Differential capacity of T cell priming in naive donors of promiscuous CD4+ T cell epitopes of HCV NS3 and Core proteins

To understand the inter-individual and virus-independent variability of CD4+ T cell responses to HCV components, we evaluated the effect on these responses of HLA II molecules in uninfected healthy donors. Using HLA II-specific binding assays, we identified, in the Core and NS3 proteins, 21 long fragments and 24 15-mer peptides that bound to four to eight of the most preponderant HLA II molecules. We then evaluated the priming capacity of eight long promiscuous peptides in 12 HLA-unrelated healthy donors. The NS3 1250-1264 peptide primed T cells in all the naive donors, while five others were stimulating in at least half of the individuals. We also report sequences that bind to multiple HLA II molecules but are weakly immunogenic. We therefore conclude that (i) broad HLA II specificity is only a prerequisite for a peptide to be stimulating in multiple individuals, and (ii) promiscuous peptides widely differ in their capacity to prime CD4+ T cells from uninfected healthy donors. We suggest that these priming differences result from inter-individual variations in the peptide-specific T cell repertoire. Interestingly, five of the most immunogenic peptides we identified correspond to frequently targeted T cell epitopes in infected patients.     

Production of infectious virus and degradation of APOBEC3G are separable functional properties of human immunodeficiency virus type 1 Vif

HIV-1 Vif regulates viral infectivity by inhibiting the encapsidation of APOBEC3G (APO3G) through proteasomal degradation of the protein. Here we compared various Vif proteins for their ability to induce APO3G degradation and rescue viral infectivity. We found that Vif expressed from proviral vectors caused relatively inefficient degradation of APO3G in HeLa cells yet was very effective in inhibiting APO3G's antiviral activity. On the other hand, Vif expressed autonomously from a codon-optimized vector caused very efficient APO3G degradation and also effectively inhibited APO3G's antiviral effects. In contrast, a Vif chimera containing an N-terminal fluorescent tag efficiently induced APO3G degradation but was unable to restore viral infectivity. The lack of a direct correlation between APO3G degradation and rescue of viral infectivity suggests that these two properties of Vif are functionally separable. Our data imply that intracellular degradation of APO3G may not be the sole activity of Vif required for the production of infectious virions from APO3G-expressing cells.     

Subfractionation and purification of intracellular granule-structures of human platelets: an improved method based on magnetic sorting

Functional analysis of intracellular structures requires isolation and purification of these cellular compartments. With regard to platelet function both delta and alpha granules are relevant target structures. However, the availability of sufficient purification protocols for these structures is rather limited and restricted to density gradient centrifugation. Because this method is time-consuming and the resulting products are often of limited purity, we designed a new purification method based on immunolabeling followed by magnetic sorting. We directly compared this new method with the conventional method of ultracentrifugation. We were able to get highly purified subcellular fractions of human platelets using several antibodies against specific markers for dense granules (LAMP2), alpha granules (P-selectin) and the plasma membrane (GPIIb/IIIa) in combination with antibody-coated magnetic beads. In the respective fractions the marker proteins used for isolation as well as further independent, structure specific markers (for example MRP4 for dense granules, von Willebrand factor (vWF) for alpha granules and protein disulfide isomerase, PDI and GPIb beta, for plasma membrane) could be detected by Western blotting. The method describes purification of membranal structures of human platelets such as the plasma membrane and both types of granules. Therefore, studies requiring highly purified material (e.g. identification of specific transporters and receptors) will benefit from these results.     

