Adenosine diphosphate ribose cyclase: An important regulator of human pathological and physiological processes.

Adenosine diphosphate ribose cyclase (ADPRC) exists widely in eukaryotes and lower metazoans cells. It can degrade nicotinamide adenine dinucleotide (NAD) into cyclic ADP ribose (cADPR) and nicotinamide, and subsequently hydrolyses cADPR to ADP ribose (ADPR). In this paper, we have summarized the relative subcellular localization of ADPRC and enzymes with ADPRC activity in organisms, related enzyme family members of ADPRC are also described. In addition, we discussed the main biological functions of ADPRC, the regulation of Ca2+ signal, the regulation of insulin and glucagon secretion, oxytocin secretion, and the effects of renal and pulmonary vasomotor tension. Finally, we expounded the relationship between ADPRC and human health and disease occurrence. It provides a theoretical basis for the targeted treatment of ADPRC as a pharmacological tool for related diseases, and has important significance in clinical diagnosis and disease intervention.

Diagnosis of paroxysmal nocturnal hemoglobinuria with flowcytometry panels including CD157: Data from the real world.

BACKGROUND: Several studies have used CD157 in white blood cells with or without proaerolysin (fluorescein-labeled proaerolysin [FLAER])-based flow cytometry assays in the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). METHODS: We designed a seven-color CD marker panel comprising FLAER, CD15, CD64, CD24, CD14, CD157, and CD45 to verify CD157's clinical applicability and diagnostic performance in a clinical setting. RESULTS: A total of 356 samples were tested. These included 43 PNH-positive samples and 313 PNH-negative samples. PNH clones confirmed by the CD157/FLAER combination were almost identical in size to the clones detected by the CD24/CD14/FLAER combination, and the accuracy of the CD157/FLAER combination was 100% in granulocytes and 99.7% in monocytes. Substitution of FLAER with CD157 resulted in 1.9% and 3.5% false-positives in granulocytes and monocytes, respectively. The accuracy was 98.3% and 96.9% in granulocytes and monocytes, respectively. Moreover, the loss of CD157 expression in granulocytes and monocytes was commonly observed in non-PNH patients. Some monocytes in non-PNH patients had weak expression of CD14 but normal expression of FLAER. In this study, PNH clones in granulocytes were always lower than those in matched monocytes. CONCLUSIONS: We performed the first prospective exploration of the clinical usefulness of FLAER and CD157 in simultaneously recognizing PNH clones in granulocytes and monocytes and verified the applicability of CD157 in substitute for both CD14 and CD24. In the conditions where FLAER is not available, substitution of FLAER with CD157 is acceptable for the identification of PNH clones under the premise of giving full attention to the potential for false-positives.

Vitamin A Deficiency Induces Autistic-Like Behaviors in Rats by Regulating the RARß-CD38-Oxytocin Axis in the Hypothalamus.

SCOPE: Vitamin A (VA) is an essential nutrient for the development of the brain. We previously found that children with autism spectrum disorder (ASD) have a significant rate of VA deficiency (VAD). In the current study, we aim to determine whether VAD is a risk factor for the generation of autistic-like behaviors via the transcription factor retinoic acid receptor beta (RARß)-regulated cluster of differentiation 38 (CD38)-oxytocin (OXT) axis. METHODS AND RESULTS: Gestational VAD or VA supplementation (VAS) rat models are established, and the autistic-like behaviors in the offspring rats are investigated. The different expression levels of RARß and CD38 in hypothalamic tissue and serum retinol and OXT concentration are tested. Primary cultured rat hypothalamic neurons are treated with all-trans retinoic acid (atRA), and recombinant adenoviruses carrying the rat RARß (AdRARß) or RNA interference virus RARß-siRNA (siRARß) are used to infect neurons to change RARß signal. Western blotting, chromatin immunoprecipitation (ChIP), and intracellular Ca2+ detections are used to investigate the primary regulatory mechanism of RARß in the CD38-OXT signaling pathway. We found that gestational VAD increases autistic-like behaviors and decreases the expression levels of hypothalamic RARß and CD38 and serum OXT levels in the offspring. VAS ameliorates these autistic-like behaviors and increases the expression levels of RARß, CD38, and OXT in the gestational VAD pups. In vitro, atRA increases the Ca2+ excitability of neurons, which might further promote the release of OXT. Different CD38 levels are induced in the neurons by infection with different RARß adenoviruses. Furthermore, atRA enhances the binding of RARß to the proximal promoter of CD38, indicating a potential upregulation of CD38 transcriptional activity by RARß. CONCLUSIONS: Gestational VAD might be a risk factor for autistic-like behaviors due to the RARß signal suppression of CD38 expression in the hypothalamus of the offspring, which improves with VAS during the early-life period. The nutritional status during pregnancy and the early-life period is important in rats.

