Effect of Multi-Strain Probiotic Supplementation on URTI Symptoms and Cytokine Production by Monocytes after a Marathon Race: A Randomized, Double-Blind, Placebo Study.

(1) Purpose: Performing strenuous exercises negatively impacts the immune and gastrointestinal systems. These alterations cause transient immunodepression, increasing the risk of minor infections, especially in the upper respiratory tract. Recent studies have shown that supplementation of probiotics confers benefits to athletes. Therefore, the objective of the current study was to verify the effects of probiotic supplementation on cytokine production by monocytes and infections in the upper respiratory tract after an acute strenuous exercise. (2) Methods: Fourteen healthy male marathon runners received either 5 billion colony forming units (CFU) of a multi-strain probiotic, consisting of 1 billion CFU of each of Lactobacillus acidophilus LB-G80, Lactobacillus paracasei LPc-G110, Lactococcus subp. lactis LLL-G25, Bifidobacterium animalis subp. lactis BL-G101, and Bifidobacterium bifidum BB-G90, or a placebo for 30 days before a marathon. Plasma cytokines, salivary parameters, glucose, and glutamine were measured at baseline, 24 h before, immediately after, and 1 h after the race. Subjects self-reported upper respiratory tract infection (URTI) using the Wisconsin Upper Respiratory Symptom Survey (WURSS-21). The statistical analyses comprised the general linear model (GLM) test followed by the Tukey post hoc and Student's t-test with p < 0.05. (3) Results: URTI symptoms were significantly lower in the probiotic group compared to placebo. The IL-2 and IL-4 plasma cytokines were lower 24 h before exercise, while the other cytokines showed no significant differences. A lower level of IL-6 produced by monocytes was verified immediately after the race and higher IL-10 at 1 h post. No differences were observed in salivary parameters. Conclusion: Despite the low number of marathoners participating in the study, probiotic supplementation suggests its capability to preserve the functionality of monocytes and mitigate the incidence of URTI.

Intestinal expression of toll-like receptor gene changes early after gastric bypass surgery and association with type 2 diabetes remission.

OBJECTIVES: Abnormal activation of toll-like receptors (TLRs) is observed in obese rodents and is correlated with local dysbiosis and increased gut permeability. These purported changes trigger systemic inflammation associated with obesity-related comorbidities, including type 2 diabetes (T2D). Roux-en-Y gastric bypass (RYGB) surgery is an effective treatment for severe obesity and known to induce changes in the gut microbiota and decrease systemic inflammation in humans. This study examined the intestinal expression of TLR-encoding genes in obese women (n = 20) treated with RYGB surgery and the relationship of these genes with T2D remission (T2Dr METHODS: Intestinal biopsies were performed before and 3 months after RYGB surgery. Partial and complete T2Dr after 1 year was assessed using the American Diabetes Association criteria. Affymetrix Human GeneChip 1.0 ST array (microarray) and TaqMan assay (real-time quantitative polymerase chain reaction) were used to analyze intestinal gene expression, and associations with systemic markers of energy homeostasis were examined. RESULTS: Patients experienced significant weight loss (P < 0.001) and altered gut TLR gene expression 3 months after surgery. The main effects were a reduction in jejunal TLR4 expression in patients with complete and partial T2Dr (P < 0.05). There was a postoperative decrease in jejunal TLR7 expression in patients with complete T2Dr that correlated inversely with high-density lipoprotein cholesterol and positively with triglyceride concentrations, but not with weight loss. CONCLUSIONS: RYGB-induced weight loss-independent changes in the expression of intestinal TLR-encoding genes in obese women and complete T2Dr that was correlated with systemic markers of energy homeostasis. The modulation of intestinal TLRs may mediate inflammatory mechanisms linked to T2Dr after RYGB surgery.

Distinct pattern of one-carbon metabolism, a nutrient-sensitive pathway, in invasive breast cancer: A metabolomic study.

