Interruption of antifungal secondary prophylaxis in AIDS-related histoplasmosis

The clinical data of 21 patients, suffering AIDS-related histoplasmosis, who were able to interrupt antifungal secondary prophylaxis, after achieving a partial restoration of the cell mediated immunity by HAART administration, are presented. They were 16 males and five females, whose ages varied between 32 and 54 years (mean = 38.5 years). All of them presented disseminated progressive forms of histoplasmosis, with multiple locations (skin, mucous membranes, liver, spleen, lymph nodes and lungs). The majority of the cases suffered other concomitant diseases (specially tuberculosis and Kaposi sarcoma), 66.6 % of the patients had less than 50 CD4+ cells/microl at the start of treatment and the average viral burden was 278,385 RNA copies/ml. The initial treatment consisted in 400 mg/day of itraconazole, by oral route, in 14 cases and the remaining seven patients were treated with amphotericin B, intravenously, at a daily dose of 0.7 mg/kg of body weight. One patient who did not tolerate amphotericin B and presented a partial response to itraconazole, was treated with posaconazole orally at a daily dose of 800 mg. Fourteen patients received oral itraconazole at a daily dose of 200 mg as a secondary prophylaxis, the remaining three patients were treated with intravenous amphotericin B, 50 mg twice a week. After HAART for an average lapse of 16.7 months (10 to 32 months), five cases showed CD4+ cells counts above 150 cells/microl and the remaining 16 presented more than 200 cells/microl; 18 of them had undetectable viral burden and all cases were asymptomatic. The follow up after secondary prophylaxis discontinuation varied between six months and six years (mean= 33.6 months). Twenty out of 21 patients (95 %) were clinically stable, without any manifestation of relapses, including two patients who abandoned HAART. One patient, who discontinued HAART, contracted a fatal bacterial pneumonia. Even though the limited number of cases, the data presented in this study seem to suggest that it is possible to interrupt antifungal secondary prophylaxis of histoplasmosis, when the patient is clinically asymptomatic and the CD4+ cells counts are above 150 cells/microl.     

Identification and verification of novel rodent postsynaptic density proteins

The postsynaptic density (PSD) is a cellular structure specialized in receiving and transducing synaptic information. Here we describe the identification of 452 proteins isolated from biochemically purified PSD fractions of rat and mouse brains using nanoflow HPLC coupled to electrospray tandem mass spectrometry (LC-MS/MS). Fluorescence microscopy and Western blotting were used to verify that many of the novel proteins identified exhibit subcellular distributions consistent with those of PSD-localized proteins. In addition to identifying most previously described PSD components, we also detected proteins involved in signaling to the nucleus as well as regulators of ADP-ribosylation factor signaling, ubiquitination, RNA trafficking, and protein translation. These results suggest new mechanisms by which the PSD helps regulate synaptic strength and transmission.     

Rapid progression to simian AIDS can be accompanied by selection of CD4-independent gp120 variants with impaired ability to bind CD4

Aspartate 368 on human immunodeficiency virus type 1 (HIV-1) gp120 forms multiple contacts with CD4; in mutagenesis studies, its replacement by asparagine and corresponding changes in simian immunodeficiency virus SIVmac (D385N) reduced binding with CD4. Nevertheless, simian immunodeficiency virus envelopes with D385N were prevalent in several studies. Extending these observations, we also found D385N to be dominant among env clones from two rhesus macaques that progressed rapidly to simian AIDS. These envelopes showed a CD4-independent phenotype as well as reduced affinity to CD4. Moreover, an adjacent change, G383R, which was frequently coselected with D385N, further decreased binding. An optical biosensor study demonstrated that the SIVmac239 gp120 bound to CD4 with kinetics similar to those of HIV-1. However, the gp120s with D385N and G383R showed a 40-fold reduction in affinity, with a drastic increase in dissociation rate, indicating an inherently unstable complex. This finding showed that rapid progression to simian AIDS may be accompanied by the selection of CD4-independent gp120 variants with impaired CD4 binding ability.     

