bluuebunny/crossref_metadata_2025 huggingface dataset:
T cell repertoire development of quasi‐public polyspecific clonal peaks in NOD mice
Authors: Marrero Idania , Dai Yang David , Sercarz Eli E
Published: June 2021

A variety of autoreactive T cells have been described in T1D with specificity for a diverse set of autoantigens. Here, we analyzed the T cell repertoires that arise spontaneously in pancreatic lymph nodes (PLN) from female NOD mice, using CDR3‐length spectroscopy. Quasi‐public expansions in Vβ5.2Jβ2.5 (168), Vβ10Jβ1.5 (90) and Vβ13Jβ2.1 (131) were found in 60% (6/10) and 50% (5/10) of 4w old NOD mice. At 10w of age, expansions in the rearrangements Vβ12Jβ2.5 (152) and Vβ5.1Jβ2.2 (198) were detected in 60% and 50% of mice, respectively. Also, expansions in the rearrangements Vβ9Jβ2.4 (97), Vβ12Jβ2.5 (152) and Vβ16Jβ2.2 (121) were detected in 50% of 16w old mice. Interestingly, at this age, we identified a dominant expansion in the clonotype Vβ12Jβ2.7 (170) in all 6 diabetic NOD mice analyzed. In contrast, no expansion of this clonotype was showed at 4w of age and only 2/10 mice showed expansions in this clonotype at 10w. Therefore, the Vβ12Jβ2.7 (170) could be considered a public T cell clone. To examine the peptide specificity, PLN T cells were expanded in vitro with anti‐CD3/anti‐CD28 beads and stimulated with peptides derived from GAD65, insulin or IGRP. Surprisingly, several of the resulting peaks were responsive to different peptides from more than one islet antigen. These dynamic repertoire shifts with age, featuring the emergence of some polyspecific T cells, warrant further detailed study. Supported by JDRF and DNRG
Contribution of BAFF and DNA‐containing Immune Complexes to the Generation of DNA‐reactive B cells
Authors: Jeganathan Venkatesh , Kawabata Daisuke , Ramanujam Meera , Davidson Anne , Grimaldi Christine , Diamond Betty
Published: June 2021

We have reported that 17β‐estradiol (E2) treatment of BALB/c mice that express a transgene‐encoded heavy chain of a nephritogenic anti‐DNA antibody (R4A) induces a lupus‐like phenotype as demonstrated by the presence of serum anti‐dsDNA antibody titers, an expansion of DNA‐reactive B cells and immune complex (IC) deposition in the kidney. We have recently shown that E2 treated mice display increased expression of BAFF mRNA. BAFF is an important regulator of B cell maturation and survival, rescuing self‐reactive B cells from anergy in mice. Furthermore, increased serum levels of BAFF have been reported in patients with SLE and RA. We propose that E2 results in the production of DNA‐containing ICs, which trigger the increased expression of BAFF, facilitating in turn the survival and activation of DNA‐reactive B cells. Consistent with this hypothesis BAFF blockade impaired the generation of anti‐DNA secreting B cells in E2 treated mice. To see if BAFF induction resulted from the action of DNA‐containing immune complexes, we administered DNAse to E2‐treated mice and demonstrated a reduction in BAFF expression. Taken together, these data demonstrate that a feedback loop exists in which DNA‐containing ICs induce expression of BAFF, which enhances the maturation and survival of DNA‐reactive B cells. Thus, targeted therapies that interfere with BAFF signals or IC formation may prove to be useful treatments for SLE.
Toll like receptors in autoimmune inflammatory demyelination: relevance to Multiple Sclerosis
Authors: Brien Kate O’ , Gran Bruno
Published: June 2021