Expansion of human SCID-repopulating cells under hypoxic conditions.

It has been proposed that bone marrow (BM) hematopoietic stem and progenitor cells are distributed along an oxygen (O2) gradient, where stem cells reside in the most hypoxic areas and proliferating progenitors are found in O2-rich areas. However, the effects of hypoxia on human hematopoietic stem cells (HSCs) have not been characterized. Our objective was to evaluate the functional and molecular responses of human BM progenitors and stem cells to hypoxic conditions. BM lineage-negative (Lin-) CD34+CD38- cells were cultured in serum-free medium under 1.5% O2 (hypoxia) or 20% O2 (normoxia) for 4 days. Using limiting dilution analysis, we demonstrate that the absolute number of SCID-repopulating cells (SRCs) increased by 5.8-fold in hypoxic cultures compared with normoxia, and by 4.2-fold compared with freshly isolated Lin-CD34+CD38- cells. The observed increase in BM-repopulating activity was associated with a preferential expansion of Lin-CD34+CD38- cells. We also demonstrate that, in response to hypoxia, hypoxia-inducible factor-1alpha protein was stabilized, surface expression of angiogenic receptors was upregulated, and VEGF secretion increased in BM Lin-CD34+ cultures. The use of low O2 levels to enhance the survival and/or self-renewal of human BM HSCs in vitro represents an important advance and could have valuable clinical implications.

ZAP-70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic leukemia.

BACKGROUND: The mutational status of immunoglobulin heavy-chain variable-region (IgVH) genes in the leukemic cells of chronic lymphocytic leukemia (CLL) is an important prognostic factor in the disease. We investigated whether the expression of ZAP-70 by CLL cells correlated with the IgVH mutational status, disease progression, and survival. METHODS: The expression of ZAP-70 was analyzed in T-cell and B-cell lines and in peripheral-blood samples from 56 patients with CLL with the use of flow cytometry, Western blotting, and immunohistochemistry. The results were correlated with the IgVH mutational status and clinical outcome. RESULTS: ZAP-70 was detected by flow-cytometric analysis in cells of T-cell lineage and in leukemic cells from 32 of 56 patients with CLL. In all patients in whom at least 20 percent of the leukemic cells were positive for ZAP-70, IgVH was unmutated, whereas IgVH mutations were found in 21 of 24 patients in whom less than 20 percent of the leukemic cells were positive for ZAP-70 (P<0.001). Concordant results were obtained when ZAP-70 expression was assessed by immunohistochemistry or Western blotting. The level of ZAP-70 expression did not change over time (median, 37 months) in sequential samples from 30 patients with CLL. Patients with Binet stage A CLL who had at least 20 percent ZAP-70-positive leukemic cells had more rapid progression and poorer survival than those with less than 20 percent ZAP-70-positive cells. CONCLUSIONS: Among patients with CLL, expression of ZAP-70, as detected by flow-cytometric analysis, correlated with IgVH mutational status, disease progression, and survival.

Evi3, a zinc-finger protein related to EBFAZ, regulates EBF activity in B-cell leukemia.