Analysis of natural variants of the human immunodeficiency virus type 1 gag-pol frameshift stem-loop structure

Human immunodeficiency virus type 1 uses ribosomal frameshifting for translation of the Gag-Pol polyprotein. Frameshift activities are thought to be tightly regulated. Analysis of gag p1 sequences from 270 plasma virions identified in 64% of the samples the occurrence of polymorphism that could lead to changes in thermodynamic stability of the stem-loop. Expression in Saccharomyces cerevisiae of p1-beta-galactosidase fusion proteins from 10 representative natural stem-loop variants and three laboratory mutant constructs (predicted the thermodynamic stability [Delta G degrees] ranging from -23.0 to -4.3 kcal/mol) identified a reduction in frameshift activity of 13 to 67% compared with constructs with the wild-type stem-loop (Delta G degrees, -23.5 kcal/mol). Viruses carrying stem-loops associated with greater than 60% reductions in frameshift activity presented profound defects in viral replication. In contrast, viruses with stem-loop structures associated with 16 to 42% reductions in frameshift efficiency displayed no significant viral replication deficit.     

Changes in the glycosylation pattern of prion protein in murine scrapie. Implications for the mechanism of neurodegeneration in prion diseases

In prion diseases, the normal prion protein (PrP(c)) undergoes a conformational change that results in the abnormal form, named scrapie prion protein (PrP(sc)). The visual system of rodents provides a relatively simple neuronal model in which the cell bodies of neurons are confined to the retina and the axons constitute the optic nerve. We investigated by Western blot the profile of PrP(c) in the optic nerve and retina of normal hamsters and mice. We found that in the optic nerve the amount of PrP(c) is significantly higher than in the retina. A less abundant non-glycosylated band was observed in retinas compared with the optic nerve and brain. Similar results were found in the gray and white matter from normal mice and hamsters. After stereotaxic injection of ME7 or 139A in the superior colliculus, a visual target area, the proportion and glycopattern of PrP changed in the retina and optic nerve throughout the course of the disease. Similar results were found in the gray and white matter at terminal stage of scrapie after injection of ME7 and 139A in the dorsal hippocampus. This is the first time that changes in the distribution and glycopattern of PrP have been described in an in vivo model of prion diseases.     

Etiology of acute diarrheas in children from the Lodz region. I. Occurrence of etiologic agents

The aim of this study was determination of the etiologic agents (bacterial, fungal or viral) of acute diarrheas in children from the ?ód? region, suffering from acute diarrhoea during the period from October 1998 to February 2001. Rotaviruses were detected by the latex test. Other microorganisms belonging to the Enterobacteriaceae, Pseudomonadaceae and Vibrionaceae families, as well as the genera Listeria, Campylobacter, Candida, Staphylococcus were cultured on standard or selective culture media according to the NDH recommendations and identification by means of API system. Acute diarrhea in 155 small children below 6 years of age from the ?ód? region were caused by rotaviruses (n = 42; 27%). Enteropathogenic strains of Escherichia coli (n = 25; 16.1%) occupied the third place after Salmonella bacteria (n = 30; 19.3%--second place). Among bacterial etiologic factors of diarrhea Campylobacter bacteria showed high frequency of occurrence (n = 22; 14.1%). The investigations enabled identification both the mixed infections (n = 25; 16.1%) and more rare etiologic agents of diarrhea. CONCLUSIONS: 1. Acute diarrhea in children from the region ?ód? were most frequently caused by rotaviruses; 2. Modern microbiological diagnostics of acute diarrhea in children should be multilateral, taking account of the mixed infections and expanding the routine search for bacteria of the genus Campylobacter.     

Assisted-selection of naturally caffeine-free coffee cultivars—characterization of SNPs from a methyltransferase gene

Breeding of caffeine-free coffee cultivars require tools for an early selection of progenies bearing this later trait. Genes from caffeine synthesis and degradation represent major targets for the development of molecular markers for assisted selection. In this study, we characterized SNPs identified on the caffeine synthase gene from AC1 mutant, a naturally caffeine-free arabica coffee plant. Molecular analysis of normal and mutant sequences indicates the occurrence of SNPs in protein domains, potentially associated with caffeine synthesis in coffee. Progenies F₂, F₁BC₁ and BC from crosses of AC mutants and elite cultivars were evaluated regarding caffeine content in grains and genomic segregation profile of selected SNPs. Genotyping analysis allowed the discrimination between homozygous and heterozygous plants. Quantification of caffeine content indicated a significant variability among progenies and a low frequency of caffeine-free plants. Statistical analyses of genotyping and phenotyping results showed significant association between presence of selected SNPs and reduced caffeine content. Moreover, this association occurs through all evaluated genetic backgrounds and generations, indicating an inheritance stability of both trait and markers. The molecular markers described here represent a successful case of assisted-selection in coffee, indicating their potential use for breeding of caffeine-free cultivars.