Multiple Sclerosis is a demyelinating disease of the central nervous system. We aim to find mechanisms by which toll like receptors (TLRs) exacerbate or suppress CNS inflammatory demyelination. Both effects have been observed in an animal model of MS ‐ TLRs 2 and 9 in exacerbating disease, TLR4 in delaying onset and TLR3 in reducing severity. We studied the response of human monocyte derived dendritic cells (MDDCs) to TLR stimuli in terms of maturation and cytokine and chemokine production. The inflammatory chemokine MCP‐1 was unregulated by TLR stimuli poly(I:C) (TLR3) and lipopolysaccharide (LPS) (TLR4). The inflammatory cytokines IL‐6 and TNF‐α were unregulated by these two stimuli and also by Pam3CSK4 (TLR1/2) and HKLM (TLR2). The effects of TLRs on MDDCs in MS patients are in progress. We also determined which cytokines and chemokines are induced by TLR stimuli in murine splenocytes. MCP‐1 was unregulated by TLR stimuli ‐ Pam3CSK, HKLM, LPS, Flagellin (TLR5), follistatin‐like 1 (TLR6) and ssRNA40 (TLR7). The anti‐inflammatory cytokine IL‐10 was unregulated by the same stimuli and also by poly(I:C) and CpG ODN1826 (TLR9). TLR9 also induced increased production of IL12p70. All TLR stimuli also induced increased T cell proliferation. The production of cytokines in response to TLR stimuli may regulate autoimmune diseases and there is potential to aid immunotherapy by targeting these TLRs and the cytokines produced.
TCR Revision As A Mechanism Of Peripheral Tolerance
Authors: Vaitaitis Gisela M , Pennock Nathan D , Baker Rocky L , Barbour Gene M , Haskins Kathryn , Wagner David H
Published: June 2021

Development of autoaggressive T cells is required to fulminate autoimmune diseases such as type I diabetes. We have demonstrated a mechanism, TCR revision, present in a subset of peripheral T cells, CD4loCD40+, that can alter those T cells to become self‐antigen reactive and that circumvents thymic selective pressures. TCR revision is induced in CD4loCD40+ T cells by engagement of CD40 and this is also responsible for expansion of those T cells in autoimmunity. We speculated that an already self‐reactive T cell could, via this mechanism, be driven to further TCR revision and skew antigen recognition away from self‐antigens, thereby inducing tolerance. To address this we used a T cell clone, BDC‐2.5, that rapidly transfers diabetes to NOD.scid animals. Here we demonstrate that if the BDC‐2.5 T cells are CD40 engaged for 4 days prior to transfer into NOD.scid mice, they no longer transfer disease. Early during CD40 stimulation the BDC‐2.5 T cells induce expression and nuclear translocation of recombination activating gene (RAG) proteins that are necessary for TCR revision. RAG‐induction is followed by altered TCR Vα expression but, strikingly, not by altered TCR Vβ expression. The data suggest that agonistic CD40 treatment could be explored as a means of inducing peripheral tolerance and, interestingly, suggest that revision of TCRβ is driven by a different mechanism than revision of TCRα
Interleukin‐18 regulates T cell differentiation and the composition of peripheral T cell subsets
Authors: Marleau Annette M. , Sarvetnick Nora
Published: June 2021

Interleukin‐18 (IL‐18) has a well‐established role in promoting type 1 cytokine responses in autoimmunity and inflammation. Here, using non‐obese diabetic (NOD) mice that are genetically deficient in IL‐18 (IL‐18KO NOD), we have identified a role for IL‐18 in regulating intra‐thymic T cell differentiation that was associated with constitutive IL‐18 synthesis and IL‐18 receptor expression during thymocyte development. IL‐18 deficiency promoted the survival of double positive thymocytes, thereby permitting increased generation of naïve T cells and enlargement of the peripheral T cell pool. Accordingly, IL‐18KO NOD mice possessed reduced numbers of homeostatically expanding T cells and the periphery displayed the hallmarks of enhanced stability, including reduced steady‐state T cell apoptosis and differentiation of fewer interferon‐gamma producing T cells. These effects were also associated with the presence of fewer effector T cells in the pancreatic lymph nodes and islets and diabetes resistance in IL‐18KO NOD mice. This study defines a previously‐unrecognized role for IL‐18 as a regulator of intra‐thymic T cell differentiation and the peripheral T cell composition, thereby affecting expansion of the niche of potentially autoreactive T cells in NOD mice.
Allogeneic neonatal blood transfusion without recipient preconditioning is a novel strategy to treat autoimmune diabetes in NOD mice
Authors: Jayaraman Sundararajan , Ajani Shahnaz , Garza Rebecca , Kwon Sung , Holterman Mark , Garza Rebecca , Kwon Sung
Published: June 2021

