bluuebunny/crossref_metadata_2025 huggingface dataset:
Ethanol increases histone H3‐K4 methylation and decreases histone H3‐K9 acetylation in conjunction with increased p16 gene expression in the normal colonic epithelial cells
Authors: Jang Hyeran , Barlev Nickolai A. , Ausman Lynne M. , Mason Joel B. , Moyer Mary P. , Choi Sang‐Woon
Published: June 2021

The objective of this study was to determine whether disturbances in one‐carbon metabolism could modify p16 gene expression through histone modifications. NCM460, human colonic epithelial cells, were incubated in media 1) deficient in folate, methionine and choline, 2) with 100μM Adox (adenosine‐2', 3′‐dialdehyde, an S‐adenosylhomocysteine hydrolase inhibitor), and 3) with 100mM ethanol, which inhibits methionine synthesis and reduces methyl group availability. Histone methylation at histone H3 lysine 4 (H3‐K4) and acetylation at lysine 9 (H3‐K9), both known to be increased in active genes, were measured by Western blotting and p16 gene expression by RT‐PCR. Ethanol increased H3‐K4 trimethylation (p=0.005) and decreased H3‐K9 acetylation (p<0.0001) in a time‐dependent fashion. These changes in histone modifications were paralleled with increased p16 expression (p=0.003). Adox decreased H3‐K9 acetylation time‐dependently (p=0.004) without any change in p16 expression. Folate/methionine/choline deficiency didn't produce any changes in this in vitro culture model. This study demonstrates that in this normal colonic cell line, ethanol, which inhibits one‐carbon metabolism, alters H3‐K4 trimethylation and H3‐K9 acetylation in conjunction with an increase in p16 expression.

Grant Funding Source

NIH R21 AA016681
Roasted licorice extracts prevent chronic high glucose‐induced accumulation of glomerular mesangial matrix
Authors: Li Jing , Choi Jung‐Suk , Cho Sung‐Hee , Kang Young‐Hee
Published: June 2021

Diabetic nephropathy (DN) characterized as glomerulosclerosis can cause renal failure and end‐stage renal disease. Expansion of glomerular mesangial matrix mainly comprised of type IV collagen is a prominent feature of DN. Enhanced expression and secretion of connective tissue growth factor (CTGF) contribute an important role in this process. The present study is to investigate whether roasted licorice extracts (rLE) at non‐toxic doses can inhibit production of high glucose‐induced type IV collagen as well as CTGF in human mesangial cells (HRMC). Serum starved HRMC were cultured in media containing 5.5 mM glucose plus 27.5 mM mannitol as an osmotic control or 33 mM glucose for 3 days in the presence of 10 µg/mL rLE. Exposure of HRMC to high glucose resulted in a significant increase in collagen type IV secretion and CTGF expression, which was reversed by rLE. These effects were much greater than raw licorice extracts that exhibited a weak inhibition when extracted in ethanol. Isoliquiritigenin, a bioactive component of rLE, appeared to attenuate high glucose‐stimulated mesangial matrix accumulation. The results demonstrate that rLE may be potential therapeutic agents for the prevention and treatment of diabetic nephropathy. Supported by Brain Korea 21 from Korea Research Foundation and the grant (R01‐2006‐000‐10896‐0) from Korea Science and Engineering Foundation.
Effect of Flavonoids and Low Level Pesticide Exposure on Copper and Zinc Homeostasis in Rats
Authors: Bebe Frederick N. , Panemangalore Myna
Published: June 2021

The protective effect of flavonoids against disease is well‐documented, but little is known of their anti‐nutritional effects and potential interaction with pesticides. Two 30/d experiments (36 male SD rats each) were conducted to determine effect of administering flavonoid mixture (FM) with concomitant exposure to a pesticide mixture (PM) or FM at varied levels on changes in concentrations of zinc and copper in tissues. Experiment1: PM=chlorpyrifos, endosulfan and thiram at 25% LD50 was dissolved in SB oil and gavage‐fed 0.1mL 5days/wk; FM in SB oil was mixed in AIN‐93M diet at 1.0 and 5.0 mM/kg diet and fed at libitum. Groups = Control (CON), PM, FM1, FM5, PM+FM1 and PM+FM5. Experiment 2: FM in 1% polyethylene glycol was gavage‐fed (0.5mL/5 days/wk), at increasing levels(mg/kg bw): Groups = CON, FM5, FM25, FM50, FM100 and FM200. Experiment 1: PM plus FM increased tissue Cu as compared to the CON by: plasma16‐37%, liver (9‐20%), kidney (13‐41%), small intestinal mucosa (IM) 41% and small intestines (SI) 77%. Experiment 2: FM fed at higher levels decreased tissue Cu by: plasma 22‐36%, liver (23%), kidney (24‐44%), IM (17‐26%), SI (58 & 77%) as compared to respective CON. Kidney zinc increased at higher concentrations of FM by 14‐34%. These results indicate that flavonoids can modify copper homeostasis depending on whether they are fed in the diet or by gavage. (USDA#KYX‐50‐03‐17H).
Whole grain consumption does not slow diabetic progression in GK rats
Authors: Youn Moonyeon , Csallany A. Saari , Gallaher Daniel D.
Published: June 2021

Whole grain consumption is inversely associated with risk of type II diabetes epidemiologically. The aim of this study was to study the effect of whole grain consumption on diabetic control and progression in an animal model of type II diabetes, the Goto‐Kakisaki (GK) rat. GK rats were fed either a whole grain‐free control diet or a whole grain‐containing diet for 5 months. Whole grain diets contained 65% whole grain flours of either wheat, barley, oat, or corn. After two months, fasting plasma glucose concentration was significantly lower with the wheat, barley, or oat diets compared to the control diet. However, glycated hemoglobin was significantly greater in the wheat group compared to other groups. At 5 months, whole grain diets did not improve glycemic control. Fasting plasma insulin and C‐Peptide levels were decreased from month 2 to month 5 indicating diabetic progression. Insulin resistance, determined by the homeostasis model assessment, was not significantly different among groups at either 2 or 5 months. However, there was a trend for improvement of insulin resistance with the barley and oat diets at 2 months (P=0.06). There were no significant differences in the total pancreatic β‐cell mass among groups. These results offer little support for improvement of diabetic control and delayed progression with consumption of whole grains. (Supported by the General Mills Bell Inst. of Health and Nutrition)