Retroviral insertions that activate proto-oncogenes are a primary cause of tumors in certain strains of mice. The AKXD recombinant inbred mice are predisposed to a variety of leukemias and lymphomas as a result of viral integration. One common insertion site, the ecotropic viral insertion site 3 (Evi3), has been implicated in most B-cell tumors in the AKXD-27 strain. The Evi3 gene encodes a zinc-finger protein with sequence similarity to the Early B-cell Factor-Associated Zinc-finger gene (EBFAZ). We show that the Evi3 gene is overexpressed in several tumors with viral insertions at Evi3, which results in the upregulation of Early B-cell Factor (EBF)-target gene expression, suggesting that Evi3 modulates EBF activity. Reconstitution of primary leukemia cells showed that these tumors express high densities of the B-cell surface proteins CD19 and CD38, which are EBF targets. Using a transactivation assay, we show that the terminal six zinc-fingers of Evi3 are required for modification of EBF activity. This is the first evidence that Evi3 expression in tumors alters the level of EBF target genes, and the first characterization of the Evi3 protein domains required for modulation of EBF activity. Further, these data imply that Evi3 misexpression initiates tumorigenesis by perturbing B-cell development via an interaction with EBF.

Mesenchymal lineage precursor cells induce vascular network formation in ischemic myocardium.

Mesenchymal lineage precursors can be reproducibly isolated from adult mammalian bone marrow and grown in culture. Immunoselection with monoclonal antibodies against STRO-1 and vascular-cell-adhesion molecule 1 (VCAM1/CD106) prior to expansion results in a 1,000-fold enrichment of mesenchymal precursors compared to standard isolation techniques. Intramyocardial injection of human STRO-1-selected precursors in an athymic rat model of acute myocardial infarction results in induction of vascular network formation and arteriogenesis coupled with global functional cardiac recovery.

Combined analysis of ZAP-70 and CD38 expression as a predictor of disease progression in B-cell chronic lymphocytic leukemia.

Prognostic predictions in B-cell chronic lymphocytic leukemia (B-CLL) at early clinical stage are based on biological disease parameters, such as ZAP-70 and CD38 protein levels, genomic aberrations as well as immunoglobulin variable heavy chain gene (IgV(H)) mutation status. In the current study, ZAP-70 and CD38 expressions were examined by flow cytometry in 252 patients with B-CLL. Cytoplasmic ZAP-70 expression in more than 20% (ZAP-70(+)) and surface CD38 expression on more than 30% (CD38(+)) of B-CLL cells were associated with an unfavorable clinical course. The levels of ZAP-70 and CD38 did not change over time in the majority of patients where sequential samples were available for analysis. Combined analysis of ZAP-70 and CD38 yielded discordant results in 73 patients (29.0%), whereas 120 patients (47.6%) were concordantly negative and 59 patients (23.4%) were concordantly positive for ZAP-70 and CD38 expression. Median treatment-free survival times in patients whose leukemic cells were ZAP-70(+)CD38(+) was 30 months as compared to 130 months in patients with a ZAP-70(-)CD38(-) status. In patients with discordant ZAP-70/CD38 results, the median treatment-free survival time was 43 months. Thus, ZAP-70 and CD38 expression analyses provided complementary prognostic information identifying three patient subgroups with good, intermediate and poor prognosis. Over-representation of high-risk genomic aberrations such as 17p deletion or 11q deletion and distribution of the IgV(H) mutation status in B-CLL discordant for ZAP-70/CD38 pointed toward a distinct biologic background of the observed disease subgroups. This finding was also supported by microarray-based gene expression profiling in a subset of 35 patients. The expression of 37 genes differed significantly between the three groups defined by their expression of ZAP-70 and CD38, including genes that are involved in regulation of cell survival and chemotherapy resistance.

High stem cell frequency in acute myeloid leukemia at diagnosis predicts high minimal residual disease and poor survival.