The efficacy of neonatal blood derived from diabetes‐resistant mice to reverse autoimmune diabetes was evaluated in NOD mice under irradiation‐free and immunosuppression‐free conditions. Transfusion of allogeneic neonatal blood containing Sca‐1+ pluripotential stem/progenitor cells significantly delayed the onset and incidence of diabetes. Neonatal blood transfusion also reduced the mononuclear cell infiltration of the islets of Langerhans. Flow cytometry and RT‐PCR analysis revealed the induction of chimerism in neonatal blood transfused NOD mice. Splenocytes from most cured NOD mice failed to transfer diabetes into immunodeficient NODscid mice, indicating deletion/suppression of diabetogenic T cells in transfused mice. Protection afforded by neonatal blood transfusion was not simply due to alterations in T regulatory subsets. Splenic T cells from both cured and unprotected mice that received neonatal blood transfusion proliferated poorly and produced lower amounts of IL‐2 and IL‐17A when challenged with allogeneic antigens in vitro. These data indicate that neonatal blood transfusion can serve as a novel strategy to induce lymphoid chimerism, T cell tolerance and deletion/suppression of diabetogenic T cells in the absence of cytoreduction or costimulatory blockade.
Requirements for the Induction of IL‐12 and IL‐18 during Listeria monocytogenes Infection
Authors: Meeks Karen D. , Sieve Amy N. , Berg Rance E.
Published: June 2021

Listeria monocytogenes (LM) is a gram‐positive, intracellular bacterium that induces the secretion of pro‐inflammatory cytokines, IL‐12 and IL‐18. LM strains either deficient in listeriolysin O (LLO−LM) or deficient in LLO but replaced with an alternate hemolysin, perfringolysin, tagged for proteasome degradation (LLO−PFO+PEST) have enhanced our ability to understand the secretion of IL‐12 and IL‐18 during cytoplasmic or noncytoplasmic invasion of LM. It is unknown which innate immune cells are secreting IL‐12 and IL‐18 during different stages of LM infection; however, we have found that neutrophils are secreting predominantly more IL‐12 one day after infection with LM. Neutrophils are associated with LM in the periarteriolar lymphoid sheath (PALS) by one day post infection. Infection of mice with LLO−LM does not induce migration of infected cells into the PALS at one day post infection whereas this migration is induced during infection with LLO−PFO+PEST LM. Interestingly, neither mutant strain induces IL‐12 or IL‐18 secretion. Studies are currently ongoing to determine how this localization is induced and whether these mutant strains can induce protective immunity.
Dendritic cells infected with Listeria monocytogenes undergo a maturation response distinct from neighboring uninfected dendritic cells
Authors: Cook Anne Stanner
Published: June 2021

Dendritic Cells (DC) are considered the sentinels of the immune system due to their ability to activate naïve T‐cells, which is dependent on their maturation state. Listeria monocytogenes (L.m.) induces a maturation response marked by costimulatory molecules, cytokines, and T cell priming in a population DC if bacteria enter the cytosol. However, microscopic examination of L.m.‐infected DC both in vitro and in vivo revealed that a larger percentage of uninfected DC expressed CD86 than infected DC. Two color flow cytometric analysis of fluorescently labeled L.m. vs. intracellular IL‐12 confirmed that the majority of infected cells failed to express IL‐12 while the majority of uninfected cells produced IL‐12. However, infected DC expressed IL‐6, TNFα, and CD40, while uninfected DC expressed CD86, CD80, and IL‐12. Based on these findings, we hypothesize that Lm infected DC undergo a different maturation response than neighboring uninfected DC. Consistent with this hypothesis, we have shown that uninfected DC can be stimulated to produce IL‐12 and CD86 when treated with supernatant from Lm‐treated DC. Additionally, uninfected DC required less antigen to stimulate naïve CD 8+ T cells than infected DC. These data illustrate the cooperative nature of the immune response to a bacterial infection.

