Distribution of HIV type 1 (HIV-1) in blood components: detection and significance of high levels of HIV-1 associated with platelets

BACKGROUND: Although inactivation of enveloped viruses transmitted by plasma derivatives has been successful, no methods for virus inactivation or removal have been established for platelet concentrates or red cell (RBC) components. Relatively little is known regarding the extent or significance of virus interactions with the cellular constituents in these components. STUDY DESIGN AND METHODS: Units of whole blood were collected from six HIV type 1 (HIV-1)-positive, asymptomatic individuals and separated into peripheral blood mononuclear cells (PBMNCs), cell-free plasma, white cell-reduced platelet concentrate, and white cell-reduced RBCs. DNA and RNA polymerase chain reaction and virus culture methods were used to study the compartmentalization of HIV-1 immediately after component preparation and after storage. RESULTS: As expected, HIV DNA and infectious virus were detected in fresh blood and in PBMNCs, and virion-associated RNA was detected in fresh plasma from all six donors. The levels of viral nucleic acids in these preparations remained relatively stable with 4 degrees C storage, whereas infectivity of PBMNCs was rapidly lost. Washed RBCs tested negative for HIV in all assays at all time points. Platelets retained high levels of HIV RNA (but not infectivity) after extensive washing, as well as after storage at 4 and 22 degrees C. High-level platelet-associated HIV-1 was also demonstrated in samples collected during early seroconversion. Periseroconversion and postseroconversion levels of platelet-associated HIV-1 correlated with the level of plasma viremia and with the rate of progression to AIDS. Cell-free virus from donor plasma and tissue culture fluid rapidly and firmly attached to platelets from noninfected donors. Infectivity of tissue culture virus bound to platelets was demonstrated in vitro. CONCLUSION: Significant levels of HIV-1 are associated with platelets during all stages of infection. Platelet-associated HIV could either mediate virus clearance or facilitate virus dissemination and expanded tropism. Finally, virus inactivation research must address virus associations with platelets.     

Evaluation of atypical cytochrome P450 kinetics with two-substrate models: evidence that multiple substrates can simultaneously bind to cytochrome P450 active sites

Some cytochrome P450 catalyzed reactions show atypical kinetics, and these kinetic processes can be grouped into five categories: activation, autoactivation, partial inhibition, substrate inhibition, and biphasic saturation curves. A two-site model in which the enzyme can bind two substrate molecules simultaneously is presented which can be used to describe all of these observed kinetic properties. Sigmoidal kinetic characteristics were observed for carbamazepine metabolism by CYP3A4 and naphthalene metabolism by CYPs 2B6, 2C8, 2C9, and 3A5 as well as dapsone metabolism by CYP2C9. Naphthalene metabolism by CYP3A4 and naproxen metabolism by CYP2C9 demonstrated nonhyperbolic enzyme kinetics suggestive of a low Km, low Vmax component for the first substrate molecule and a high Km, high Vmax component for the second substrate molecule. 7, 8-Benzoflavone activation of phenanthrene metabolism by CYP3A4 and dapsone activation of flurbiprofen and naproxen metabolism by CYP2C9 were also observed. Furthermore, partial inhibition of 7, 8-benzoflavone metabolism by phenanthrene was observed. These results demonstrate that various P450 isoforms may exhibit atypical enzyme kinetics depending on the substrate(s) employed and that these results may be explained by a model which includes simultaneous binding of two substrate molecules in the active site.     

BiP maintains the permeability barrier of the ER membrane by sealing the lumenal end of the translocon pore before and early in translocation

Secretory proteins are cotranslationally translocated across the mammalian ER membrane through an aqueous pore in the translocon while the permeability barrier is maintained by a tight ribosome-membrane junction. The lumenal end of the pore is also blocked early in translocation. Extraction of soluble lumenal proteins from microsomes and reconstitution with purified proteins demonstrate, by fluorescence collisional quenching, that BiP seals the lumenal end of this pore. BiP also seals translocons that are assembled but are not engaged in translocation. These ribosome-free translocons have smaller pores (9-15 A diameter versus 40-60 A in functioning translocons) and are generated when ribosomes dissociate from functioning translocons with large pores. BiP therefore maintains the permeability barrier by sealing both nontranslocating and newly targeted translocons.     