Grant Funding Source

General Mills Bell Inst. of Health and Nutrition
The effects of portabella mushrooms on bone mineral density and body composition in mice without and with collagen induced arthritis.
Authors: Traore Djibril , Petersorn Sandra , Lim Yin , Alexander Heather , Sharp Dean , Clarke Stephen , Lucas Edralin , Smith Brenda , Kuvibidila Solo
Published: June 2021

Certain exotic mushrooms contain beta‐glucan that has been shown to modulate immunity and provide health benefits. However, little information is available on the health benefits of portabella mushrooms (PM). The objective of the study was to assess the effects of PM on indices of nutritional status in mice without and with collagen‐induced arthritis (CIA). Eight‐week old, DBA female mice were fed either the AIN76 (baseline) diet or the same diet fortified with 5% lyophilized PM (20/group) for 6 weeks. To induce CIA, 50% of mice received 100 ug bovine collagen type II intradermally followed by 50 ug LPS 20 days later. Lean and fat mass, bone mineral density, and various organ weights were assessed by standard techniques 10 days after LPS injection. Between 90‐100% of mice that received collagen developed mild‐moderate paw swelling. In PM and baseline fed mice, CIA reduced body weight and thymus weights by 13%‐47%, doubled spleen weights, and increased relative weights of heart, liver, and kidney by 20‐24.5% for PM and 22‐28% for baseline diet (p <0.05). Non‐CIA and CIA mice fed PM diet had higher percent of body fat (32.6% & 25.8%) than those fed baseline diet (25% & 21.9%) (p <0.05). Compared to baseline diet, PM attenuated thymus atrophy (13% vs 47%) and loss of lean mass (10% vs 17%) after CIA induction (p<0.05). PM slightly (4‐5%) increased bone mineral content but not bone mineral density in CIA and non‐CIA mice. Data suggest that PM increase the percent of body fat which may attenuate muscle protein loss and minimize thymus atrophy hence T cell numbers in rheumatoid arthritis. Supp.: Grant # 580790706 (Mushroom Council/USDA/NutriCore) and funds from Oklahoma State University.

Grant Funding Source

The Mushroom Council/USDA
Freeze‐dried strawberry powder lowers total cholesterol and LDL‐cholesterol in females with abdominal adiposity and dyslipidemia
Authors: WILKINSON MARCI , SIMMONS BRANDI , BETTS NANCY , BASU ARPITA
Published: June 2021

Strawberry flavonoids are potent antioxidants and anti‐inflammatory agents and have been shown to reduce cardiovascular mortality in prospective studies. In this feeding study we tested the hypothesis that freeze‐dried strawberry powder (FSP) will lower fasting lipids and biomarkers of oxidative stress compared to baseline. Females (n=16) with abdominal adiposity (BMI = 30, waist circumference >35 inches) and elevated lipids (total cholesterol >200 mg/dL, or triglycerides > 150mg/dL) or low high‐density lipoproteins (HDL <50mg/dL) were fed 2 cups of the strawberry drink daily. Each cup had 25g of FSP blended in water. Subjects made 3 days/week visits for 4 weeks. Fasting blood draws, anthropometrics and blood pressure measurements were done at baseline and 4 weeks. FSP significantly reduced total cholesterol and low‐density lipoproteins (LDL)‐cholesterol levels at 4 weeks versus baseline {195.25±6.23mg/dL vs. 205.56±7.18 mg/dL (p=0.017), and 114.93±4.83 mg/dL vs. 124.27±6.37 mg/dL (p= 0.038), respectively}. Oxidized‐LDL showed a decreasing trend at 4 weeks (p=0.123). Thus, freeze‐dried strawberry powder exerts hypocholesterolemic effects and lowers CVD risk factors in obese women. (Funded by CHES, OSU)
Responsiveness of ARNT‐deficient mouse hepatoma (BPRc1) cells transfected with HIF‐1beta to oxidative stress and antioxidants
Authors: KIM Jong‐Sang , LEE In Ae , SEO Ji‐Yeon , KIM Hyo Jung , Garcia Annalene , LIM Ji‐Sun , Yoon Park Jung Han
Published: June 2021

Our previous study showed that phosphoglycerate kinase 1 (PGK1) may be a potential biomarker for oxidative stress. However, the expression of hypoxia inducible factors (HIFs) known as a major upstream regulator of glycolytic enzymes including PGK1 was little affected by hydrogen peroxide but significantly downregulated by antioxidants. Furthermore, PGK1 level in BPRc1 cells lacking HIF‐1beta remained unchanged regardless of treatment of either hydrogen peroxide or delphinidin, suggesting the importance of heterodimer formation between HIF‐1alpha and HIF‐1beta in the regulation of PGK1 expression. PGK1, therefore, appears to be a reliable protein biomarker for oxidative stress or antioxidant potential, and its expression seems to be mediated by the intracelluar level of HIF‐1alpha. To confirm the role of HIF‐1beta in cellular response to oxidative stress and antioxidant, we constructed HIF‐1beta cDNA cloning vector and expression vector using pcDNA3.1/myc‐His construction. The full‐length cDNA of mHIF‐1beta was cloned from mouse HIF‐1beta by RT‐PCR using primers corresponding to the sequence BC012870. The transfectants generated by transfecting BPRc1 with HIF‐1beta will be used to evaluate whether BPRc1 cells insensitive to oxidative stress and antioxidants restore their responsiveness to intracellular redox status by introduction of HIF‐1beta gene or not.
Effects of prebiotic Quercetin on proliferation and beta‐catenin expression in colon cancer cell lines
Authors: Marzo Florencio , Alfaro Maria , Jauregui Patricia , Benito Ivan , Santidrian Santiago , Urdaneta Elena
Published: June 2021

Colon cancer incidence is increased in the occidental countries, prevention is very important to reduce the incidence of this disease. Functional foods could play a modulating function on colon cancer development. The aim of our study was to study the cellular and molecular effects of quercetin on the proliferation of human HT‐29 colon cells.

