Polymorphisms of Pyrimidine Pathway Enzymes Encoding Genes and HLA-B*40∶01 Carriage in Stavudine-Associated Lipodystrophy in HIV-Infected Patients

Purpose

To assess in a cohort of Caucasian patients exposed to stavudine (d4T) the association of polymorphisms in pyrimidine pathway enzymes and HLA-B*40∶01 carriage with HIV/Highly active antiretroviral therapy (HAART)-associated lipodystrophy syndrome (HALS).

Methods

Three-hundred and thirty-six patients, 187 with HALS and 149 without HALS, and 72 uninfected subjects were recruited. The diagnosis of HALS was performed following the criteria of the Lipodystrophy Severity Grading Scale. Polymorphisms in the thymidylate synthase (TS) and methylene-tetrahydrofolate reductase (MTHFR) genes were determined by direct sequencing, HLA-B genotyping by PCR-SSOr Luminex Technology, and intracellular levels of stavudine triphosphate (d4T-TP) by a LC-MS/MS assay method.

Results

HALS was associated with the presence of a low expression TS genotype polymorphism (64.7% vs. 42.9%, OR = 2.43; 95%CI: 1.53–3.88, P<0.0001). MTHFR gene polymorphisms and HLA-B*40∶01 carriage were not associated with HALS or d4T-TP intracellular levels. Low and high expression TS polymorphisms had different d4T-TP intracellular levels (25.60 vs. 13.60 fmol/10⁶ cells, P<0.0001). Independent factors associated with HALS were(OR [95%CI]: (a) Combined TS and MTHFR genotypes (p = 0.006, reference category (ref.): ‘A+A’; OR for ‘A+B’ vs. ref.: 1.39 [0.69–2.80]; OR for ‘B+A’ vs. ref.: 2.16 [1.22–3.83]; OR for ‘B+B’ vs. ref.: 3.13, 95%CI: 1.54–6.35), (b) maximum viral load ≥5 log10 (OR: 2.55, 95%CI: 1.56–4.14, P = 0.001), (c) use of EFV (1.10 [1.00–1.21], P = 0.008, per year of use).

Conclusion

HALS is associated with combined low-expression TS and MTHFR associated with high activity polymorphisms but not with HLA-B*40∶01 carriage in Caucasian patients with long-term exposure to stavudine.     

Disregulated influenza A virus-specific CD8+ T cell homeostasis in the absence of IFN-gamma signaling

Recent studies indicate that IFN-gamma may influence both the expansion and the trafficking of virus-specific CD8+ CTL, though the effects are not necessarily consistent for different models of viral and bacterial disease. Influenza A virus infection of mice deficient for IFN-gamma (IFN-gamma(-/-)) or deficient for the IFN-gamma receptor 1 (IFNGR1(-/-)) was, when compared with the wild-type (WT) B6 controls, associated with increased Ag-specific CD8+ T cell counts in the spleen and mediastinal lymph nodes. At the same time, fewer of these CTL effectors were found in the bronchoalveolar lavage population recovered from the IFN-gamma(-/-) mice. Comparable effects were observed for WT mice treated with a neutralizing IFN-gamma-specific mAb. Transfer of WT memory Thy1.1(+) CD8+ populations into Thy1.2+ B6 IFN-gamma(-/-) or IFNGR1(-/-) mice followed by intranasal virus challenge demonstrated both that IFN-gamma produced by the host was important for the regulation of Ag-specific CTL numbers and that IFN-gamma was likely to act directly on the T cells themselves. In addition, the prevalence of CTLs undergoing apoptosis in spleen was lower when measured directly ex vivo for IFN-gamma(-/-) vs WT B6 mice. The present analysis is the first comprehensive demonstration that IFN-gamma signaling can differentially regulate both Ag-specific CTL homeostasis in secondary lymphoid organs and trafficking to a site of virus-induced pathology.     

