Prevention of graft-versus-host-disease with preserved graft-versus-leukemia-effect by ex vivo and in vivo modulation of CD4(+) T-cells.

This is the first report showing that an epitope-specific ex vivo modulation of an allogeneic hematopoietic stem cell graft by the anti-human CD4 antibody MAX.16H5 IgG1 simultaneously facilitates the anti-tumor capacity of the graft (Graft-versus-leukemia effect, GvL) and the long-term suppression of the deleterious side effect Graft-versus-host-disease (GvHD). To distinguish and consolidate GvL from GvHD, the anti-human CD4 antibody MAX16.H5 IgG1 was tested in murine GvHD and tumor models. The survival rate was significantly increased in recipients receiving a MAX.16H5 IgG1 short-term (2 h) pre-incubated graft even when tumor cells were co-transplanted or when recipient mice were treated by MAX.16H5 IgG1 before transplantation. After engraftment, regulatory T-cells are generated only supporting the GvL effect. It was also possible to transfer the immune tolerance from GvHD-free recipient chimeras into third party recipient mice without the need of reapplication of MAX.16H5 IgG1 anti-human CD4 antibodies. These findings are also benefical for patients with leukemia when no matched related or unrelated donor is available and provides a safer allogeneic HSCT, which is more effective against leukemia. It also facilitates allogeneic (stem) cell transplantations for other indications (e.g., autoimmune-disorders).

Allogeneic bone marrow grafts with high levels of CD4(+) CD25(+) FoxP3(+) T cells can lead to engraftment failure.

Regulatory CD4(+) CD25(+) FoxP3(+) T cells (T(regs) ) suppress immunological reactions. However, the effect of adding T(regs) to hematopoietic stem cell grafts on recovery and graft versus host disease (GvHD) is unknown. T(regs) from splenocytes of C57Bl/6 and Balb/c wild-type mice were isolated by MACS separation and analyzed by flow cytometry. Using a murine syngeneic transplantation model that clearly distinguishes between donor and host hematopoiesis, we showed that co-transplantation of bone marrow cells (BMCs) with high levels of T(regs) leads to a 100% survival of the mice and accelerates the hematopoietic recovery significantly (full donor chimerism). In allogeneic transplantation, bone marrow and T(regs) co-transplantation were compared to allogeneic bone marrow transplantation with or without the addition of splenocytes. Survival, leukocyte recovery, chimerism at days -2, 19, 33, and 61 for murine CD4, human CD4, HLA-DR3, murine CD3, murine CD8, murine Balb/c-H2K(d) , murine C57Bl/6-H2K(b) , and GvHD appearance were analyzed. Allogeneic bone marrow transplantation requires the addition of splenocytes to reach engraftment. Mice receiving grafts with bone marrow, splenocytes and high levels of allogeneic T(regs) died within 28 days (hematopoietic failure). Here, we show also detailed flow cytometric data reagarding analysis of chimerism after transplantation in unique murine hematopoietic stem cell transplantation models. Our findings showed that the syngeneic co-transplantation of CD4(+) , CD25(+) , FoxP3(+) T-cells and BMCs induced a stimulating effect on reconstitution of hematopoiesis after irradiation. However, in the allogeneic setting the co-transplantation of T(regs) aggravates the engraftment of transplanted cells.

A real-time PCR for the detection and characterisation of Aspergillus species.

An early diagnosis of an invasive fungal infection is essential for the initiation of a specific antifungal therapy and to avoid unnecessary discontinuation of a baseline therapy for haematological or oncological diseases. A real-time PCR assay for the detection and strain identification of Aspergillus species from culture strains was evaluated. DNA preparation was evaluated in contaminated culture media, urine and serum. A LightCycler PCR to differentiate various Aspergillus species was established. A real-time PCR assay for the detection of Aspergillus species was improved and was able to detect and differentiate medically important Aspergillus spp. The sensitivity of the test was <10 plasmid equivalents/assay. The real-time PCR assay is a useful tool for the rapid identification of Aspergillus species and might be useful as an early diagnostic tool to detect an invasive fungal infection. A real-time PCR protocol was improved by generating plasmid standards, additional generation of melting curves for species identification and the correlation between the melting temperature and the nucleotide exchanges within the used 18S rRNA gene region.

Characterization of murine non-adherent bone marrow cells leading to recovery of endogenous hematopoiesis.