Research supported by NIH/NIAD grant 1R01AI057770‐01A1
A high‐throughput genetic system for identifying enhanced altered peptide ligands of tumor associated antigens: implications for enhancing adoptive immunotherapy
Authors: Abdul‐Alim C. Siddiq , Li Yongqing , Yee Cassian
Published: June 2021

Cancer immunotherapy offers a promising approach for eradicating metastatic tumors that fail to fully respond to existing therapies. Unfortunately, efforts are frequently hampered by tolerance mechanisms that act to prevent autoimmunity. A potential means for circumventing these mechanisms is the use of altered peptide ligands (APL). APLs are mutant T cell epitopes that can either enhance or dampen a T cell response. Existing methods for identifying effective APLs are either limited in scope or are expensive and technically restrictive. The objective of this study is to develop a comprehensive genetic approach for identifying enhanced APLs of CTL epitopes. With this procedure every point mutant of a CTL epitope can be generated and screened, and the effective APL easily identified. In this study we utilize saturation mutagenesis to independently mutagenize every codon of Mart‐126–35 A27L. Recombinant mini‐gene products of the APLs are cross‐presented to Mart‐126–35 ‐specific CTL clones via dendritic cells in a 96‐well based assay format. Here we identify three novel superagonist peptides of Mart‐126–35, which can generate higher frequencies of Mart‐126–35 ‐specific CTL from peripheral blood leukocytes of melanoma patients. This work has implications for the identification of APLs that may prove clinically beneficial in generating tumor‐specific CTLs for use in adoptive T cell immunotherapy.
Highly pure, viable and functional intact human Natural Killer (NK) cells isolated in a single isolation step using Dynabeads® FlowCompTM Technology
Authors: Schjetne Karoline Western , Kullman Anette , Møller Anne Sophie , Kierulf Bente , Lycke kirsten , Karlsson Malin , Neurauter Axl
Published: June 2021

BACKGROUND

NK cells contribute to a variety of innate immune responses to viruses, tumors and allogeneic cells. In humans, NK cell express CD56. However, this surface marker is not exclusive for NK cells and separation technology focusing on this marker will potentially result in contamination of non‐NK cells or involve several isolation steps. Here we present a single step, positive isolation protocol for human NK cells.

METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from healthy individuals. NK‐specific antibody conjugated to a modified biotin was added and incubated at 2–8 °C. In the next step modified streptavidin coated Dynabeads were added and placed in the magnet after incubation at room temperature. After resuspension with FlowComp release buffer the bead‐free cells were analyzed on flow cytometry and tested in cytotoxicity assays.

RESULTS: Highly pure human CD56+CD3− NK cells were isolated from healthy blood donors using Dynabeads® FlowComp™ Human NK Cell Kit. Using the single step positive isolation protocol, very few CD3+ NKT/T cells were present after isolation and viability was >95%. Isolated NK cells were used in functional assays where LAMP‐1 expression was assayed by flow cytometry and where cytotoxicity was measured in a 51Cr release assay using K562 as target cells. We demonstrate that isolated NK cells were activated and kill target cells.
Expression and Purification of native TL1A (TNFSF15) to generate TL1A Aptamers.
Authors: Majid Ayaz Mahmood , Deyev Vadim , Podack Eckhard R
Published: June 2021

The TNF‐like cytokine TL1A (TNFSF15) has gained significant immunological interest since its characterization as a ligand for TNFRSF25. We have shown that TL1A is intimately involved in the pathogenesis of inflammatory lung disease. Moreover, TL1A up‐regulation on macrophages and lymphocytes has been implicated in human inflammatory bowel disease (IBD) and rheumatoid arthritis.

The pMAL‐2 protein fusion and purification system (New England Biolabs) was initially invented for obtaining recombinant protein targets in E. coli. We have devised a strategy to utilize that system in eukaryotic cells to express native TL1A. Since active TL1A is proposed to be in a soluble form, an expression cassette containing the IgK secretion signal, maltose binding protein (MBP) and human TL1A (hTL1A) was created in a pBMG‐Neo plasmid. Transfection and selection of NIH 3T3 cells or 293SF suspension cells resulted in active secretion of MBP‐hTL1A as shown by an ‘in‐house’ TL1A ELISA, and MBP‐based Western blots. Secreted MBP‐TL1A proteins were purified on Amylose columns under serum free conditions and analyzed by SDS‐PAGE or Agilent protein electrophoresis. Furthermore, an enterokinase cleavage site allowed cleavage of hTL1A from MBP. Chromatography techniques were applied to further investigate native TL1A monomers and trimeric complexes. These targets will be utilized to generate TL1A aptamers using SELEX based or capillary electrophoresis applications. Selected aptamers may allow further understanding of TL1A immunology and provide useful immunotherapeutic interventions.