Impact of dietary fatty acids on oocyte quality and development in lactating dairy cows

The purpose of this study was to examine the effects of level of rumen inert fatty acids on developmental competence of oocytes in lactating dairy cows. Estrous cycles were synchronized in 22 cows on a silage-based diet supplemented with either low (200 g/day) or high (800 g/day) fat. A total of 1051 oocytes were collected by ultrasound-guided ovum pickup (OPU) in seven sessions/cow at 3-4 day intervals. Oocytes were matured, fertilized, and cultured to the blastocyst stage in vitro. Embryo quality was assessed by differential staining of Day 8 blastocysts. The high-fat diet reduced numbers of small and medium follicles. There was no effect on the quality of oocytes (grades 1-4) or cleavage rate. However, high fat significantly improved blastocyst production from matured (P < 0.005) and cleaved (P < 0.05) oocytes. Blastocysts from the high-fat group had significantly more total, inner cell mass and trophectoderm cells than the low-fat group (P < 0.05). Regression analysis showed negative effects of milk yield (P < 0.001), dry matter intake (P < 0.001), metabolizable energy intake (P < 0.005), and starch intake (P < 0.001) on blastocyst production in the low-fat group but not in the high-fat group. Within the low-fat group, blastocyst production was negatively related to growth hormone (P < 0.05) and positively related to leptin (P < 0.05). The low-fat group had higher nonesterified fatty acids than the high-fat group (P < 0.05). In conclusion, higher milk yields were associated with reduced developmental potential of oocytes in cows given a low-fat diet. Provision of a high-fat diet buffered oocytes against these effects, resulting in significantly improved developmental potential.     

Contribution of horizontally acquired genomic islands to the evolution of the tubercle bacilli

The contribution of horizontal gene transfer (HGT) to the evolution of Mycobacterium tuberculosis -- the main causal agent of tuberculosis in humans -- and closely related members of the M. tuberculosis complex remains poorly understood. Using a combination of genome-wide parametric analyses, we have identified 48 M. tuberculosis chromosomal regions with atypical characteristics, potentially due to HGT. These specific regions account for 4.5% of the genome (199 kb) and include 256 genes. Many display features typical of the genomic islands found in other bacteria, including residual material from mobile genetic elements, flanking direct repeats, insertion in the vicinity of tRNA sequences, and genes with putative or documented virulence functions. Southern blotting analysis of nine of these 48 regions confirmed their presence in "Mycobacterium prototuberculosis," the ancestral species of the M. tuberculosis complex. Finally, our results strongly suggest that the ancestor of the tubercle bacilli was an environmental bacillus that exchanged genetic material with other bacterial species, including Proteobacteria in particular, present in its surroundings. This study describes a rational approach to searching for mycobacterial virulence genes, and highlights the importance of dissecting gene transfer networks to improve our understanding of mycobacterial pathogenicity and evolution.     

Reversible acetylation regulates vascular endothelial growth factor receptor-2 activity

The tyrosine kinase receptor vascular endothelial growth factor receptor 2 （VEG FR2） is a key regulator of angiogenesis. Here we show that VEGFR2 is acetylated in endothelial cells both at four lysine residues forming a dense cluster in the kinase insert domain and at a single lysine located in the receptor activation loop. These modifications are under dynamic control of the acetyltransferase p300 and two deacetyiases HDAC5 and HDAC6. We demonstrate that VEGFR2 acetylation essentially regulates receptor phosphorylation. In par- ticular, VEGFR2 acetylation significantly alters the kinetics of receptor phosphorylation after ligand binding, allowing receptor phos- phoryiation and intraceUular signaling upon proLonged stimulation with VEGF. Molecular dynamics simulations indicate that acetylation of the lysine in the activation loop contributes to the transition to an open active state, in which tyrosine phosphorylation is favored by better exposure of the kinase target residues. These findings indicate that post-translational modification by acetyiation is a critical mechanism that directLy affects VEGFR2 function.     

