Establishing phenotypic features associated with morbidity in human T-cell lymphotropic virus type 1 infection

The human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HT). Although it is widely believed that virus infection and host immune response are involved in the pathogenic mechanisms, the role of the immune system in the development and/or maintenance of HT remains unknown. We performed an analysis of the peripheral blood leukocyte phenotype for two different subcohorts of HTLV-1-infected individuals to verify the existence of similar immunological alterations, possible laboratory markers for HT. The leukocyte population balance, the activation status of the T lymphocytes, and the cellular migratory potential of T lymphocytes, monocytes, and neutrophils were evaluated in the peripheral blood of HTLV-1-infected individuals classified as asymptomatic individuals, oligosymptomatic individuals, and individuals with HT. Data analysis demonstrated that a decreased percentage of B cells, resulting in an increased T cell/B cell ratio and an increase in the CD8+ HLA-DR+ T lymphocytes, exclusively in the HT group could be identified in both subcohorts, suggesting its possible use as a potential immunological marker for HT for use in the laboratory. Moreover, analysis of likelihood ratios showed that if an HTLV-1-infected individual demonstrated B-cell percentages lower than 7.0%, a T cell/B cell ratio higher than 11, or a percentage of CD8+ HLA-DR+ T lymphocytes higher than 70.0%, this individual would have, respectively, a 12-, 13-, or 22-times-greater chance of belonging to the HT group. Based on these data, we propose that the T cell/B cell ratios and percentages of circulating B cells and activated CD8+ T lymphocytes in HTLV-1-infected patients are important immunological indicators which could help clinicians monitor HTLV-1 infection and differentiate the HT group from the asymptomatic and oligosymptomatic groups.     

Purification of the paraflagellar rod of the trypanosomatid Herpetomonas megaseliae and identification of some of its minor components

The paraflagellar rod (PFR) is a component of the flagellar cytoskeleton of trypanosomatid protozoa, representing a filamentous structure that runs alongside the common 9 + 2 microtubular axoneme. The high degree of ultrastructural complexity and organization of the PFR suggests that it might be formed by numerous biochemical components. However, biochemical analysis of the PFR has revealed, to date, a modest degree of complexity in what concerns both major and minor PFR proteins. In this paper the preparation of purified PFR fractions by a combination of conventional cell-fractionation procedures, non-ionic detergent treatment and limited proteolysis is described. Comparative SDS-PAGE analysis of the different purification steps indicates that the purified PFR fractions possess high amounts of the well-known major PFR proteins (77 and 83 kDa). Also, bands of 147, 139, 129 and 122 kDa are clearly enriched in such fractions and may correspond to minor PFR components. A slight enrichment in a specific fraction of a doublet of bands of 181/188 kDa suggest the participation of these proteins in the composition of the bridges between the PFR and the axoneme.     

Evaluation of the Determine Rapid Syphilis TP assay using sera

The Abbott Determine Rapid Syphilis TP assay is a treponemal test that can be used in resource-poor settings that lack laboratory facilities. However, this test has not been extensively evaluated. We measured its sensitivity and specificity by using stored serum specimens (n = 567) from all persons who tested Treponema pallidum hemagglutination assay (TPHA) positive (n = 250) or TPHA indeterminate (n = 17) in the year 2001 and the first 300 patients in 2001 who tested TPHA negative at the Evandro Chagas Research Institute in Rio de Janeiro, Brazil. This rapid assay was independently interpreted by three different observers. With TPHA results as the reference, sensitivity ranged between readers from 95.6 to 98.4% and specificity ranged from 97.3 to 95.7%. There was little interreader variability in the interpretation of results, with approximately 98% agreement for all reader combinations. Of samples from persons with human immunodeficiency virus (HIV) infection (n = 198), sensitivity was 96.9 to 99.2% and it was 94.4 to 96.3% among HIV-negative persons (n = 127). Specificity was 92.4 to 95.5% among HIV-positive persons and 97.2 to 100% among HIV-negative persons. We found this test to have high sensitivity and specificity and little interreader variability, indicating that it may be easily used in resource-poor settings without laboratory facilities. Further studies are needed using this test on whole blood and under the clinical conditions for which it is intended.     