PURPOSE: In CD34-positive acute myeloid leukemia (AML), the leukemia-initiating event originates from the CD34(+)CD38(-) stem cell compartment. Survival of these cells after chemotherapy may lead to minimal residual disease (MRD) and subsequently to relapse. Therefore, the prognostic impact of stem cell frequency in CD34-positive AML was investigated. EXPERIMENTAL DESIGN: First, the leukemogenic potential of unpurified CD34(+)CD38(-) cells, present among other cells, was investigated in vivo using nonobese diabetic/severe combined immunodeficient mice transplantation experiments. Second, we analyzed whether the CD34(+)CD38(-) compartment at diagnosis correlates with MRD frequency after chemotherapy and clinical outcome in 92 AML patients. RESULTS: In vivo data showed that engraftment of AML blasts in nonobese diabetic/severe combined immunodeficient mice directly correlated with stem cell frequency of the graft. In patients, a high percentage of CD34(+)CD38(-) stem cells at diagnosis significantly correlated with a high MRD frequency, especially after the third course of chemotherapy. Also, it directly correlated with poor survival. In contrast, total CD34(+) percentage showed no such correlations. CONCLUSIONS: Both in vivo data, as well as the correlation studies, show that AML stem cell frequency at diagnosis offers a new prognostic factor. From our data, it is tempting to hypothesize that a large CD34(+)CD38(-) population at diagnosis reflects a higher percentage of chemotherapy-resistant cells that will lead to the outgrowth of MRD, thereby affecting clinical outcome. Ultimately, future therapies should be directed toward malignant stem cells.

Role of the second-messenger cyclic-adenosine 5'-diphosphate-ribose on adrenocorticotropin secretion from pituitary cells.

We examined the role of the second-messenger cyclic-ADP-ribose (cADPR) on the regulation of ACTH secretion using AtT20 corticotroph tumor cell line. We found that the cADPR antagonist, 8-Br-cADPR, substantially diminished the secretion of ACTH induced by CRH and potassium in these cells, whereas xestospongin C, an inositol 1,4,5-triphosphate receptor antagonist, had no effect. In addition, the cADPR agonist, 3-deaza-cADPR, augmented ACTH secretion. The presence of the components of the cADPR system, namely ryanodine receptor, CD38, and cADPR itself, was determined in AtT20 cells. Furthermore, we observed that antagonists of the ryanodine channel and cADPR system can decrease the potassium-induced Ca2+ transients in these cells. These results suggest that cADPR is a second messenger in pituitary cells and regulates ACTH secretion by a mechanism dependent on activation of the ryanodine channel by extracellular Ca2+.

The maturation of myeloma cells correlates with sensitivity to chemotherapeutic agents.

We analyzed both morphologic and phenotypic findings of myeloma cells before and after chemotherapy in 21 patients with multiple myeloma. The morphologic analysis was based on the Greipp classification, and phenotypic analysis was performed by 3-color flow cytometry using the CD38 plasma gating method (Marrow plasma 38). Results with flow cytometry using a combination of MPC1, CD49e, and CD45 supported the morphologic findings for the myeloma cells. Treatment with 3 or 4 cycles of VAD (vincristine, doxorubicin, and dexamethasone) therapy was effective in reducing the total numbers of myeloma cells, but the proportion of immature myeloma cells increased after this treatment. However, the immature myeloma cells were reduced by high-dose melphalan (HD-Mel) therapy followed by autologous stem cell transplantation (ASCT). High-dose cyclophosphamide treatment for stem cell harvesting did not show an effect on the residual immature myeloma cells after VAD treatment. In addition, thalidomide was not effective in reducing the numbers of immature myeloma cells. These results suggest that VAD (3 or 4 cycles) therapy plus HD-Mel followed by ASCT is a reasonable treatment for multiple myeloma and that Marrow plasma 38 analysis is a useful method for monitoring the response of multiple myeloma to chemotherapy.

Karyotyping, immunophenotyping, and apoptosis analyses on human hematopoietic precursor cells derived from umbilical cord blood following long-term ex vivo expansion.

