bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for B cell antigen receptor (BCR)‐driven, BAFF‐facilitated positive selection within a unique population of late transitional B cells
Authors: Meyer‐Bahlburg Almut , Andrews Sarah F , Yu Karl OA , Porcelli Steven , Rawlings David J
Published: June 2021

In addition to negative selection, several lines of evidence suggest that positive selection may also play an important role in shaping the naïve mature B cell repertoire.

We have characterized a late transitional (T2) B cell subset with a distinct phenotypic, functional, and gene expression profile. Adoptive transfer experiments identify this subset as an immediate precursor of both follicular mature (FM) and marginal zone (MZ) B cells. Intriguingly, in contrast to the largely quiescent early transitional (T1) and FM B cells, a high percentage of T2 B cells are actively cycling as demonstrated by cell cycle analysis, measurement of kappa‐deleting recombination excision circles (KRECS) and in vivo BrdU labeling. Proliferation of this subset is BAFF‐dependent, consistent with the highest levels of BAFF‐R expression among all splenic B cells. Finally, M167 BCR idiotypic‐specific B cells are first selected by self‐antigen within this subset leading, ultimately, to preferential enrichment within the MZ compartment.

In summary, our data indicate that late transitional B cells represent a bipotent precursor for mature splenic B cells; and can function as a target for BCR‐driven, BAFF‐facilitated homeostatic signaling. These combined findings suggest that this small pool of cells may operate at a previously unknown selection point important for establishment of the mature B repertoire. Funded by NIH 5RO1HD37091
NF90 and NF45 Regulate Interleukin‐13 (IL13) Gene Transcription in Human T Cells
Authors: Kiesler Patricia , Haynes Paul , Shi Lingfang , Kao Peter , Wysocki Vicki , Vercelli Donata
Published: June 2021

The Th2 cytokine IL13 is a critical effector of allergic inflammation. We previously showed human IL13 gene expression is paralleled by chromatin remodeling, leading to the formation of multiple DNase I hypersensitive (HS) sites throughout the locus. The distal IL13 promoter harbors two HS sites, HS4 and HS5, detected in both naïve and polarized CD4+ T helper cells. We now show HS4, which is located ~1.5 kb upstream of the IL13 ATG, acts as a cis‐regulatory element that enhances transcription driven by the IL13 promoter in transiently transfected, activated Jurkat T cells. The enhancing activity mapped to the 3’half of HS4 (HS4‐3’), a region which binds Nuclear Factor‐90 (NF90) and NF45 as demonstrated by DNA‐affinity chromatography and tandem mass spectrometry. The NF90/45 binding motif in HS4‐3’ was further mapped and dissected by gel shift analysis. Chromatin immunoprecipitation confirmed recruitment of NF90 and NF45 to the HS4‐containing region in the endogenous IL13 locus upon T cell stimulation. Moreover, stable overexpression of NF90 and NF45 increased HS4‐mediated enhancement of IL13 transcription by 3‐fold and 4‐fold, respectively. Collectively, our results identify NF90 and NF45 as important regulators of human IL13 transcription in response to T cell activation.
CXCL10+ T cells and NK cells assist in the recruitment and activation of CXCR3+ and CXCL11+ leukocytes during Mycobacterium avium paratuberculosis‐mediated colitis
Authors: Singh Udai P , Singh Rajesh , Singh Shailesh , Karls Russell K , Quinn Frederick , Lillard James W
Published: June 2021

Recently, our laboratory suggested that spontaneous colitis in IL‐10−/− mice is driven in part by antigens conserved in Mycobacterium species. In the present study, we show that serum from inflammatory bowel disease (IBD) patients contain significantly higher levels of Mycobacterium avium paratuberculosis‐specific IgG1 and IgG2 antibodies (Abs), serum amyloid A (SAA) as well as CXCR3 ligands than serum from healthy donors. To study the cellular mechanisms of Mycobacterium‐induced colitis, pathogen free IL‐10−/− mice were given heat‐killed or live M. avium paratuberculosis. The number of mucosal T cells, neutrophils, NK/NKT cells that expressed TNFα, IFN‐γ, and/or CXCL10 were significantly higher in mice that received live Mycobacterium than other groups. The number of mucosal CXCR3+, CXCL9+, CXCL11+ and/or IFN‐γ+ dendritic cells (DCs) were also significantly higher in M. avium paratuberculosis‐challenged mice as compared to control mice. Together, the data show that Mycobacterium‐dependent host responses can drive colitis in IL‐10−/− mice.
A Universal T Cell Vaccine Against Influenza A
Authors: Tai Wendy Man‐Ting , Gilbert Brian Edward , Corry David Brian
Published: June 2021

A bivalent influenza virus (INFV) A vaccine composed of purified HA and NA has been available for INFV protection. However, this vaccine requires annual re‐administration. We have developed a novel T cell‐based vaccine that obviates this need. Our trivalent vaccine, is comprised of highly conserved peptides derived from H1N1 M2, HA, and NP peptides formulated with the TLR ligands (TLRLs), MPL, TDM and Poly (I:C) in a liposomal matrix.

The tripeptide vaccine (50 μl) was given IN to WT mice on d‐21, ‐14, and ‐7 prior to challenge. Additional groups of WT and Rag1−/− mice received only TLRL once ‐2d prior to challenge. All animals were challenged on d0 with LD90 INFV A H3N2. Survival, weight loss, and symptom scores were monitored. On d0, tissues were collected and assessed for secretion of peptide specific IFN‐γ. In addition, lung cells were assessed for NP tetramer+ CD8 cells and also lung histopathology.