The diagnostic value of thrombopoietin level measurements in thrombocytopenia

It has been reported that blood trombopoietin (TPO) levels can discriminate between thrombocytopenia due to increased platelet destruction and decreased platelet production. With our TPO ELISA and a glycocalicin ELISA we analysed a large group of patients in detail and could confirm and amplify the above notion in detail. TPO levels were determined in plasma from 178 clinically and serologically well-defined thrombocytopenic patients: 72 patients with idiopathic autoimmune thrombocytopenia (AITP), 29 patients with secondary AITP, 5 patients with amegakaryocytic thrombocytopenia and 72 patients who suffered from various diseases (46 in whom megakaryocyte deficiency was not and 26 in whom it was expected). In addition, we measured the level of glycocalicin as a marker of total body mass of platelets. In all patients with primary AITP and secondary AITP, TPO levels were within the normal range or in some (n = 7) cases only slightly increased. The level of glycocalicin was not significantly different from that of the controls (n = 95). The patients with amegakaryocytic thrombocytopenia had strongly elevated TPO levels and significantly decreased glycocalicin levels. Similarly, among the 72 thrombocytopenic patients with various disorders, elevated TPO levels were only found in patients in whom platelet production was depressed. The mean level of glycocalicin in these patients was decreased compared to that in controls and patients with AITP, but was not as low as in patients with amegakaryocytic thrombocytopenia. In conclusion, all patients with depressed platelet production had elevated levels of circulating TPO, whereas the TPO levels in patients with an immune-mediated thrombocytopenia were mostly within the normal range. Therefore, measurement of plasma TPO levels provides valuable diagnostic information for the analysis of thrombocytopenia in general. Moreover, treatment with TPO may be an option in AITP.     

The Australian brushtail possum (Trichosurus vulpecula) gonadotrophin alpha-subunit: analysis of cDNA sequence and pattern of expression

Substantial evidence has accumulated to suggest that in the near future implementation of the veto-cell-suppressor concept in the treatment of kidney allograft recipients might lead to the establishment of life-long specific allograft tolerance in the absence of further immunosuppressive therapy. Veto suppression prevents the generation of antigen-specific T-helper and cytotoxic T lymphocytes in vitro provided that the T-lymphocyte precursors specifically recognize antigenic peptides associated with the major histocompatibility complex molecules class II and class I, respectively, expressed on the surface of the veto-active cell. Data from a large number of experimental and clinical studies strongly indicate that veto-active cells function in vivo and are capable of preventing allograft rejection. Thus, donor-cell-mediated veto activity is the most likely explanation for the well-known graft tolerizing effect of pretransplant donor blood transfusions in kidney graft recipients. A prerequisite for a veto-active environment in vivo is the establishment of lymphoid microchimerism, in which veto-active donor and recipient cells mutually downregulate potential alloaggression.     

The syndrome of thrombosis, thrombocytopenia, and recurrent spontaneous abortions associated with antiphospholipid antibodies: Hughes syndrome

aPL-associated thrombosis (Hughes syndrome) is widely recognized as a major cause of organ damage in autoimmune diseases. Beginning with the first symposium in 1984, international aPL symposia have facilitated research on aPL antibodies, and the clinical standardization of aPL tests. It is hoped that the present symposium will continue this tradition, because much remains to be learnt about the origin and pathogenicity of aPL antibodies. In addition, new insights are needed for more effective therapies to be developed.     

Interactions among iron, calcium, phosphorus and magnesium in the nutritionally iron-deficient rat

We studied the development of nutritional iron deficiency 0, 10, 20, 30 and 40 days after the intake of a semisynthetic diet lacking iron (diet 0) and the possible interactions with calcium, phosphorus and magnesium in both control rats and rats after 40 days of iron deficiency. During this period, iron deficiency was found to produce stress in the rats, as evidenced by high levels of cortisol in the serum. High levels of parathyroid hormone (PTH) were also found. There was a considerable increase in the absorption of calcium, phosphorus and magnesium, but the phosphorus and magnesium balance decreased and that of calcium remained practically unchanged, although there was an increase in calcium urinary elimination. Despite the noticeable degree of bone demineralization, which was evident in the femur, serum levels of calcium, phosphorus and magnesium remained constant. The present study shows that severe nutritional ferropenic anaemia provokes significant alterations in the metabolism of calcium, phosphorus and magnesium. We conclude that these alterations should be taken into account in the treatment of this pathology, given its prevalence and the fact that it may exacerbate other pathologies, particularly those related to the metabolism of calcium and phosphorus.