Biomedical subjects
Publications and source records attributed to B Polack.
Bovine immunodeficiency virus (BIV) is a lentivirus whose serologic prevalence is worldwide. Little is known about its impact on animal health status, pathogenesis and mode of transmission. Understanding BIV biology implies isolation of new viral strains and long-term studies on experimentally-infected cows and surrogate hosts such as rabbits.
Multiple coagulation disorders are unusual. We report here a combination of haemophilia B Leyden with type 1 von Willebrand disease (vWD) affecting different members of the same family. Haemophilia B Leyden was due to a -6 G-->A mutation within the promoter of the factor IX gene and was responsible for a mild haemophilia in the father of the proband. The proband and her sister (age 4 and 6) exhibited a twofold lower level of factor IX activity (0.4 IU/ml) than the paternal grandmother (0.95 IU/ml). The differences in F IX levels in the three carriers of the same -6 G-->A mutation suggest the implication of an age-related mechanism responsible for the increase in factor IX plasma level. Haemophilia B Leyden patient and carriers suffered also from a mild von Willebrand disease. The diagnosis of this associated type 1 vWD was performed by assaying plasma von Willebrand factor together with multimer electrophoretic studies and DDAVP test. The inheritance of this vWD was investigated by haplotype analysis of the vWF gene. Individuals affected by such an association are actually asymptomatic, but per- and post-operative bleeding risk remains to be evaluated.
We have studied the infection by the bovine immunodeficiency-like virus (BIV) in three experimentally infected calves, by polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR), from the peripheral blood mononuclear cells (PBMC). Two primer pairs located in the gag and pol regions of the viral genome allowed to detect the viral genomic DNA by PCR, as well as the unspliced genomic viral RNA transcript, by RT-PCR. We also present the evidence of the presence in peripheral blood mononuclear cells (PBMCs) of a mRNA transcript of the regulatory trans-activator tat gene, according to the splicing pattern of the viral genome, by use of reverse transcription followed by nested PCR. The active expression of the virus in these animals was further assessed by the sequential rescue of the virus from unstimulated PBMCs in cell culture, from 4 weeks until 15 months following the infection.
A total of 458 eight blood cell counts, accompanied by blood film reviews of the same samples, were performed with an electronic cell counter and with the QBC. In the great majority of cases, the QBC gave fast and accurate control of the flags from the electronic counter, thus avoiding the necessity for manual validation with its associated risks of infection and contaminations. Furthermore, QBC non readability could be related to microcytosis and hypochromia and hence point to possible cases of congenital or acquired haemoglobinopathy.
Bovine leukemia virus (BLV), an oncovirus related to human T-cell leukemia virus type I, causes a B-cell lymphoproliferative syndrome in cattle, leading to an inversion of the T-cell/B-cell ratio and, more rarely, to a B-cell lymphosarcoma. Sheep are highly sensitive to BLV experimental infection and develop B-cell pathologies similar to those in cattle in 90% of the cases. BLV tropism for B cells has been well documented, but the infection of other cell populations may also be involved in the BLV-induced lymphoproliferative syndrome. We thus looked for BLV provirus in other leukocyte populations in sheep and cattle by using PCR. We found that while B cells harbor the highest proviral load, CD8+ T cells, monocytes, and granulocytes, but not CD4+ T cells, also bear BLV provirus. As previously described, we found that persistent lymphocytosis in cows is characterized by an expansion of the CD5+ B-cell subpopulation but we did not confirm this observation in sheep in which the expanded B-cell population expressed the CD11b marker. Nevertheless, BLV could be detected both in bovine CD5+ and CD5- B cells and in sheep CD11b+ and CD11b- B cells, indicating that the restricted BLV tropism for a specific B-cell subpopulation cannot explain its expansion encountered in BLV infection. Altogether, this work shows that BLV tropism in leukocytes is wider than previously thought. These results lead the way to further studies of cellular interactions among B cells and other leukocytes that may intervene in the development of the lymphoproliferative syndrome induced by BLV infection.