Altered cell metabolism is a hallmark of cancer and critical for its development. Particularly, activation of one-carbon metabolism in tumor cells can sustain oncogenesis while contributing to epigenetic changes and metabolic adaptation during tumor progression. We assessed whether increased one-carbon metabolism activity is a metabolic feature of invasive ductal carcinoma (IDC). Differences in the metabolic profile between biopsies from IDC (n = 47) and its adjacent tissue (n = 43) and between biopsies from different breast cancer subtypes were assessed by gas spectrometry in targeted (Biocrates Life Science ® ) and untargeted approaches, respectively. The metabolomics data were statistically treated using MetaboAnalyst 4.0, SIMCA P+ (version 12.01), Statistica 10 software and t test with p < 0.05. The Cancer Genome Atlas breast cancer dataset was also assessed to validate the metabolomic profile of IDC. Our targeted metabolomics analysis showed distinct metabolomics profiles between IDC and adjacent tissue, where IDC displayed a comparative enrichment of metabolites involved in one-carbon metabolism (serine, glycine, threonine, and methionine) and a predicted increase in the activity of pathways that receive and donate carbon units (i.e., folate, methionine, and homocysteine). In addition, the targeted and untargeted metabolomics analyses showed similar metabolomics profiles between breast cancer subtypes. The gene set enrichment analysis identified different transcription-related functions between IDC and non-tumor tissues that involved one-carbon metabolism. Our data suggest that one-carbon metabolism may be a central pathway in IDC and even in general breast tumors, representing a potential target for its treatment and prevention.

Potential premalignant status of gastric portion excluded after Roux en-Y gastric bypass in obese women: A pilot study.

We evaluated whether the excluded stomach (ES) after Roux-en-Y gastric bypass (RYGB) can represent a premalignant environment. Twenty obese women were prospectively submitted to double-balloon enteroscopy (DBE) with gastric juice and biopsy collection, before and 3 months after RYGB. We then evaluated morphological and molecular changes by combining endoscopic and histopathological analyses with an integrated untargeted metabolomics and transcriptomics multiplatform. Preoperatively, 16 women already presented with gastric histopathological alterations and an increased pH (≥4.0). These gastric abnormalities worsened after RYGB. A 90-fold increase in the concentration of bile acids was found in ES fluid, which also contained other metabolites commonly found in the intestinal environment, urine, and faeces. In addition, 135 genes were differentially expressed in ES tissue. Combined analysis of metabolic and gene expression data suggested that RYGB promoted activation of biological processes involved in local inflammation, bacteria overgrowth, and cell proliferation sustained by genes involved in carcinogenesis. Accumulated fluid in the ES appears to behave as a potential premalignant environment due to worsening inflammation and changing gene expression patterns that are favorable to the development of cancer. Considering that ES may remain for the rest of the patient's life, long-term ES monitoring is therefore recommended for patients undergoing RYGB.

Fecal bile acid profile after Roux-en-Y gastric bypass and its association with the remission of type 2 diabetes in obese women: A preliminary study.

OBJECTIVE: To assess the influence of Roux-en-Y gastric by-pass (RYGB) on fecal bile acid (BA) profile and its relationship with postoperative remission of type 2 diabetes (T2D). METHODS: Fecal samples were collected 3 and 12 months after RYGB from diabetic obese women who were responsive (n = 12) and non-responsive (n = 8) to postoperative remission of T2D. Fecal BA profile was accessed by liquid chromatography coupled to tandem mass spectrometry in a targeted approach. RESULTS: Relative to pre-operative levels, a total of 10 fecal BA profiles decreased after RYGB (ANOVA, p ≤ 0.05) with significant fold-changes for glycochenodeoxycholic, glycocholic, taurocholic, and taurochenodeoxycholic acids at 3-months postoperatively, and for glycochenodeoxycholic, glycocholic and taurocholic acids at 12 months postoperatively (Benjamini-Hochberg, p ≤ 0.05). Postoperative changes in fecal BA were different between responsive and non-responsive women, with a significant reduction in more sub-fractions of BA in responsive women than in non-responsive women, and a marked difference in the temporal behavior of cholic acid (CA) and chenodeoxycholic acid (CDCA), thus reflecting changes in CA/CDCA ratio, and tauroursodeoxycolic (TUDCA) levels between these responsiveness groups (ANOVA, p ≤ 0.05). CONCLUSION: RYGB induces a marked reduction in the concentration of fecal BA, which is heterogeneous according to T2D responsiveness.

Reduced intestinal FADS1 gene expression and plasma omega-3 fatty acids following Roux-en-Y gastric bypass.