By means of flow cytometry, CD34+/CD38- hematopoietic stem cells (HSC) were collected from umbilical cord blood (UCB) of 10 healthy women at the time of delivery and cultivated in stem-cell culture media supplemented with cell growth stimulating factors (IL-3, IL-6, GM-CSF, EPO, IGF-1, and SCF) for long periods. Apoptotic status, cell surface marker expression, and karyotypes of the cultured UCB-derived CD34+/CD38- stem-cells were investigated by flow cytometry and GTG-banding methods. The UCB-derived CD34+/CD38- stem-cells were able to divide and proliferate in vitro for at least 6 months. They did not show significantly increased apoptosis following ex vivo expansion for 20 and 32 days, respectively, in 2 cases and retained the same cell surface marker expression pattern (i.e., CD34+ and CD38-) in the majority of the cells of 2 cases following 20 and 37 days of incubation, respectively. In another 2 cases, chromosome analysis showed no evidence of numerical and structural abnormalities in the CD34+/CD38- stem-cells obtained after 20 and 43 days in culture, respectively. Our findings indicated that UCB-derived CD34+/CD38- stemcells are able to maintain their basic biologic and genetic characteristics after dividing and proliferating in vitro for a long period of time. UCB-derived HSC following ex vivo expansion can serve as a reliable resource for hematopoietic precursor cells transplantation.

Plasmacellular differentiation in extranodal marginal zone B cell lymphomas of the ocular adnexa: an analysis of the neoplastic plasma cell phenotype and its prognostic significance in 136 cases.

AIM: To determine (a) the expression of plasma cell related antigens in extranodal marginal zone B cell lymphomas (EMZL) of the ocular adnexa; and (b) the prognostic value of plasmacellular differentiation in these tumours. METHODS: A consecutive case series of 136 ocular adnexal EMZL obtained from three ocular pathology centres over 20 years was analysed retrospectively. An extensive immunohistochemical panel, including the plasma cell related antigens VS38c, CD38, CD138, multiple myeloma oncogene-1-protein (MUM1/IRF4), and CREB binding protein (CBP) was performed. EMZL were defined as "plasmacellular differentiated" on the basis of morphological features, evidence of cytoplasmic immunoglobulin, negativity for BSAP/PAX5, and expression of at least one of the investigated plasma cell related antigens. Controls included normal or hyperplastic lymphatic tissues. Detailed clinical data were collected for most patients, and compared with the results of immunohistochemistry. The end points considered for statistical analysis were development of local tumour recurrence, development of systemic disease, and lymphoma related death. RESULTS: 57 (42%) of the 136 ocular adnexal EMZL showed a plasmacellular differentiation; 45 of these plasmacytoid cases were primary tumours. In contrast with most admixed normal plasma cells, which displayed co-expression of MUM1/IRF4, Vs38c, CD38, CD138, and CBP, the plasmacellular differentiated EMZL tumour cells demonstrated co-expression of all five plasma cell related antigens in only six of 57 (11%) plasmacellular differentiated ocular adnexal EMZL. The most commonly expressed plasma cell related antigen was MUM1/IRF4, immunoreactivity being seen in 56/57 (98%) plasmacellular differentiated EMZL examined. Although the association of plasmacellular differentiation in primary ocular adnexal EMZL and disseminated disease was statistically significant on univariate analysis (p = 0.042), this was weaker on multivariate analysis. CONCLUSION: Plasmacellular differentiated tumour cells in EMZL demonstrate an aberrant immune profile for plasma cell related antigens when compared with normal plasma cells. On multivariate analysis, plasmacellular differentiation in ocular adnexal EMZL was not significantly associated with local recurrence, the development of systemic disease, or with lymphoma related death.

Preferential expression of the vasoactive intestinal peptide (VIP) receptor VPAC1 in human cord blood-derived CD34+CD38- cells: possible role of VIP as a growth-promoting factor for hematopoietic stem/progenitor cells.

