Efficient Pseudotyping of Different Retroviral Vectors Using a Novel,Codon-Optimized Gene for Chimeric GALV Envelope

The Gibbon Ape Leukemia Virus envelope protein (GALV-Env) mediates efficient transduction of human cells, particularly primary B and T lymphocytes, and is therefore of great interest in gene therapy. Using internal domains from murine leukemia viruses (MLV), chimeric GALV-Env proteins such as GALV-C_4070A were derived, which allow pseudotyping of lentiviral vectors. In order to improve expression efficiency and vector titers, we developed a codon-optimized (co) variant of GALV-C_4070A (coGALV-Env). We found that coGALV-Env mediated efficient pseudotyping not only of γ-retroviral and lentiviral vectors, but also α-retroviral vectors. The obtained titers on HEK293T cells were equal to those with the classical GALV-Env, whereas the required plasmid amounts for transient vector production were significantly lower, namely, 20 ng coGALV-Env plasmid per 10⁶ 293T producer cells. Importantly, coGALV-Env-pseudotyped γ- and α-retroviral, as well as lentiviral vectors, mediated efficient transduction of primary human T cells. We propose that the novel chimeric coGALV-Env gene will be very useful for the efficient production of high-titer vector preparations, e.g., to equip human T cells with novel specificities using transgenic TCRs or CARs. The considerably lower amount of plasmid needed might also result in a significant cost advantage for good manufacturing practice (GMP) vector production based on transient transfection.     

Gonarthroseprävention

With § 3 of the German occupational disease ordinance (Berufskrankheitenverordnung, BKV), accident insurers have an instrument at their disposal which has served successfully for many years to preserve work places. In the case of early intervention, good efficiency can be achieved by the use of reasonable means. Experience with skin diseases has demonstrated precisely this. Relevant §3 BKV measures are also possible for gonarthrosis (OD gonarthrosis, OD 2112), assuming that clear medical evidence reliably identifying disease characteristics are compiled for physicians and accident insurers. Suggestions are presented here.     

Complement-dependent and complement-independent cytotoxicity of polyclonal antithymocyte globulins in chronic lymphocytic leukemia

OBJECTIVE: Despite important progress in its management, chronic lymphocytic leukemia (CLL) remains incurable with standard therapies. Allogeneic stem cell transplantation (SCT) is a potentially curative therapy for patients with CLL. Polyclonal antithymocyte (or anti-T-cell) globulins (ATGs) are used for conditioning in allogeneic SCT mainly due to their anti-T-cell activity. ATGs however, contain antibodies targeting antigens expressed on various hematopoietic cells including B cells. METHODS: We assessed anti-CLL activity of two commercially available ATG preparations at clinically relevant concentrations (10-100 microg/ml) in CLL samples from 16 patients. Cytotoxicity was determined by staining with 7-amino-actinomycin D (7-AAD), annexin V and flow cytometry. RESULTS: Both ATG preparations induced marked complement-independent dose-dependent cytotoxicity in all samples. Addition of complement strongly enhanced the cytotoxic effect of both ATG preparations significantly. ATG-induced complement-dependent cytotoxicity (CDC) was at least as high as that observed with Alemtuzumab. Both ATGs enhanced the cytotoxic effect of Fludarabine. CONCLUSION: ATG is an effective agent against CLL in vitro. We suggest that this potential be taken into consideration when developing stem cell transplantation protocols for patients with CLL.     

Kostenanalyse bei Frakturheilungsstörungen

Trauma und Berufskrankheit - Frakturheilungsstörungen in Form von Pseudarthrosen und Infekten sind seltene, aber ernst zu nehmende Komplikationen im berufsgenossenschaftlichen Heilverfahren,...     

Autologous stem cell transplantation followed by a dose-reduced allograft induces high complete remission rate in multiple myeloma

We evaluated toxicity, engraftment, chimerism, graft-versus-host disease (GVHD), and response to a dose-reduced allograft after cytoreductive autografting in 17 patients with advanced stage II/III multiple myeloma (MM). After autografting with melphalan (200 mg/m2) the patients received after a median interval of 119 days (range 60-210) a dose-reduced regimen consisting of fludarabine (180 mg/m2), melphalan (100 mg/m2), and antithymocyte globulin (3 x 10 mg/kg) followed by allografting from related (n = 7), mismatched related (n = 2), or unrelated (n = 8) donors to induce a graft-versus-myeloma effect. After dose-reduced allografting all patients became neutropenic (< 0.2 x 10(9)/L) for at least 8 days. All patients engrafted with a median time for leukocyte (> 1 x 10(9)/L) and platelet (> 20 x 10(9)/L) counts of 16 (range, 11-24) and 23 days (range, 12-43), respectively. Complete donor chimerism was detected after a median of 30 days (range, 19-38). Acute GVHD stage II occurred in 4 patients (25%) and grade III GVHD in 2 patients (13%). Chronic GVHD developed in 40% of the patients, but only 1 patient experienced extensive chronic GVHD requiring further immunosuppressive therapy. Two patients died of alveolar hemorrhage and pneumonia, resulting in a day 100 mortality rate of 11%. The rate of complete remission with negative immunofixation increased from 18% after autografting to 73% after allografting. After a median follow-up of 17 months after autologous and 13 months after allogeneic transplantation 13 patients are alive and 12 of them free of relapse or progression. The tandem auto-allotransplant protocol is highly active and provides rapid engraftment with complete donor chimerism and tolerable toxicity.     

CD133 marks a stem cell population that drives human primary myelofibrosis

Primary myelofibrosis is a myeloproliferative neoplasm characterized by bone marrow fibrosis, megakaryocyte atypia, extramedullary hematopoiesis, and transformation to acute myeloid leukemia. To date the stem cell that undergoes the spatial and temporal chain of events during the development of this disease has not been identified. Here we describe a CD133⁺ stem cell population that drives the pathogenesis of primary myelofibrosis. Patient-derived circulating CD133⁺ but not CD34⁺CD133⁻ cells, with a variable burden for JAK2^V617F mutation, had multipotent cloning capacity in vitro. CD133⁺ cells engrafted for up to 10 months in immunocompromised mice and differentiated into JAK2-V617F⁺ myeloid but not lymphoid progenitors. We observed the persistence of human, atypical JAK2-V617F⁺ megakaryocytes, the initiation of a prefibrotic state, bone marrow/splenic fibrosis and transition to acute myeloid leukemia. Leukemic cells arose from a subset of CD133⁺ cells harboring EZH2^D265H but lacking a secondary JAK2^V617F mutation, consistent with the hypothesis that deregulation of EZH2 activity drives clonal growth and increases the risk of acute myeloid leukemia. This is the first characterization of a patient-derived stem cell population that drives disease resembling both chronic and acute phases of primary myelofibrosis in mice. These results reveal the importance of the CD133 antigen in deciphering the neoplastic clone in primary myelofibrosis and indicate a new therapeutic target for myeloproliferative neoplasms.