HIV is associated with endothelial activation despite ART, in a sub-Saharan African setting.

Objective: To study the relationship between endothelial dysfunction, HIV infection, and stroke in Malawians. Methods: Using a cross-sectional design, we measured plasma levels of intercellular adhesion molecule-1 (ICAM-1), plasminogen activator inhibitor-1 (PAI-1), vascular endothelial growth factor (VEGF), and soluble thrombomodulin (sTM) in stroke patients and controls, stratified by HIV status. These biomarkers were measured using ELISA. After dichotomization, each biomarker was used as the dependent variable in a multivariable logistic regression model. Primary independent variables included HIV and stroke status. Adjustment variables were age, sex, hypertension, diabetes mellitus, tobacco and alcohol consumption, personal/family history of stroke, antiretroviral therapy status, and hypercholesterolemia. Results: Sixty-one stroke cases (19 HIV+) and 168 controls (32 HIV+) were enrolled. The median age was 55 years (38.5-65.0) for controls and 52 years (38.0-73.0) for cases (p = 0.38). The median CD4+ T-cell count was 260.1 cells/mm3 (156.3-363.9) and 452 cells/mm3 (378.1-527.4) in HIV-infected cases and controls, respectively. HIV infection was independently associated with high levels of ICAM-1 (OR = 3.6, 95% CI: 1.3-10.6, p = 0.018) in controls but not in stroke cases even after excluding patients with a viral load >1,000 RNA copies/mL (OR = 4.1, 95% CI: 1.3-13.1, p = 0.017). There was no association between the clinical profiles of HIV-positive controls or HIV-positive stroke and high levels of PAI-1, VEGF, and sTM. Conclusions: HIV infection is associated with endothelial activation despite antiretroviral treatment. Our findings underscore the need for larger clinical cohorts to better understand the contribution of this perturbation of the endothelial function to the increasing burden of cardiovascular diseases in sub-Saharan Africa.

A metabolite profiling method for diagnosis of precancerous cervical lesions and HPV persistence.

AIM: With the advent of rapid metabolic profiling techniques and of portable mass spectrometers we examined whether cells distinguished by their cytology and persistence of human papillomavirus infection, could be easily differentiated by their metabolite profile. MATERIALS & METHODS: Direct injection electrospray mass spectrometry was used in a nontargeted double-blind experiment. Samples were collected from women diagnosed with one of two grades of cervical cytology and exhibiting either human papilloma virus persistence or clearance. Cell extracts were prepared using a DNA extraction procedure and the resulting supernatant, normally discarded, was analyzed. Data were interpreted using principal component analysis. RESULTS: The results indicate strongly that a simple metabolite profiling method could be used to rapidly identify women at increased risk of cervical cancer.

Methyl Donor Status Influences DNMT Expression and Global DNA Methylation in Cervical Cancer Cells.

BACKGROUND: Methyl donor status influences DNA stability and DNA methylation although little is known about effects on DNA methyltransferases. The aim of this study was to determine whether methyldonor status influences DNA methyltransferase (Dnmt) gene expression in cervical cancer cells, and if so, whether there are associated effects on global DNA methylation. MATERIALS AND METHODS: The human cervical cancer cell line, C4 II, was grown in complete medium and medium depleted of folate (FM+) and folate and methionine (FM). Growth rate, intracellular folate, intracellular methionine and homocysteine in the extracellular medium were measured to validate the cancer cell model of methyl donor depletion. Dnmt expression was measured by qRT PCR using relative quantification and global DNA methylation was measured using a flow cytometric method. RESULTS: Intracellular folate and methionine concentrations were significantly reduced after growth in depleted media. Growth rate was also reduced in response to methyl donor depletion. Extracellular homocysteine was raised compared with controls, indicating disturbance to the methyl cycle. Combined folate and methionine depletion led to a significant downregulation of Dnmt3a and Dnmt3b; this was associated with an 18% reduction in global DNA methylation compared with controls. Effects of folate and methionine depletion on Dnmt3a and 3b expression were reversed by transferring depleted cells to complete medium. CONCLUSIONS: Methyl donor status can evidently influence expression of Dnmts in cervical cancer cells, which is associated with DNA global hypomethylation. Effects on Dnmt expression are reversible, suggesting reversible modulating effects of dietary methyl donor intake on gene expression, which may be relevant for cancer progression.