Primitive hematopoietic progenitor cells such as severe combined immunodeficiency- repopulating cells and long-term culture-initiating cells are enriched in CD34+CD38- cells derived from various stem cell sources. In this study, to elucidate the features of such primitive cells at the molecular level, we tried to isolate genes that were preferentially expressed in umbilical cord blood (CB)-derived CD34+CD38- cells by subtractive hybridization. The gene for VPAC1 receptor, a receptor for the neuropeptide vasoactive intestinal peptide (VIP), was thereby isolated and it was shown that this gene was expressed in both CD34+CD38- and CD34+CD38+ CB cells and that the expression levels were higher in CD34+CD38- CB cells. Next, we assessed the effects of VIP on the proliferation of CD34+ CB cells using in vitro culture systems. In serum-free single-cell suspension culture, VIP enhanced clonal growth of CD34+ CB cells in synergy with FLT3 ligand (FL), stem cell factor (SCF), and thrombopoietin (TPO). In serum-free clonogenic assays, VIP promoted myeloid (colony-forming unit-granulocyte/macrophage (CFU-GM)) and mixed (CFU-Mix) colony formations. Furthermore, in Dexter-type long-term cultures, VIP increased colony-forming cells at week 5 of culture. These results suggest that VIP functions as a growth-promoting factor of CB-derived hematopoetic progenitor cells.

Membrane-bound delta-4 notch ligand reduces the proliferative activity of primitive human hematopoietic CD34+CD38low cells while maintaining their LTC-IC potential.

To examine the role of the Notch ligand Delta-4 on hematopoietic stem cells, human CD34+CD38low cord blood cells were cocultured on S17 cells transduced with transmembrane Delta-4 (mbD4/S17) or an empty vector (C/S17). By the end of a 3-week culture, mbD4/S17 induced a 25-fold reduction in nucleated cell production, as compared to C/S17, by maintaining a higher proportion of cells in G0/G1 phase. A specific retention of a high proportion of CD34+ cells throughout the culture was observed with mbD4/S17, contrary to C/S17. Although mbD4/S17 promoted expansion of cells with the phenotype of committed lymphoid precursors (CD34+CD7+CD45RA+), these cells still retained their myeloid differentiation potential. mbD4/S17 maintained a higher LTC-IC frequency in output CD34+ cells, compared to C/S17, as in the subsets of cells having completed the same number of divisions on mbD4/S17. A Delta4-Fc protein (extracellular part of human Delta4 fused to Fc human IgG1 portion), immobilized on plastic, also reduced cell production and retained the LTC-IC potential. Transplantation of cells grown on mbD4/S17 into NOD/SCID mice showed no significant enhancement of the long-term repopulating ability. Thus, Delta4 appears to inhibit hematopoietic stem cell proliferation, in association with the maintenance of short-term lymphoid and myeloid repopulation capacity.

The symmetry of initial divisions of human hematopoietic progenitors is altered only by the cellular microenvironment.

OBJECTIVE: We examined if cellular elements or adhesive ligands were able to alter asymmetric divisions of CD34(+)/CD38(-) cells in contrast to soluble factors at a single cell level. MATERIALS AND METHODS: After single cell deposition onto 96-well plates, cells were cocultured for 10 days with the stem cell supporting cell line AFT024, fibronectin (FN), or bovine serum albumin (BSA). The divisional history was monitored with time-lapse microscopy. Subsequent function for the most primitive cells was assessed using the myeloid-lymphoid-initiating cell (ML-IC) assay. Committed progenitors were measured using colony-forming cells (CFC). RESULTS: Only contact with AFT024 recruited significant numbers of CD34(+)/CD38(-) cells into cell cycle and increased asymmetric divisions. Although most ML-IC were still identified among cells that have divided fewer than 3 times, a significant number of ML-IC shifted into the fast-dividing fraction after exposure to AFT024. The increase in ML-IC frequency was predominantly due to recruitment of quiescent and slow-dividing cells from the starting population. Increase in CFC activity induced by AFT024 was found only among rapidly dividing cells. CONCLUSIONS: For the first time, we have demonstrated that asymmetric divisions can be altered upon exposure with a stem cell-supporting microenvironment. For the primitive subset of cells (ML-IC), this was predominantly due to recruitment into cell cycle and increased rounds of cycling without loss of function. Exposure to AFT024 cells also increased proliferation and asymmetric divisions of committed CFC. Hence direct communication between hematopoietic progenitors with stroma cells is required for maintaining self-renewal potential.