Vitamin E affects cell death in adult rat dentate gyrus

We have previously reported the presence of dying cells in the granule cell layer (GCL) of adult rat dentate gyrus (DG), where neurogenesis occurs. In particular, we found that cell death in the GCL increased in vitamin E deficiency and decreased in vitamin E supplementation. These findings were regarded as related to changes in neurogenesis rate, which in turn was influenced by vitamin E availability; a neuroprotective effect of vitamin E on cell death was also proposed. In order to verify this latter hypothesis, we have studied cell death in all layers of DG in vitamin E-deficient and vitamin E-supplemented rats and in control rats at different ages, using TUNEL and nick translation techniques. The phenotype of TUNEL-positive cells was characterized and the existence of dying BrdU-positive cells was investigated. Dying cells with neuronal phenotype were observed throughout the DG in all experimental groups. The number of TUNEL-positive cells decreased from juvenile to adult age. A higher number of TUNEL-positive cells in vitamin E-deficient rats and a lower number in vitamin E-supplemented rats, with respect to age-matched controls, were found; moreover, in these groups, TUNEL-positive cells had a different percentage distribution in the different layers of the DG. Our results confirm the occurrence of cell death in DG, demonstrate that cell death affects neuronal cells and support the hypothesis that the effect of vitamin E on cell death is not related to neurogenesis.     

Olfactomotor activity during imagery mimics that during perception

Neural representations created in the absence of external sensory stimuli are referred to as imagery, and such representations may be augmented by reenactment of sensorimotor processes. We measured nasal airflow in human subjects while they imagined sights, sounds and smells, and only during olfactory imagery did subjects spontaneously enact the motor component of olfaction--that is, they sniffed. Moreover, as in perception, imagery of pleasant odors involved larger sniffs than imagery of unpleasant odors, suggesting that the act of sniffing has a functional role in creating of olfactory percepts.     

Genetic unmasking of epigenetically silenced tumor suppressor genes in colon cancer cells deficient in DNA methyltransferases

Hypermethylation associated silencing of the CpG islands of tumor suppressor genes is a common hallmark of human cancer. Here we report a functional search for hypermethylated CpG islands using the colorectal cancer cell line HCT-116, in which two major DNA methyltransferases, DNMT1 and DNMT3b, have been genetically disrupted (DKO cells). Using two molecular screenings for differentially methylated loci [differential methylation hybridization (DMH) and amplification of inter-methylated sites (AIMS)], we found that DKO cells, but not the single DNMT1 or DNMT3b knockouts, have a massive loss of hypermethylated CpG islands that induces the re-activation of the contiguous genes. We have characterized a substantial number of these CpG island associated genes with potentially important roles in tumorigenesis, such as the cadherin member FAT, or the homeobox genes LMX-1 and DUX-4. For other genes whose role in transformation has not been characterized, such as the calcium channel alpha1I or the thromboxane A2 receptor, their re-introduction in DKO cells inhibited colony formation. Thus, our results demonstrate the role of DNMT1 and DNMT3b in CpG island methylation associated silencing and the usefulness of genetic disruption strategies in searching for new hypermethylated loci.     

Association of Bordetella pertussis with host immune cells in the mouse lung

Mouse models are frequently used to study immunity and pathogenesis to Bordetella pertussis infection. To improve the understanding of the mouse infection model, the influx of host cells and B. pertussis localisation in the lungs were evaluated. Furthermore, the roles of filamentous hemagglutinin (FHA) and fimbriae (Fim) in these processes were determined. B. pertussis infection stimulated the recruitment of polymorphonuclear granulocytes (PMN), alveolar macrophages, and lymphocytes. As determined by double immunofluorescence staining, 2 hr after infection most B. pertussis were free in the alveolar space, some were attached to alveolar epithelia, and some were associated with and phagocytosed by PMN. After 3 days, most bacteria were associated with and phagocytosed by macrophages, some by PMN. B. pertussis was shown not to be ingested by epithelial cells or associated with interstitial macrophages. B. pertussis mutants lacking expression of FHA or Fim were associated with and phagocytosed by the same cell types as parental bacteria. The Fim mutant, however, induced a more severe inflammation, and was cleared faster from the lungs compared to the parental strain and the FHA mutant. These results suggest that Fim does not affect bacterial localisation in the mouse lung, but does influence host immune mechanisms. Possibly, Fim may exert an anti-inflammatory function and thereby inhibit killing by macrophages.     