Elucidation of bacteria found in car interiors and strategies to reduce the presence of potential pathogens

The human microbiome is influenced by a number of factors, including environmental exposure to microbes. Because many humans spend a large amount of time in built environments, it can be expected that the microbial ecology of these environments will influence the human microbiome. In an attempt to further understand the microbial ecology of built environments, the microbiota of car interiors was analyzed using culture dependent and culture independent methods. While it was found that the number and type of bacteria varied widely among the cars and sites tested, Staphylococcus and Propionibacterium were nearly always the dominant genera found at the locations sampled. Because Staphylococcus is of particular concern to human health, the characteristics of this genus found in car interiors were investigated. Staphylococcus epidermidis, S. aureus, and S. warnerii were the most prevalent staphylococcal species found, and 22.6% of S. aureus strains isolated from shared community vehicles were resistant to methicillin. The reduction in the prevalence of pathogenic bacteria in cars by using silver-based antimicrobial surface coatings was also evaluated. Coatings containing 5% silver ion additives were applied to steering wheels, placed in cars for five months and were found to eliminate the presence of culturable pathogenic bacteria recovered from these sites relative to controls. Together, these results provide new insight into the microbiota found in an important built environment, the automobile, and potential strategies for controlling the presence of human pathogens.     

Obesity in Spanish Schoolchildren: Relationship with Lipid Profile and Insulin Resistance

This article reports cross‐sectional data from a total of 1048 children, 6 to 8 years of age, categorized by presence or absence of obesity, who participated in a voluntary survey of cardiovascular risk factors in Spain over the period of 1998 to 2000, to establish the relationship between obesity and its metabolic consequences at this age. The prevalence of obesity and overweight were 9.4% and 15.7%, respectively, in boys and 10.5% and 18.0%, respectively, in girls. We observed that, in both sexes, obese children had higher triglycerides and lower high‐density lipoprotein‐cholesterol levels than non‐obese children. No differences were found in plasma glucose or low‐density lipoprotein‐cholesterol levels between normal and obese children. However, we observed that insulin levels and the homeostasis model assessment for insulin resistance were significantly (p < 0.001) higher in obese children of both sexes but that free fatty acid levels were lower in obese children than in nonobese children, with a statistical significance in girls (0.72 ± 0.30 vs. 0.61 ± 0.16 mEq/liter). In summary, our survey found some metabolic consequences of obesity similar to those found in adults (elevated triglycerides, insulin, and the homeostasis model assessment for insulin resistance, and lower high‐density lipoprotein‐cholesterol). However, other features (glucose, total cholesterol, low‐density lipoprotein‐cholesterol, and free fatty acid levels) were found to behave differently, indicating that the association of obesity with risk factors seems to change as the children age and may depend on the chronology of sexual maturation.     

Inactivation of Lipoamide Dehydrogenase by Cobalt(II) and Iron(II) Fenton Systems: Effect of Metal Chelators, Thiol Compounds and Adenine Nucleotides

Fe(II)-and Co(II)-Fenton systems (FS) inactivated the lipoamide reductase activity but not the diaphorase activity of pig-heart lipoamide dehydrogenase (LADH). The Co(II) system was the more effective as LADH inhibitor. Phosphate ions enhanced the Fe(II)-FS activity. EDTA, DETAPAC, DL-histidine, DL-cysteine, glutathione, DL-dithiothreitol, DL-lipoamide, DL-thioctic acid, bathophenthroline, trypanothione and ATP, but not ADP or AMP, prevented LADH inactivation. Reduced disulfide compounds were more effective protectors than the parent compounds. Mg ions counteracted ATP protective action. Glutathione and DL-dithiothreitol partially restored the lipoamide dehydrogenase activity of the Fe(II)-FS-inhibited LADH. DL-histidine exerted a similar action on the Co(II)-FS-inhibited enzyme. Ethanol, mannitol and benzoate did not prevent LADH inactivation by the assayed Fenton systems and, accordingly, it is postulated that site-specific generated HO'radicals were responsible for LADH inactivation. With the Co(II)-FS, oxygen reactive species other than HO, might contribute to LADH inactivation.