduction in oligodendrocyte number in vivo and we find that the expression of these miRNAs in primary cultures of oligodendrocyte precursor cells promotes cell proliferation by influencing Akt signaling. Together, these results suggest that the miRNA pathway is essential in determining oligodendroglial cell number and that the miR-17-92 cluster is crucial in this process.

Extracorporeal photopheresis (ECP) is an important second-line therapy for graft-versus-host disease. A central therapeutic mechanism is the induction of immune tolerance through apoptosis in patient’s leukocytes, caused by ex vivo incubation with 8-methoxypsoralen (8-MOP) and subsequent UVA irradiation.We hypothesized that different 8-MOP incubation times and an additional 8-MOP removal step could influence the apoptosis kinetics of leukocytes in general and in particular could lead to different apoptotic levels in the leukocyte subpopulations. After 8-MOP/UVA treatment of human leukocytes, cells were cultured and the percentage of annexin V positive cells from several leukocyte subpopulations was determined. Only regulatory T cells (Tregs) were relatively resistant to 8-MOP/UVA induced apoptosis. When cells were incubated for 30 minutes with 8-MOP prior to UVA exposure, higher percentages of annexin V positive cells were detected on day 1 and day 2 after treatment. Removal of 8-MOP after UVA exposure caused no significant changes in the apoptosis kinetics during the 72 h culture period compared with unwashed cells. The results of our in vitro study indicate that it could be possible to adjust the apoptosis kinetics via modulation of the 8-MOP incubation time. In further in vivo studies it should be elucidated to which extent different apoptosis kinetics influence the therapeutic effect of ECP since steady-state apoptosis levels are probably important for establishing a long lasting immune tolerance. Furthermore we found that Tregs, according to their well-known tolerogenic function, are more resistant to apoptosis after 8-MOP/UVA treatment compared to GvHD inducing T cell populations.

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Background and Objective Extracorporeal photopheresis (ECP) is an important immune tolerance inducing therapy for graft-versus-host disease (GvHD). However, a sufficient number of ECP cycles cannot be performed in patients with severe GvHD and contraindications for apheresis. Allogeneic sources of leucocytes for use as ECP treatment would be of great benefit. Therefore, this study aimed to test the therapeutic potential of novel sources of leucocytes for ECP. Materials and MethodsGraft-versus-host disease mice were treated with ECP using therapeutic cells from different allogeneic sources. Splenocytes were incubated with 8-methoxypsoralen (8-MOP), irradiated with UVA light and injected into GvHD mice as a model for ECP. ResultsThe therapy with 8-MOP/UVA-treated cells from healthy mice of the bone marrow transplantation (BMT) donor strain reduced the GvHD symptoms, at least in a model of chronic GvHD. In the acute GvHD model, 8-MOP/UVA-treated cells from the BMT donor or recipient strain did not show significant improvements in GvHD symptoms or survival time. Pre-activation of cells by mixed lymphocyte reactions before 8-MOP/UVA treatment also failed to result in significant differences in survival time or GvHD score. In contrast, ECP with third-party 8-MOP/UVA-treated cells from a HLA-mismatched donor resulted in a mean survival time of 37 days compared to 21 days in the control group.Conclusion In our analysis of novel allogeneic leucocyte sources for ECP, we could demonstrate that the source of the 8-MOP/UVA-treated cells is crucial. The underlying immunologic effect of allogeneic 8-MOP/UVA-treated cells needs to be investigated in future studies.