Identification and characterization of 13 new mutations in mucopolysaccharidosis type I patients

In this study we have investigated a group of 29 Brazilian patients, who had been diagnosed with the lysosomal storage disorder, Mucopolysaccharidosis type I (MPS-I). MPS I is caused by a deficiency in the lysosomal hydrolase, alpha-L-iduronidase. Ninety percent of the MPS I patients in this study were genotyped and revealed 10 recurrent and thirteen novel IDUA gene mutations. Eight of these new mutations and three common mutations W402X, P533R, and R383H were individually expressed in CHO-K1 cells and analyzed for alpha-L-iduronidase protein and enzyme activity. A correlation was observed between the MPS I patient clinical phenotype and the associated mutant alpha-L-iduronidase protein/enzyme activity expressed in CHO-K1 cells. This was the first time that Brazilian MPS I patients had been thoroughly analyzed and highlighted the difficulties of mutation screening and clinical phenotype assessment in populations with high numbers of unique mutations.     

Relief of cough and nasal symptoms associated with allergic rhinitis by mometasone furoate nasal spray.

BACKGROUND: Cough commonly occurs as a symptom of seasonal allergic rhinitis (SAR), an inflammatory condition of the nasal mucous membranes that results in rhinorrhea, nasal stuffiness/congestion, nasal itching, and sneezing. Mometasone furoate nasal spray (MFNS, Nasonex, Schering, Kenilworth, NJ), an anti-inflammatory nasal corticosteroid, has been shown to be safe and effective in reducing the nasal inflammation of SAR. OBJECTIVE: To examine the effectiveness of MFNS in relieving SAR-associated cough, in addition to nasal symptoms. METHODS: This was a multicenter, randomized, double-blind study. Patients 12 years of age or older with > or = 1-year history of SAR symptoms, positive skin test to a prevailing seasonal allergen, moderate nasal symptoms, and moderate cough were treated for 14 days with MFNS 200 microg daily (n = 122) or placebo (n = 123). RESULTS: The group treated with MFNS showed significant improvement in the daytime cough severity score at endpoint compared with placebo (P = 0.049). Improvement in the nighttime cough severity score showed a trend in favor of MFNS treatment. Treatment with MFNS significantly improved total nasal symptoms in both the daytime and nighttime compared with placebo at endpoint (P < or = 0.017). Overall daytime symptom scores (cough + total nasal) improved significantly compared with placebo at endpoint (P = 0.005). Overall nighttime symptom scores improved significantly compared with placebo at endpoint (P = 0.028). Treatments were well tolerated, with no significant differences in the incidence of adverse events. CONCLUSIONS: MFNS is effective and well tolerated in the treatment of daytime cough associated with SAR.     

Levels of Cadmium, Mercury, and Lead in Magellanic Penguins (Spheniscus magellanicus) Stranded on the Brazilian Coast

Cadmium (Cd), mercury (Hg), and lead (Pb) were determined in samples of liver and breast muscles of first-year Magellanic penguins (Spheniscus magellanicus), from two different areas on the Brazilian coast, 35 on the Rio de Janeiro coast and 12 on the Rio Grande do Sul coast. In both areas, Cd concentrations in muscle samples were <0.025 μg/g. However, the Cd and Hg concentrations found in liver and Hg concentrations found in muscle showed a significant difference between the two regions. The geometric mean of the concentrations was higher in the specimens from Rio de Janeiro (Cd—6.8 μg/g; Hg—liver, 1.6 μg/g, and muscle, 0.4 μg/g wet weight) than in those from Rio Grande do Sul (Cd—2.3 μg/g; Hg—liver, 0.9 μg/g, and muscle, 0.1 μg/g wet weight). The site differences could be related to differences in diet influenced by geographic factors. Brazil’s southeastern coast is highly urbanized, and its coastal waters are contaminated by the waste of agricultural and industrial activities. There is a lack of information on the levels of heavy metals in S. magellanicus, however, their wide distribution and top position in the trophic chain make the use of stranded specimens an attractive source of information for monitoring heavy metals in the South Atlantic coast.