Structural and kinetic properties of adenylyl sulfate reductase from Catharanthus roseus cell cultures.

A cDNA encoding a plant-type APS reductase was isolated from an axenic cell suspension culture of Catharanthus roseus (Genbank/EMBL-databank accession number U63784). The open reading frame of 1392 bp (termed par) encoded for a protein (Mr=51394) consisting of a N-terminal transit peptide, a PAPS reductase-like core and a C-terminal extension with homology to the thioredoxin-like domain of protein disulfide isomerase. The APS reductase precursor was imported into pea chloroplasts in vitro and processed to give a mature protein of approximately 45 kDa. The homologous protein from pea chloroplast stroma was detected using anti:par polyclonal antibodies. To investigate the catalytical function of the different domains deleted par proteins were purified. ParDelta1 lacking the transit sequence liberated sulfite from APS (Km 2.5+/-0.23 microM) in vitro with glutathione (Km 3+/-0.64 mM) as reductant (Vmax 2.6+/-0.14 U mg-1, molecular activity 126 min-1). ParDelta2 lacking the transit sequence and C-terminal domain had to be reconstituted with exogenous thioredoxin as reductant (Km 15. 3+/-1.27 microM, Vmax 0.6+/-0.014 U mg-1). Glutaredoxin, GSH or DTT were ineffective substitutes. ParDelta1 (35.4%) and parDelta2 (21. 8%) both exhibited insulin reductase activity comparable to thioredoxin (100%). Protein disulfide isomerase activity was observed for parDelta1.

Marrow engraftment of hematopoietic stem and progenitor cells is independent of Galphai-coupled chemokine receptors.

The mechanism of hematopoietic stem and progenitor cell (HSPC) homing to hematopoietic organs after transplantation is still poorly understood. There is evidence that HSPC homing is a multistep process involving integrins and other adhesion molecules as well as stimulation of cytokine and chemokine receptors, similar to the process of lymphocyte recirculation and leukocyte emigration. This study examined the effect of pertussis toxin (PT), an inhibitor of signaling by many Galphai protein-coupled chemokine receptors, on engraftment of HSPC. An in vitro incubation of total bone marrow cells in PT-supplemented media prior to transplantation into lethally irradiated syngeneic mice resulted in an increase in marrow repopulation and a parallel decrease of colony-forming unit-spleen (CFU-S) on day 13. PT treatment of Rh(low)Lineage(neg)Sca-1pos cells prior to transplant resulted in delayed spleen cell engraftment, but no observable difference in the bone marrow cellularity compared to animals transplanted with untreated cells. FACS analysis of hematopoietic organs revealed that myeloid cell recovery in the bone marrow was unaffected by PT treatment of HSPC. However, a reduced myeloid cell recovery in the spleen and an increased B lymphoid recovery in both the spleen and the bone marrow were observed in recipients of PT-treated grafts relative to untreated grafts. To test the hypothesis that PT inhibits proliferation rather than engraftment of HSPC in the spleen, the effect of PT on cytokine-stimulated proliferation of HSPC was tested. Although an inhibition of the growth of microcolonies in response to interleukin 6 as a single cytokine could be observed after PT treatment, colony growth of HSPC after steel factor or steel factor + interleukin 6 stimulation was unaffected by PT. This study demonstrates that bone marrow, but not splenic, recovery after HSPC transplantation is independent of PT-sensitive mechanisms. It is likely that PT inhibits spleen cell recovery by disrupting a Galphai-coupled homing receptor expressed by HSPC. These studies support the hypothesis that distinct mechanisms regulate splenic vs bone marrow engraftment of HSPC, and that B lymphocyte progenitors and HSPC can utilize a PT-resistant homing mechanism to localize in hematopoietic tissues after transplantation.

Modulation of hematopoietic stem/progenitor cell engraftment by transforming growth factor beta.