HT‐29 cells were obtained from the European Collection of Cell Culture (EACC) and cultured in Dulbecco's modified Eagle's medium supplemented. At 80% confluence, cells were plated in 96‐well plates at 4x104cells/well and then were incubated in serum‐free medium in the presence or absence of various concentrations of quercetin (50µM‐ 125µM) during 72h. Cell proliferation was measured by MTT assay. â‐Catenin plays an important role in the Wnt signaling pathway. The expression of â‐catenin in cells treated was analysed by western blott.

Quercetin inhibited the proliferation of HT‐29 cells, being the cells treated with quercetin presented a significant decrease (p<0.001) in growth cells compared with control group. â‐catenin expression in the cells treated with quercetin during 48h is lower compared with cells treated during 24h and control group. This data suggests that quercetin shown beneficial effects related to cell proliferation, showing an inhibitory effect.

This work was supported by Spanish Education and Science Ministry (AGL2006‐10296).
Antimutagenicity and anticancer effect of Korean mistletoe (viscum album coloratum) in HT‐29 human colon carcinoma cells
Authors: Park Kun‐Young , Kim Boh‐Kyung , Bak Soon‐Sun , Kim So‐Hee
Published: June 2021

The antimutagenic and anticancer effects of Korean mistletoe (Viscum album coloratum) were investigated under in vitro and cellular system using HT‐29 human colon carcinoma cells. The water extract from the mistletoe showed antimutagenity against mutagenicities induced by aflatoxin B1 and N‐methyl‐N′‐nitro‐N‐nitrosoguanidine in Salmonella typhimurium TA100 as concentration dependent‐manner. Moreover, under HT‐29 cellular model, the mistletoe extract led significant decreases in cell viability in MTT assay and growth inhibition test. The results of DAPI staining showed that mistletoe extract induced apoptosis in HT‐29 cells and the ratio of apoptotic body increased as dose dependent. Mistletoe‐induced apoptosis in HT‐29 cells was associated with the decreased expression of the anti‐apoptotic Bcl‐2 and a concomitant increase of the pro‐apoptotic Bax protein and mRNA. In addition, mistletoe treatment induced marked accumulation of tumor suppressor p53 and an induction of cyclin‐dependent kinase inhibitor p21. The present study suggests that Korean mistletoe extract would have the chemopreventive effects.
Kinetic modeling and biological system dynamics with Microsoft Excel: how to estimate parameters and solve equations for problems in nutrition
Authors: Hargrove James L. , Smith Jeffrey W.
Published: June 2021

Microsoft Excel and similar freeware are powerful tools for mathematical and statistical analysis that are widely available but underutilized in nutritional research and teaching. For example, it is desirable to establish dose‐response curves and study oral dosing, bioavailability, metabolism and tissue effects of dispensable and indispensable nutrients and phytochemicals. We show how to use the Solver function to estimate parameters that provide best fits of data to specific equations. In addition, we have developed cell formulas and/or used Excel's accessory program, Visual Basic for Applications, to develop integrators based on methods of Euler and Runge‐Kutta that solve finite difference equations by iterative methods. With this approach, one may solve equations developed for kinetics and dynamics of specific nutrients or phytochemicals, and address modeling topics that range from changes in gene expression to effects on growth or deficiency conditions. This abstract will compare results of solving an analytical equation for approach to steady state of a single compartment with the same equation as solved by iterative, numerical methods. We will then show how to link several compartments and set up a systems model. In this manner, Microsoft Excel can perform simulation, analysis and modeling much as specialty modeling software does, but without the cost of an additional site license.
Using a Model of Human Energy Metabolism to Estimate Energy Costs of Turnover
Authors: Johnson Heidi , Keim Nancy , Calvert Chris
Published: June 2021

The goal of this research is to use a computational model of nutrient digestion and metabolism in a human to predict energy use and quantify effects of increased futile cycling on energy metabolism. The model is composed of 6 state variables representing amino acids, body protein, visceral protein, glucose, triglyceride, and fatty acids. Differential equations represent carbohydrate, amino acid, and fatty acid uptake and output by tissues. The model estimates metabolizable energy, heat production (HP), respiratory quotient, P/O ratio, energy balance and glucose and fatty acid oxidation based on ATP creation and use, O2 use, CO2 production, heats of combustion, and nutrient flux. Protein deposition is a function of maximum genetic potential for protein accretion. Adipose deposition is a function of energy balance. To examine effects of increasing protein and adipose turnover, protein turnover increased from 2 to 14 %/day and adipose turnover increased from 1.5 to 3.0 %/day for a 70 kg male. Increasing protein turnover increased HP from 11.2 to 20.5 MJ/day, increased glucose oxidation from 1.88 to 2.29 mole/day, increased fatty acid oxidation from 0.132 to 0.873 mole/day and decreased body weight by 6 kg over a 30 day simulation. Increasing adipose turnover decreased HP from 10.8 to 10.7 MJ/day, decreased glucose oxidation from 2.18 to 2.04 mole/day, increased fatty acid oxidation from 0.0200 to 0.0543 mole/day, and decreased body weight by 4.5 kg over a 30 day simulation. Therefore the model is able to predict body weight loss due to increased heat production from increased energy use as a result of changes in muscle and adipose turnover. Since humans are genetically diverse with a range of basal metabolic rates, activity levels and diets, a computational model will quantify diversity and predict metabolic function for a wide range of individuals.
Secondary effects of 'Duffy Antigen Receptor for Chemokines' (DARC) blockade on inflammation and hemostasis
Authors: Singbartl Kai , Schmolke Mirco , Bishop Jeffrey , Zarbock Alexander
Published: June 2021

Introduction

Unlike typical chemokine receptors, DARC is presented on red blood and endothelial cells but not on leukocytes. We have previously shown that DARC‐deficient mice (DARC‐/‐) display protection from LPS‐induced kidney injury and lower serum CXC‐chemokine levels. The latter one implies changes in CXC chemokine receptor 2 (CXCR2) expression on neutrophils (PMN). Here, we sought to compare CXCR‐2 homeostasis and its biological effects in wild‐type (WT) and DARC‐/‐ mice.

