Glycoprotein IIb-IIIa and RGD(S) are not important for fibronectin- dependent platelet adhesion under flow conditions

Previous studies have indicated that activated blood platelets interact with fibronectin through binding of fibronectin to the glycoprotein IIb- IIIa complex (GPIIb-IIIa). The cell attachment site of fibronectin with its crucial arg-gly-asp(-ser) [RGD(S)]sequence is involved in these bindings. We studied the importance of these interactions for the fibronectin dependence of platelet adhesion under flow conditions. An RGDS-containing hexapeptide (GRGDSP) was compared with a nonreactive control peptide (GRGESP). The GRGDSP-peptide inhibited thrombin-induced aggregation and adhesion under static conditions at 0.1 mmol/L. This concentration had no effect on platelet adhesion to nonfibrillar collagen type I in flow. GRGDSP at 1 mmol/L had a significant inhibitory effect at 1,500 s-1, but not at the lower shear rates of 800 and 300 s-1 where platelet adhesion is also fibronectin dependent. On the matrix of cultured human umbilical vein endothelial cells, 1 mmol/L GRGDSP had no effect on platelet adhesion. The relation between GPIIb- IIIa and fibronectin dependence was investigated with platelets of a patient with Glanzmann's thrombasthenia and monoclonal antibodies to GPIIb-IIIa using endothelial cell matrix (ECM) as a surface. Platelets of normal controls or a patient with Glanzmann's thrombasthenia showed a similar inhibition of adhesion in the presence of fibronectin-free plasma after the ECMs had been preincubated with antifibronectin F(ab')2 fragments. Incubation of platelets with anti-GPIIb-IIIa showed inhibition of platelet adhesion at high shear rates. Dependence on fibronectin for platelet adhesion was still observed even though separate experiments had shown that these anti-GPIIb-IIIa antibodies could block binding of radiolabeled fibronectin to thrombin-activated platelets. These data suggest the existence of another binding system for the interaction of platelets with fibronectin that may only appear when fibronectin is present on a surface.     

High-dose intravenous immunoglobulin modifies complement-mediated in vivo clearance

The mechanism of effect of high-dose intravenous immunoglobulin (IVIG) therapy in immune cytopenias is incompletely known. One of the leading theories ascribes the short-term effects of IVIG to the competition of infused IVIG for Fc receptors, thereby inhibiting IgG-mediated clearance. Using a system independent of IgG-Fc receptor interactions, we examined another potential mechanism of IVIG action. Guinea pigs were infused with a human IVIG preparation at 600 mg/kg/day for two consecutive days. Parallel groups of animals were treated with the same volume and/or concentration of saline and albumin. Clearance of IgM- sensitized guinea pig erythrocytes, which is wholly complement dependent, was significantly retarded in animals treated with high-dose IVIG. The effect was specific for IVIG, since human albumin (as a second foreign protein) failed to change the clearance of IgM- sensitized guinea pig erythrocytes. Experiments in which IVIG-treated animals were subjected to pre- and posttreatment clearance studies revealed heterogeneity among individual animals in respect to their response to IVIG infusions. Decrease of available plasma complement components did not account for the effect, since both C3 and CH50 values remained unchanged after IVIG treatment, despite rising levels of IVIG in sera of treated animals. The results of in vitro C3 uptake studies and the effect of IVIG on clearance of preopsonized cells suggest that IVIG produces a kinetic depression of C3 uptake and modifies the process of complement fragment deposition on erythrocytes. A generalized effect on mononuclear phagocytes is less likely but cannot be wholly ruled out. These studies establish another potential mechanism of IVIG action and suggest extension of its use to other complement-mediated diseases.     

Hemolytically inactive C5b67 complex: an agonist of polymorphonuclear leukocytes

The activity of hemolytically inactive C5b67, designated iC5b67, was evaluated as an agonist for functional responses of human polymorphonuclear leukocytes (PMN). C5b67 was formed from purified human complement components and decayed in phosphate-buffered saline (PBS) until it had no lytic activity for sheep erythrocytes in a standard assay. iC5b67, at nanomolar concentrations, stimulated PMN chemotaxis and Ca2+ fluxes, but inhibited superoxide production and failed to upregulate CR1 and CR3. There was no significant contamination of the iC5b67 with C5a to explain these results. Neither isolated C5b6 nor C7 alone exhibited the activities of iC5b67, while insolubilized anti-C7 could remove the PMN agonist activity from the iC5b67 preparation. Binding studies to define a specific receptor for iC5b67 on PMN were hampered by the very hydrophobic nature of the ligand. 125I-iC5b67, by contrast to hemolytically active 125I-C5b67, was unable to insert in erythrocytes, suggesting that iC5b67 need not insert in the PMN membrane to induce signaling. Two lines of evidence suggest that iC5b67 and C5a and FMLP share common steps in intracellular signaling (1) pretreatment of PMN with iC5b67 deactivates PMN for C5a- and FMLP-induced chemotaxis; and (2) pretreatment of PMN with pertussis toxin inhibits iC5b67-induced chemotaxis. Thus, iC5b67 has important effects on the activity of PMN and G-proteins and Ca2+ are involved in the signaling.     