Tumour suppressor gene methylation and cervical cell folate concentration are determinants of high-risk human papillomavirus persistence: a nested case control study.

BACKGROUND: Persistent infection with one or more high-risk human papillomavirus [HR-HPV] types increases the risk of intraepithelial neoplasia and cervical cancer. A nested case-control study was conducted to investigate the importance of cervical cell folate concentration and tumour suppressor gene methylation as risk factors for HR-HPV persistence. METHODS: Cervical cell samples from 955 women with HR-HPV infection and normal, borderline or mild dyskaryosis were retrieved from the archive of a population-based screening trial. Women were classified as cases or controls, reflecting the presence or absence [respectively] of any HR-HPV infection at a follow-up clinic at least 6 months from baseline. Cervical cell folate concentration and promoter methylation of five tumour suppressor genes were measured in independent samples from cases and controls. RESULTS: A higher cervical cell folate concentration [P = 0.015] was an independent predictor of infection at follow-up, together with infection with HPV-16 or infection with multiple HR-HPV types. Methylation of the tumour suppressor gene DAPK was associated with a 2.64-fold [95% CI, 1.35-5.17] increased likelihood of HPV infection whilst CDH1 methylation was associated with a 0.53-fold [95% CI, 0.331-0.844] likelihood of HR-HPV infection at follow-up. When considering women with normal or abnormal cytology, the predictive effect of higher cervical cell folate was only seen in women with mild cytology [P = 0.021]; similarly the effect of DAPK methylation was seen in women with mild or borderline cytology [P < 0.05]. CONCLUSIONS: Higher cervical cell folate concentration and promoter methylation of the tumour suppressor gene, DAPK, in women with cervical cell dyskaryosis, are associated with increased risk of HR-HPV persistence.

A vitamin B-12 supplement of 500 µg/d for eight weeks does not normalize urinary methylmalonic acid or other biomarkers of vitamin B-12 status in elderly people with moderately poor vitamin B-12 status.

Plasma vitamin B-12 is the most commonly used biomarker of vitamin B-12 status, but the predictive value for low vitamin B-12 status is poor. The urinary methylmalonic acid (uMMA) concentration has potential as a functional biomarker of vitamin B-12 status, but the response to supplemental vitamin B-12 is uncertain. A study was conducted to investigate the responsiveness of uMMA to supplemental vitamin B-12 in comparison with other biomarkers of vitamin B-12 status [plasma vitamin B-12, serum holotranscobalamin (holoTC), plasma MMA] in elderly people with moderately poor vitamin B-12 status. A double-blind, placebo-controlled, randomized 8-wk intervention study was carried out using vitamin B-12 supplements (500 µg/d, 100 µg/d, and 10 µg/d cyanocobalamin) in 100 elderly people with a combined plasma vitamin B-12 <250 pmol/L and uMMA ratio (µmol MMA/mmol creatinine) >1.5. All biomarkers had a dose response to supplemental vitamin B-12. Improvements in plasma vitamin B-12 and serum holoTC were achieved at cobalamin supplements of 10 µg/d, but even 500 µg/d for 8 wk did not normalize plasma vitamin B-12 in 8% and serum holoTC in 12% of people. The response in uMMA was comparable with plasma MMA; 15-25% of people still showed evidence of metabolic deficiency after 500 µg/d cobalamin for 8 wk. There was a differential response in urinary and plasma MMA according to smoking behavior; the response was enhanced in ex-smokers compared with never-smokers. uMMA offers an alternative marker of metabolic vitamin-B12 status, obviating the need for blood sampling.

Determinants of urinary methylmalonic acid concentration in an elderly population in the United Kingdom.