Results

Before LPS stimulation, DARC‐/‐ showed 3x higher CXCR2 expression on PMN than WT (*). Afterwards, DARC‐/‐ revealed greater CXCR2 down‐regulation than WT (*), suggesting desensitization of PMN in DARC‐/‐. Compared to WT, DARC‐/‐ showed impaired PMN recruitment in the lung and full protection from ALI (*). Adoptive transfer of PMN proved that these effects were solely due to PMN from DARC‐/‐. DARC‐/‐ revealed prolonged bleeding time and decreased TEG amplitude (*), suggesting impaired platelet‐platelet or platelet‐PMN interactions. Conclusions : Alteration in serum CXC‐chemokine levels and subsequent changes in CXCR2 homeostasis appear to have profound anti‐inflammatory and anti‐hemostatic effects.
Regulation of Cell Adhesion by the Physical Properties of Fibrin(ogen) Substrates
Authors: Yermolenko Ivan , Fuhrmann Alexander , Podolnikova Nataly , Magonov Sergei , Ros Robert , Ugarova Tatiana
Published: June 2021

The physical properties of substrates are crucial to the regulation of cell adhesion through integrin‐mediated signaling. The matrices formed of fibrin(ogen) represent the natural substrates whose variable rigidity may control adhesion of blood cells. Binding of fibrinogen to the surface of fibrin gel prevents firm adhesion of leukocytes and platelets creating the anti‐adhesive layer. Also, fibrinogen immobilized on surfaces at high density supports weak adhesion whereas at low density it is highly adhesive. To understand the mechanism underlying differential cell adhesion, we have characterized the structural and physical properties of surfaces prepared by deposition of different concentrations of fibrinogen using atomic force microscopy and force spectroscopy. Fibrinogen deposited at high density results in the multilayered material characterized by low rupture forces. In contrast, immobilization of fibrinogen at low density produces a single layer in which molecules are directly attached to the solid surface resulting in higher rupture forces. Consistent with their distinct physical properties, low, but not high density fibrinogen induces strong intracellular signaling and cell spreading. The data suggest that multilayered fibrinogen prevents cell adhesion by modifying the physical properties of surfaces which results in reduced force generation and insufficient signaling.

HL63199
Biomarker profiling in elderly patients with acute hip fracture: modulation by prophylactic dosage of enoxaparin
Authors: Knesek Michael , Litinas Evangelos , Lasssen Michael , Borris Lars , Hopkinson William , Hoppensteadt Debra , Fareed Jawed
Published: June 2021

Hip fracture (HF) is common in the elderly and associated with a high risk of venous thromboembolism. Activation of inflammation and coagulation results in generation of various biomarkers. This study is designed to identify biomarkers in elderly HF patients using protein chip array methods. Plasma samples from elderly patients included in a randomized HF study comparing enoxaparin (40 mg o.d.s.c.) and UFH (5000 IU b.i.d.s.c.) were collected prior to and at 1, 3, 5 and 7 days post‐surgery. 37 samples were analyzed using surface enhanced laser desorption ionization‐time of flight mass spectroscopy to identify biomarkers with molecular weights <150 kDa. Plasma from 29 healthy volunteers and pooled plasma from total knee/hip replacement (TKR/THR) patients containing an 11.9 kDa biomarker were used as quality controls. In healthy individuals, no unique peaks were noted in comparison to normal human plasma. 8.1% of plasmas obtained prior to HF surgery exhibited unique biomarkers. On day 1 post‐surgery, 83% showed a distinct peak at 11.9 kDa. On day 3, 94% patients showed the presence of this biomarker at its strongest intensity. 55% showed this biomarker on day 5 and 12.5% on day 7. Consistent with earlier findings, these results demonstrate that HF in the elderly is associated with generation of a unique biomarker at 11.9 kDa. Unlike the TKR/THR patients, the elderly HF patients exhibited sustained generation of this marker.
Fibrinokinetic Deficit in Chronic Kidney Disease and End Stage Renal Disease Patients Contributes to the Hemostatic Abnormalities
Authors: Adiguzal Cafer , Bansal Vinod , Cunanan Josephine , Litinas Evangelos , Hoppensteadt Debra , Fareed Jawed
Published: June 2021

Increased bleeding occurs in patients with chronic kidney disease (CKD) or end stage renal disease (ESRD) despite a normal coagulation profile and fibrinogen level and may be due to defects in fibrin formation. The fibrinokinetic profile of CKD (n=50) and ESRD patients on hemodialysis and healthy normals was measured. Citrated plasma was supplemented with thrombin and CaCl2. The rate of fibrin formation was measured by monitoring optical density (OD) at 405 nM. After reaching steady state, urokinase was added to measure the fibrinolytic profile. Fibrinokinetic profiles for normals showed strong clot formation (average OD = 1.2±0.3;range 0.7‐1.4). In CKD patients a much weaker clot was formed (average OD of 0.21±0.13;range 0.05‐0.41). Pre‐dialysis samples from ESRD patients showed a weaker fibrinokinetic profile reaching near normal levels with a clot density of 1.3±0.4 (range 0.8‐1.6). CKD patients' plasma exhibited a much stronger fibrinolytic index compared to the normal population (80% vs. 20% lysis). In ESRD patients, clot lysis was weaker compared to the CKD patients. These results are contrary to previous observations that dense clot resistant to fibrinolysis is formed in CKD and ESRD patients. Maintenance hemodialysis results in improving the fibrinokinetic and fibrinolytic profile in the ESRD patients and may contribute to improved hemostasis in ESRD patients.
Neutrophil Adhesion to Periostin is Mediated by Integrins
Authors: Reaves Titus A. , Liu Yuan , Norris Russell , Borg Thomas , Lebel David P.
Published: June 2021