M2/TLRL and NP/TLRL immunizations provoked marked inflammation of the lung airways. Immunization with the tripeptide vaccine resulted in 100% survival and minimal weight loss and elevation of symptom scores relative to naïve controls (0% survival). M2 and NP specific IFN‐γ responses were observed predominantly in the lungs. Staining of lung cells with NP tetramer demonstrated ≈5% positive T cells. IN vaccination with TLRL ‐2d prior to challenge also protected WT and Rag1−/− mice against lethal challenge.
HLA A2 restricted HIV specific mucosal and systemic immunity induced with secreted heat shock protein gp96‐Ig
Authors: Strbo Natasa , Pahwa Savita , Kolber Michael , Fisher Eva , Franchini Genoveffa , Podack Eckhard
Published: June 2021

Heat shock protein gp96 is the primary chaperone of the endoplasmic reticulum and a natural adjuvant for priming the innate and adaptive immune system. We have generated a secreted gp96‐fusion protein (gp96‐Ig) that mediated strong, antigen specific CD8‐CTL expansion in vivo.

The aim of this study was to generate and test gp96‐Ig secreting and HIV‐antigen expressing cells as vaccines for HIV.

Murine NIH‐3T3 or human 293 cells were co‐transfected with gp96‐Ig and HIVgag‐gfp cDNAs. To ensure high level of expression of HIV antigen transfected cells were sorted for gag‐gfp+ cells and level of gp96‐Ig secretion was determined by ELISA. We used H2Dd‐HLA A2 transgenic mice and immunize them with 3T3‐HIV‐gag‐gfp‐gp96‐Ig. The frequency of the murine gag‐specific CD8 CTL response was quantified with HLA A2 tetramers loaded with HIV‐gag‐peptide (SLYNTVATL). HLA A2 tetramer specific CD8 cells were found in the peripheral blood, spleen, peritoneal cavity as well as at mucosal sites (Peyer's Patches, intraepithelial compartment and lamina propria) within five days after intraperitoneal injection of 3T3‐HIV‐gag‐gfp‐gp96‐Ig.

Our results imply that allogeneic cells secreting gp96‐Ig with associated HIV gag‐peptides work well as vaccines and are inducing both mucosal and systemic immunity.

This study was supported by NIH/NIAID grants AI073234 and AI068515
Human Hepatocytes are Capable of Expressing B7 Family Inhibitory Ligands
Authors: Kassel Rachel , Taylor Nicholas A , Hahn Young S
Published: June 2021

Hepatitis C virus (HCV) establishes persistent infection in 70% of acutely infected individuals. Hepatocytes, the primary site of infection, potentially contribute to the impaired T cell responses that characterize progression to chronic infection. We hypothesize that HCV‐infected hepatocytes express PD and related ligands that signal to CD8+ T cells; CD8+ T cells have been reported to up‐regulate PD‐1 in chronically infected patients. To this end, expression of B7 family members and MHC Class I were examined on Huh 7.5 cells and primary hepatocytes following culture in conditions mimicking the HCV‐infected liver environment. Wild type Huh 7.5 cells (a hepatoma cell line) as well as those containing full‐length (FL) or the non‐structural region of HCV were cultured in the presence or absence of IFN‐γ for 24‐48 hours. Huh 7.5 cells responded to IFN‐γ; they expressed PD‐L1 and MHC Class I following stimulation. Huh 7.5 cells expressed B7‐H3 independent of IFN‐γ stimulation and did not inducibly express PD‐L2. There was no significant difference in expression of B7‐H1, B7‐H3, or MHC Class I between wild‐type Huh 7.5 and those containing FL or sub‐genomic HCV constructs cultured +/– IFN‐γ. Importantly, cultured, non‐transformed primary hepatocytes expressed MHC Class I, B7‐H1, B7‐DC, and B7‐H3. This suggests that hepatocytes are capable of expressing negative stimulatory molecules under normal conditions and modulating expression under inflammatory conditions, resulting in impaired T cell responses. HCV infection may utilize this tolerogenic predisposition of the liver to establish chronic infection.
Chronic infection affects development of protective memory CD4 T cells in murine malaria
Authors: Stephens Robin , Langhorne Jean
Published: June 2021

Both malaria infections and vaccines are reported to induce only short‐lived immunity; possibly due to lack of T cell memory, as both T cell cytokines and B cell help are critical. Using a T cell receptor transgenic mouse, where CD4 T cells recognize Plasmodium chabaudi, Merozoite Surface Protein‐1 (1157‐1171/I‐Ed); we investigated development of CD4 T cell memory and its part in protection against infection and pathology in chronic and drug‐treated infection. We observed that specific memory cells, as defined by surface phenotype (CD44hi, CD25−, IL7‐Rαhi, CD62Lhi/lo) and cytokine production (IFNγhi, IL‐2hi) accumulated after one infection, and that although they were rapidly activated in a second infection, they did not proliferate faster than in a first infection. In a RAG transfer model, we observed that memory T cells from a drug‐limited infection allowed clearance of a challenge infection faster than naïve cells, but can increase pathology. Interestingly, chronic infection changed both memory phenotype and protective capacity of specific CD4 cells and maintained a memory population with a phenotype similar to effector cells (CD44hi, IL7Rα−) that protected no better than naïve CD4 T cells. (MRC)
Nutrition transition in Panama: Adult overweight and obesity are linked to urbanization, ethnicity and poverty status
Authors: Bermudez Odilia I. , Gao Xiang , Valdes Victoria
Published: June 2021

