[NiFe]‐hydrogenases generally carry the bimetallic Ni‐Fe reaction center on their largest subunit. The [NiFeSe]‐hydrogenase Vhu from Methanococcus voltae has an unusual subunit composition. Some of the amino acids participating in the formation of the reaction center are within a separate, very small subunit, called VhuU. It consists of only 25 amino acids and contains the selenocysteinyl residue, a ligand to the Ni atom. We have tested whether the special configuration of the Vhu‐hydrogenase is of particular biochemical relevance. We have constructed a fusion subunit derived from the VhuA and VhuU subunits by generating a gene fusion which was inserted into the chromosome of M. voltae by gene replacement. The enzyme was purified and shown to be as active as the wild‐type enzyme. M. voltae carries the genetic information for four different [NiFe]‐hydrogenases. In addition to the Vhu‐hydrogenase, a second selenium‐containing enzyme, Fru, has been purified. Two selenium‐free enzymes, Vhc and Frc, are homologues of Vhu and Fru, respectively. Their gene groups, vhc and frc are transcribed only upon selenium depletion. The selenium‐containing subunit VhuU has been implicated in their negative regulation. However, cells containing the fusion hydrogenase still exhibited normal regulation of the vhc and frc promoter activities as tested in reporter gene constructs. This indicates that the free VhuU polypeptide is not required for the negative regulation of the vhc or frc genes.
Profound depletion of T and B cells is a fundamental prerequisite for haploidentical transplantation and allows to minimize GvHD despite HLA incompatibility. However, posttransplant recovery of donor derived T cells is delayed after various graft manipulation procedures and may result in severe infections. Methods to improve this recovery are of importance. Here we present immune reconstitution data in patients who received CD3/CD19 depleted stem cells in combination with melphalan based or standard conditioning regimens. 32 patients with ALL(n=14), AML/MDS n=17), CML(n=1) were included. T- and B-cells were directly depleted using antiCD3/antiCD19 coated magnetic microbeads and the CliniMACS™ device. The patients received either TBI or Bu i.v. and OKT3(n=9) or a reduced intensity conditioning ("RIC": Mel 140 mg/m2, Flud 160 mg/m2, TT 10 mg/kg, OKT3, n=23). Absolute numbers of lymphocyte subsets per microliter on day 90 were compared within these both groups and with a historical control group (patients with leukemias who received CD34 selected grafts and TBI or Bu based standard conditioning regimen in combination with ATG, n=28). CD3+4+, CD3+8+ and total numbers of CD3+ of patients after CD3/CD19 depletion were significantly higher in the RIC-group than in the TBI/Bu-group (mean: 85.83 vs. 38.84; 133.46 vs. 19.69; 270.27 vs. 63.99; p<0.05, t-test). Comparison with the whole CD34 historical group showed a faster recovery of CD3+4+ in patients with CD3/19 depletion and RIC (104.26 vs. 54.22; p=0,034) but no significant difference in CD3+8+ and CD3+. Furthermore, subgroups of the CD34 historical population were compared: patients with CD3/19 depletion and RIC had a significantly faster recovery of CD3+4+, CD3+8+ and CD3+ than CD34 patients with TBI (104.26vs. 25.48; 133.46vs. 43.17; 270.27 vs. 65.86; p<0.05) but had no advantage over CD34 patients with non-TBI conditioning. the type of graft manipulation appeared to have an influence on the speed of CD4+ recovery (CD3/19 depletion>CD34 selection). Moreover, the use of TBI had a negative impact on all T cell subsets: patients with TBI had a slower recovery than patients with non-TBI conditioning, independent from graft manipulation procedures and probably due to thymic damage. Thus, the use of RIC-protocols in combination with CD3/CD19 depletion may help to speed up the immune recovery after haploidentical transplantation. Further studies are warranted to evaluate the risk of relapse.
Ziel: Ziel der Studie war der Vergleich verschiedener Applikationsformen des Ultraschallkontrastverstärkers Levovist® hinsichtlich Verstärkungsdauer und Verstärkungsintensität bei Patienten mit Raumforderungen in Leber oder Pankreas. Patienten und Methode: 7 Patienten mit Raumforderungen der Leber oder des Pankreas wurden dreimal prospektiv mittels kontrastverstärkter Power-Doppler-Sonographie (Levovist® 8 ml, 400 mg/ml) untersucht: 1. mit einer Bolusinjektion über den Hauptkanal, 2. über das Zuspritz-Ventil einer Venenverweilkanüle und 3. mit kontinuierlicher Infusion. Die erzielte Verstärkung wurde im zeitlichen Verlauf von drei voneinander unabhängigen Untersuchern semiquantitativ beurteilt. Ergebnisse: Bei gleicher Levovist®-Dosis konnte für die Infusion eine signifikant verlängerte Verstärkungsdauer von 9 : 43 min extra- und 7 : 34 min intratumoral gegenüber 6 : 01 min extra- und 4 : 54 min intratumoral bei der Bolusinjektion über den Hauptkanal (extratumoral p = 0,0156; intratumoral p = 0,0313) nachgewiesen werden. Die Verstärkungsintensität war unter der Infusion verringert. Die Bolusinjektion über das Zuspritz-Ventil führte zu einer geringeren Verstärkungsdauer und -intensität als die Injektion über den Hauptkanal. Schlußfolgerung: Mit der kontinuierlichen Infusion von Levovist® sind gegenüber der Bolusinjektion eine signifikante Verlängerung der Verstärkungsdauer und eine geringere Verstärkungsintensität nachweisbar. Die Applikation über das Zuspritz-Ventil führt nicht zu einer optimalen Kontrastverstärkung und ist daher nicht zu empfehlen.
Pediatric patients with relapsed metastatic tumors have a poor prognosis and new treatment strategies are warranted. We present preliminary results of a pilot study, evaluating the feasibility and toxicity of transplantation of haploidentical T and B cell depleted grafts with high numbers of NK cells. 6 patients with relapsed metastatic neuroblastomas (n = 4), rhabdomyosarcoma (n = 1) or Ewing's sarcoma (n = 1) after previous autologous transplantation received CD3/CD19 depleted grafts from mismatched family donors with a median number of 16 × 106/kg stem cells, 167 × 106/kg Natural Killer cells and only 5.4 × 104/kg residual T cells. A melphalan-based, reduced intensity conditioning was used. Despite pretransplant chemotherapy, patients entered transplantation with significant tumor burden. Primary engraftment occurred in 6/6 patients. One patient had secondary graft failure. Hematopoietic recovery was rapid (ANC > 0.5 × 109/L: 11 days (9-12); independence from platelet substitution: 8 days (7-11)). Four patients had acute GvHD grade II, limited chronic GvHD was observed in 2 patients. No transplant-related mortality and only low toxicity occurred. Four patients died from progression, two patients are alive. Overall median survival time is 6 months (2-11) to date. Analysis of posttransplant NK cell function revealed stable cytotoxic activity against K562 targets, whereas activity against neuroblastoma targets was low. Stimulation with cytokines and use of appropriate antibodies clearly enhanced specific lysis in vitro. In summary, these preliminary results indicate the feasibility and low toxicity even in intensively pre-treated patients with neuroblastomas/sarcomas. This approach may form the basis for posttransplant immunomodulation and other therapeutic strategies. Further experience is warranted to evaluate the method.