Human CD34(+) and CD34(+)CD38(-) hematopoietic progenitors in sickle cell disease differ phenotypically and functionally from normal and suggest distinct subpopulations that generate F cells.

OBJECTIVE: Sickle cell disease (SCD) is remarkable for stress erythropoiesis. We investigated the progenitor populations contributing to erythroid stress. MATERIALS AND METHODS: We characterized hematopoietic progenitor cells in sickle bone marrow and sickle peripheral blood from patients with SCD compared to those in normal bone marrow. RESULTS: There were increased proportions of sickle bone marrow and sickle peripheral blood CD34(+) cells that coexpressed glycophorin A (GlyA), normally expressed late during erythroid differentiation when CD34 is down-regulated. Remarkably, increased numbers of CD34(+)CD38(-) hematopoietic progenitor cells from sickle bone marrow (p < 0.03) and sickle peripheral blood (p < 0.004) coexpressed GlyA, compared to normal bone marrow CD34(+)CD38(-) hematopoietic progenitor cells. At a molecular level, even the sickle bone marrow and sickle peripheral blood CD34(+)CD38(-) hematopoietic progenitor cells not expressing GlyA by fluorescence-activated cell sorting or reverse transcriptase-polymerase chain reaction expressed the erythroid-specific gene GATA-1, unlike normal bone marrow, suggesting desynchronized erythroid gene expression in the SCD hematopoietic progenitor cells. We also generated red blood cells in vitro from GlyA(+) and GlyA(-)CD34(+) cells. GlyA(+)CD34(+) produced more F cells (p < 0.02) and had lower clonogenicity (p < 0.01) and erythroid expansion potential. Increased F cells were generated only from sickle CD34(+) hematopoietic progenitor cells (p < 0.04), as occurs in vivo. CONCLUSION: Stress erythropoiesis in SCD has been postulated to accelerate erythropoiesis and production of F cells. Thus, CD34(+)CD38(-) expressing GlyA may represent the "stress progenitor" population. This is the first study characterizing CD34(+) and CD34(+)CD38(-) hematopoietic progenitor cells in sickle bone marrow, comparing them to sickle peripheral blood and normal bone marrow and using them to generate sickle red blood cells that recapitulate F cell production observed in vivo. We identified a unique population of GlyA(+)CD34(+) cells in SCD, which is in an accelerated erythroid differentiation pathway, has not down-regulated CD34 antigen expression, and predominantly generates F cells.

Immunological and virological study of enfuvirtide-treated HIV-positive patients.

OBJECTIVE: To evaluate the predictive value and evolution of immunological and virological parameters related to HIV entry and pathogenesis in patients receiving enfuvirtide (ENF) plus an optimized regimen. METHODS: A phase III clinical trial substudy of ENF in 22 patients measured virus coreceptor use and sensitivity to ENF, levels of chemokines, cytokines and chemokine receptors, CD38 and HLA-DR expression as markers of T cell activation and ex vivo cell death at baseline and at week 32. RESULTS: Treatment including ENF reduced HIV viral load (P < 0.001) and increased the CD4 cell count in patients that responded (RP) to treatment (n = 14). Significant (P < 0.05) increases were noted in the RP group in CXCR4 and CCR5 expression in CD4 cells without major differences in chemokine and interleukin-7 levels. A decrease in CD38 expression in the absence of HLA-DR changes was observed in CD4 cells. Apoptosis of peripheral blood mononuclear cells was significantly reduced in the RP group. Coreceptor use or ENF sensitivity of virus isolated at baseline was not associated with virus resistance or response to treatment, which appeared to be related to the activation state (HLA-DR expression) of CD4 cells at baseline. CONCLUSION: The outcome of ENF-containing treatment could not be associated with HIV coreceptor use at baseline. CD4 cell activation and viral drug resistance were the only markers of treatment response. Changes induced by ENF-containing regimen were seen in HIV coreceptor expression, including an increase in CCR5+CD4+ cells, a decrease in CD38 T cells and a concomitant reduction of T cell apoptosis.