This study used public data from the 2003 Living Standards Measurement Study (LSMS) in Panama to examine associations of overweight (owt, body mass index: BMI≥25) and obesity (ob, BMI≥30), with age, sex, education, poverty status, area of residency and ethnicity (urban: U, rural non‐indigenous: RN, and rural indigenous: RI) and geo‐political regions, using logistic regression models in a total sample of 14,486 subjects (51% men) with adjustments for sample weights and survey design. Owt/ob (52% in the total group), were higher among U (36%/20%) than RN (30%/15%) and RI (30%/7%) subjects. More obesity was observed among females than males (Odds Ratio: OR=1.6), among older vs. younger adults, with declines after age 80. Those in extreme (OR=0.5) or non‐extreme poverty (OR=0.6) were less likely to be obese, as compared to the nonpoor. Comparing results from the LSMS with those from a 1982 national survey, we found that while height changed little for both men (+1.9 cms) and women (+0.5 cm), weight increased in 15% and 14% in men and women, respectively, with mean BMI moving from 22 to 25 for men, and 23 to 26 for women and obesity rates from 4%/8% to 14%/22%, in men and women, respectively. The severity of the overweight and obesity problem in Panama is showing some of the effects of the nutrition transitions that had affected other countries.
Are food price changes a way to affect the diet of Chinese poor: shifts over time for the poor and the rich
Authors: Jones‐Smith Jessica Claire , Popkin Barry M
Published: June 2021

With over a third of adults overweight and rises in all nutrition‐related noncommunicable diseases, China must to find ways to shift toward a healthier diet and reduce energy imbalance. This study uses longitudinal data on 11,316 Chinese adults followed from 1991–2004. Random effects mixed models were used to model the relationships among food consumption, price and income over time. A logit analysis of the decision whether or not to consume, and conditional on consumption, an OLS analysis of the quantity consumed, examined the association between price and intake of 5 key foods (rice, pork, cabbage, chicken, eggs). Own‐ and cross‐price elasticities were examined as were time‐ and income‐price interactions. An increase in the price of rice, pork or eggs was associated with a significant decrease in the likelihood of consuming each food, particularly for the poor. For both the poor and rich these price elasticities became smaller over time as incomes increased and prices continued to fall. For more basic foodstuffs, cabbage and chicken, the price increase was associated with a decrease in quantity of each food consumed in the poor, but not the rich. Significant effects of food price changes on consumption of substitute and complementary foods were seen (e.g. an increase in the price of pork was associated with a significant increase in likelihood of consuming cabbage (β =.39; p<.0005), eggs (β = 0.13; p=.03), and chicken (β =.34; p=.001). We use techniques to account for the pricing effects on consumption of all products and provide insights into potential targets for changing price subsidies to improve the nutrient profile of the poor.
Diet quality of Americans in 1994–1996 and 2001–2002 as measured by the Healthy Eating Index‐2005
Authors: Guenther Patricia M. , Juan WenYen , Reedy Jill , Britten Patricia , Lino Mark , Carlson Andrea , Hiza Hazel H. , Krebs‐Smith Susan M.
Published: June 2021

The Healthy Eating Index‐2005 (HEI‐2005) is a measure of how well diets comply with the 2005 Dietary Guidelines for Americans. HEI‐2005 scores for the U.S. population were estimated from national surveys conducted in 1994–96 (n=15,011) and 2001–02 (n=9,032) to assess the quality of diets prior to the release of the 2005 Guidelines. Long‐term intakes of the various dietary components assessed by the HEI‐2005 were estimated by using the population ratio method, which is the best available proxy for the long‐term mean HEI‐2005 scores for a group. In 1994–96 and in 2001–02, scores were below the maximum for all components, except for Total Grains and Meat and Beans. The following components had the lowest scores in 2001–02: Whole Grains (20% of the maximum); Dark Green and Orange Vegetables and Legumes (28%); Sodium (41%); and Calories from Solid Fat, Alcohol, and Added Sugar (38%). Scores for Total Fruit, Whole Fruit, Total Vegetables, Milk, Oils, and Saturated Fat were 62–68% of their maximums. Compared with 1994–96, scores had improved significantly for Sodium, Milk, and Oils and declined for Total Vegetables and Whole Grains. To improve diet quality, Americans should increase their intake of fruits, vegetables, whole grains, and fat‐free or low‐fat milk and milk products; choose more nutrient‐dense forms of foods, that is, foods low in solid fats and free of added sugars; and lower their intake of saturated fats and sodium.
Leucine oxidation in its excess in growing rats: Proposal of the method to determine safe intake level for leucine
Authors: Matsumoto Hideki , Kodama Riho , Amao Michiko , Miura Makoto , Kimura Takeshi , Sakai Ryosei
Published: June 2021

Animals have ability to adapt to various dietary intake levels of protein and amino acids (AA) by controlling enzymatic activities. Therefore, we hypothesized that leucine (Leu) intakes within its catabolic capacity are safe levels, and investigated Leu‐oxidizing capacity in growing rats.

Male F344 rats (N=88) were accustomed to 9h of feeding of diets containing graded doses of additional Leu (0–30%) for 14 days. After 15h of fasting period, rats were given diets in which parts of additional Leu were replaced with L‐[1–13C]Leu. Breath CO2, plasma Leu and plasma . α‐ketoisocaproate (KIC) after 6h feeding were analyzed using IRMS, AA analyzer and GCMS, respectively. Rates of Leu oxidation (decarboxylation of KIC) were calculated from the rates of 13CO2 production and 13C‐enrichments of plasma KIC. Plasma Leu concentrations and rates of Leu oxidation increased depending on the dosage of additional Leu. However, higher Leu load (20–30%) did not increase the oxidation rates further. Correlation analysis of these two parameters resulted in two linear regression segments that met at a breakpoint (0.8 mM of plasma Leu), suggesting saturation of its catabolic pathway above the breakpoint. The breakpoint occurred at 5 to 6% of additional Leu, which was less than no observed adverse effect level (NOAEL) of Leu in growing rats. Thus, determination of metabolic capacity would be one of keys to estimates safe intake level of Leu.
Citrulline modulates muscle protein synthesis via S6R and P70S6kinase1 activation in old malnourished rats
Authors: Moinard Christophe , Le Plenier Servane , Perret Christine , Cynober Luc
Published: June 2021

Introduction:

It has been observed that citrulline (CIT) is an activator of muscle protein synthesis in aged malnourished rats. However, the underlying mechanism is not known. The rate of protein synthesis is regulated in part by amino acids (i.e. leucine [LEU]) at the level of the mammalian Target Of Rapamycin (mTOR) pathway. The aim of this work is to evaluate the ability of CIT to modulate mTOR pathway in a model of aged malnourished rats.

