Factor XIIIa formation promoted by complexing of alpha-thrombin, fibrin, and plasma factor XIII

Fibrin polymers (des A,B fibrinogen) reduced the concentration of alpha- thrombin required for 50% activation of plasma factor XIII (a2b2 tetramer) by approximately 100-fold. In the presence of fibrin, the amount of gamma-thrombin required for activation was not affected. Catalytically inactive i-Pr2P- and D-Phe-Pro-Arg-CH2-alpha-thrombin were found to inhibit over 95% of the activation by alpha-thrombin in the presence of fibrin. Unlike plasma factor XIII, the concentration of alpha-thrombin required for 50% activation of platelet factor XIII (a2 dimer) was lower, and the activation was not enhanced by fibrin. However, when the a2 platelet factor XIII was incubated with purified b- chains, the alpha- and gamma-thrombin concentrations required for activation increased tenfold and reached levels similar to those required for activation of the plasma factor XIII. When fibrin was present, the alpha-thrombin concentrations needed for activation of the a2b2 complexes were reduced, and the presence of fibrin had no effect on gamma-thrombin cleavage of the a2b2 complexes. Therefore, the b- chains must inhibit a-chain cleavage by alpha-thrombin in the absence of fibrin. These results imply that the formation of a cocomplex involving alpha-thrombin, fibrin, and plasma factor XIII causes some conformational change in plasma factor XIII such that the b-chains no longer inhibit cleavage of the a-chains.     

Role of stromal cells and macrophages in fibronectin biosynthesis and matrix assembly in human long-term marrow cultures

Fibronectin is a major component of the extracellular matrix of adherent layers of human long-term marrow cultures where it may stabilize the extracellular matrix network and provide adhesion sites for primitive hemopoietic cells. This study was devised to analyze the role of adherent cell populations in fibronectin synthesis, matrix assembly, and degradation. In cultures performed under the conditions described by Gartner and Kaplan, immunoprecipitation after metabolic labeling showed that adherent cells synthesized a fibronectin variant comprising the EDa domain and lacking the EDb one. Vascular smooth muscle-like stromal cells were the cell subset responsible for this synthesis. Once synthesized by stromal cells, EDa+fibronectin was secreted into the supernatant and incorporated into the extracellular matrix. The cumulation in the extracellular matrix was predominant by weeks 5 and 6 of culture, when a decrease in the stromal cell intracytoplasmic content of fibronectin was observed. Stromal cells from a transformed cell line, L2Ori-, were also able to synthesize the EDa+fibronectin variant, although for these cells the assembly into the extracellular matrix was partly impaired. Besides stromal cells, other cell types participated in fibronectin synthesis: early-adhering granulomonocytic cells and macrophages appearing later in culture were able to synthesize an EDa-, EDb- fibronectin variant, clearly distinct from the EDa+ variant produced by stromal cells. Studies on cultures in which macrophage growth was stimulated at the expense of stromal cells by adding granulocyte-macrophage colony-stimulating factor (50 ng/mL) to the culture medium showed a striking decrease in amounts of fibronectin measured in the adherent layer. This decrease was caused by a lack of incorporation of fibronectin in the extracellular matrix, disclosing a major difference between stromal cells and macrophages in terms of matrix assembly. This study confirms the similarity between stromal cells and vascular smooth muscle cells, because in vivo subendothelial intimal aortic smooth muscle cells and cultured smooth muscle cells from the aortic media express the EDa+, EDb- fibronectin variant. Furthermore, our results suggest that the level of fibronectin in adherent layers is regulated by stromal cells and macrophages. The balance between these two cell populations may therefore be crucial for the local control of hemopoiesis by regulating the extracellular fibronectin available for the adhesion of hematopoietic cells. Our data indicate that it may be essential to study the adhesion of stem cells to EDa+, EDb- fibronectin instead of EDa-, EDb- soluble fibronectin, as found in human plasma.     

Combinations of recombinant colony-stimulating factors are required for optimal hematopoietic differentiation in serum-deprived culture

Previous in vitro investigations on enriched human hematopoietic progenitors have led to the conclusion that the purified recombinant multipoietins, interleukin 3 (IL-3) and granulocyte-macrophage colony- stimulating factor (GM-CSF) can alone induce the formation of colonies from a variety of multipotent and lineage committed progenitors. Since fetal calf serum was included in these cultures and itself might contain growth factors or other cofactors, we re-examined the actions of the CSFs in serum-deprived conditions. Results show that both the multipoietins are inadequate stimuli of colony formation. At maximal concentrations IL-3 alone induces only 25% of the granulocyte and macrophage colony-forming units (CFU-G and CFU-M) produced by a T-cell conditioned medium that contains a mixture of CSFs. When IL-3 was added at the initiation of the cultures and erythropoietin (ep), G-CSF, or M- CSF added on day 3, almost full recovery of erythroid, granulocytic, and monocytic colonies, respectively, was obtained. Similar results were obtained with GM-CSF except that fewer erythroid colonies were recovered at high concentrations, and almost maximal CFU-M proliferation could be induced. These results show that in serum- deprived conditions, the multipoietins must be combined with lineage specific CSFs for full progenitor expression.     

