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

Serotonin regulates mitochondrial biogenesis and function in rodent cortical neurons via the 5-HT2A receptor and SIRT1–PGC-1α axis

Mitochondria in neurons, in addition to their primary role in bioenergetics, also contribute to specialized functions, including regulation of synaptic transmission, Ca2+ homeostasis, neuronal excitability, and stress adaptation. However, the factors that influence mitochondrial biogenesis and function in neurons remain poorly elucidated. Here, we identify an important role for serotonin (5-HT) as a regulator of mitochondrial biogenesis and function in rodent cortical neurons, via a 5-HT2A receptor-mediated recruitment of the SIRT1–PGC-1α axis, which is relevant to the neuroprotective action of 5-HT. We found that 5-HT increased mitochondrial biogenesis, reflected through enhanced mtDNA levels, mitotracker staining, and expression of mitochondrial components. This resulted in higher mitochondrial respiratory capacity, oxidative phosphorylation (OXPHOS) efficiency, and a consequential increase in cellular ATP levels. Mechanistically, the effects of 5-HT were mediated via the 5-HT2A receptor and master modulators of mitochondrial biogenesis, SIRT1 and PGC-1α. SIRT1 was required to mediate the effects of 5-HT on mitochondrial biogenesis and function in cortical neurons. In vivo studies revealed that 5-HT2A receptor stimulation increased cortical mtDNA and ATP levels in a SIRT1-dependent manner. Direct infusion of 5-HT into the neocortex and chemogenetic activation of 5-HT neurons also resulted in enhanced mitochondrial biogenesis and function in vivo. In cortical neurons, 5-HT enhanced expression of antioxidant enzymes, decreased cellular reactive oxygen species, and exhibited neuroprotection against excitotoxic and oxidative stress, an effect that required SIRT1. These findings identify 5-HT as an upstream regulator of mitochondrial biogenesis and function in cortical neurons and implicate the mitochondrial effects of 5-HT in its neuroprotective action.Fil: Fanibunda, S. E.. International Centre Of Theoretical Science. Tata Institute Of Fundamental Research; España. Kasturba Health Society; IndiaFil: Deb, Sukrita. International Centre Of Theoretical Science. Tata Institute Of Fundamental Research; EspañaFil: Maniyadath, Babukrishna. International Centre Of Theoretical Science. Tata Institute Of Fundamental Research; EspañaFil: Tiwari, Praachi. International Centre Of Theoretical Science. Tata Institute Of Fundamental Research; EspañaFil: Ghai, Utkarsha. International Centre Of Theoretical Science. Tata Institute Of Fundamental Research; EspañaFil: Gupta, Samir. International Centre Of Theoretical Science. Tata Institute Of Fundamental Research; EspañaFil: Figueiredo, Dwight. International Centre Of Theoretical Science. Tata Institute Of Fundamental Research; EspañaFil: Weisstaub, Noelia V.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Neurociencia Cognitiva. Fundación Favaloro. Instituto de Neurociencia Cognitiva; ArgentinaFil: Gingrich, Jay A.. Columbia University; Estados UnidosFil: Vaidya, Ashok D.B.. Kasturba Health Society; IndiaFil: Kolthur Seetharam, Ullas. International Centre Of Theoretical Science. Tata Institute Of Fundamental Research; EspañaFil: Vaidya, Vidita A.. International Centre Of Theoretical Science. Tata Institute Of Fundamental Research; Españ

Surfing the spectrum - what is on the horizon?

Diagnostic imaging techniques have evolved with technological advancements - but how far? The objective of this article was to explore the electromagnetic spectrum to find imaging techniques which may deliver diagnostic information of equal, or improved, standing to conventional radiographs and to explore any developments within radiography which may yield improved diagnostic data. A comprehensive literature search was performed using Medline, Web of Knowledge, Science Direct and PubMed Databases. Boolean Operators were used and key-terms included (not exclusively): terahertz, X-ray, ultraviolet, visible, infra-red, magnetic resonance, dental, diagnostic, caries and periodontal. Radiographic techniques are primarily used for diagnostic imaging in dentistry, and continued developments in X-ray imaging include: phase contrast, darkfield and spectral imaging. Other modalities have potential application, for example, terahertz, laser doppler and optical techniques, but require further development. In particular, infra-red imaging has regenerated interest with caries detection in vitro, due to improved quality and accessibility of cameras. Non-ionising imaging techniques, for example, infra-red, are becoming more commensurate with traditional radiographic techniques for caries detection. Nevertheless, X-rays continue to be the leading diagnostic image for dentists, with improved diagnostic potential for lower radiation dose becoming a reality

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

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

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

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