OBJECTIVE: To investigate if cell cycle progression plays a role in modulating the engraftment potential of mouse hematopoietic stem and progenitor cells (HSPC). MATERIALS AND METHODS: HSPC were isolated from adult mouse bone marrow, cultured in vitro under conditions promoting cell cycle arrest, and subsequently were evaluated for cell cycle status, clonogenic activity, and transplant potential. RESULTS In the presence of steel factor (STL) as a survival cytokine, transforming growth factor beta (TGF-beta) increased the G0/G1 fraction of cycling progenitor cells (Rh(high)) after a 20-hour culture. Clonogenic activity of quiescent long-term repopulating (Rh(low)) HSPC was unaffected by this culture, whereas clonogenic potential of Rh(high) cells decreased by about 30%. In competitive repopulation assays, Rh(low) cells cultured in STL + TGF-beta engrafted better than cells cultured in STL alone. However, culture in STL + TGF-beta did not overcome the failure of Rh(high) cells to engraft after transplant. We also utilized a two-stage culture system to first induce proliferation of Rh(low) HSPC by a 48-hour culture in STL + interleukin 6 + Flt-3 ligand, followed by shifting the culture to STL + TGF-beta for 24 hours to induce cycle arrest. A competitive repopulation assay demonstrated a relative decrease in repopulating potential in cultures that were cycle arrested compared to those that were not. CONCLUSION: Cell cycle progression by itself cannot account for the decrease in repopulating potential that is observed after ex vivo expansion. Other determinants of engraftment must be identified to facilitate the transplantation of cultured HSPC.

Severe acute graft-versus-host disease after T-cell depleted allogeneic stem cell graft from a second donor caused by persisting T-cells from the first donor.

HLA disparity is a major risk factor for graft rejection and GVHD. We report a patient with CML (accelerated phase) who underwent allogeneic SCT from a mismatched unrelated donor and developed acute GVHD. With immunosuppression, GVHD symptoms improved but graft rejection occurred. After a second conditioning regimen, the patient received a second graft from a haploidentical related donor. Engraftment occurred, but the patient died from GVHD and pulmonary aspergillosis. Chimeric analysis revealed that all leukocytes were of donor 2 origin apart from CD3+ T cells, which were 100% donor 1 type. Thus, in spite of intensified immunosuppression and successful transplantation of a second graft, T cells from the first donor persisted and induced severe acute GVHD.

Successful haematopoietic stem cell transplantation from a matched unrelated donor following three graft failures from HLA-mismatched related donors.

Graft rejection and graft failure are serious complications after allogeneic stem cell transplantation (SCT). We report a patient with CML in first chronic phase who finally engrafted with a transplant from an HLA-identical unrelated donor after graft failures from two related HLA-mismatched sibling donors. After failure of one BM and two PBSC grafts from two 1 HLA-antigen mismatched related donors, the patient was finally successfully transplanted from a subsequently identified HLA-identical unrelated donor (donor 3). At 5 years post transplant, the patient is in complete cytogenetic and molecular remission.

Avascular necrosis of bone following allogeneic stem cell transplantation: MR screening and therapeutic options.

With improvement in long-term survival after allogeneic stem cell transplantation, late complications with significant morbidity are of increasing importance. We retrospectively analysed 272 recipients of an allogeneic stem cell transplant for the development of osteonecrosis. The incidence among allograft recipients was 6.3% (17/272) for the whole patient population, and 11.8% (17/144) for long-term survivors. All patients were treated with high-dose prednisolone, 16 for severe acute or extensive chronic graft-versus-host disease (GVHD) and one patient for graft rejection. The mean age at time of diagnosis was 33 years (range 16-45) and the mean time from transplant to diagnosis of osteonecrosis was 13 months. Osteonecrosis was diagnosed by magnetic resonance (MR) imaging, which allows early detection of osteonecrosis and assessment of stage. At the time of diagnosis, eight patients had stage I, three patients stage II, three patients stage III and three patients stage IV osteonecrosis according to MR criteria. All but one patient had involvement of the femoral head. The median total dosage of prednisolone at the time of diagnosis was 189 mg/kg (single manifestation 150 mg/kg; multiple manifestations 313 mg/kg) with a total range of 13-555 mg/kg. Six patients were treated by conservative means, 77 patients underwent surgery (three core decompression, eight joint replacement). MR screening of patients receiving high-dose steroids might help to detect osteonecrosis at an early stage and thus prevent progression by early intervention, for example, by core decompression.

Treatment of acute graft-versus-host disease with PUVA (psoralen and ultraviolet irradiation): results of a pilot study.