Material and methods:

Thirty aged (19 month‐old) rats were submitted to dietary restriction (50 % of the spontaneous food intake) during 12 weeks. They were then randomized in 3 groups: 10 rats (R group) were killed and 20 other were refed (at 90 % of the spontaneous food intake) for 1 week with a standard diet (R+NEAA) or a CIT‐supplemented diet (5g/kg/d) (R+CIT). The refed groups received isonitrogenous nutrition thanks to addition of non‐essential AA (NEAA) to the control group. An additional group was an absolute control group: healthy old rats fed ad libitum (AL)

Animals were sacrificed at postabsorptive state, and the tibialis (T) muscle was removed. P70S6K1 and S6R phosphorylation were determined by Western Blot.

Results:

Conclusion:

For the first time the ability of a non proteic amino acid to activate mTOR pathway is demonstrated. This suggest that CIT could be a major player in the control of nitrogen homeostasis.

This work was supported by funds from the Ministry of Research and Technology
Validity of the Latin American and Caribbean Household Food Security Scale (ELCSA) in South Haiti
Authors: Perez‐Escamilla Rafael , Dessalines Michael , Finnigan Mousson , Hromi‐Fiedler Amber , Pachón Helena
Published: June 2021

We tested the validity of ELCSA in a convenience sample of 153 women with children under five in South Haiti. ELCSA was applied to the women in Creole, contained 16 items and used a reference period of 3 months. Cronbach's alpha was 0.92. Based on affirmative response prevalence, the items referring to child hunger tended to be the most severe. However, social unacceptability of procuring food was the most severe item. This question asked ‘Was there any time during the past 3 months when you had to do something that you would have preferred not to do (such as begging or sending the children to work) to be able to get food?’ There were no food secure households in the sample, 44% were food insecure (FI), 49% were very FI, and 7% were extremely FI. Criterion validity was strong. Those reporting having good/very good health ranged from 38.8% among those FI to 9.1% among those extremely FI (p=0.02). Households with children who had recently had malaria were more likely to be very/extremely FI than households where the index child had been free of malaria (82.0% vs. 37.1%, p<0.001). Additional factors associated with very/extreme FI (p<0.05) were: female‐headed household, lack of electricity at home, no land ownership, and poorer dietary quality. Results suggest that ELCSA is a valid tool for assessing household FI in rural Haiti. Funded by CIDA (7034161) through a grant to the Centro Internacional de Agricultura Tropical (CIAT).
Blood mercury level and fish intake in Korean school‐aged children
Authors: Cho H.C. , Kang D.M. , Kim S.Y. , SA K.J. , Lee C.G. , Lim J.H. , Jee Y.K. , Ha M.N. , Hong S.J. , Hong Y.C. , Kwon H.J. , Oh S.Y.
Published: June 2021

There has been a notable promotion of fish consumption based on the importance of fish as a part of a healthy diet. However, the balance between health benefits and risks, due to the intake of chemical contaminants, is not well characterized. In the present study, we analyzed data on blood mercury level and dietary intakes of 1,983 urban children aged 7 to 8 year in Korea. Dietary intakes were assessed by a semi‐quantitative food frequency questionnaire and blood mercury level was analyzed by the cold vapor generation method. A cut of point of 2μ/L of blood mercury level was used to categorize children into high (n = 934) and low (n = 1,049) groups. After adjusting for possible confounding variables, intakes of animal iron, zinc, and n3‐fatty acids were positively associated with high blood mercury level. In food group analyses, more frequent consumptions of white and blue fishes, shell fishes, processed and fermented fishes were observed in the high mercury group than the low mercury group. These results showed blood mercury level is strongly associated with fish consumption independent of type of fish. (Supported by a grant from the Ministry of Environment, Korea in 2006)
School Meal Participation and Obesity Among Children
Authors: Gleason Philip Michael
Published: June 2021

Understanding the role of school meal program participation in children's risk of obesity is critical to improving the weight status of America's children. The most complete and up‐to‐date data to address these issues is from the nationally representative data collected in the 2005 third School Nutrition Dietary Assessment Study (SNDA‐III). In this study, we will use SNDA‐III data to examine whether participation in the National School Lunch Program (NSLP) or School Breakfast Program (SBP) affects children's body mass index (BMI) or risk of obesity. This relationship will be estimated through multivariate analysis, with a wide range of variables controlling for confounding factors, including demographic and socioeconomic factors, physical activity, and parents’ perceptions of their child's dietary habits. To test the sensitivity of the results, we also estimate a propensity score model and instrumental variables model. Finally, we examine potential explanations for any observed effects of program participation on BMI, including children's overall food energy intake, intake of macronutrients such as fat and cholesterol, and patterns of food consumption over the course of a day.
Three Dimensional (3D) Body Imaging for Assessment of Body Volume
Authors: Freeland‐Graves Jeanne H , Xu Bugao , Pepper M Reese , Dai Zhaoli , Yu Wurong , Cahill Jodi M
Published: June 2021

