Successful outcomes achieved in assisted reproduction cycles using sperm with high levels of high DNA stainability.

The sperm chromatin structure assay (SCSA) has been proposed as a useful addition to the battery of tests routinely used to explore semen quality and hence to give an indication of the likelihood of a successful pregnancy. As usually performed at present, the assay yields two main sperm variables, the DNA fragmentation index (DFI) and the high DNA stainability (HDS). In the present study 275 patients undergoing 215 in vitro fertilization (IVF) and 215 intracytoplasmic sperm injection (ICSI) cycles were studied with the purpose of defining the clinical significance of HDS in IVF and ICSI cycles. Using the Spearman correlation test there were no significant statistical relationships between %HDS and fertilization rate, rate of embryo growth, blastocyst rate, implantation rate, or live birth rate. Rate of pregnancy loss showed a negative relationship significant at the 0.05 level which is unexplained. It is not known whether the normal practice of using processed sperm for fertilization plays any part in this lack of a negative effect of HDS level upon the stages of the cycle. A total of 16 patients with HDS levels >28% had an average live birth rate of 47.8% and an average pregnancy loss of 8.7%, which compared favourably with the group of patients as a whole.

Pro- and antiinflammatory cytokines in threatened miscarriages.

OBJECTIVE: The purpose of this study was to evaluate circulating and intracellular levels of Th1 and Th2 cytokines in women with threatened miscarriage (TM) and subsequent outcome. STUDY DESIGN: Plasma levels of tumor necrosis factor (TNF)-receptors 1 and 2, TNFα, interferon gamma (IFNγ), and interleukins (IL) -6 and -10 were measured by flow cytometric bead assays in 80 women with TM: 53 women with normal outcome and 27 women who miscarried. Fluorescent antibody labeling was also performed on whole blood in a subgroup of 27 women of TM: 16 women with normal outcome and 11 women who miscarried. RESULTS: Monocyte expression of TNFα and circulating levels of TNFα, IFNγ, IL-10, IL-6, and TNF-R1 were significantly lower, whereas circulating levels of TNFα/IL-10, IFNγ/IL-10, and TNFα/IL-6 ratios were significantly higher, in women with TM who subsequently miscarried, compared with the women with normal outcome. CONCLUSION: An increased Th1 type of immune response, which was similar to that observed in preterm delivery, was found in TM cases that were complicated by a subsequent miscarriage.

Relationship between FLT3 mutation status, biologic characteristics, and response to targeted therapy in acute promyelocytic leukemia.

The prognostic significance of FLT3 mutations in acute promyelocytic leukemia (APL) is not firmly established and is of particular interest given the opportunities for targeted therapies using FLT3 inhibitors. We studied 203 patients with PML-RARA-positive APL; 43% of the patients had an FLT3 mutation (65 internal tandem duplications [ITDs], 19 D835/I836, 4 ITD+D835/I836). Both mutations were associated with higher white blood cell (WBC) count at presentation; 75% of the patients with WBC counts of 10 x 10(9)/L or greater had mutant FLT3. FLT3/ITDs were correlated with M3v subtype (P < .001), bcr3 PML breakpoint (P < .001), and expression of reciprocal RARA-PML transcripts (P = .01). Microarray analysis revealed differences in expression profiles among patients with FLT3/ITD, D835/I836, and wild-type FLT3. Patients with mutant FLT3 had a higher rate of induction death (19% vs 9%; P = .04, but no significant difference in relapse risk (28% vs 23%; P = .5) or overall survival (59% vs 67%; P = .2) at 5 years. In in vitro differentiation assays using primary APL blasts (n = 6), the FLT3 inhibitor CEP-701 had a greater effect on cell survival/proliferation in FLT3/ITD+ cells, but this inhibition was reduced in the presence of ATRA. Furthermore, in the presence of CEP-701, ATRA-induced differentiation was reduced in FLT3/ITD+ cells. These data carry implications for the use of FLT3 inhibitors as frontline therapy for APL.

Generation of potent antitumor CTL from patients with multiple myeloma directed against HM1.24.

PURPOSE: The purpose of this work was to test the suitability of the HM1.24 antigen as a CTL target for immunotherapy of patients with multiple myeloma. EXPERIMENTAL DESIGN: Antigen-specific T cells were generated from patients with multiple myeloma using stimulation with protein-pulsed dendritic cells and tested in ELISPOT and CTL assays. RESULTS: HM1.24-primed T cells responded selectively to HM1.24-loaded autologous peripheral blood mononuclear cells (PBMC) in an IFN-gamma ELISPOT assay (median, 342; range, 198-495 IFN-gamma-producing cells/10(5) cf. unloaded PBMC median, 98; range, 7-137; P < 0.05, n = 5) and also to autologous malignant plasma cells (MPC; median, 227; range, 153-335; P < 0.05 when compared with the response to allogeneic MPC median, 57; range, 22-158; n = 5). HM1.24-primed T cells lysed autologous MPC (at 20:1 E/T ratio: median, 48% specific killing; range, 23-88%; at 10:1 E/T ratio: median, 43%; range, 15-80%; n = 12) but not allogeneic MPC. Lysis of autologous MPC was inhibited by anti-MHC class I but not anti-MHC class II antibodies and was blocked by Concanamycin A. Lysis of autologous MPC was blocked by competition with autologous HM1.24-transfected dendritic cells (10:1 ratio with autologous MPC). Unmanipulated, or control plasmid-transfected dendritic cells had no effect on lysis of autologous MPC. CONCLUSION: Our results indicate that HM1.24 is a promising target for immunotherapy of multiple myeloma.

