CD34 modulates the trafficking behavior of hematopoietic cells in vivo

The CD34 surface antigen has been recognized as a marker of hematopoietic stem cells (HSCs) and is widely used for HSC selection as well as for quality control in HSC transplantation. CD34 has been implicated in cytoadhesion signaling, and its expression has been suggested to reflect the activation state of hematopoietic progenitor cells. However, the function of CD34 remains essentially unknown. Here we analyzed the effects of ectopic CD34 expression in vivo in a bone marrow transplantation model. We transduced murine bone marrow stem cells with retroviral vectors encoding either murine full-length or the alternative splice product truncated CD34. Transduced cells were transplanted into syngeneic, marrow ablated hosts. For comparison, "control" animals received either enhanced green fluorescent protein (eGFP)-transduced or mock-transduced cells. Six months post-transplantation, transduced differentiated blood cells ectopically expressing murine CD34 showed decreased migration from peripheral blood to both bone marrow and thymus, an effect that was more pronounced with full-length CD34 than with the truncated variant. In contrast, no influence of transgene expression on trafficking of differentiated blood cells was seen in the eGFP control group. Our data indicate that CD34 expression in mature blood cells has a suppressive effect on cellular trafficking to hematopoietic stroma organs, thereby supporting a modulating role of the CD34 molecule in cytoadhesion.     

Bone marrow mesenchymal stromal cells remain of recipient origin after allogeneic SCT and do not harbor the JAK2V617F mutation in patients with myelofibrosis

The close association of the myeloproliferative neoplasms with the activating non-receptor tyrosine kinase JAK2V617F mutation is well established. To further clarify the pathomechanisms of this mutation in patients with myelofibrosis, we performed screening with quantitative real-time PCR for the respective mutation in in vitro expanded bone marrow (BM) mesenchymal stromal cells (MSCs) and compared the results with BM/peripheral blood (PB). Eight patients with primary/secondary myelofibrosis were investigated before (n = 4) or after allogeneic stem cell transplantation (n = 4). All patients had systemic evidence of the JAK2V617F mutation in BM/PB (mutation ratios 0.2–23.5) at the time of investigation in contrast to negative results in the MSCs (n = 7) or a very low (0.004) mutation ratio (n = 1) which was probably due to hematopoietic contamination. The four patients post-transplant had systemic donor chimerism between 96.5 and 100% in BM/PB, while MSCs showed no evidence of donor-specific alleles. In conclusion, in myelofibrosis, the JAK2V617F mutation is restricted to hematopoietic cells, and cannot explain the stromal alterations being observed in this disorder. Further, the MSCs remain of recipient origin after allogeneic SCT, which might contribute to the increased risk of graft dysfunction or failure in myelofibrosis patients after allogeneic transplantation.     

Rehabilitationsmanagement

The contract to supply the best possible care of our insured persons cannot be fulfilled except by fusing the individual phases of rehabilitation with the objective of a holistic and comprehensive rehabilitation. One of the instruments that could be used to achieve this in practice is rehabilitation management, which is an option especially for insured persons whose prognosis for reintegration is poor. Following validation of the diagnosis and the activity profile a rehabilitation plan is set up, which should be documented in writing; this plan specifies the main important objectives, and the breakdown of smaller objectives derived from these, defined in terms of action and time, for the payer and the persons to be rehabilitated. There are then continuous checks to see that the plan is adhered to or, if necessary, adapted to suit changed circumstances. As well as the rehabilitation plan, early personal contact with those involved is of key importance in rehabilitation management. Since this strategy is now applied by various accident insurance institutions, coordination is necessary to achieve an effect seen by outsiders as uniform.     

Denosumab mimics the natural decoy receptor osteoprotegerin by interacting with its major binding site on RANKL

Bone homeostasis critically relies on the RANKL-RANK-OPG axis which can be targeted by the fully human monoclonal antibody denosumab in conditions with increased bone resporption such as bone metastases. The binding site and therefore the molecular mechanism by which this antibody inhibits RANKL has not been characterized so far. Here, we used random peptide phage display library screenings to identify the denosumab epitope on RANKL. Alignments of phage derived peptide sequences with RANKL suggested that this antibody recognized a linear epitope between position T233 and Y241. Mutational analysis confirmed the core residues as critical for this interaction. The spatial localization of this epitope on a 3-dimensional model of RANKL showed that it overlapped with the major binding sites of OPG and RANK on RANKL. We conclude that denosumab inhibits RANKL by both functional and molecular mimicry of the natural decoy receptor OPG.     