Techniques for evaluation of body volume are limited due to high cost, subject burden and/or inaccuracy. This research compared a small‐scale 3D photonic scanning imager to hydrodensitometry (HD) in order to conduct preliminary assessments of its validity. A secondary goal was to assess reproducibility of the 3D body imager. Women (25.4±9.3 yr) visited the laboratory on one occasion after a 4 hour fast and avoidance of alcohol and caffeine for 10 hours. Subjects had a mean Body Mass Index (BMI) of 23.9±4.6 kg/m2, ranging from 15.9 to 35.2 kg/m2. Results from the mean of ten scans of 3D body imaging were significantly related to the median of the highest three of ten measurements of HD (corrected for lung volume) of total volume (r=0.99, p<0.01). Reproducibility of repeated 3D scans of 20 times, with relaxation of body between the scans, had a coefficient of variation of 0.5%. Body scans taken before and after drinking 1 liter of water were highly correlated (r=0.99, p<0.05). These results suggest that 3D body imaging assessment is independent of hydration status and a valid measure of body volume. Supported by NIH 1 R21 DK081206‐01.
Alternate‐day fasting reduces the ratio of visceral to subcutaneous fat by modulating adipose tissue triglyceride metabolism in mice
Authors: Varady Krista A , Roohk DJ , Vissa Madhav , Hellerstein Marc
Published: June 2021

BACKGROUND: Calorie restriction (CR) regimens reduce visceral adiposity in non‐obese animals. The effects of alternate‐day fasting (ADF) regimens on fat distribution are not known.

OBJECTIVE: To investigate the effects of ADF on body fat distribution in mice, and to determine the lipid dynamics underlying redistribution of fat among depots.

METHODS: In a 2‐week study, 24 male C57BL/6J mice were randomized to 1 of 2 groups: ADF‐100% (24 h of 100% CR, alternating with 24 h ad libitum feeding) or control (ad libitum fed everyday).

RESULTS: Body weight did not differ between groups. The epidydimal (visceral) fat depot of the ADF‐100% group was 31% smaller (P < 0.01), while the inguinal (subcutaneous) fat depot was 45% larger (P < 0.01), compared to controls. Absolute TG synthesis in the ADF‐100% group was higher (P < 0.05) in inguinal fat and lower (P < 0.05) in epidydimal fat than in controls. No differences in net lipolysis were observed between groups in either fat pad. Net TG accumulation in ADF‐100% mice was higher (P < 0.05) in inguinal fat and lower (P < 0.05) in epidydimal fat, relative to controls. Epidydimal fat cells were 56% smaller (P < 0.05) in the ADF‐100% group, post‐treatment, relative to controls.

CONCLUSION: These findings suggest that short‐term ADF redistributes adipose TG from visceral to subcutaneous depots by augmenting TG synthesis in inguinal fat, while lowering it in epidydimal fat.
Type of dietary protein regulates gene expression pattern in adipose tissue
Authors: Vazquez‐Vela Maria‐Eugenia Frigolet , Torres Nimbe , Tovar Armando R
Published: June 2021

Dietary protein plays an important role in the regulation of lipogenesis in liver and secretion of insulin by pancreas, however it is not known whether the type of protein exerts an effect in adipose tissue functionality. Thus, the aim of this study was to analyze the gene expression pattern of adipose tissue in obese rats fed casein or soy protein diet. Results indicate that adipose tissue from retroperitoneal fat pads of rats fed soy protein presented a significant decrease in the expression of leptin, tumor necrosis factor alpha and its receptor, angiotensinogen, genes involved in inflammatory response, genes of signaling mediators and genes involved in cholesterol metabolism compared with rats fed casein diet. These results suggest that soy protein improves adipose tissue functionality in a model of diet induced obesity. This work was supported by grant # 46135M, CONACYT.
Cigarette smoke induces cyclooxygenase‐2 expression in human tracheal smooth muscle cells
Authors: Yang Ya‐Ling , Kou Yu‐Ru , Yang Chuen‐Mao
Published: June 2021

Induction of COX‐2 expression by cigarette smoke (CS) may play an important role in airway inflammation. Here, the roles of mitogen‐activated protein kinases (MAPKs) and nuclear factor‐κB (NF‐κB) pathways for CS‐induced COX‐2 expression were investigated in human smooth muscle cells (HTSMCs). CS‐induced expression of COX‐2 protein and mRNA was inhibited by inhibitors of MEK1/2 (U0126), p38 (SB202190), and NF‐κB (helenalin). The involvement of p42/p44 MAPK and p38 in these responses was further confirmed by that transfection with small interference RNAs (siRNA) of p42, and p38 attenuated CS‐induced COX‐2 expression. Consistently, CS‐stimulated phosphorylation of p42/p44 MAPK and p38 was attenuated by pretreatment with U0126 or SB202190, respectively. In addition, CS‐stimulated translocation of NF‐κB into the nucleus and degradation of IκB‐α was blocked by helenalin, U0126, or SB202190. Moreover, CS‐induced responses were inhibited by pretreatment with antioxidant reagents such as N‐aceytlcysteine, apocynin, diphenyleneiodonium chloride, and pyrrolidine dithiocarbamate, indicating the involvement of ROS generation in CS‐induced COX‐2 expression and MAPKs phosphorylation. Taken together, these results suggest that in HTSMCs, activation of p42/p44 MAPK and p38 pathways converged to NF‐κB translocation is essential for CS‐induced COX‐2 gene expression. These results provide new insight into the mechanisms by which CS may promote inflammatory responses in the airway disease.
LACTOBACILLUS RHAMNOSUS SUPPRESSES EPITHELIAL APOPTOSIS BY UPREGULATING CYTOPROTECTIVE GENES IN THE IMMATURE GUT
Authors: Lin Patricia Lanee Wei , Nasr Tala R. , Berardinelli Andrew , Kumar Amrita , Neish Andrew S.
Published: June 2021

Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in preterm infants. Recently, inappropriate intestinal epithelial apoptosis has been implicated in its pathogenesis. Clinical trials suggest that probiotics may reduce NEC, and in vitro studies suggest that the probiotic Lactobacillus rhamnosus (LGG) may act by suppressing apoptosis in cultured epithelia. Here, we aimed to determine whether and how LGG may prevent NEC by reducing apoptosis in the developing gut in vivo. We fed 2 week old preweaned mice (maturity expected at 3 weeks) media with or without LGG (4h) and harvested small intestines for ex vivo apoptotic induction. LGG significantly reduced epithelial apoptosis as measured by staining for cleaved caspase 3 and TUNEL. To delineate mechanism, we used cDNA microarray which revealed that LGG upregulated a battery of known and candidate cytoprotective genes. The dual specificity phosphatase, DUSP3 was the most highly upregulated gene by microarray and qRT‐PCR. Further, we confirmed that oral LGG increased intestinal epithelial DUSP3 in immature mice by immunohistochemistry. DUSPs have been implicated in anti‐apoptotic, cytoprotective responses in a variety of systems by inhibiting MAPK signaling. These studies indicate that probiotics such as LGG may augment intestinal host defenses in the developing intestine by stimulating anti‐apoptotic, cytoprotective responses.
The anti‐inflammatory cytokine IL‐19 suppresses VSMC activation by attenuation of proliferative and inflammatory gene expression
Authors: Cuneo Anthony Albert , Autieri Michael
Published: June 2021

The development of multiple vascular diseases are inflammatory in nature. Interleukin‐19 (IL‐19) is a recently described member of the IL‐10 family of anti‐inflammatory cytokines. Nothing has been reported on the expression or mechanism(s) of IL‐19 effects in vascular cells. We have found that IL‐19 is not expressed in quiescent vascular smooth muscle cells (VSMC) or normal arteries, but is induced in VSMC by inflammatory cytokines and in arteries by injury. IL‐19 is anti‐proliferative for human coronary artery VSMC, and reduces inflammation‐stimulated expression of proliferative and inflammatory gene proteins, including Cyclins B and D, CDK, IL‐8, and COX2. IL‐19 transiently reduces expression of HuR, an inflammatory gene mRNA stabilizing transcription factor. Actinomycin D transcription blockade, and quantitative RT‐PCR assessment of mRNA shows that IL‐19 treatment reduced abundance of these mRNAs to 50–60% of untreated controls. (P<0.001 for all genes). IL‐19 inhibits serine phosphorylation of HuR, which is necessary for its cytoplasmic translocation. Since the mRNA stabilizing function of HuR is linked to its cytoplasmic translocation, this indicates that not only does IL‐19 decrease HuR accumulation, but also its activity. Together, these data indicate that one potential mechanism whereby IL‐19 inhibits VSMC proliferation and gene expression is by a reduction in expression and function of HuR.
Septic shock: A role for RGS proteins?
Authors: Hendriks‐Balk Mariëlle , Tjon‐Atsoi Moenira , Mathy Marie‐Jeanne , Hajji Najat , Michel Martin , Peters Stephan , Alewijnse Astrid
Published: June 2021

The profound hypotension in septic shock patients is difficult to treat as they display depressed vascular responses to α‐adrenergic agonists. Bacterial lipopolysaccharide (LPS) is the main trigger for most of the cardiovascular alterations occurring in septic shock. Recently, LPS‐induced cardiac failure was found to be associated with upregulation of the Regulator of G protein Signalling (RGS) proteins RGS4 and RGS16 (Cardiovasc Res 53:156, 2002).

In this study we investigate the effects of LPS exposure on vascular contractility in general and the role of RGS proteins in the LPS‐induced vascular alterations.

Exposure of rat aortic rings to various LPS concentrations (1, 3, 10, 30 μg ml−1) for 22 hours had differential effects on the contractile responses to agonists at four distinct G‐protein coupled receptors. Phenylephrine‐ and angiotensin II‐induced contraction was reduced whereas serotonin‐induced contraction was enhanced. The endothelin‐1‐induced contraction was unaffected. Concomitantly, LPS treatment increased the RGS16 mRNA expression level both in aortic rings and cultured vascular smooth muscle cells (VSMCs) but RGS2, RGS3, RGS4 and RGS5 was unaffected. Regulation of RGS16 mRNA in VSMCs was also time‐ and concentration‐dependent. The changes in RGS16 mRNA might contribute to the differential regulation of the contractile responses in an LPS model of septic shock.
Evolution of Ca2+ signals with the development of nano‐domains in smooth muscle cells of the rabbit inferior vena cava
Authors: Dai Jiazhen Minnie , Kuo Kuo‐Hsing , Lee Cheng‐Han , van Breemen Cornelis
Published: June 2021

We have previously shown that phenylephrine(PE)‐induced oscillatory Ca2+ signals underlie excitation‐contraction coupling in adult rabbit inferior vena cava (IVC). These oscillations are caused by waves of regenerative Ca2+ release, which are maintained by sarcoplasmic reticulum (SR) reloading via junctions formed between plasma membrane and SR (PM‐SR). Here we present evidence that these junctions together with oscillatory Ca2+ signaling develop post‐natal. We examined the agonist‐induced Ca2+ signal in in situ vascular smooth muscle cells (VSMC) of the neonatal rabbit IVC in relation to force generation. PE elicits a biphasic Ca2+ signal with an initial Ca2+ transient followed by a steady state plateau averaging 25 % of the peak value. In contrast to the adult, the plateau phase of PE‐induced Ca2+ signal and tonic contraction in the VSMC of neonatal rabbit IVC was abolished by nifedipine. Employing electron microscopy, we found that VSMC in neonatal rabbit IVC posses less junctional SR, 6±1%, as compared with the adult rabbit IVC, 18±1%. Our results suggest that complex structures, defining a nano‐domain (~20 nm) and composition, including clusters of Ca2+ and Na+ transport proteins, of the PM‐SR junctions develops during the neo‐natal period and that this correlates with the appearance of oscillatory Ca2+ signaling in VSMC. This study is supported by funding from the Canadian Institute of Health Research.
Up‐regulation of the non‐ neurogenic cholinergic system in the aorta of spontaneously hypertensive rats
Authors: Tang Eva Hoi Ching , Vanhoutte Paul M
Published: June 2021