Defect in B cell function in HTLV III/LAV positive hemophilia patients

The capacity of the peripheral blood lymphocytes (PBL) to generate an antibody response in vitro T cell-dependent antigen ovalbumin was studied in 12 severe hemophilia patients who were otherwise in good health. PBL from four of 12 patients were not capable of generating such a response after stimulation in vitro, whereas all controls were normal. This negative plaque-forming cell (PFC) response coincided with the presence of antibodies directed toward human T-lymphotropic virus III/lymphadenopathy-associated virus (HTLV-III/LAV). Only one patient with antibodies against HTLV-III/LAV had a normal PFC response. The negative PFC response was not due to a deficient T helper cell activity, nor to an excessive T suppressor cell function. However, in the peripheral blood of these four patients, the presence of activated B cells that are refractory to antigen-specific T helper cell signals and secrete specific antibodies spontaneously could be demonstrated. Most of the patients showed a hyperimmunoglobulinemia. No correlation between the T4/T8 ratio and the level of the PFC response was demonstrable. From the data obtained in these investigations we raise the hypothesis that infection with HTLV-III/LAV in hemophilia patients will lead to in vivo (pre)activation of B cells that results in unresponsiveness or decreased response to antigen-specific signals.     

The growth of Rauscher erythroleukemia cells is mediated by autocrine production of a factor with biological activity similar to interleukin- 3

Under serum-deprived and chemically defined culture conditions, the growth of Rauscher erythroleukemia cells is mediated by an autocrine mechanism. The growth-promoting activity is produced by fresh or irradiated cells and resembles the activity of interleukin-3 (IL-3) in its ability to sustain colony formation from three of four IL-3- dependent cell lines and to induce formation of granulocyte/macrophage (GM) colonies and, in the presence of erythropoietin (Ep), of erythroid bursts and mixed erythroid colonies. IL-3, IL-1, IL-4, IL-6, GM colony- stimulating factor (GM-CSF), G-CSF, M-CSF, Ep, and media conditioned by concanavalin A-stimulated mouse spleen cells or phytohemagglutinin- stimulated LBRM 33 cells were unable to induce proliferation of the Rauscher erythroleukemia cells. Northern analysis of total and polyA- selected RNA extracted from untreated Rauscher cells or from cells 24 hours after irradiation showed the presence of message for M-CSF but not for IL-3, IL-1, GM-CSF, or G-CSF. The production of IL-6 was excluded by a sensitive bioassay. These results indicate that the autocrine growth of the Rauscher cell line is mediated by a growth factor different from IL-3, but with similar biological activity. Activation of the expression of such a growth factor during viral infection may contribute to the generation of leukemic cells that have the property to grow in vitro and generate Rauscher erythroleukemia cell lines.     

Human platelets secrete chemotactic activity for eosinophils

Thrombin-stimulated platelets liberate factors that induce chemotaxis of eosinophils and raise their cytosolic Ca2+ content ([Ca2+]i). The sources of this activity are the dense- and alpha-granules because inhibition of prostaglandin endoperoxide/thromboxane A2 formation and the platelet-activating factor receptor-antagonist WEB 2086 have no effect. Platelets from patients with Storage-Pool Deficiency show about 60% of the normal chemotactic activity with little effect on [Ca2+]i, whereas completely degranulated platelets fail to affect eosinophils. In concentrations secreted by the platelets, adenosine diphosphate (ADP), and platelet factor 4 have no effect, whereas adenosine triphosphate (ATP) induces a strong chemotactic response and increases [Ca2+]i. However, apart from ATP other modulating factors must be involved as platelet releasates induce more chemotaxis than ATP alone. Thus, platelets secrete factors that activate eosinophils and may contribute to inflammatory and allergic processes.     

Cognitive outcomes at ages seven and nine years in South African children from the children with HIV early antiretroviral (CHER) trial: a longitudinal investigation