BACKGROUND & AIMS: Roux-en-Y gastric bypass (RYGB) limits food ingestion and may alter the intestinal expression of genes involved in the endogenous synthesis of polyunsaturated fatty acids (PUFAs). These changes may decrease the systemic availability of bioactive PUFAs after RYGB. To study the impact of RYGB on the dietary ingestion and plasma concentration of PUFAs and on the intestinal expression of genes involved in their endogenous biosynthesis in severely obese women with type 2 diabetes. METHODS: Before, and 3 and 12 months after RYGB, obese women (n = 20) self-reported a seven-day dietary record, answered a food frequency query and provided plasma samples for alpha-linolenic (ALA), eicosapentaenoic (EPA), docosahexaenoic (DHA) and arachidonic (ARA) acid assessment by gas chromatography. Intestinal biopsies (duodenum, jejunum and ileum) were collected through double-balloon endoscopy before and 3 months after RYGB for gene expression analysis by microarray (Human GeneChip 1.0 ST array) and RT-qPCR validation. RESULTS: Compared to the preoperative period, patients had decreased intakes of PUFAs, fish and soybean oil (p < 0.05) and lower plasma concentrations of ALA and EPA (p < 0.001) 3 and 12 months after RYGB. FADS1 gene expression was lower in duodenum (RT-qPCR fold change = -1.620, p < 0.05) and jejunum (RT-qPCR fold change = -1.549, p < 0.05) 3 months following RYGB, compared to before surgery. CONCLUSION: RYGB decreased PUFA ingestion, plasma ALA and EPA levels, and intestinal expression of FADS1 gene. The latter encodes a key enzyme involved in endogenous biosynthesis of PUFAs. These data suggest that supplementation of omega-3 PUFAs may be required for obese patients undergoing RYGB. Clinical Trial Registry number and website: www.clinicaltrials.gov - NCT01251016; Plataforma Brasil - 19339913.0.0000.0068.

Tissue-specific methylation profile in obese patients with type 2 diabetes before and after Roux-en-Y gastric bypass.

Eating habits, lifestyles, and exposure to specific environmental factors can greatly impact the risk of developing type 2 diabetes (T2D), influence the genome epigenetically, and affect the expression of genes, including genes related to glycemic control, at any stage of life. The epigenetic mechanism underlying obesity and T2D pathogenesis remains poorly understood. Conventional strategies for the treatment of obesity and its comorbidities often have poor long-term adherence, and pharmacological interventions are limited. Bariatric surgery is the most effective current option to treat severe obesity, and Roux-en-Y gastric bypass (RYGB) is the most applied technique worldwide. Epigenetic changes differ depending on the approach used to treat obesity and its associated comorbidities (clinical or surgical). Compared to primary clinical care, bariatric surgery leads to much greater loss of body weight and higher remission rates of T2D and metabolic syndrome, with methylation profiles in promoter regions of genes in obese individuals becoming similar to those of normal-weight individuals. Bariatric surgery can influence DNA methylation in parallel with changes in gene expression pattern. Changes in clinical biomarkers that reflect improvements in glucose and lipid metabolism after RYGB often occur before major weight loss and are coordinated by surgery-induced changes in intestinal hormones. Therefore, the intestine methylation profile would assist in understanding the mechanisms involved in improved glycemic control after bariatric surgery. The main objectives in this area for the future are to identify epigenetic marks that could be used as early indicators of metabolic risk, and to develop treatments able to delay or even reverse these epigenetic changes. Studies that provide the "human epigenetic profile" will be of considerable value to identify tissue-specific epigenetic signatures and their role in the development of chronic diseases. Further studies should apply methods based on global analysis of the genome to identify methylated sites associated with disease and epigenetic marks associated with the remodeling response to bariatric surgery. This review describes the main epigenetic alterations associated with obesity and T2D and the potential role of RYGB in remodeling these changes.

Phase angle obtained by bioelectrical impedance analysis independently predicts mortality in patients with cirrhosis.

AIM: To evaluate the prognostic value of the phase angle (PA) obtained from bioelectrical impedance analysis (BIA) for mortality prediction in patients with cirrhosis. METHODS: In total, 134 male cirrhotic patients prospectively completed clinical evaluations and nutritional assessment by BIA to obtain PAs during a 36-mo follow-up period. Mortality risk was analyzed by applying the PA cutoff point recently proposed as a malnutrition marker (PA ≤ 4.9°) in Kaplan-Meier curves and multivariate Cox regression models. RESULTS: The patients were divided into two groups according to the PA cutoff value (PA > 4.9°, n = 73; PA ≤ 4.9°, n = 61). Weight, height, and body mass index were similar in both groups, but patients with PAs > 4.9° were younger and had higher mid-arm muscle circumference, albumin, and handgrip-strength values and lower severe ascites and encephalopathy incidences, interleukin (IL)-6/IL-10 ratios and C-reactive protein levels than did patients with PAs ≤ 4.9° (P ≤ 0.05). Forty-eight (35.80%) patients died due to cirrhosis, with a median of 18 mo (interquartile range, 3.3-25.6 mo) follow-up until death. Thirty-one (64.60%) of these patients were from the PA ≤ 4.9° group. PA ≤ 4.9° significantly and independently affected the mortality model adjusted for Model for End-Stage Liver Disease score and age (hazard ratio = 2.05, 95%CI: 1.11-3.77, P = 0.021). In addition, Kaplan-Meier curves showed that patients with PAs ≤ 4.9° were significantly more likely to die. CONCLUSION: In male patients with cirrhosis, the PA ≤ 4.9° cutoff was associated independently with mortality and identified patients with worse metabolic, nutritional, and disease progression profiles. The PA may be a useful and reliable bedside tool to evaluate prognosis in cirrhosis.