Despite the intense interest in the effect of acetylcholine in the vascular system, no evidence has been provided suggesting that endothelial cells are directly innervated by cholinergic neurons. The present study was designed to test whether or not the rat aorta contain the biochemical apparatus needed to synthesize, transport, store and degrade acetylcholine, and if so, whether or not differences in the expression of these components exist between preparations from normotensive Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Gene expressions were quantified by real‐time PCR. The mRNA expression for the neuronal choline acetyltransferase (which synthesizes acetylcholine from the substrates acetyl CoA and choline), vesicular acetylcholine transporter (which specializes in the trafficking of acetylcholine) and acetylcholine esterase (which breakdowns acetylcholine) were expressed in the aorta of both WKY and SHR. The expression of these enzymes was significantly higher in the latter. These data suggest that acetylcholine synthesis is not limited to the cholinergic nerves, but that the cholinergic transmitter is released directly from the aorta in an autocrine fashion to modulate local blood flow. The enhanced vascular synthesis of acetylcholine in the hypertensive rat may reflect a compensatory response to correct for endothelial dysfunction. This work is funded by the RGC – HKU.
Characterization of the vasoactive properties of S1P receptors in rat aorta
Authors: Alewijnse Astrid E , Jongsma Maikel , Mathy Marie‐Jeanne , van Loenen Pieter B , Hajji Najat , Michel Martin C , Peters Stephan L
Published: June 2021

In the cardiovascular system, the bioactive lipid sphingosine‐1‐phosphate (S1P) exerts its vasoactive effects predominantly via the S1P1, S1P2 and S1P3 receptor which are differentially expressed in vascular smooth muscle cells (VSMCs) and endothelial cells (ECs).We have explored the contribution of these three receptors to vascular reactivity in rat aorta and to related signaling mechanisms in isolated VSMCs and ECs using some new S1P receptor ligands. In rat aortic rings, all agonists at the S1P1 and/or S1P3 receptor induced endothelium‐dependent vasorelaxation in pre‐contracted preparations. In rat aortic ECs, which showed a high mRNA expression of the Gi‐coupled S1P1 receptor, all ligands decreased the forskolin‐induced cAMP accumulation whereas no major changes in [Ca2+]i were observed. In VSMCs, which express mRNA for all three S1P receptors, predominantly Gq‐coupled S1P2 receptor signaling was observed, resulting in increases in [Ca2+]i.In conclusion, the compounds tested in this study can act on both ECs and VSMCs from rat aorta. S1P1 receptor stimulation in ECs leads to Gi activation probably resulting in relaxation via activation of PI3K/Akt and subsequent NO production. Stimulation of mainly the S1P2 receptor in VSMCs results in calcium elevation, a response typically linked to contraction. For agonists devoid of S1P2 activity, the net effect observed on aortic tone is thus vasorelaxation.
Protection of mouse pulmonary arteries from hypoxia‐induced apoptosis: Cross talk between phosphoinositide 3‐kinase (PI3K) and ATP‐sensitive potassium (KATP) channels
Authors: Bodiga Sreedhar , Falck John R , Zhang Rong , Gao Ying , Jacobs Elizabeth R , Medhora Meetha M
Published: June 2021

We have demonstrated that epoxyeicosatrienoic acids (EETs), the cytochrome P450 epoxygenase metabolites of arachidonic acid, inhibit both intrinsic and extrinsic pathways of apoptosis of human endothelial cells, through activation of phosphatidyl inositol 3‐kinase (PI3K). EETs are also potent sarcolemmal and mitochondrial ATP‐sensitive K+ (KATP) channel activators. In this study, we examined the effects of EETs on hypoxia/reoxygenation (HR)‐induced apoptosis (as determined by measuring increase in caspase 3 activity) in mouse pulmonary arteries maintained ex vivo, by organ culture. Mouse pulmonary arteries subjected to HR for 8/16 h demonstrated increased apoptosis. This was inhibited by pretreatment with either 14,15‐EET (300 nM), the non‐specific KATP channel opener pinacidil (100 microM) or the mitochondrial KATP channel opener, diazoxide (100 microM). Pretreatment with the PI3K inhibitor, wortmannin abolished the protective effect of EET, pinacidil or diazoxide. There was no cumulative effect of wortmannin and glibenclamide when applied together, on the apoptotic indices during HR. Thus, cross‐talk between PI3K and KATP channels, two targets of signaling by EET, mediate protection of the pulmonary vasculature. Funding: NIH/NHLBI 69996 (MMM), 68627 (ERJ), 49294 (ERJ), GM 31278 (JRF).
Mechanisms of Pulmonary Tolerance in Female Mice to the Cytotoxicant Naphthalene
Authors: Sutherland Katherine , Edwards Patricia C , Van Winkle Laura S
Published: June 2021