IntroductionMany children living with HIV (CLWH) display impaired cognition. Although early combination antiretroviral therapy (ART) produces improved cognitive outcomes, more long‐term outcome data are needed. After concluding the Children with HIV Early antiRetroviral (CHER) trial in 2011, we investigated cognitive performance, at seven and nine years of age. Participants had been randomized to deferred ART (ART‐Def; n = 22); immediate time‐limited ART for 40 weeks (ART‐40W; n = 30) and immediate time‐limited ART for 96 weeks (ART‐96W; n = 18). We also recruited HIV‐exposed uninfected (CHEU; n = 28) and HIV‐unexposed (CHU; n = 35) children.MethodsData were collected between May 2012 and December 2017. Mixed‐model repeated‐measures ANOVAs assessed differences over time between CLWH (ART‐40W, ART‐96W and ART‐Def) and CHIV‐ CHEU and CHU between ART‐Early (ART‐40W and ART‐96W), ART‐Def, CHEU and CHU; and between ART‐40W, ART‐96W, ART‐Def, CHEU and CHU.ResultsAll comparisons found significant effects of Time for most outcome variables (better scores at nine than at seven years; ps < 0.05). The first ANOVAs found that for (a) motor dexterity, CLWH performed worse than CHIV‐ at seven years (p < 0.001) but improved to equivalence at nine years, (b) visual‐spatial processing and problem solving, only CLWH (p < 0.04) showed significant performance improvement over time and (c) working memory and executive function, CLWH performed worse than CHIV‐ at both seven and nine years (p = 0.03 and 0.04). The second ANOVAs found that for (a) working memory, CHU performed better than ART‐Early and CHEU (p < 0.01 and <0.04), and (b) motor dexterity, ART‐Def performed worse than ART‐Early, CHEU and CHU at seven years (p = 0.02, <0.001 and <0.001 respectively) but improved to equivalence at nine years (ps > 0.17). Similarly, for motor dexterity, ART‐Def performed worse than ART‐96W, CHEU and CHU at seven years (p < 0.04, <0.001 and <0.001) but improved to equivalence at nine years (ps > 0.20).ConclusionsAlthough neurocognitive developmental trajectories for treatment groups and controls were largely similar (i.e. performance improvements from 7 to 9), all ART‐treated children, regardless of treatment arm, remain at risk for cognitive deficits over early school ages. Although the nature of these deficits may change as cognitive development proceeds, there are potential negative consequences for these children’s future learning, reasoning and adaptive functioning.     

Role of matrix metalloproteinase, tissue inhibitor of metalloproteinase and tumor necrosis factor-α single nucleotide gene polymorphisms in inflammatory bowel disease

AIM: To study the (functional) relevance of single nucleotide polymorphisms (SNPs) in genes encoding matrix metalloproteinases (MMP)-1, -2, -3, -9, tissue inhibitors of metalloproteinases (TIMP)-1, -2 and tumor necrosis factor (TNF)-α in the etiopathogenesis of inflammatory bowel diseases (IBD), that may enhance susceptibility and/or disease severity.METHODS: Genomic DNA from 134 Crohn's disease (CD), 111 ulcerative colitis (UC) patients and 248 control subjects was isolated from resected intestinal tissue or blood. Allelic composition at SNP loci was determined by PCR-RFLP or tetra primer ARMS PCR.RESULTS: The TIMP-1 genotype ∏ in women and T in men at SNP +372 T/C was found to increase CD susceptibility (39% vs 23.8%, P = 0.018 and 67.9% vs 51.6%, P = 0.055, respectively), while women with this genotype were less prone to development of fistulae during follow-up (41.4% vs 68.3%, P = 0.025). Male IBD or CD patients carrying the TIMP-1 +372 T-allele expressed lower levels of TIMP-1 in surgically resected macroscopically inflamed tissue (0.065 ＜ P ＜ 0.01). The 5TST genotype at MMP-3 SNP -1613 5T/6T increased the chance of stenotic complications in CD during followup (91.2% vs 71.8%, P = 0.022) but seemed to protect against colonic involvement of this disease at first endoscopic/radiologic examination (35.3% vs 59.5%, P = 0.017).CONCLUSION: Allelic composition at the examined SNPs in genes coding for TIMP-1 and MMP-3 affect CD susceptibility and/or phenotype, i.e., fistulizing disease,stricture pathogenesis and first disease localisation.These findings reinforce the important role of these proteins in IBD.     

Alpha globin gene number: population and restriction endonuclease studies

Restriction endonuclease analysis was used to test a proposed genetic model using alpha-globin gene number to account for the observed distributions of the proportions of hemoglobin (Hb) S in sickle cell trait. In a subsample of specimens collected during a population survey in India, these studies confirmed that the postulated genotype was present in 22 of the 23 individuals examined. In the study population, the number of alpha-globin genes explains about 90% of the variance in the proportion of HbS in sickle cell trait (r2 = 0.895, p less than 10(- 10)).     

Molecular characterization of a novel form of (A gamma delta beta)zero thalassemia deletion in a Chinese family

We have identified and molecularly characterized a novel deletion in the beta-globin gene cluster that is associated with elevated fetal hemoglobin in the adult. The propositus is a homozygote from the Yunnan province of China. The deletion spans about 90 kb of DNA and removes the A gamma, delta, and beta-globin genes. The 5' breakpoint of the deletion is located about 0.13 kb upstream from the A gamma-globin gene, whereas the 3' breakpoint is located about 66 kb downstream from the beta-globin gene, about 13 kb upstream from the breakpoint of the Chinese (A gamma delta beta)zero-thalassemia. Heterozygotes for this Yunnanese form of (A gamma delta beta)zero-thalassemia express between 9% and 17% of fetal hemoglobin, whereas the homozygote present with a mild anemia (Hb = 10.7 g/dl). Comparison of the sites of 3' breakpoints of the Yunnanese and the Chinese (A gamma delta beta)zero-thalassemia mutants is compatible with the hypothesis that an enhancer element is located between the 3' breakpoints of these two mutants. Juxta-position to the G gamma gene of this element may be responsible for the efficient gamma-gene expression in the Yunnanese mutant.