Neutrophil (PMN) localization at the lumenal epithelium surface is a key histological feature of the large intestine of inflammatory bowel disease ((IBD), Crohn's Disease and Ulcerative Colitis) patients. Extracellular matrix proteins (ECM) can affect PMN recruitment and other functions. Periostin, an ECM protein secreted by fibroblasts during cellular injury and tissue repair, was investigated as an adhesive molecule for PMN. Immunofluorescence reveals that periostin similarly to the subepithelial fibroblasts is confined to the base of the crypt epithelium. Flow Cytometry and adhesion results show that intestinal fibroblasts and T84 epithelial cells adhere to periostin and express periostin on the cell surface. Anti‐αvβ3 and anti‐αvβ5 (known integrin receptors for periostin) antibodies inhibit periostin adhesion to fibroblasts and T84 cells. In particular, anti‐αvβ3 and anti‐αvβ5 antibodies inhibit 50% of adhesion. However, anti‐CD11b antibodies inhibit 95% of this adhesion. PMN surface expression of periostin is reduced when PMN are exposed to microbial products and anti‐periostin antibodies inhibit PMN adhesion to fibrinogen (a known ligand for CD11b, a precursor to fibrin, and important in the inflammatory response) by 85%. Thus, PMN adhesion to periostin can be modulated by αvβ3, αvβ5 and CD11b integrins and may play a role in PMN‐epithelial interactions in IBD.
Salmonella enterica serovar Typhimurium flagellin modulates CD4+ T cell apoptosis in Peyer's patches and spleen
Authors: Fournier Benedicte Marie , Gewirtz Andrew , Williams Ifor , Neish Andrew
Published: June 2021

Salmonella enterica serovar Typhimurium, a Gram‐negative pathogen, causes human gastroenterocolitis. In this study, we examined the role of flagellin in the apoptosis of Peyer's patch (PP) cells during Salmonellosis. Apoptosis is a crucial mechanism that eliminates cells without releasing noxious components. We used both enterocolitis and systemic typhoid fever models in C57BL/6 mice, with various isogenic Salmonella strains. In the enterocolitis model, an aflagellate C−B− mutant increased apoptosis of PP CD4+ T cells at 12 h post‐infection compared to the wild‐type strain, whereas the aflagellate mutant slightly decreased cell death at 24 h. In the typhoid model, the aflagellate mutant modulated CD4+ T cell apoptosis differently in PP and spleen on day 4 post‐infection. As ICAM‐1 is a costimulatory molecule involved in T cell activation, we examined whether the expression of this adhesion molecule was modified in the absence of flagellin. Interestingly, the aflagellate mutant modified ICAM‐1 expression on macrophages only in PPs but not in mesenteric lymph nodes (MLN). We also observed that the absence of flagellin elicits an increase of macrophages in PPs and MLN as well as an increased bacterial load in these organs. In conclusion, flagellin seems to be involved in the apoptosis of CD4+ T cells in several organs. However, its role appears to depend on the model, the organ and the stage of the infection.
B cells compartement in centenarian offspring and old people
Authors: Bulati Matteo , Colonna‐Romano Giuseppina , Vitello Salvatore , Pellicanò Mariavaleria , Vasto Sonya , Candore Giuseppina , Caruso Calogero
Published: June 2021

Immunosenescence is considered a major contributory factor to the increased frequency of morbidity and mortality among elderly. Immunosenescence studies focus mainly on T cells, although B cells are affected too. In our studies we analyzed B cells in two classes of individuals: old people (O), (age range 70‐85 years) and centenarian offspring (CA). Our data showed difference in number of B cells from O and CA. B cells were analyzed according to the expression of sIgD and CD27. B cells were classified as follows: IgD+CD27−(naïve),IgD+CD27+(unswitched‐memory),IgD−CD27+ (switched ‐memory) and IgD−CD27−(double negative, DN). As expected, in both cohorts we observed a marked decreased of B cell count although within the B cell population, CA do not behave as O subjects. Indeed, in CA , naïve B cells are more abundant whereas DN B cells don't show the typical increase that we have previously demonstrated in healthy elderly donors. So, B cells of the CA seem to be more similar to that of young respect to the old one. B cell subsets changes could represent a hallmark of immunosenescence and could be used as a biomarker of human life span, potentially useful for the evaluation of anti‐ageing treatment.

This work was supported by grants from the Italian Ministry of Education, University and Research (MIUR, ex 40%)
Comparison of histochemical methods for murine eosinophil detection in a RSV vaccine‐enhanced inflammation model
Authors: Griffin Michelle Ann , Castilow Elaine M , Varga Steven M , Meyerholz David K
Published: June 2021