Gastrointestinal Transcriptomic Response of Metabolic Vitamin B12 Pathways in Roux-en-Y Gastric Bypass.

OBJECTIVES: Vitamin B12 (B12) deficiency after Roux-en-Y gastric bypass (RYGB) is highly prevalent and may contribute to postoperative complications. Decreased production of intrinsic factor owing to gastric fundus removal is thought to have a major role, but other components of B12 metabolism may also be affected. We evaluated changes in the expression levels of multiple B12 pathway-encoding genes in gastrointestinal (GI) tissues to evaluate the potential roles in contributing to post-RYGB B12 deficiency. METHODS: During double-balloon enteroscopy, serial GI biopsies were collected from 20 obese women (age, 46.9±6.2 years; body mass index, 46.5±5.3 kg/m2) with adult-onset type 2 diabetes (fasting plasma glucose ≥126 mg/dl; hemoglobin A1c≥6.5%) before and, at the same site, 3 months after RYGB. Gene expression levels were assessed by the Affymetrix Human GeneChip 1.0 ST microarray. Findings were validated by real-time quantitative PCR (RT-qPCR). RESULTS: Gene expression levels with significant changes (P≤0.05) included: transcobalamin I (TCN1) in remnant (-1.914-fold) and excluded (-1.985-fold) gastric regions; gastric intrinsic factor (GIF) in duodenum (-0.725-fold); and cubilin (CUBN) in duodenum (+0.982-fold), jejunum (+1.311-fold), and ileum (+0.685-fold). Validation by RT-qPCR confirmed (P≤0.05) observed changes for TCN1 in the remnant gastric region (-0.132-fold) and CUBN in jejunum (+2.833-fold). CONCLUSIONS: RYGB affects multiple pathway-encoding genes that may be associated with postoperative B12 deficiency. Decreased TCN1 levels seem to be the main contributing factor. Increased CUBN levels suggest an adaptive genetic reprogramming of intestinal tissue aiming to compensate for impaired intestinal B12 delivery.

Docosahexaenoic Acid Modulates a HER2-Associated Lipogenic Phenotype, Induces Apoptosis, and Increases Trastuzumab Action in HER2-Overexpressing Breast Carcinoma Cells.

In breast cancer, lipid metabolic alterations have been recognized as potential oncogenic stimuli that may promote malignancy. To investigate whether the oncogenic nature of lipogenesis closely depends on the overexpression of HER2 protooncogene, the normal breast cell line, HB4a, was transfected with HER2 cDNA to obtain HER2-overexpressing HB4aC5.2 cells. Both cell lines were treated with trastuzumab and docosahexaenoic acid. HER2 overexpression was accompanied by an increase in the expression of lipogenic genes involved in uptake (CD36), transport (FABP4), and storage (DGAT) of exogenous fatty acids (FA), as well as increased activation of "de novo" FA synthesis (FASN). We further investigate whether this lipogenesis reprogramming might be regulated by mTOR/PPARγ pathway. Inhibition of the mTORC1 pathway markers, p70S6 K1, SREBP1, and LIPIN1, as well as an increase in DEPTOR expression (the main inhibitor of the mTOR) was detected in HB4aC5.2. Based on these results, a PPARγ selective antagonist, GW9662, was used to treat both cells lines, and the lipogenic genes remained overexpressed in the HB4aC5.2 but not HB4a cells. DHA treatment inhibited all lipogenic genes (except for FABP4) in both cell lines yet only induced death in the HB4aC5.2 cells, mainly when associated with trastuzumab. Neither trastuzumab nor GW9662 alone was able to induce cell death. In conclusion, oncogenic transformation of breast cells by HER2 overexpression may require a reprogramming of lipogenic genetic that is independent of mTORC1 pathway and PPARγ activity. This reprogramming was inhibited by DHA.

Lipid raft disruption by docosahexaenoic acid induces apoptosis in transformed human mammary luminal epithelial cells harboring HER-2 overexpression.