Graft-versus-host disease (GvHD) still belongs to the major challenges after allogeneic hematopoietic stem cell transplantation (HSCT). Immune-suppressive therapy against GvHD is a double-edged sword due to risk of infections and relapse. The ability to adapt prophylactic treatment according to the probability of severe GvHD would be an essential advantage for the patients. We analyzed different biomarkers for their potential to predict the development of GvHD in 28 patients who underwent allogeneic HSCT. Blood was taken once directly after hematopoietic engraftment. In this study, patients were monitored for 12 months after HSCT for the occurrence of acute GvHD or acute/chronic GvHD overlap syndrome. Soluble IL-2 receptor and CD4/CD8 T cell ratio were independently associated with the occurrence of GvHD in the observation period. However, the largest area under the receiver operating characteristic curve with 0.90 was observed when a 5-parameter biomarker score based on CD4 T cells, CD8 T cells, CD19 CD21 precursor B cells, CD4/CD8 T cell ratio, and soluble IL-2 receptor was used to predict GvHD. In addition, CD8 T cell levels above 2.3% of all mononuclear cells after engraftment may predict relapse-free survival at least for 12 months. In summary, we found a new biomarker panel for prediction of GvHD which is featured by established laboratory assays and high statistical significance. In order to introduce the biomarker panel into routine clinical protocols, we suggest performing a larger multi-center study.

BackgroundGraft-versus-host disease (GvHD) is a major challenge after hematopoietic stem cell transplantation but treatment options for patients are still limited. In many cases first-line treatment with glucocorticoids is not successful. Among second-line therapies the extracorporeal photopheresis (ECP) is frequently performed, due to induction of selective tolerance instead of general immunosuppression. However, for some patients with severe acute GvHD the leukapheresis step of the ECP procedure is physically exhausting and limits the number of ECP cycles.MethodsWe hypothesized that leukocytes from healthy cell donors could be used as a replacement for ECP leukocytes gained from the GvHD patient. For this purpose we used a well established mouse model of acute GvHD. The ECP therapy was based on cells with the genetic background of the initial donor of the stem cell transplantation. As a precondition we developed a protocol representing conventional ECP in mice equivalent to clinical used ECP setup.ResultsWe could demonstrate that conventional, clinically derived ECP setup is able to alleviate acute GvHD. By using leukocytes obtained from healthy mice with the bone marrow donor’s genetic background we could not observe a statistically significant therapeutic effect.ConclusionsConventional human ECP setup is effective in the mouse model of severe acute GvHD. In addition we could not prove that ECP cells from healthy mice with bone marrow donor’s genetic background are as effective as ECP cells derived from GvHD mice. Based on our findings, new questions arise for further studies, in which the cellular characteristics for ECP mediated immune tolerance are a matter of investigation.

Spontaneous Rh blood group changes are a striking sign, reported to occur mainly in patients with hematologic disorders. Upon routine blood grouping, 2 unrelated individuals showed unexplained mixed red cell phenotype regarding the highly immunogenic c antigen (RH4), clinically relevant for blood transfusion and fetomaternal incompatibility. About half of their red cells were c-positive, whereas the other half were c-negative. These apparently hematologically healthy females had no history of transfusion or transplantation, and they tested negative for chimerism. Genotyping of flanking chromosome 1 microsatellites in blood, finger nails, hair, leukocyte subpopulations, and erythroid progenitor cells showed partial loss of heterozygosity encompassing the RHD/RHCE loci, spanning a 1p region of 26.7 or 42.4 Mb, respectively. Remarkably, in one case this was detected in all investigated tissues, whereas in the other, exclusively myeloid cells showed loss of heterozygosity. Both carried the RhD-positive haplotypes CDe and the RhD-negative haplotype cde . RHD/RHCE genotypes of single erythroid colonies and dual-color fluorescent in situ hybridization analyses indicated loss of the cde haplotype and duplication of the CDe haplotype in the altered cell line. Accordingly, red cell C antigen (RH2) levels of both propositae were higher than those of heterozygous controls. Taken together, the Rhc phenotype splitting appeared to be caused by deletion of a part of 1p followed by duplication of homologous stretches of the sister chromosome. In one case, this phenomenon was confined to myeloid stem cells, while in the other, a pluripotent stem cell line was affected, demonstrating somatic mosaicism at different stages of ontogenesis.

Our findings on an in vitro assessment show major improvements in red blood cell integrity and platelet function of suction blood when using the TCSS compared to a conventional suction system. These results reflect a significant benefit for autologous re-transfusion. We suggest testing the TCSS in surgery for clinical evaluation.