Five histochemical methods previously reported for eosinophil detection were quantitatively and qualitatively compared in an established murine RSV vaccine‐enhanced inflammation model. Nonspecific neutrophil staining was evaluated in tissue sections of neutrophilic lesions and bone marrow from respective animals. Eosinophils had granular red to orange‐red cytoplasmic staining, depending on the method, whereas neutrophils had a more homogenous cytoplasmic pattern. Nonspecific background staining of similar coloration was variably seen in arterial walls and erythrocytes. Astra Blue/Vital New Red (AB/VNR), Congo Red, Luna, Modified Hematoxylin & Eosin (Mod HE), and Sirius Red techniques were all effective (p<0.001) in detecting increased eosinophil recruitment compared to controls; however, differences in absolute eosinophil quantification were seen. AB/VNR had the best specificity for differentiating eosinophils and neutrophils, but had a significantly reduced ability to enumerate eosinophils. The Luna stain had excessive non specific staining of tissues and a reduced enumeration of infiltrating eosinophils making it suboptimal. For multiple parameters such as eosinophil detection, specificity, and contrast with background tissues, the Sirius Red followed by Congo Red and Mod HE methods were useful, each with their own staining qualities. Supported by NIH and UI Department of Pathology.
Asbestos bodies in lung tissue are associated with pathologic features and patient outcome for epithelial but not non‐epithelial mesothelioma treated by extrapleural pneumonectomy
Authors: Richards William G , Godleski John , Katler Marshall , Mueller Jordan , Sugarbaker David
Published: June 2021

We examined patient survival and pathologic tumor features in relation to asbestos body (AB) counts in lung tissue from patients with malignant pleural mesothelioma (MPM).

Lung tissue was digested with Clorox® by standard methods. AB counts were made using phase contrast light microscopy. Kaplan‐Meier estimates of overall survival were compared using logrank tests for dichotomized AB count in WHO epithelial (E) and non‐epithelial (NE) MPM subgroups. Association of AB count with reported pathological features was assessed using Fisher's Exact test.

From 1988 to 2005, 365 E (72% male; median age 57) and 194 NE patients (90% male; median age 60) were treated. Of these, 234 (64%) E and 142 (73%) NE had AB evaluation. Median AB count for E was 265/g (wet weight) vs. 1084/g for NE and 20/g control AB count. Among E patients, 105 with AB≤200/g had 27‐mo. median survial vs 17.1 mo. for 129 with >200 AB/g (p=0.0003). By contrast, survival for 32 NE with AB≤200/g (9.5 mo.) did not differ from 109 NE with >200 AB/g (12.7 mo.;p=0.58). AB counts above median were significantly associated with involvement of lung parenchema (p=0.0055) and intralobar fissure (p=0.0176) for E but not NE. No relation of AB to diaphragm, pericardium or chest wall involvement was evident.

We conclude that AB has prognostic significance in MPM and that E and NE represent distinct disease entities.

Supported by the International Mesothelioma Program
Anti‐inflammatory benefits of an antibiotic via modulation of neutrophil and macrophage function: The example of tulathromycin
Authors: Fischer Carrie Dawn , Beatty Jennifer K , Morck Douglas W , Lucas Merlin J , Robb Edward J , Buret Andre G
Published: June 2021

The pathogenesis of bacterial‐induced inflammatory diseases is due, in part, to the host immune response. The ability of an antibiotic to modulate immune cell function during infection may confer anti‐inflammatory benefits. Tulathromycin (TUL), an antibacterial agent for bovine respiratory disease, is a novel model for investigating immuno‐modulating effects of antibiotics. Studies have shown that TUL exerts pro‐apoptotic effects in bovine neutrophils (PMN). Nuclear factor‐¿B (NF¿B), a transcription factor implicated in the onset of inflammation, is known to inhibit apoptosis. The effects of TUL on macrophage function are unclear.

Aims

To investigate the mechanisms of TUL‐induced PMN apoptosis and to determine the effects of TUL on bovine macrophage (MØ) function.

Results

ELISA revealed TUL‐induced PMN apoptosis is caspase‐3 and ‐8 dependent. Western blotting showed TUL reduces phosphorylation of NF¿B inhibitor, I¿B, in zymosan‐stimulated PMN. Light microscopy suggested TUL stimulates phagocytosis of apoptotic PMN in MØ. ELISA and Greiss reaction showed TUL reduced secreted levels of pro‐inflammatory interleukin‐8 and nitric oxide in E. coli lipopolysaccharide‐challenged MØ.

Conclusion

Modulation of PMN and MØ function by TUL is associated with inhibition of NF¿B signaling. These findings illustrate novel mechanisms through which an antibiotic may deliver anti‐inflammatory benefits.
Haplotypes encompassing the PSMA6 and KIAA0391 Gene Cluster confer risk for Myocardial Infarction
Authors: Dzimiri Nduna , Muiya Paul , Mohamed Gamal , Alsmadi Osama , Al‐Saud Haya , Al‐Mohanna Futwan , Alshahid Maie , Meyer Brian
Published: June 2021

The proteasomal alpha subunit type 6 (PSMA6) gene is a multi‐catalytic endopeptidase believed to play an important role in regulating inflammatory processes, largely contributory to cardiovascular‐related events. It constitutes part of the KIAA0391 gene, which has similarly been implicated in some cardiovascular events. However, the involvement of this gene cluster in myocardial infarction (MI) remains poorly understood. We genotyped, using molecular beacons, five single nucleotide polymorphisms (SNPs) within this gene cluster in 1135 Saudi individuals with MI and 866 MI‐free subjects. The rs4981283 denoted as 5 [O.R. (95% C.I.) = 1.16 (1.01‐1.33); p=0.035] conferred risk, while the other four SNPs, rs8008319 (1), rs7157492 (2), rs1038990 (3), and rs12878391 (4) were not associated with MI. In contrast, three haplotypes constructed from these SNPs, consisting of 1T‐2G‐3C‐4G‐5A (χ2=4.19; p = 0.05), 1A‐2A‐3C‐4A‐5A (χ2 = 7.69; p = 0.010) and 1T‐2G‐3C‐4A‐5G (χ2 = 10.39; p = 0.020) were identified as risk factors for MI. No confounding effect of gender, age, type 2 diabetes mellitus, hyperlipidaemia, hypertension or obesity was observed on the relationships of the individual SNPs with the disorder. Therefore, our study identified three haplotypes on chromosome 14q13.2 encompassing the KIAA0391 and PSMA6 genes as conferring risk for MI. The results also point to haplotyping as a more robust tool than the individual SNP genotyping in assessing the role of gene polymorphisms in disease.
HIV protease inhibitors induce ER stress and apoptosis in human endothelial cells
Authors: Sun Lixin , Wu Xudong , Cao Risheng , Zha Weibin , Pecora Beth S , Gurley Emily C , Studer Elaine , Hylemon Phillip B. , Pandak William M. , Zhang Luyong , Hu Yiqiao , Wang Guangji , Zhou Huiping
Published: June 2021