In the present paper, three multiparametric coagulation instruments were evaluated with regard to chronometric tests for aPTT (CK-Prest and automated APTT), PT (Recombiplastin and Thromborel), fibrinogen and factors of the prothrombin complex. Analysis of within-run precision and linearity and comparative studies showed the analytical performances of the instruments to differ according to the reagents used and emphasized the difficulty of finding the best compromise between instrument and reagent. On the basis of this study, the mechanical instrument appeared to be more versatile than the optical machines. This conclusion could however be modified after further evaluation of the recent new generation coagulation instruments.
We evaluated and compared three automated blood cell counters, Coulter STKS, Sysmex NE 8000 and Technicon H-2. These perform both a complete blood cell count and a full white cell differential count. Carry-over was found to be acceptable, except for the leucocyte count by the Sysmex NE 8000 (1.5%; p < 0.001). Linearity over a wide concentration range for all of the measured parameters, haemoglobin, red blood cells, white blood cells and platelets, was excellent (r > 0.98). Within-run precision was verified with 22 samples covering a wide range of cell counts by repeated analysis (n = 20) of the same sample. Coefficients of variation (CV) were acceptable, < 4% for values within the normal range. The CVs for eosinophils and basophils were less good but without clinical impact. Analysis of 270 samples showed that the Coulter counter indicated the presence of atypical cell populations more frequently (19%; p = 0.02). Message of qualitative abnormalities displayed by the three analysers were often discordant. These three blood cell counters differed in their sensitivity and positive predictive value for detecting abnormal blood cells rather than in their specificity and negative predictive value. Other operating aspects of three instruments that are important in haematological practice are documented.
Monocytes are active elements of the host response against Plasmodium falciparum. They are able to express tissue factor and trigger the extrinsic pathway of blood coagulation the activation of which remained unclear in malaria. Our aim was to assess the tissue factor expression of purified blood monocytes stimulated by cultured Plasmodium falciparum-infected erythrocytes. Malaria parasite induced an early generation of tissue factor with a peak between 8 and 12 h of stimulation. Maximum expression was observed for parasitemia ranging from 1 to 2%. Plasmodium falciparum culture supernatants had the same effect showing the existence of a soluble factor able to induce the tissue factor expression. These data, demonstrating an activation of the tissue factor pathway by the malaria parasite, emphasize thrombin generation. Therefore, thrombin could participate in malaria pathology either in the microcirculatory blockade via platelet and fibrinogen activation or as a mitotic.
Toxoplasma gondii alone does not induce tumor necrosis factor alpha (TNF-alpha) secretion by human monocytes and macrophages. Nevertheless, sera from infected patients with high titers of specific immunoglobulin G antibodies against T. gondii induce TNF-alpha secretion, which is significantly higher than the corresponding induction by negative sera (P less than 0.05). After incubation with the positive serum, parasites also induce secretion of this cytokine, but TNF-alpha levels are lower (11.4 to 71.8%) than those obtained with positive serum alone. Therefore, this secretion seems to be elicited in part by antibody-T. gondii complexes and/or another unidentified factor(s), probably different from lipopolysaccharide, interleukin-1, TNF-alpha, and gamma interferon. In this study, monocytes secreted more TNF-alpha into the culture fluid than macrophages did (P less than 0.05), and no correlation was observed between secretion of this cytokine by the monocytes and the intracellular multiplication of the parasites, evaluated by [3H]uracil incorporation. Sera from patients with other infections diseases did not induce secretion of TNF-alpha; however, serum free of antibodies to T. gondii, obtained from patients with leishmaniosis, also stimulated secretion of the cytokine.
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Platelets are not restricted to hemostasis and thrombosis since they are involved in defence mechanisms especially in parasitic diseases. They express cytotoxic functions in vitro and in vivo against extra-cellular and intra-cellular parasites. The toxic process is mediated either by specific antibodies or directly by the parasite itself. As some metabolic pathways seem to be implicated both in hemostasis and in parasiticidal activity, primary hemostasis and cytotoxicity may be considered as specialized inflammatory responses.
