Inflammation Markers in Adipose Tissue and Cardiovascular Risk Reduction by Pomegranate Juice in Obesity Induced by a Hypercaloric Diet in Wistar Rats

Pomegranate juice (Punica granatum) has been used since ancient times in traditional medicine (Unani Medicine, Ayurveda); its main compounds are anthocyanins and ellagic acid, which have anti-inflammatory, antioxidant, hepatoprotective, and cardiovascular health effects. The objective was to evaluate the effect of pomegranate juice on inflammation, blood pressure, and vascular and physiological markers associated with obesity induced by a high-fat diet in a murine model. The results show that pomegranate juice reduces the concentration of low-density lipoprotein cholesterol (cLDL) 39% and increases the concentration of high-density lipoprotein cholesterol (cHDL) by 27%, leading to a 12%–18% decrease in the risk of cardiovascular diseases (CVD). In addition to reducing blood pressure by 24%, it also had an antiatherogenic effect by decreasing sE-selectin levels by 42%. On the other hand, the juice significantly increased adiponectin levels in adipose tissue, decreased levels of inflammation markers (tumor necrosis factor-α (TNF-α), plasminogen activator inhibitor-1 (PAI-1), interleukin-17A (IL-17A), interleukin-6 (IL-6), interleukin-1β (IL-1β)), and inhibited the monocyte chemoattractant protein-1 (MCP-1). Pomegranate juice requires clinical studies to prove its immunoregulatory and therapeutic effects on cardiovascular and atherogenic risks

Features of antibody responses after SARS-COV-2 infection in healthcare workers in the first wave of COVID-19 pandemic in Mexico City

Objective To investigate the antibody response to SARS-CoV-2 and identify associated factors in frontline and second-line healthcare workers (HCWs) at a large hospital in Mexico City during the first wave of COVID-19 pandemic. Methods This was a cross-sectional study of HCWs returning to work following mandatory isolation after recovering from COVID-19. Immunoglobulin (Ig) M and IgG antibodies elicited by SARS-CoV-2 were semiquantitatively measured using densitometric analysis of band intensities in lateral flow assay (LFA) devices. The mean pixel intensity (dots-per-inch [dpi]) of each band on the LFA was considered a measure of antibody titre. Results Of the 111 HCWs involved in the study, antibody responses were detected in 73/111 (66%) participants. Severe COVID symptoms was associated with old age. No differences in IgM intensity were observed between men and women, but IgG intensity was significantly higher in men than in women. Second-line HCWs produced a higher IgG intensity than firstline HCWs. The IgG intensity was high in severe cases. Conclusions For HCWs who may acquire SARS-CoV-2 infection, it is necessary to establish a routine program for detection of the virus to avoid risk of infection and spread of COVID-19

Immunological and Functional Characterization of RhoGDI3 and Its Molecular Targets RhoG and RhoB in Human Pancreatic Cancerous and Normal Cells

<div><p>RhoGDI proteins have been implicated in several human cancers; changes in their expression levels have shown pro- or anti-tumorigenic effects. Pancreatic Ductal Adenocarcinoma (PDAC) is a complex pathology, with poor prognosis, and most patients die shortly after diagnosis. Efforts have been focused on understanding the role of RhoGDI's in PDAC, specially, RhoGDI1 and RhoGDI2. However, the role of RhoGDI3 has not been studied in relation to cancer or to PDAC. Here, we characterized the expression and functionality of RhoGDI3 and its target GTPases, RhoG and RhoB in pancreatic cell lines from both normal pancreatic tissue and tissue in late stages of PDAC, and compared them to human biopsies. Through immunofluorescences, pulldown assays and subcellular fractionation, we found a reduction in RhoGDI3 expression in the late stages of PDAC, and this reduction correlates with tumor progression and aggressiveness. Despite the reduction in the expression of RhoGDI3 in PDAC, we found that RhoB was underexpressed while RhoG was overexpressed, suggesting that cancerous cells preserve their capacity to activate this pathway, thus these cells may be more eager to response to the stimuli needed to proliferate and become invasive unlike normal cells. Surprisingly, we found nuclear localization of RhoGDI3 in non-cancerous pancreatic cell line and normal pancreatic tissue biopsies, which could open the possibility of novel nuclear functions for this protein, impacting gene expression regulation and cellular homeostasis.</p></div

Cancerous and non-cancerous pancreatic cell lines show different expression patterns of RhoGDI3 protein.

<p>(A) Immunofluorescence microscopy analysis of RhoGDI3 protein (green); 58 kDa protein, Golgi apparatus marker (Red) and Nuclei (DAPI, blue) was performed on hTERT-HPNE (upper panel), BxPC3 (middle panel) and PANC-1 (bottom panel) cells lines. (B) Representative Immunoblot using antibodies anti-RhoGDI3, anti-RhoGDI2 and anti-GAPDH were used as loading control. Total lysates from hTERT-HPNE, BxPC3 and PANC-1 cell lines were analyzed. (C) Densitometric analysis of the bands detected in the Western blots of RhoGDI3 (n = 3) of protein extracts from all three cell lines, the data was normalized to GAPDH. Densitometric analysis was determined with Image Lab software. Values are means ± SEM, **P<0.005 (Anova-test). Scale bar 20μm.</p

Nuclear localization of RhoGDI3 in rhEGF treated hTERT-HPNE cells.