BACKGROUND: An age-related deterioration of vitamin B-12 status has been well documented. The early detection of deficiency may prevent the development of serious clinical symptoms, but plasma vitamin B-12 concentration is known to be an imperfect measure of vitamin B-12 status. Urinary methylmalonic acid (MMA) may be a more informative biomarker of vitamin B-12 status; however, biochemical, dietary, and other lifestyle determinants are not known. OBJECTIVE: We identified determinants of urinary MMA concentrations in free-living men and women aged ≥65 y in the United Kingdom. DESIGN: A cross-sectional study in 591 men and women aged 65-85 y, with no clinical evidence of vitamin B-12 deficiency, was conducted to determine the demographic, clinical, and lifestyle determinants of urinary MMA concentration expressed as the ratio of micromoles of MMA to millimoles of creatinine (uMMA ratio). RESULTS: Twenty percent of subjects had plasma vitamin B-12 concentrations <200 pmol/L. Seventeen percent of the variation in the uMMA ratio could be explained by plasma holotranscobalamin and sex; total vitamin B-12 intake and measures of renal function and gastric function made only a small contribution to the model. The uMMA ratio was lower in people with moderately impaired renal function. CONCLUSIONS: Plasma holotranscobalamin and sex were the most important determinants of uMMA ratio in elderly people with no clinical diagnosis of renal impairment. This biomarker might underestimate vitamin B-12 deficiency in a population in which renal impairment is prevalent. This trial was registered at www.controlled-trials.com as ISRCJN83921062.

Folate status and aberrant DNA methylation are associated with HPV infection and cervical pathogenesis.

Aberrant DNA methylation is a recognized feature of human cancers, and folate is directly involved in DNA methylation via one-carbon metabolism. Previous reports also suggest that folate status is associated with the natural history of human papillomavirus (HPV) infection. A cross-sectional study was conducted to test the hypothesis that folate status and aberrant DNA methylation show a progressive change across stages of cervical pathology from normal cells to cervical cancer. Additionally, we postulated that a gene-specific hypermethylation profile might be used as a predictive biomarker of cervical cancer risk. DNA hypermethylation of seven tumor suppressor genes, global DNA hypomethylation, systemic folate status, and HPV status were measured in 308 women with a diagnosis of normal cervix (n = 58), low-grade cervical intraepithelial neoplasia (CIN1; n = 68), high-grade cervical intraepithelial neoplasia (CIN2, n = 56; and CIN3, n = 76), or invasive cervical cancer (ICC; n = 50). Lower folate status was associated with high-risk HPV infection (P = 0.031) and with a diagnosis of cervical intraepithelial neoplasia or invasive cervical cancer (P < 0.05). Global DNA hypomethylation was greater in women with invasive cervical cancer than all other groups (P < 0.05). A cluster of three tumor suppressor genes, CDH1, DAPK, and HIC1, displayed a significantly increased frequency of promoter methylation with progressively more severe cervical neoplasia (P < 0.05). These findings are compatible with a role for folate in modulating the risk of cervical cancer, possibly through an influence over high-risk HPV infection. DAPK, CDH1, and HIC1 genes are potential biomarkers of cervical cancer risk.

Transcriptional responses of Escherichia coli to S-nitrosoglutathione under defined chemostat conditions reveal major changes in methionine biosynthesis.

Nitric oxide and nitrosating agents exert powerful antimicrobial effects and are central to host defense and signal transduction. Nitric oxide and S-nitrosothiols can be metabolized by bacteria, but only a few enzymes have been shown to be important in responses to such stresses. Glycerol-limited chemostat cultures in defined medium of Escherichia coli MG1655 were used to provide bacteria in defined physiological states before applying nitrosative stress by addition of S-nitrosoglutathione (GSNO). Exposure to 200 microm GSNO for 5 min was sufficient to elicit an adaptive response as judged by the development of NO-insensitive respiration. Transcriptome profiling experiments were used to investigate the transcriptional basis of the observed adaptation to the presence of GSNO. In aerobic cultures, only 17 genes were significantly up-regulated, including genes known to be involved in NO tolerance, particularly hmp (encoding the NO-consuming flavohemoglobin Hmp) and norV (encoding flavorubredoxin). Significantly, none of the up-regulated genes were members of the Fur regulon. Six genes involved in methionine biosynthesis or regulation were significantly up-regulated; metN, metI, and metR were shown to be important for GSNO tolerance, because mutants in these genes exhibited GSNO growth sensitivity. Furthermore, exogenous methionine abrogated the toxicity of GSNO supporting the hypothesis that GSNO nitrosates homocysteine, thereby withdrawing this intermediate from the methionine biosynthetic pathway. Anaerobically, 10 genes showed significant up-regulation, of which norV, hcp, metR, and metB were also up-regulated aerobically. The data presented here reveal new genes important for nitrosative stress tolerance and demonstrate that methionine biosynthesis is a casualty of nitrosative stress.