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The platelet glycoprotein GPIIb/IIIa and the vitronectin receptor (VNR) are alpha beta-heterodimeric proteins and share the same beta-subunit. By performing swainsonine treatment and digestion with endoglycosidase H (Endo H), we showed that the heavy chains of GPIIb and VNR alpha are glycosylated by complex-type oligosaccharide chains, and provided the first evidence for the presence of one complex carbohydrate residue on their light chains. The proteolytic cleavage of pro-GPIIb and the acquisition of Endo H-resistance are independent events occurring in the same Golgi compartment. We demonstrated the Endo H-sensitivity of GPIIIa and VNR beta in all cellular systems tested. In addition, this beta-subunit is differently glycosylated according to whether it is associated with GPIIb or VNR alpha, one carbohydrate chain being processed to the complex type on GPIIIa, but not on VNR beta.
Blood platelets are involved in Plasmodium falciparum malaria pathology as shown by thrombocytopenia and increased plasma level of two alpha granule proteins: beta thromboglobulin (beta TG) and platelet factor 4 (PF4). In this study we demonstrate that Plasmodium falciparum parasitized erythrocytes activate directly the secretion of beta TG and PF4 by human platelets. This secretion is related to parasitemia and occurs immediately after contact. Treatment of parasited erythrocytes by trypsin and diffusion chamber experiments suggest that platelet activation is triggered by parasitic substances shed on erythrocyte membrane and released in the culture medium.
Human endothelial cells express a membrane glycoprotein alpha beta heterodimer similar to the human platelet glycoprotein IIb-IIIa complex (GPIIb-IIIa). This noncovalently associated complex is the vitronectin receptor (VNR). These two receptors belong to the cytoadhesin family and share the same beta subunit. They express different recognition specificities: platelet GPIIb-IIIa is a receptor for fibrinogen, fibronectin, and von Willebrand factor (vWF), whereas VNR is a receptor for vitronectin, and is possibly a receptor for fibrinogen and vWF. We analyzed the biosynthesis of the endothelial cell VNR. Our data show that VNR alpha is a two-chain protein which is biosynthesized as a single-chain precursor: the pro-VNR alpha. Pro-VNR alpha forms a complex with VNR beta, and this association occurs prior to the Golgi-mediated processing of the oligosaccharide side chains. Mature VNR beta is glycosylated by not fully processed oligosaccharide side chains because it remains endoglycosidase H (endo H) sensitive, even when the complex is expressed on the cell surface. This characteristic appears as a common feature for the members of the cytoadhesin family. These results indicate that although VNR and GPIIb-IIIa are biosynthesized in different cells, their expression is controlled by similar mechanisms, providing further support for the concept that the cytoadhesin family constitutes a distinct group of adhesion receptors.
Platelets take an active part in immunological processes as well as in haemostasis, especially in the host-parasite relationship. Our aim is to assess the growth of Plasmodium falciparum, cultured in human erythrocytes in the presence of fresh washed human platelets, since thrombocytopaenia is frequently observed during malarial infections. Our results show that platelets induce a dose-related growth inhibition of P. falciparum. Both proliferation and maturation of intraerythrocytic stages of the parasite are inhibited. This growth inhibition is triggered by the parasite itself as neither specific antibodies nor any other components are needed to activate platelets. Activated platelets are directly toxic since complement is not involved. Furthermore, inhibition is not mediated by erythrocyte lysis or by toxic oxygen metabolites. Platelets induce an inhibition of P. falciparum growth, at least in vitro, although the importance of their role played in vivo in malarial immunity has yet to be evaluated.
The only sensitive and convenient assay to assess the biological activity of low molecular weight heparins (LMWHs) is based on the potentiation of activated factor Xa inhibition. Several procedures for measuring the socalled anti Xa activity have been proposed. In this collaborative study including eight laboratories, we have used four different assays (three amidolytic and one clotting based methods) for measuring the anti Xa activity of ex vivo samples obtained after injecting three different LMWHs. The dispersion of the results obtained by calibration against standard heparin could be reduced by using any of the three LMWHs for calibration. A coefficient of variation less than 0.20 between values obtained in different laboratories using a variety of methods seems acceptable. However it is necessary to refer to a common international standard for expressing the results in units and to define, for each of the three products, the therapeutic range.