Endothelial dysfunction/injury represents a key step in the early stage of atherogenesis. Recent studies have shown that HIV protease inhibitors (PIs) induce endothelial dysfunction, but the underlying molecular mechanism remains unclear. Our previous studies have shown that HIV PI‐induced ER stress and activation of the unfolded protein response (UPR) represents an important cellular mechanism by which HIV PIs disrupt lipid metabolism and induce inflammation in hepatocytes and macrophages. The aim of this study was to determine if HIV PIs also induce ER stress and apoptosis in human endothelial cells.

Methods

Human umbilical vein endothelial cells (HUVECs) were treated with individual HIV PIs for various time periods. The mRNA and protein levels of ER stress responsive genes were measured by real‐time RT‐PCR and Western blot analysis, respectively. The apoptosis was detected by fluorescence microscopy using Annexin V‐FITC/propidium iodide. The protein levels of TNF‐α, IL‐6, and MCP‐1 were determined by ELISA.

Results

Both lopinavir and ritonavir dose‐dependently activated the UPR, increased IL‐6 and MCP‐1 expression, and induced apoptosis in HUVECs. But amprenavir had no significant effect.

Conclusions

HIV PI‐induced ER stress response and activation of the UPR may represent a critical molecular mechanism underlying HIV PI‐associated endothelial dysfunction and atherogenesis.
Gp130 cytokines stimulate proteasomal degradation of tyrosine hydroxylase in sympathetic neurons
Authors: Shi Xiao , Habecker Beth A
Published: June 2021

Alterations in cardiac sympathetic transmission after myocardial infarction contribute to ventricular arrhythmias and sudden cardiac death. Tyrosine hydroxylase (TH) content, the rate‐limiting enzyme in norepinephrine synthesis, is depleted in the left ventricle after myocardial infarction, but elevated in other parts of heart. At the same time that TH content per nerve fiber is decreased, inflammatory cytokines including leukemia inhibitory factor, cardiotrophin‐1 and interleukin 6 are elevated in the damaged ventricle after myocardial infarction. These cytokines signal through the gp130 receptor complex, and preliminary data in cultured sympathetic neurons suggests that activation of gp130 stimulates TH degradation. We used cultured sympathetic neurons to investigate the effect of gp130 cytokines on TH content. Inhibiting proteasome activity by lactacystin prevented cytokine‐induced TH protein loss in cultured sympathetic neurons. These data suggest that gp130 cytokines have a direct effect on TH protein stability, providing a potential mechanism for the local depletion of TH in the left ventricle after myocardial infarction. Additional preliminary data suggest the absence of gp130 receptor in sympathetic neurons prevented depletion of TH protein in the left ventricle after myocardial infarction. Supported by: Steinberg graduate Fellowship (XS), NIH HL68321 (BAH)
Hypoxia‐inducible Factor 1α is Critically Involved in Metallothionein Protection against Diabetic Cardiomyopathy
Authors: Feng Wenke , Xue Wanli
Published: June 2021

The purpose of this study was to examine whether or not MT affects HIF‐1α activity in the heart of diabetic mice and in the cardiac cells leading to the regulation of angiogenic factors and glycolytic enzymes. Diabetes decreased cardiac VEGF and HK‐II protein level in mice; both cardiac‐specific MT and HIF‐1α over‐expression could relieve this suppression. Further analysis showed that MT over‐expression increased HIF‐1α protein level in diabetic heart. MT rescues HIF‐1α transcriptional activity leading to normalization of VEGF and HK‐II in cardiomyocytes under diabetic conditions. (Supported in part by an American Diabetes Association Grant 07‐07‐JF‐23)
Cleavage of glycogen synthase kinase‐3beta by matrix metalloproteinase‐2 enhances its kinase activity
Authors: Kandasamy Arulmozhi D , Schulz Richard
Published: June 2021

Matrix metalloproteinase (MMP)‐2 is localized to the sarcomere of cardiomyocytes and contributes to myocardial ischemia‐reperfusion injury by degrading sarcomeric and cytoskeletal proteins. Oxidative stress mediates its damaging effects through activation of MMP‐2 within the cardiomyocyte and subsequent proteolysis of susceptible intracellular targets. Glycogen synthase kinase (GSK)‐3β is involved in growth, death & metabolism and susceptible to proteolytic cleavage. We determined whether GSK‐3β is a proteolytic substrate of MMP‐2 and how this cleavage affects its kinase activity.

Incubation of MMP‐2 and GSK‐3β resulted in a time‐ and concentration‐dependant cleavage of GSK‐3β with significant increase in GSK‐3β kinase activity. Dephosphorylation of GSK‐3β with λ‐phosphatase prevented MMP‐2 mediated augmented kinase activity. H2O2 treated H9c2 cardiomyoblasts showed increased levels and activity of MMP‐2 accompanied by significant reduction in GSK‐3β protein levels and increased kinase activity.