Impaired bone marrow homing of cytokine-activated CD34+ cells in the NOD/SCID model.

The reduced engraftment potential of hematopoietic stem/progenitor cells (HSPCs) after exposure to cytokines may be related to the impaired homing ability of actively cycling cells. We tested this hypothesis by quantifying the short-term homing of human adult CD34+ cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) animals. We show that the loss of engraftment ability of cytokine-activated CD34+ cells is associated with a reduction in homing of colony-forming cells (CFCs) to bone marrow (BM) at 24 hours after transplantation (from median 2.8% [range, 1.9%-6.1%] to 0.3% [0.0%-0.7%]; n = 3; P < .01), coincident with an increase in CFC accumulation in the lungs (P < .01). Impaired BM homing of cytokine-activated cells was not restored by using sorted cells in G0G1 or by inducing cell cycle arrest at the G1/S border. Blocking Fas ligation in vivo did not increase the BM homing of cultured cells. Finally, we tested cytokine combinations or culture conditions previously reported to restore the engraftment of cultured cells but did not find that any of these was able to reverse the changes in homing behavior of cytokine-exposed cells. We suggest that these changes in homing and, as a consequence, engraftment result from the increased migratory capacity of infused activated cells, leading to the loss of selectivity of the homing process.

The "stomatin" gene and protein in overhydrated hereditary stomatocytosis.

In overhydrated hereditary stomatocytosis (OHSt), Coomassie- and silver-stained polyacrylamide gels show an apparently complete deficit of the 32-kDa membrane protein, stomatin. We have used an antistomatin antibody to examine peripheral blood films, bone marrow, splenic tissue, and hepatic tissue from these patients by immunocytochemistry. This technique revealed that, in fact, some red cells did show positive stomatin immunoreactivity; and consistent with this result, Western blot analysis of the red cell membranes confirmed that about one twentieth to one fiftieth of the normal amount of stomatin was in fact present. Flow cytometry, combining immunoreactive quantitation of stomatin expression with thiazole orange staining for reticulocytes, showed that in OHSt, it was the young cells that had more stomatin. Magnetic-activated cell separation studies, using beads to which an anti-transferrin receptor antibody was conjugated, confirmed that in OHSt there was a correspondence between expression of stomatin and the transferrin receptor. Immunocytochemistry and Western blotting revealed that in OHSt patients, the protein was present in spleen, liver, neutrophils, platelets, monocytes, and about 50% of the peripheral lymphocytes, with the same distribution as in healthy controls. Neither Southern blots, nor direct sequencing of multiple subclones of the cDNA, nor sequencing of amplicons from genomic DNA revealed any significant abnormality in stomatin gene sequence in these patients. The deficiency of stomatin from red cells appears to be due to a loss of stomatin from these red cells on maturation in the bone marrow and in the circulation.

The upregulation of CC chemokine receptor 7 and the increased migration of maturing dendritic cells to macrophage inflammatory protein 3beta and secondary lymphoid chemokine is mediated by the p38 stress-activated protein kinase pathway.

Using the p38 stress-activated protein kinase (p38SAPK) inhibitor, SB203580, increased responsiveness of monocyte-derived dendritic cells (MoDCs) to secondary lymphoid chemokine (SLC) and macrophage inflammatory protein 3beta (MIP3beta), following lipopolysaccharide-induced MoDC maturation, was shown to be mediated by the p38SAPK pathway. This was due to the complete abrogation of upregulation of CC chemokine receptor 7, the receptor for MIP3beta/SLC. Once mature, MoDCs utilized both the p38SAPK and phosphoinositide-3 kinase pathways to migrate in response to SLC or MIP3beta. These findings have implications for the mechanism of action of p38SAPK inhibitors, currently in use in clinical trials for patients with autoimmune diseases.

The T-lineage-affiliated CD2 gene lies within an open chromatin environment in acute promyelocytic leukemia cells.

The nature of hemopoietic progenitors subject to leukemic transformation in acute myeloid leukemia (AML) has not been clearly defined. To address this issue, we have used DNase I hypersensitivity assays to study the chromatin structure surrounding the T-lineage-affiliated CD2 gene in the acute promyelocytic subtype of AML (APL). Upstream and downstream flanking regions of CD2 were found to be hypersensitive to DNase I in primary APL blasts, with an identical pattern of hypersensitive sites to those detected in cells of T-lineage. All of the sites were confirmed to be inaccessible to DNase I in B-lineage leukemia cells. The demonstration of T-cell-associated chromatin features in primary APL blasts has implications for the origin of APL that may arise in more primitive progenitors than previously considered to be the case.