Anti-platelet autoantibodies from ITP patients recognize an epitope in GPIIb/IIIa deduced by complementary hydropathy.

Idiopathic thrombocytopenic purpura (ITP) is a frequent platelet disorder due to the presence of anti-platelet autoantibodies. Recently a fibronectin/fibrinogen receptor in platelets, integrin GPIIb/IIIa, has been implicated as the antigen in chronic ITP. To examine the epitopes involved in the autoimmune response against GPIIb/IIIa we have used concepts from the complementary hydropathy principle. We used the peptide Trp-Thr-Val-Pro-Thr-Ala, WTVPTA (deduced from the complementary nucleotide sequence to that which codes for the Arg-Gly-Asp, RGD, domain in fibronectin), to test the immunologic activity of ITP sera. Sera from 31 patients with clinically defined ITP were tested in ELISA for reactivity towards WTVPTA and affinity purified GPIIb/IIIa. Seventeen sera (57%) reacted strongly with the glycoprotein complex, five of which reacted with the peptide. By affinity chromatography of one of these sera, we were able to show that antibodies that bind to the peptide are within the population that binds to GPIIb/IIIa. Liquid phase competition experiments revealed that binding of ITP serum to WTVPTA was inhibited only by a hydropathically compatible peptide. Our data indicate that autoantibodies can bind to hydropathically generated antigenic determinants and thus, render these peptides clinically important as diagnostic tools.     

Intracellular lectin-binding sites in symbiont-bearingCrithidia species

Crithidia oncopelti, C. deanei, andC. desouzai are flagellates of the Trypanosomatidae family that present bacterium-like endosymbionts in their cytoplasm. Direct and indirect lectin-gold labeling techniques were used at the electron microscopic level in Lowicryl K4M-embedded cells to demonstrate the presence of intracellular lectin-binding sites. We used the lectinsUlex europaeus I, Griffonia simplicifolia II, Ricinus communis I, Arachis hypogaea, G. simplicifolia I, Wistaria floribunda, Limulus polyphemus, andCanavalia ensiformis, which recognize -l-fucose, - and -N-acetylglucosamine, -galactose and -N-acetylgalactosamine, -galactose, -galactose, -N-acetylgalactosamine, sialic acid and -d-mannose, and -d-glucose residues, respectively. The nucleus was the cellular structure most frequently labeled by the lectins. The Golgi complex was seldom labeled, whereas the endoplasmic reticulum and the flagellar pocket presented a large number of binding sites. Symbionts had their two unit membranes weakly labeled by the different lectins but displayed no labeling of the space between the membranes.     

The association of socioeconomic status and use of crack/cocaine with unprotected anal sex in a cohort of men who have sex with men in Rio de Janeiro, Brazil.

To evaluate the relation between illicit drug use, sexual practices, and socioeconomic status, we analyzed data from the baseline interview of a cohort of 675 men who have sex with men conducted from 1994 to 1999 in Rio de Janeiro, Brazil. Bivariate analyses of factors associated with crack/cocaine use with sex revealed that men who reported crack/cocaine use were significantly ( p <.05) more likely than men who did not report drug use to be unemployed (42.7% vs. 29.1%), to have an income of <$250 per month (70.7% vs. 60.9%), to have <8 years of education (69.5% vs. 50.9%), to report bisexual activity (81.7% vs. 41.7%), and to engage in commercial sex (72.0% vs. 37.9%). Multivariate analysis of factors associated with unprotected anal sex with casual male partners in the last 6 months demonstrated that the following variables were associated with this outcome: an income <$250 per month (adjusted odds ratio [AOR] = 1.73, 95% confidence interval [CI]: 1.04-2.87), less than 8 years of education (AOR = 2.21, CI: 1.38-3.53), a greater sense of vulnerability (AOR = 2.58, CI: 1.54-4.33), a willingness to participate in vaccine trials (AOR = 1.91, CI: 1.20-3.05), and use of crack/cocaine (AOR = 1.91, CI: 1.05-3.46). Our findings suggest that HIV prevention programs for these men need to address drug use and how drug use may influence sexual behaviors.     

