HIV gp120 Binds to Mannose Receptor on Vaginal Epithelial Cells and Induces Production of Matrix Metalloproteinases

BACKGROUND: During sexual transmission of HIV in women, the virus breaches the multi-layered CD4 negative stratified squamous epithelial barrier of the vagina, to infect the sub-epithelial CD4 positive immune cells. However the mechanisms by which HIV gains entry into the sub-epithelial zone is hitherto unknown. We have previously reported human mannose receptor (hMR) as a CD4 independent receptor playing a role in HIV transmission on human spermatozoa. The current study was undertaken to investigate the expression of hMR in vaginal epithelial cells, its HIV gp120 binding potential, affinity constants and the induction of matrix metalloproteinases (MMPs) downstream of HIV gp120 binding to hMR. PRINCIPAL FINDINGS: Human vaginal epithelial cells and the immortalized vaginal epithelial cell line Vk2/E6E7 were used in this study. hMR mRNA and protein were expressed in vaginal epithelial cells and cell line, with a molecular weight of 155 kDa. HIV gp120 bound to vaginal proteins with high affinity, (Kd = 1.2±0.2 nM for vaginal cells, 1.4±0.2 nM for cell line) and the hMR antagonist mannan dose dependently inhibited this binding. Both HIV gp120 binding and hMR exhibited identical patterns of localization in the epithelial cells by immunofluorescence. HIV gp120 bound to immunopurified hMR and affinity constants were 2.9±0.4 nM and 3.2±0.6 nM for vaginal cells and Vk2/E6E7 cell line respectively. HIV gp120 induced an increase in MMP-9 mRNA expression and activity by zymography, which could be inhibited by an anti-hMR antibody. CONCLUSION: hMR expressed by vaginal epithelial cells has high affinity for HIV gp120 and this binding induces production of MMPs. We propose that the induction of MMPs in response to HIV gp120 may lead to degradation of tight junction proteins and the extracellular matrix proteins in the vaginal epithelium and basement membrane, leading to weakening of the epithelial barrier; thereby facilitating transport of HIV across the vaginal epithelium

Lifestyle, dietary and treatment adherence pattern of uncontrolled diabetics in coastal Karnataka, India

Background: Diabetes Mellitus shows a rising trend in India, driven by a combination of factors like sedentary lifestyle, unhealthy diet and tobacco use. The cornerstone for interventions to reduce this is lifestyle modification. Aim & Objective: This study aims to determine lifestyle behaviours among uncontrolled diabetics in rural South India. Settings and Design: This is a pilot study conducted as part of a community trial which enrolled uncontrolled diabetics (Glycosylated haemoglobin, HbA1C of 7% or more) selected from baseline survey of 2 RBS readings. Methods and Material: The sociodemographic details, lifestyle habits and treatment adherence of eligible participants were recorded with a validated questionnaire. Statistical analysis used: Data was compared among 2 groups of poor glycaemic control using Chi square test. Results: There was no significant association of age or gender with HbA1C levels. Majority were non-smokers, non-alcoholics and did not exercise. Higher proportions of those with hospital admissions, longer duration of disease and less frequent check-ups had poor control; but these were not statistically significant. Dietary control was inadequate. However, there were no significant association of dietary habits with poor control. Conclusions: Although overall adherence to medication and follow up was satisfactory, lifestyle modification is not being sufficiently followed

Clinical experience with the use of Fluorescence In Situ Hybridization on uncultured cells for prenatal diagnosis.

The aim of the study was to prospectively evaluate the usefulness and limitations of Fluorescence In Situ Hybridization (FISH) on uncultured cells for prenatal diagnosis of numeric aneuploidies of chromosomes 13, 18, 21, X and Y. Hundred prospectively selected pregnant women that were at high risk of giving birth to an abnormal child were offered prenatal diagnosis by FISH after appropriate counseling. Fetal tissue was obtained by chorionic villus sampling (n=26), amniocentesis (n=62) and or fetal blood sampling (n=12) and processed for FISH using commercial probes. Six cases were excluded initially owing to maternal blood contamination or inadequate sample. FISH results were available in 98% of cases, in 2% of cases there was FISH failure. Of the remaining 92 cases, chromosome aneuploidy was detected in eleven cases. FISH was found extremely valuable in cases presenting with fetal abnormalities detected on ultrasonography and also for rapid screening of aneuploidies in cases of abnormal triple marker test. But as the diagnosis is limited to only a small number of chromosomes, appropriate evaluation of the cases with counseling regarding the limitations of FISH is mandatory before offering this test for prenatal diagnosis

Acute and chronic effects of aspirin on hematological parameters and hepatic ferritin expression in mice

OBJECTIVE: To examine the acute and chronic effects of aspirin on peripheral blood and bone marrow counts and hepatic ferritin expression in mice. MATERIAL AND METHODS: Adult male albino mice were orally administered aspirin at a dose of 600 mg/kg thrice daily for 7 days or 150 mg/kg once daily for 6/7 days up to 25 weeks. At the end of the experiment the red and white blood cell counts, hemoglobin, and packed cell volume were estimated. Bone marrow films were studied to estimate the rate of erythropoiesis and leucopoiesis. Expression of liver ferritin was tested by immunohistochemistry. RESULTS: Acute or chronic doses of aspirin reduced the RBC count, hemoglobin and other red cell indices as compared to controls. The WBC counts were higher in the treated animals as compared to the untreated animals. Both the treatment regimens appeared to suppress the rate of erythropoiesis in the marrow, while the rate of leucopoiesis appeared to increase in the marrow of the treated animals. Aspirin treatment did not significantly affect the expression of ferritin in the liver. CONCLUSION: Aspirin in either acute or chronic doses induces anemia associated with leucocytosis in mice; the anemia does not seem to be induced due to alterations in iron metabolism. The drug appears to use multiple targets which affect red cell production and maturation processes

HIV gp120 binding to vaginal hMR and its affinity constants.