In HER-2-overexpressing breast cells, HER-2 receptors exist on the cell surface as monomers, homodimers and heterodimers. For signal activation and transduction to occur, HER-2 must be localized to lipid rafts. Therefore, we hypothesized that the amount of lipid rafts on the cell membrane would be a factor in HER-2 signaling. To test this, we used HB4a (an untransformed human mammary epithelial cell line) and HB4aC5.2 cells. HB4aC5.2 cells are HB4a derivatives that have been transfected with five copies of pJ5E.c-ErbB-2 and express approximately 900 times more HER-2 than HB4a cells. In these cells, HER-2 overexpression was accompanied by increased lipid rafts in cell membranes, a hyperactivation of downstream Akt and ERK1/2 proteins, and an increased rate of cell growth compared to HB4a. In addition, HER-2 overexpression was associated with an increased activation of FASN, a key enzyme involved in cellular lipogenesis. Its final product, palmitate, is frequently used to synthesize lipid rafts. We further hypothesized that treatment with docosahexaenoic acid (DHA), an omega-3 fatty acid, would disrupt the lipid rafts and lead to a growth arrest. In HB4aC5.2 cells, but not HB4a cells, we found that DHA treatment disrupted lipid raft; inhibited HER-2 signaling by decreasing activation of Akt, ERK1/2 and FASN proteins; and induced apoptosis. Although little is known about lipid rafts, our data support the idea that disturbances in these microdomains induced by DHA may represent a useful tool for controlling the signaling initiated by HER-2 receptors and its therapeutic potential in the treatment of HER-2 positive breast cancer.

Gene expression profile in response to doxorubicin-rapamycin combined treatment of HER-2-overexpressing human mammary epithelial cell lines.

HER-2-positive breast cancers frequently sustain elevated AKT/mTOR signaling, which has been associated with resistance to doxorubicin treatment. Here, we investigated whether rapamycin, an mTOR inhibitor, increased the sensitivity to doxorubicin therapy in two HER-2-overexpressing cell lines: C5.2, which was derived from the parental HB4a by transfection with HER-2 and SKBR3, which exhibits HER-2 amplification. The epithelial mammary cell line HB4a was also analyzed. The combined treatment using 20 nmol/L of rapamycin and 30 nmol/L of doxorubicin arrested HB4a and C5.2 cells in S to G(2)-M, whereas SKBR3 cells showed an increase in the G(0)-G(1) phase. Rapamycin increased the sensitivity to doxorubicin in HER-2-overexpressing cells by approximately 2-fold, suggesting that the combination displayed a more effective antiproliferative action. Gene expression profiling showed that these results might reflect alterations in genes involved in canonical pathways related to purine metabolism, oxidative phosphorylation, protein ubiquitination, and mitochondrial dysfunction. A set of 122 genes modulated by the combined treatment and specifically related to HER-2 overexpression was determined by finding genes commonly regulated in both C5.2 and SKBR3 that were not affected in HB4a cells. Network analysis of this particular set showed a smaller subgroup of genes in which coexpression pattern in HB4a cells was disrupted in C5.2 and SKBR3. Altogether, our data showed a subset of genes that might be more robust than individual markers in predicting the response of HER-2-overexpressing breast cancers to doxorubicin and rapamycin combination.

Complementarity of Subjective Global Assessment (SGA) and Nutritional Risk Screening 2002 (NRS 2002) for predicting poor clinical outcomes in hospitalized patients.

BACKGROUND & AIMS: We evaluated the ability of Nutritional Risk Screening 2002 (NRS 2002) and Subjective Global Assessment (SGA) to predict malnutrition related to poor clinical outcomes. METHODS: We assessed 705 patients at a public university hospital within 48 h of admission. Logistic regression and number needed to screen (NNS) were calculated to test the complementarity between the tools and their ability to predict very long length of hospital stay (VLLOS), complications, and death. RESULTS: Of the patients screened, 27.9% were at nutritional risk (NRS+) and 38.9% were malnourished (SGA B or C). Compared to those patients not at nutritional risk, NRS+, SGA B or C patients were at increased risk for complications (p=0.03, 0.02, and 0.003, respectively). NRS+ patients had an increased risk of death (p=0.03), and SGA B and C patients had an increased likelihood of VLLOS (p=0.008 and p<0.0001, respectively). Patients who were both NRS+ and SGA C had lower estimates of NNS than patients who were NRS+ or SGA C only, though their confidence intervals did overlap. CONCLUSIONS: The concurrent application of SGA in NRS+ patients might enhance the ability to predict poor clinical outcomes in hospitalized patients in Brazil.