Synthesis and Characterization of Hyperbranched Polyethylenes Made with Nickel‐α‐Diimine Catalysts

The polymerization of ethylene in the presence of 1,4‐bis(2,6‐diisopropylphenyl)acenaphthenediiminenickel(II) dichloride ( 1 ) and methylaluminoxane (MAO) gives hyperbranched polyethylene (HBPE) in appropriate reaction conditions. The system 1 /MAO is active in solvents like toluene or hexane at temperatures as high as 80 °C and ethylene pressures ranging from 1 to 15 atm. The polyethylenes obtained show high molecular weights (up to 467 kg · mol^?1) and more than 218 branches per 1 000 backbone carbon atoms, qualifying these materials as hyperbranched. Dynamic‐mechanical thermal analysis (DMTA) of these materials shows high β‐transitions, directly related to the branch content of these polyethylenes.

DMTA analysis of polyethylenes obtained with 1 /MAO at 0, 30, and 50 °C (corresponding to entries 1, 2 and 3).     

Platelet-activating factor receptor deficiency delays elimination of adult worms but reduces fecundity in Strongyloides venezuelensis-infected mice

We describe the parasitological kinetics and histopathological and immunological alterations in platelet-activating factor receptor-deficient (PAFR(-/-)) and wild-type mice after a single Strongyloides venezuelensis infection (subcutaneous inoculation of 500 L3 larvae). There was no difference in the numbers of worms that reached and became established in the small intestines of PAFR(-/-) and wild-type mice. However, at 12 days after infection, significantly more worms were recovered from PAFR(-/-) mice. Although PAFR(-/-) infected mice showed a delay in elimination of adult worms, worms established in the small intestine of these mice produced a significantly lower number of eggs due to a reduction in worm fecundity. There were also significant reductions in the number of circulating and tissue eosinophils and tumor necrosis factor levels in the small intestines of PAFR(-/-) mice infected for 7 days compared to the number and level in wild-type mice. Histological analysis confirmed the reduced inflammatory process and revealed that the PAFR(-/-) mice had a smaller number of goblet cells. The concentrations of the type 2 cytokines interleukin-4 (IL-4), IL-5, and IL-10 were lower in small intestine homogenates and in supernatants of antigen-stimulated lymphocytes from spleens or mesenteric lymph nodes of PAFR(-/-) mice than in the corresponding preparations from wild-type mice. Thus, in S. venezuelensis-infected PAFR(-/-) mice, decreased intestinal inflammation is associated with enhanced worm survival but decreased fecundity. We suggest that although a Th2-predominant inflammatory response decreases worm survival, the worm may use factors produced during this response to facilitate egg output and reproduction. PAFR-mediated responses appear to modulate these host-derived signals that are important for worm fecundity.     

Characterization of the hamster FancG/Xrcc9 gene and mutations in CHO UV40 and NM3

The human FANCG/XRCC9 gene, which is defective in Fanconi anemia complementation group G (FA-G) cells, was first cloned by genetic complementation of the mitomycin C (MMC) sensitivity of CHO mutant UV40. The CHO NM3 mutant was subsequently assigned to the same complementation group. The parental AA8 CHO cells are hemizygous at the FancG locus, and we identified frameshift mutations that result in N-terminal truncations of the protein in both UV40 and NM3. Hypersensitivity to DNA cross-linking agents, such as MMC, typically characterizes FA cells. By introducing the native CHO FancG gene into mutant NM3, we demonstrate that hamster FancG fully corrects the 3-fold sensitivity to methyl methanesulfonate (MMS) as well as the 10-fold sensitivity to MMC, whereas resistance to ionizing radiation did not increase appreciably. In contrast, hamster cDNA transformants showed incomplete correction for both MMC and MMS sensitivity. The constitutively expressed FancG protein is present in the cytoplasmic, nuclear and chromatin fractions. FancG protein levels and subcellular localization do not change appreciably as a function of cell cycle position. Our results are consistent with roles of FancG in both the nuclear and cytoplasmic compartments to maintain genomic stability in response to various genotoxic agents.