Acute graft-versus-host disease (aGVHD) is a frequent and major complication after allogeneic stem cell transplantation. For many years psoralen and ultraviolet (UV)-A light have been used in the treatment of chronic cutaneous graft-versus-host disease, but few patients have received PUVA therapy for aGVHD. We assessed 20 patients who received PUVA therapy for acute cutaneous GVHD (grade 2-4). Seven patients showed additional organ manifestations (liver, gut). To better quantify the cutaneous lesions, a new scoring system was introduced: intensity of erythema (0-3) x %body surface + size of bullae (4-5) x %body surface affected. All patients received prednisolone and PUVA for treatment of aGVHD. Fifteen patients (75%), 12 with manifestations restricted to the skin, responded by score classification (average time to a 50% score reduction: 39 days) and reduction of the dosage of prednisolone (average time to a 50% prednisolone reduction: 35 days). PUVA treatment was well tolerated and might play a role in the therapy of acute cutaneous GVHD.

[Could the outcome of therapy of deep thrombophlebitis be improved with a combination of surgical thrombectomy and regional fibrinolysis?]. / Können die Behandlungsergebnisse bei tiefer Thrombophlebitis verbessert werden durch Kombination von chirurgischer Thrombektomie und regionaler Fibrinolyse?

A new mode of treatment of extensive acute and subacute deep venous thrombosis of the lower extremities is introduced. For this purpose the beneficial effects of surgical thrombectomy and of thrombolysis with streptokinase are combined during the course of a single surgical intervention. Rapid-flow regional perfusion is the vehicle used for administration of streptokinase and probably represents the third arm of this therapeutic approach by adding a hemodynamic wash-out effect. Because the thrombolytic agent is rinsed out of the circuit at the end of regional perfusion the usual side effects and contra-indications of this drug are avoided. Early and late results of this treatment are assessed clinically and with repeat venograms in a group of six unselected patients. Highly satisfactory results were obtained in four patients with complete anatomical and functional restoration of deep veins along their entire length in three cases. It is felt that continued use of this method is warranted.

[Haptoglobin in the course of surgery with the heart lung machine (author's transl)]. / Haptoglobin im Verlauf von Operationen mit der Herz-Lungen-Maschine

In 20 patients, who underwent open heart surgery, the results of quantitavely and semi-quantitavely determined haptoglobin (hp) were evaluated. A rapid method (Rapi tex-Hp-Test, Behring-Werke) was used. Five minutes after the heart lung machine was cut off hp decreased slightly, and increased about 200% at the 4th day after operation, compared to the preoperative value. These changes were less pronounced in patients with prosthetic heart values. Blood transfusions did not influence the results. There is a significant correlation between the results of hp determined quantitatively and semi-quantitatively.

Expression of CD27 on murine hematopoietic stem and progenitor cells.

Hematopoietic stem cells (HSC) are defined by self-renewal and multilineage differentiation potentials. In order to uncover the genetic program of HSC, we utilized high-density arrays to compare gene expression in highly purified mouse HSC and their mature progeny. One molecule specifically expressed in immature cells is CD27, a member of the TNF receptor family previously shown to play roles in lymphoid proliferation, differentiation, and apoptosis. We show here that the CD27 protein is expressed by about 90% of cells in a purified HSC population. Interestingly, the CD27pos cells are enriched for cells with short-term hematopoietic activities (colony forming potential in vivo and in vitro), while the minority CD27neg population is more effective in clonal long-term transplantation.

Phenotypic distinction and functional characterization of pro-B cells in adult mouse bone marrow.

A lymphoid-committed progenitor population was isolated from mouse bone marrow based on the cell surface phenotype Thy-1.1(neg)Sca-1(pos)c-Kit(low)Lin(neg). These cells were CD43(pos)CD24(pos) on isolation and proliferated in response to the cytokine combination of steel factor, IL-7, and Flt3 ligand. Lymphoid-committed progenitors could be segregated into more primitive and more differentiated subsets based on expression of AA4.1. The more differentiated subset generated only B lymphoid cells in 92% of total colonies assayed, lacked T lineage potential, and expressed Pax5. These studies have therefore defined and isolated a B lymphoid-committed progenitor population at a developmental stage corresponding to the initial expression of CD45R.