<p>(a) HIV gp120 binding and its inhibition to immunopurified hMR from human vaginal proteins and Vk2/E6E7 proteins. Results are mean ± SE, n = 3. *Statistically significant (p<0.05) when compared with controls. ^#Statistically significant (p<0.05) when compared with HIV gp120 treated cells. Saturation Isotherm of HIV gp120 binding to hMR immunopurified from (b) human vaginal proteins and (c) Vk2/E6E7 proteins at concentrations indicated on the X axis. Affinity constants, Kd = 2.9±0.4 nM and Kd = 3.2±0.6 nM for human vaginal proteins and Vk2/E6E7 proteins respectively. Results are a representative of three independent experiments.</p

Expression of hMR in vaginal cells.

<p>(a) Purity of vaginal epithelial cells: RT-PCR for lymphocyte marker CD3 (lane 2, 5 9), macrophage/monocyte marker CD14 (lanes 3, 6, 10) and dendritic cell marker CD11c (lanes 4, 7 and 11) using RNA isolated form PBMCs (lane 2, 3, 4), epithelial cells (lanes 5, 6, 7) and Vk2/E6E7 (lanes 9, 10, 11). Lanes 8 and 12 - positive control (18S rRNA) from vaginal epithelial cells and Vk2/E6E7 cells respectively. Lane 1 is 100 bp ladder. (b) RT-PCR for hMR mRNA: Lane 1: 100 bp DNA ladder. Lanes 2 and 6: Amplification of hMR in RNA derived from vaginal epithelial cells and Vk2/E6E7 cells respectively using hMR specific primers (201 bp product). Lanes 4 and 5: positive control (18S rRNA) from vaginal epithelial cells and Vk2/E6E7 cells respectively. Lane 3: negative control (no template). (c) Western blot for hMR protein: Lane 1: human vaginal protein lysate and lane 2: Vk2/E6E7 protein lysate were probed with goat polyclonal anti-hMR serum (1∶1000) and HRP conjugated donkey anti-goat secondary antibody (1∶5000), and detected by chemiluminescence. Lane 3: human vaginal protein lysate and lane 4: Vk2/E6E7 protein lysate were probed with normal goat serum (negative controls) and detected as in lanes 1 and 2.</p

Effect of HIV gp120 on MMP production.

<p>(a) Effect of increasing concentrations of HIV gp120 on mRNA expression of MMP-9 and MMP-2 in Vk2/E6E7 cells. (b) Effect of increasing concentrations of HIV gp120 on MMP-9 and MMP-2 activity. (c) Representative zymograms of HIV gp120 treated Vk2/E6E7 cells (upper panel); lane 1: untreated cells, lane 2: 0.083 nM of HIV gp120, lane 3: 0.83 nM of HIV gp120, lane 4: 83 nM of HIV gp120. Lower panel represents effect of anti-hMR antibody on activity of MMP-9 and MMP-2; lane 1: Cells treated with HIV gp120 (83 nM), lane 2: HIVgp120 (83 nM) + isotype IgG, lane 3: HIV gp120 (83 nM) + anti-hMR IgG, lane 4: untreated cells. (d) Effect of anti-hMR antibody on expression of HIV gp120 induced MMP-9 mRNA. (e) Effect of anti-hMR antibody on MMP-9 activity. (a) and (d) values are mean ± SE of relative expression normalized to 18S rRNA from 3 independent experiments. (b) and (e) are mean ± SE of MMP activity expressed as fold change over unstimulated cells from 3 independent experiments. *Statistically significant (p<0.05) when compared with controls. ^#Statistically significant (p<0.05) when compared with HIV gp120 treated cells.</p

Binding of HIV gp120 to vaginal proteins and its inhibition by mannan or the hMR blocking antibody.

<p>Saturation isotherm of HIV gp120 HRP conjugated binding at the indicated concentrations on the X axis to (a) vaginal protein lysates (Kd = 1.2±0.2 nM) and (b) vaginal epithelial cell line Vk2/E6E7 protein lysates (Kd = 1.4±0.2 nM). Results are representative of three experiments. Inhibition of HIV gp120 binding using mannan in (c) vaginal protein lysates or (d) Vk2/E6E7 protein lysates. Mannan or sucrose concentrations in ug/ml are plotted on a logarithimic scale. Experiments were repeated in triplicates. Binding of HIV gp120 to (e) vaginal protein lysates or (f) Vk2/E6E7 protein lysates in the presence of increasing concentrations of hMR blocking antibody or isotype matched IgG antibody. Results are mean ± SE, n = 3. ^*Statistically significant (p<0.05) when compared with cells treated with HIV gp120 in the absence of antibody.</p

Localization of HIV gp120 binding and hMR in vaginal epithelial cells.

<p>Vaginal epithelial cells (a and e) and Vk2/E6E7 cells (b and f) incubated with FITC labeled HIV gp120 (green fluorescence) and counter stained with propidium iodide (red fluorescence). Vaginal epithelial cells (c and g) and Vk2/E6E7 cells (d and h) probed with hMR antibody (green fluorescence) and counter stained with propidium iodide (red fluorescence). <i>Inset</i> on (a) and (b) represents vaginal epithelial cells and Vk2/E6E7 cells respectively, incubated with excess unlabelled gp120. <i>Inset</i> on (c) and (d) represents negative control (-ve), for vaginal epithelial cells and Vk2/E6E7 cells respectively labeled with IgG antibody FITC labeled. Images (a and c) are at 400× magnification, (b and d) are at 630× magnification. (e–h): Images captured at 1260× magnification and further digitally magnified.</p