Non-adherent bone marrow-derived cells (NA-BMCs) are a mixed cell population that can give rise to multiple mesenchymal phenotypes and that facilitates hematopoietic recovery. We characterized NA-BMCs by flow cytometry, fibroblast colony-forming units (CFU-f), real-time PCR, and in in vivo experiments. In comparison to adherent cells, NA-BMCs expressed high levels of CD11b(+) and CD90(+) within the CD45(+) cell fraction. CFU-f were significantly declining over the cultivation period, but NA-BMCs were still able to form CFU-f after 5 days. Gene expression analysis of allogeneic NA-BMCs compared to bone marrow (BM) indicates that NA-BMCs contain stromal, mesenchymal, endothelial cells and monocytes, but less osteoid, lymphoid, and erythroid cells, and hematopoietic stem cells. Histopathological data and analysis of weight showed an excellent recovery and organ repair of lethally irradiated mice after NA-BMC transplantation with a normal composition of the BM.

Modelling hematological parameters after total body irradiation.

PURPOSE: The time- and dose-dependent reconstitution of hematopoiesis after radiation exposure is strongly related to the stem cell population and can be used to predict hematological parameters. These parameters allow further insight into the hematopoietic system and might lead to the development of novel stem cell transplantation models. MATERIALS AND METHODS: CD4-/- C57Bl/6 mice, transgenic for human CD4 and HLA-DR3, were irradiated in a single (3, 6, 8 and 12 Gy) and fractionated (6 × 1 Gy, 6 × 1.5 Gy, 6 × 2 Gy; twice daily) dose regimen. Blood was analyzed weekly for red blood cells (RBC), hemoglobin concentration (Hb), hematocrit (HCT) and white blood cells (WBC). Organ and tissue damage after irradiation were examined by histopathology. RESULTS: The recovery curves for RBC, Hb, HCT and WBC showed the same velocity (< 1 week) for all radiation doses (3-12 Gy) starting at different, dose-dependent times. The only dose-dependent parameter was defined by the beginning of the recovery process (dose-dependent shift) and higher doses were related to a later recovery of the hematopoietic system. The RBC, Hb and HCT recovery was followed by a saturation curve reaching a final concentration independent of the radiation dose. Histological analysis of the bone marrow in the single dose cohort showed a dose-dependent reduction of the cellularity in the bone marrow cavities. The fractioned radiation dose cohort resulted in a regeneration of all bone marrow cavities. CONCLUSION: Specific functions were developed to describe the reconstitution of hematological parameters after total body irradiation.

Combined bendamustine, prednisone and bortezomib (BPV) in patients with relapsed or refractory multiple myeloma.

INTRODUCTION: Bortezomib (Velcade) is a proteasome inhibitor that has shown important clinical efficacy either as a single agent or in combination with other cytostatic agents in multiple myeloma (MM). In the present protocol, bortezomib was combined with other active substances like bendamustine and prednisone (BPV), in order to assess the efficacy and toxicity of the combination therapy in patients with relapsed or refractory MM. METHODS: Between January 2005 and December 2011, 78 patients with relapsed or refractory MM were treated with bendamustine 60 (-120) mg/m(2) on days 1 and 2, bortezomib 1.3 mg/m(2) on days 1, 4, 8 and 11, and prednisone 100 mg on days 1, 2, 4, 8 and 11. The median number of prior therapies was 2 with a wide range of 1-9. Thirty-three patients had pre-existing severe thrombocytopenia and/or neutropenia (WHO grade 3 or 4). RESULTS: A median number of two (range 1-7) BPV treatment cycles were given to the patients. The majority of the patients (n = 54; 69 %) responded after at least one cycle of chemotherapy with 3 CR, 10 nCR, 10 VGPR and 31 PR. Median PFS and OS for patients without severe hematological toxicities due to previous treatments (n = 45) were 11 and 50 months, respectively. Outcome for these patients was significantly better than that for patients with severe hematological toxicities (grade 3 or 4, n = 33) with a PFS, and OS of 3 months (p < 0.05) and 5 months (p < 0.001), respectively. The regimen was well tolerated with few significant side effects in patients without severe hematological toxicities due to previous treatments. These results indicate that the combination of bortezomib, bendamustine and prednisone is well tolerated in patients with relapsed or refractory MM.