Influence of anti-thymocyte globulin as part of the conditioning regimen on immune reconstitution following matched related bone marrow transplantation

The aim of this work was to analyze the influence of anti-thymocyte globulin (ATG) as part of the conditioning regimen on immune reconstitution following matched related bone marrow transplantation. The rate and pattern of the recovery of total lymphocytes, natural killer (NK) cells, and several T and B cell subsets were determined in 38 patients for more than 2 years following BMT. We compared two patient groups: the first comprised 19 patients after matched related BMT without ATG prevention for graft-versus-host disease (GVHD) and the second contained 19 patients after matched related BMT with ATG treatment for GVHD prophylaxis. We observed impaired immune reconstitution in the ATG group in comparison with the non-ATG group, indicating a significant influence of ATG on immune recovery for several months after BMT.     

Antithymocyte globulin induces ex vivo and in vivo depletion of myeloid and plasmacytoid dendritic cells

We investigate the in vivo and ex vivo effect of rabbit antithymocyte globulin (ATG) on myeloid dendritic cells (MDCs) and plasmacytoid dendritic cells (PDCs). After incubation with ATG and complement, the mean number of MDCs and PDCs decreases from 3,168 to 739 x 10/mL (P=0.004) and from 5,314 to 790 x 10/mL (P=0.01), respectively. In vivo ATG given as part of the conditioning regimen before allogeneic stem-cell transplantation induces a stronger reduction of circulating MDCs and PDCs than chemotherapy alone (reduction: 100% vs. 78%-98%). These data show that ATG induces depletion of circulating MDCs and PDCs, which might be in addition to the T-cell depletion a further mechanism to reduce graft-versus-host disease after allogeneic stem-cell transplantation.     

T lymphocytes as targets of gene transfer with Moloney-type retroviral vectors

Peripheral T lymphocytes are a target of choice for many gene therapeutic strategies. Retrovirus-mediated transduction allows genomic integration and long-term expression of transgenes in target cells. Over many years, low transduction efficiency into primary T lymphocytes has limited clinical application of existing protocols. Recently, gene transfer rates > 50% have been achieved facilitating clinical studies. More attention is thus being focused on the ability of gene-modified cells to carry out innate as well as conferred functions in vivo and the influence of culture conditions, retroviral vector and host response thereon.     

Sensitive balance of suppressing and activating effects of mesenchymal stem cells on T-cell proliferation

The human leukocyte antigen-independent immune-modulatory potential of human mesenchymal stem cells (hMSC) makes them a promising candidate for clinical cell therapy. A better understanding of their "immune-privileged" status is therefore of high priority. Here we used Ki-67-antigen staining to estimate T-cell alloreactivity in mixed lymphocyte cultures in the presence of hMSC as second or third party. We found that the allostimulatory activity of mesenchymal stem cells (MSC) leading to an increased T-cell proliferation and interleukin-2 secretion is measurable only at low MSC/effector ratios (< or =0.1:1). Moreover, this stimulating effect could be efficiently suppressed by MSC-conditioned medium. This suggests that the "immune-privileged" status of MSC exists only when MSC-mediated downregulation of immune cell activation can overrule their own allostimulatory potential. Thus the "immune-privileged" state of MSC represents a sensitive balance of suppressing and activating effects, which should be considered in a clinical setting with limited cell amounts.     

Farber disease: clinical presentation, pathogenesis and a new approach to treatment

Background  

Farber Disease is an autosomal-recessively inherited, lysosomal storage disorder caused by acid ceramidase deficiency and associated with distinct clinical phenotypes. Children with significant neurological involvement usually die early in infancy, whereas patients without or only mild neurological findings suffer from progressive joint deformation and contractures, subcutaneous nodules, inflammatory, periarticular granulomas, a hoarse voice and finally respiratory insufficiency caused by granuloma formation in the respiratory tract and interstitial pneumonitis leading to death in the third or fourth decade of live. As the inflammatory component of this disorder is caused by some kind of leukocyte dysregulation, allogeneic hematopoietic stem cell transplantation can restore a healthy immune system and thus may provide a curative option in Farber Disease patients without neurological involvement. Previous stem cell transplantations in two children with severe neurological involvement had resulted in a disappointing outcome, as both patients died of progressive deterioration of their neurological status. As a consequence, stem cell transplantation does not appear to be able to abolish or even reduce the neurotoxic effects of the abundant ceramide storage in the brain.