<p>Subcellular fractionation was performed after cells were treated with rhEGF (marked above the images as 0, 2 and 10 rhEGF Min). Nuclear (N) and cytosolic (C) fractions from hTERT-HPNE (A), BxPC3 (B) and PANC-1 (C) cell lines were obtained and analyzed by immunoblotting, using anti-RhoGDI3, anti-RhoG, anti-RhoB antibodies. Anti-histone H3 antibody was used as a nuclear control and anti-Aldolase B antibody was used as a cytosol control. 20 μg of cell lysates were loaded.</p

The expression of RhoG and RhoB proteins is altered in cancerous pancreatic cell lines.

<p>Immunofluorescence microscopy analysis of RhoGDI3 protein (green); RhoG (A) and RhoB (B) (Red) and Nuclei (DAPI, blue) of hTERT-HPNE (upper panel), BxPC3 (middle panel) and PANC-1 (bottom panel) cells lines. Representative Immunoblot using antibodies anti-RhoG (C), anti RhoB (D), GAPDH was used as loading control. Total lysates from hTERT-HPNE, BxPC3 and PANC-1 cell lines were analyzed. Total amount of RhoG (E) and RhoB (F) proteins was normalized to GAPDH (n = 3). Immunoblot densitometric analysis was performed with Image Lab software. Values are means ± SEM, **P<0.005, *P<0.005 (Anova-test). Scale bar 10μm.</p

Nuclear localization of RhoGDI3 in normal pancreatic tissue.

<p>Immunofluorescence microscopy staining of RhoGDI3 (green) and RhoG (red) was carried out on human pancreatic normal (A) and moderate (aggressiveness) PDAC biopsies (C). (B) Magnification and lateral view of the immunofluorescence of RhoGDI3 and RhoG, to evidence nuclear localization in human pancreatic normal tissue. Arrowheads denote the localization of RhoGDI3 and RhoG into the nuclei. Scale bar 10 = μm.</p

Activation of RhoG GTPase in hTERT-HPNE, BxPC3 and PANC-1 pancreatic cell lines.

<p>Cells were starved 6 hours and confronted with rhEGF for the period of 0, 2 and 10 minutes (Marked as 0, 2 and 10). Fluorescence microscopic staining of RhoG (green) was carried out in hTERT-HPNE (A), BxPC3 (B) and PANC-1 (C) cells lines. To show the cytoskeleton reorganization, F-Actin was stained with rhodamine phalloidin. Measurement of RhoG activity was performed using RhoG pulldown assay. Immunoblots for RhoG, Rac-1 and GAPDH proteins for hTERT-HPNE (D), BxPC3 (E) and PANC-1 (F) are shown. To quantify the amount of RhoG-GTP and bound-Rac-1 through the temporal course, densitometric analysis was performed using Image Lab software, hTERT-HPNE (G), BxPC3 (H) and PANC-1 (I). For comparison of RhoG activity, the total amount of RhoG in cell lysates was normalized to total RhoG. GAPDH was used as a protein loading control. ELMO1-GST beads coomassie are shown as beads loading control. Arrowheads denote the localization of RhoG into the peripheral membrane; boxes with number represent the number of cells with this phenotype. Scale bar 100 μm.</p

GTPase RhoB shows differential activation in PDAC cell lines.

<p>Cells were starved for 6 hours and treated with rhEGF for a period of 0, 2 and 10 minutes (Marked as 0, 2 and 10 min). An immunofluorescence microscopy analysis of RhoB (green) was carried out on hTERT-HPNE (A), BxPC3 (B) and PANC-1 (C) cells lines. To show the cytoskeleton reorganization, F-Actin was stained with rhodamine phalloidin. Measurement of RhoB activity was performed using RhoB pulldown assay. Immunoblots for RhoB and GAPDH, as loading control for hTERT-HPNE (D), BxPC3 (E) and PANC-1 (F) cell lines are shown. To quantify the amount of RhoB-GTP, densitometric analysis (n = 3) was performed using Image Lab software for samples of hTERT-HPNE (G), BxPC3 (H) and PANC-1 (I) cell lines. For comparison of RhoB activity, GTP-RhoB was normalized to total RhoB. GAPDH was used as a protein loading control. Coomassie of RBD-GST beads are shown as beads loading control. Arrowheads denote the localization of RhoB into the peripheral membrane; boxes with number represent the quantity of cells per field with this phenotype. Scale bar = 100 μm.</p