Exposure to air pollution has been linked to pulmonary diseases. Naphthalene (NA), an abundant polycyclic aromatic hydrocarbon in tobacco smoke and urban air, is a model toxicant for air pollution effects in the lung. Chronic exposures to compounds like NA can have both toxic and conditioning effects. Tolerance is the emergence of a resistant cell phenotype after prior exposure. Research in male mice has found the development of tolerance to NA involves upregulation of gamma‐glutamylcysteine synthetase, the rate limiting enzyme in glutathione synthesis. Tolerance has not been studied in females. Females have sex differences in airway epithelial responses and in the prevalence of airway diseases. To address female specific airway responses, mice were injected with a tolerizing regimen of NA and gene and protein expression of enzymes involved in NA bioactivation and detoxification were assessed. We found decreased gene/protein expression for CYP2F2, a P450 that metabolizes NA to a toxic epoxide, as well as increased gene/protein expression of glutathione s‐transferase pi, an enzyme involved in detoxification of the epoxide, in the airways of NA tolerant female mice. We conclude that mechanisms contributing to the development of NA tolerance in female mice include changes in airway epithelial specific metabolizing and detoxifying enzymes. Funded by NIH ES013066, ES012720, TRDRP 14RT‐0132, EPA RD‐832414
The overlooked contribution of Flavin‐containing Monoxygenases in the determination of total metabolic flux
Authors: Kousba Ahmed A , Stewart Joshua , Soll Richard , Martin Michael
Published: June 2021

Purpose: The purpose of the current study was to assess the relative contribution of flavin‐containing monoxygenases (FMO) and CYP‐mediated pathways with respect to total metabolic flux of selected compounds. Methods: The contribution of FMO and CYP in hepatic metabolic flux was assessed in human liver microsomes in the presence of CYP and FMO isoform specific inhibitors. In addition, FMO heat sensitivity was also employed in liver microsomal evaluation. The reaction phenotyping was further refined using individual human recombinant CYP and FMO isoforms. The metabolic flux through each identified pathway was normalized to the content of the individual CYP and FMO isoform in human liver microsomes and expressed as % Total Normalized Rate (%TNR). Results: A marked contribution of FMO in the total metabolic flux of the selected compounds (~50%) was observed. The normalization of the metabolic rates to the CYP and FMO isoform contents suggests that CYP3A4 and FMO3 are the main isoforms responsible for the compounds in vitro metabolism (87–99%). Conclusions: While FMO isoforms play an important role in drug metabolism, most literature based approaches for reaction phenotyping primarily evaluate CYP‐mediated metabolism. In the current study, CYP and FMO demonstrated comparable contribution total metabolic flux of the selected compounds. The marked contribution of FMO metabolism of the selected compounds may provide an advantageous property for minimizing the potential for adverse drug‐drug interactions. FMO isoforms are not readily induced nor inhibited by drugs or environmental chemicals.
Symadex™, a FLT3 kinase inhibitor, is metabolized by aldehyde oxidase
Authors: Goodman Keith , Duncan Kenneth , Locniskar Ann , Ajami Alfred
Published: June 2021

Symadex™ (C‐1311) is a potent inhibitor of FMS‐like tyrosine kinase 3 (FLT3). This imidazoacridinone class compound has started development for oncology indications and is being investigated as a therapy for autoimmune diseases. A major circulating metabolite is an oxygenated species (+16 amu) that is found in plasma and urine after oral and intravenous administration.

Aldehyde oxidase (AO) is a molybdenum‐containing enzyme present in liver that catalyzes the oxidation of a wide variety of nitrogen containing heterocycles. Data are presented from in vitro incubations with microsomes, liver cytosol, inhibitors specific for AO, and an isotopic labelling experiment to confirm the role of AO in the oxidation of Symadex™.

The proposed metabolite was synthesized and its structure verified by NMR. It was shown to have similar chromatography, spectroscopy, and MS/MS fragmentation compared to the in vitro and in vivo derived metabolite. The structure was consistent with an oxidation catalyzed by AO.

This non‐CYP mediated transformation is important for the metabolism of imidazoacridinones and likely plays a significant role in the metabolism of other tyrosine kinase inhibitors with imidazole and related susceptible heterocyclic scaffolds. This work presents a comprehensive investigation into this novel route of drug metabolism.
Denitrase activity of macrophages reverses nitrosylation of smooth muscle calcium channel
Authors: Kang Minho , Ross Gracious R , Akbarali Hamid I
Published: June 2021

We have recently shown that smooth muscle calcium currents are reduced during colonic inflammation due to nitration of tyrosine residues of the carboxy terminus of Cav1.2b (JPET, 322, 2007). Nitrotyrosylation prevents src kinase regulation of the calcium channel. The objective of the present study was to determine if nitration of the c‐terminus could be reversed by the denitrase activity from activated macrophages. The c‐terminus fragment (1809–2138) of the human Cav1.2b was subcloned into pGEX‐6P‐3 to generate GST fusion protein, GST‐CT. Nitrotyrosylation of GST‐CT was induced by treating with peroxynitrite (150 μM) plus SIN‐1 (100 μM) for 1 hour and confirmed by Western blot using anti‐nitrotyrosine antibody. Following nitration, GST‐CT was run of SDS‐PAGE, transferred to PVDF membrane and treated with cell lysates from lysophosphatidic (LPS) activated macrophages, RAW 264.7 for 4 hours at 370C. The expression of GST‐CT was not affected by this treatment as confirmed by c‐terminus specific antibody, anti‐CT. Immunoblot with anti‐nitrotyrosine antibody however, showed marked decrease in nitrotyrosylated GST‐CT. Nitrotyrosylation was not reversed by heat inactivated macrophage cell lysates. These data suggest that nitration of the tyrosine residue within the c‐terminus of Cav1.2b may be reversed by denitrase activity of LPS‐activated macrophages. Supported by NIH DK46367