Fragment D-dimer levels: an objective marker of vaso-occlusive crisis and other complications of sickle cell disease

Although abnormalities in coagulation tests have been reported during vaso-occlusive crises in patients with sickle cell disease, objective, readily performed laboratory tests that document the occurrence of this complication have not been available. We examined the relationship between fibrin D-dimer levels and the occurrence of complications in patients with sickle cell disease, using a commercially available latex bead agglutination assay. The patients were either asymptomatic, hospitalized for vaso-occlusive crisis, or had other complications of sickle cell disease including leg ulcers, chronic cholecystitis, aseptic necrosis, joint pain and infection. Fifty-seven percent of 187 samples on 96 patients had elevated levels of fibrin D-dimer. Ninety percent of 75 samples from asymptomatic patients were negative for fibrin D-dimer (less than 1 microgram/ml) but 97% of 29 samples from patients with vaso-occlusive crisis and 85% of 83 samples from patients with other complications of sickle cell disease were positive. In serial studies, worsening or amelioration in clinical complications were reflected in increasing or decreasing levels of fibrin D-dimer, respectively. The molecular species of fibrin identified by the latex agglutination test was shown to be fragment D-dimer by successive immunoprecipitation and protein blot analysis. We conclude that the complications of sickle cell disease, including vaso-occlusive crisis, result in the production of fibrin D-dimer, and its detection may be used as a marker for the presence of the complication.     

Increased HbF in sickle cell anemia is determined by a factor linked to the beta S gene from one parent

Members of 7 large families, containing 20 patients with sickle cell anemia (SS) characterized by high levels of fetal hemoglobin (HbF), were studied using immunofluorescence to count F cells and a radioimmunoassay to measure small amounts of HbF. In five of these families, one of the sickle cell trait (AS) parents had a much higher HbF and F-cell count than the other; in one family, both parents had a marked increase in HbF and F cells; in the remaining family, HbF and F cells were at borderline values in both parents. Seven of 14 AS siblings, but only 1 of 8 normal hemoglobin (AA) siblings, also had HbF and F-cell counts above the "normal" range. It seems that a factor for increased F cells, linked to the beta S gene of one parent, is segregating in these families and is responsible for the greatly increased HbF and F cells in the SS subjects. HbF per F cell in AS parents and siblings was the same as that of normal AA subjects, whereas in the SS offspring it was greatly increased, suggesting that it was the result of marrow hyperplasia associated with their hemolytic anemia. The similarity of this "increased F-cell gene" to heterocellular hereditary persistence of fetal hemoglobin (HPFH). Swiss type, is discussed, and it is suggested that it may control the persistent synthesis of HbF in sickle cell anemia by its presence in early infancy.     

Neonatal mortality in a referral hospital in Cameroon over a seven year period: trends,associated factors and causes

Background

The fourth Millennium Development Goals targets reduction of the mortality rate of under-fives by 2/3 by the year 2015. This reduction starts with that of neonatal mortality representing 40% of childhood mortality. In Cameroon neonatal mortality was 31% in 2011.

Objectives

We assessed the trends, associated factors and causes of neonatal deaths at the Yaounde Gynaeco-Obstetric and Pediatric Hospital.

Methods

The study was a retrospective chart review. Data was collected from the hospital records, and included both maternal and neonatal variables from 1^st January 2004 to 31^st December 2010.

Results

The neonatal mortality was 10%. Out-borns represented 49.3% of the deceased neonates with 11.3% born at home. The neonatal mortality rate followed a downward trend dropping from 12.4% in 2004 to 7.2% in 2010. The major causes of deaths were: neonatal sepsis (37.85%), prematurity (31.26%), birth asphyxia (16%), and congenital malformations (10.54%). Most (74.2%) of the deaths occurred within the first week with 35% occurring within 24hours of life. Mortality was higher in neonates with birth weight less than 2500g and a gestational age of less than 37 weeks. In the mothers, it was high in single parenthood, primiparous and in housewives and students..

Conclusion

There has been a steady decline of neonatal mortality since 2004.Neonatal sepsis, prematurity, birth asphyxia and congenital malformations were the major causes of neonatal deaths. Neonatal sepsis remained constant although at lower rates over the study period.     