The role of cyclic-ADP-ribose-signaling pathway in oxytocin-induced Ca2+ transients in human myometrium cells.

Human myometrial contraction plays a fundamental role in labor. Dysfunction of uterine contraction is an important cause of labor progression failure. Although the mechanisms controlling uterine contraction are not completely understood, intracellular Ca2+ mobilization plays an important role during uterine contraction. Several mechanisms of intracellular Ca2+ mobilization are present in smooth muscle, but in the human uterus, only 1,4,5-trisphosphate-induced Ca2+ release has been studied extensively. Ryanodine receptor channels are present in myometrium. We determined the role of the cyclic ADP-ribose (cADPR)-signaling pathway in oxytocin-induced intracellular Ca2+ [(Ca2+)i] transients in human myometrial cells. We found that oxytocin-induced Ca2+ transient is dependent on several sources of Ca2+, including extracellular Ca2+ and intracellular Ca2+ stores. In addition, we found that both the 1,4,5-trisphosphate- and the cADPR-induced Ca2+ releasing systems are important for the induction of [Ca2+]i transients by oxytocin in human myometrial cells. Furthermore, we investigated TNFalpha regulation of oxytocin-induced [Ca2+]i transients, CD38 cyclase activity, and CD38 expression in human myometrial cells. We found that oxytocin-induced [Ca2+]i transients were significantly increased by 50 ng/ml TNF. Similarly, CD38 mRNA levels, CD38 expression, and cyclase activity were increased by TNFalpha, thus increasing cADPR levels. We propose that a complex interaction between multiple signaling pathways is important for the development of intracellular Ca2+ transients induced by oxytocin and that TNFalpha may contribute for the myometrium preparation for labor by regulating the cADPR-signaling pathway. The observation that the cADPR-signaling pathway is important for the development of intracellular Ca2+ transients in human myometrial cells raises the possibility that this signaling pathway could serve as a target for the development of new therapeutic strategies for abnormal myometrial contraction observed during pregnancy.

CM1 ligation initiates apoptosis in a caspase 8-dependent manner in Ramos cells and in a mitochondria-controlled manner in Raji cells.

Burkitt lymphoma (BL) is a tumor with the characteristics of germinal center B cells. We previously reported that the CM1 (centrocyte/-blast marker 1) molecule is expressed only in germinal center B cells, specifically, in a subpopulation of centroblasts and centrocytes. In the present study, we investigated the apoptosis induced by anti-CM1 in the Ramos and Raji human BL cell lines. The Ramos is protected from apoptosis by the crosslinking of sIgM and the calcium ionophore by the ligation of CD40 with anti-CD40 monoclonal antibodies (mAb) or soluble CD40 ligand (sCD40L). In this investigation on the effect of CM1 on apoptosis in BL cell lines, we found that cellular signaling by CM1 induces apoptosis and decreases cell viability, in BL cell lines cultured for 24 hours with protein-G agarose beads conjugated anti-CM1 mAb. Stimulation by CD40 ligated with sCD40L protected Raji cells from CM1-induced apoptosis, but did not protect Ramos cells. Furthermore, after anti-CM1 mAb stimulation, CD95 expression was upregulated and CD40 expression was unaltered or slightly decreased in Ramos cells, whereas CD95 was downregulated and CD40 was slightly upregulated in Raji cells. The engagement of CD40 by sCD40L enhanced CD95 expression, but the level of CM1 expression was unchanged in Ramos. However, sCD40L downregulated both CD95 and CM1 expression in Raji. In addition, the caspase-8 specific inhibitor blocked CM1-induced apoptosis in Ramos cells, but not in Raji cells. Increased mitochondrial membrane permeabilization was observed only in Raji cells. Moreover, the effector caspase inhibitor, z-DEVD, blocked CM1-mediated apoptosis in both cell lines. We found that CM1-induced apoptosis is achieved via different initiation pathways, which are cell-type dependent.