The data indicate that GSK‐3β is a target of MMP‐2 and that its cleavage by MMP‐2 enhances GSK‐3β kinase activity. GSK‐3β dephosphorylation studies suggest that MMP‐2 may cleave off the inhibitory serine‐9 residue. This MMP‐2 mediated augmentation of GSK‐3β kinase activity may contribute to cardiac dysfunction as a result of myocardial oxidative stress injury. This work was supported by CIHR.
The role of CMKLR1a in the differentiation and function of C2C12 myoblasts
Authors: Issa Mark E , Goralski Kerry B
Published: June 2021

Chemerin is a novel adipokine that exerts its effects via the G‐protein coupled receptor CMKLR1. Recent studies have shown that CMKLR1 plays an important role in the differentiation and function of cultured adipocytes and osteoblasts. We hypothesize that CMKLR1 plays a functional role in the differentiation of myoblasts due to their common mesenchymal ancestry with adipocytes and osteoblasts. In the present study we show that CMKLR1 mRNA is expressed in mouse skeletal muscle. In C2C12 mouse myoblasts, the expression of CMKLR1 increases 3‐fold with the differentiation of those cells into multinucleated myotubules. Consistent with the mRNA expression immunodetectable CMKLR1 is present in the myotubules. Abolishing CMKLR1 expression using adenoviral‐delivered shRNA: impairs the differentiation of C2C12 myoblasts into mature myotubules; inhibits the expression of muscle specific transcription factors myogenin and myoD, and reduces the expression of glucose transporter‐4 and the insulin receptor. We conclude that CMKLR1 signaling is active in skeletal muscle cells and that this pathway acts as a regulator of myogenesis. This work was supported by operating grants from CIHR, NSHRF, PEF and DMRF.
HYPOGLYCEMIC EFFECT OF POLYSACCHARIDE ENRICHED EXTRACT OF ASTRAGALUS MEMBRANACEUS IN DIET INDUCED INSULIN RESISTANT C57BL/6J MICE AND ITS POTENTIAL MECHANISM
Authors: Mao Xianqing , Zou Feng , Wang Nian , Ouyang Jingping
Published: June 2021

To further confirm the hypoglycemic effect of APS and to investigate its possible mechanism. Diet‐induced insulin resistant C57BL/6J mice treated with or without APS (orally, 700 mg/kg/d) for 8 weeks were analyzed and compared. Simultaneously, an insulin resistant C2C12 cell model and an ER stressed HepG2 cell model were established and incubated with or without APS (200 μg/ml) for 24 hours respectively. Systematic insulin sensitivity was measured with an insulin‐tolerance test(ITT)and an homeostasis model assessment (HOMA IR) index. Metabolic stress variation was analyzed for biochemical parameters and pathological variations. The expression and activity of protein tyrosine phosphatase 1B (PTP1B) was measured by immunoprecipitation and Western blot. The ER stress response was analyzed through XBP1 transcription and splicing by real‐time PCR. APS could alleviate insulin resistance and ER stress induced by high glucose in vivo and in vitro respectively. APS enhanced adaptive capacity of the ER and promoted insulin signaling by the inhibition of the expression and activity of PTP1B. We have shown that APS has beneficial effects on insulin resistance and hyperglycemia. The mechanism is related to the alleviation of ER stress and insulin resistance under hyperglycemia conditions.
Role of calcium‐activated potassium channels with small conductance in bradykinin‐induced vasodilation of porcine retinal arterioles
Authors: Dalsgaard Thomas , Kroigaard Christel , Bek Toke , Simonsen Ulf
Published: June 2021

This study investigated the role of calcium‐activated potassium channels of small (SKCa) and intermediate (IKCa) conductance in bradykinin(BK)‐induced relaxation in porcine retinal arterioles. Retinal arterioles (diameter ~112 µm) were mounted in microvascular myographs for isometric tension recordings. The arterioles were contracted with U46619 and concentration‐response curves for BK and NS309, an opener of SKCa‐ and IKCa‐channels, were obtained. BK and NS309 induced concentration‐dependent relaxations. In vessels without endothelium, BK relaxation was abolished and NS309 relaxation was attenuated. Inhibition of NO synthase and/or cyclooxygenase markedly reduced BK and NS309 relaxation. Inhibition of NO synthase and cyclooxygenase together with oxyhemoglobin abolished BK relaxation and attenuated NS309 relaxation. Blocking of SKCa‐channels in the absence or presence of indomethacin or blocking of SKCa‐ and IKCa‐channels markedly reduced BK and NS309 relaxation, whereas blocking of IKCa‐channels had no significant effect. In porcine retinal arterioles, NO and prostaglandins were found to be involved in BK and NS309 relaxation. Blocking of SKCa‐channels alone, and in the presence of indomethacin, reduced BK and NS309 relaxation, suggesting that SKCa‐channels are involved in NO relaxation and may have an essential role in retinal arterial endothelial function.
State of ClCa phosphorylation affects the pharmacology of niflumic acid in rabbit pulmonary artery smooth muscle cells
Authors: Wiwchar Michael , Greenwood Ian A.
Published: June 2021

Ca2+ dependent Cl− currents (IClCa) are important modulators of membrane excitability in vascular smooth muscle cells. The underlying channel (ClCa) appears to be negatively regulated by CaMKII‐induced phosphorylation, and up regulated by the phosphatase calcineurin. Niflumic acid (NFA), considered the most potent blocker of IClCa in many cell types, both blocks and paradoxically enhances IClCa in pulmonary artery (PA) myocytes. This study aimed to determine if the state of channel phosphorylation affects the pharmacology of NFA in dispersed rabbit PA myocytes. IClCa was evoked by 500 nM free Ca2+. The ability of NFA to inhibit IClCa was greatly reduced in conditions promoting phosphorylation. NFA also appeared to have a dual effect on IClCa when applied at low concentrations (0.1‐10 μM), enhancing IClCa in the presence of 5 mM ATP, while blocking IClCa in cells dialyzed with10 µM KN‐93 (CaMKII inhibitor) and no ATP. Analysis of the voltage‐dependence of interaction of NFA with IClCa showed that while the maximal level of block at positive potentials and stimulation at negative potentials were similar, dephosphorylation shifted the voltage at which conversion from block to stimulation occurs by more than ‐80 mV. These results show that the state of phosphorylation has a profound influence on the interaction of NFA with ClCa channels, which may explain the wide range of sensitivities to NFA reported in many studies.