Bone marrow infarction in sickle cell anemia: correlation with hematologic profiles

Bone marrow infarction was investigated by 99mTc-sulfur colloid imaging in 42 patients with sickle cell anemia (SS) over a period of 2 yr. Marrow defects were demonstrated in 28 patients (66.6%), and in 15 (aged 19--52 yr), they were matched by roentgenographic evidence of medullary bone infarction. Repeated images showed no change in the size or site of these defects. Among 13 patients (aged 6--32 yr), all in crisis when initially examined, marrow defects were not associated with roentgenographic changes, and in many cases, repeated images showed resolution or decrease in size of the defects in 3--6 mo, even if the limb had been swollen and the marrow defect large. Among 14 patients (aged 18--36 yr), all asymptomatic at the time of study, no defects were found. Comparison of hematologic variables revealed a higher mean hemoglobin and hematocrit level among those with marrow infarcts (p less than 0.0001). High levels of HbF, or the presence of alpha- thalassemia, did not protect against marrow infarction. Pulmonary fat embolism was not observed. 99mTc-sulfur colloid marrow imaging was considered to provide more useful information in the initial management of bone pain and swelling in sickle cell crisis than either roentgenographs or conventional 99mTc-methyldiphosphate bone images.     

Human erythrocytes bearing electroinserted CD4 neutralize infection in vitro by primary isolates of human immunodeficiency virus type 1

Human erythrocytes bearing electroinserted full-length CD4 (RBC-CD4) can bind and fuse with a laboratory strain of human immunodeficiency virus type 1 (HIV-1) or with T cells infected by HIV-1. Here we show that RBC-CD4 neutralize primary HIV-1 strains in an assay of cocultivation of peripheral blood mononuclear cells (PBMC) from HIV-1- infected persons with uninfected PBMC. RBC-CD4 inhibited viral p24 core antigen accumulation in these cocultures up to 10,000-fold compared with RBC alone. Viral p24 accumulation was inhibited equally well when measured in culture supernatants or in call extracts. The inhibition was dose-dependent and long-lived. Two types of recombinant CD4 tested in parallel were largely ineffective. The neutralization of primary HIV- 1 by RBC-CD4 in vitro was demonstrated in PBMC cultures from 21 of a total of 23 patients tested at two independent sites. RBC-CD4 may offer a route to blocking HIV-1 infection in vivo.     

Characterization of a new monoclonal antibody (PG-M3) directed against the aminoterminal portion of the PML gene product: immunocytochemical evidence for high expression of PML proteins on activated macrophages, endothelial cells, and epithelia

PG-M3 is a new monoclonal antibody (MoAb) specifically directed against a peptide sequence located in the aminoterminal region of the human PML protein. PML gene fuses with the retinoic acid receptor alpha (RAR alpha) gene during the t(15; 17) chromosomal translocation of acute promyelocytic leukemia (APL). The epitope recognized by PG-M3 is species-specific and fixative-resistant and is shared by most PML isoforms and PML/RAR alpha fusion proteins. PML is consistently located within the nucleus, although a minority of cells (about 20%), both in vitro and in vivo, show positivity for PML also in the cytoplasm. The nuclear staining pattern of PG-M3 varies from speckled (cells other than APL) to micropunctate (APL cells). Although two physiologically expressed PML isoforms are detectable by immunocytochemistry only or predominantly in the cytoplasm of transfected cells, the cytoplasmic localization of PML is a property also shared by the PML isoforms that predominantly localize to the nuclei. Immunohistologic analysis of normal human tissues with the PG-M3 MoAb showed variable PML expression, with the highest levels of the protein in postmitotic, differentiated cell types, such as endothelial cells, epithelia, and tissue macrophages, especially activated ones. In keeping with this in vivo finding, PML appears strongly upregulated in the U937 promonocyte cell line after exposure to agents that induce monocyte/macrophage activation (interferon gamma) or maturation (vitamin D3 and transforming growth factor beta 1).     

Total lymphoid irradiation and cyclophosphamide as preparation for bone marrow transplantation in severe aplastic anemia

A new combination of total lymphoid irradiation and cyclophosphamide was used prior to bone marrow transplantation in an attempt to achieve decreased rejection rates and graft-versus-host disease. Nine previously transfused patients with severe aplastic anemia received marrow from an HLA-identical, MLC-compatible sibling following this preparative regimen. There were no episodes of graft rejection, and only one patient developed graft-versus-host disease. Of the 9 patients, 7 (78%) are surviving with a median follow-up of 400 days. The excellent results of this pretransplant combination of total lymphoid irradiation and cyclophosphamide warrants application of this regimen to a larger series of patients.