This work was supported by NIH grants; AI061807 and AI068515.
Childhood overweight prevention programs: Evidence for success
Authors: Doak Colleen , Solerno Lucila , Visscher Tommy L.S. , Renders Carry M. , Seidell Jaap C.
Published: June 2021

Background: A previous review showed 67% of childhood obesity interventions to be effective. Aim: This study is an update of that review, focusing on interventions to prevent childhood overweight. Methods: Included are diet or physical activity related interventions, targeting children 6 to 19 years of age, evaluated using anthropometric outcomes, and published in a peer review journal. Excluded are treatment programs, interventions targeting high risk children, and pilot studies. Intervention effectiveness is defined by statistically significant differences for BMI or skin‐fold outcomes compared to the control. Results: Forty interventions met the inclusion and exclusion criteria, 17 were new studies. From this total, 31 (78%) were effective, mostly short term. Nineteen studies reported both BMI and skin‐fold outcomes, 10 were effective by skinfolds (53%) and 7 were effective by BMI (37%). Seven studies were effective only for girls, two only for boys. Conclusions: The differences in BMI versus skin‐folds outcomes could be due to increased physical activity or measurement error. However, the difference in BMI versus skin‐folds explains why Cochrane reviews, focusing on BMI measures, draw less optimistic conclusions. Interventions may need to target boys and girls differently. Overall, most interventions are effective in the short term.
Food insecurity and poor produce intake are associated with food bank use in British Columbia, Canada
Authors: Kevesdy Ashley E , Holben David H , Holcomb John P
Published: June 2021

Adult clients from 4 food banks in British Columbia (Surrey, Richmond, TriCities, Nanaimo) were surveyed for differences between food security (FS), dietary perceptions, and produce intake & behaviors. Of 1,064 invited, 496 (49.8%) completed the study. Overall, 95.3%, 94.5%, & 45.1% were food secure at household, adult, and child levels. FS (ANOVA, p=0.017; Tukey HSD, Surrey<Richmond), adult FS (ANOVA, p=<0.001; Tukey HSD, Surrey<Richmond), and perceived control of produce intake (ANOVA, p=0.006; Tukey HSD, Nanaimo0.05). For all clients, adult FS was significantly related to poorer perceived diet quality (rho=0.290, p<.001), poorer vegetable (r=0.159, p=0.001), fruit (r=0.159, p=0.001), & total produce (r=0.180, p<0.001) intakes, and perceived barriers to produce intake (rho=−0.247, p<0.001). Food bank users are characterized by food insecurity and poor produce intake. Improved availability of produce, nutrition education, and programs that improve economic self‐sufficiency are needed.
Formative Assessment of Physical Activity Interests and Barriers
Authors: Palmer Debrah M. , Ryan Kelly M.
Published: June 2021

To combat obesity, which is disproportionately high among limited‐resource audiences, nutrition educators are being called upon to recommend increased physical activity. To ensure valid, culturally appropriate and behaviorally‐focused recommendations are made, research is needed on the target audiences’ activity preferences, practical considerations related to increasing activity, and barriers to exercise. This project's aim was to gather pilot data on these matters via four focus groups with adults from a low‐income, urban community in which study participants were primarily Hispanic and Black. Those who exercised predicatably mentioned activities such as walking and playing basketball; however, many participants considered the physical labor they performed at work and at home to constitute sufficient activity. Lack of acclimation to New Jersey's climate was frequently mentioned as a barrier to achieving physical activity goals, as were long work hours, childcare issues, physical disablities, and lifestyle conflicts like smoking. Traditional guidance fails to account for these considerations, thus strong physical activity advice will require additional research and message tailoring to meet audience needs, and account for their activity norms and obstacles. Funding was provided by Rutgers University and the NJ Agricultural Experiemnt Station.
Contribution of Mucosal Maltase‐Glucoamylase to Mouse Small Intestinal Starch α‐Glucogenesis and Total Glucose Metabolism
Authors: Nichols Buford L , Quezada‐Calvillo Roberto , Ao Zihua , Hamaker Bruce R , Marini Juan , Jahoor Farook
Published: June 2021

Digestion of starch requires four mucosal maltases; sucrase and isomaltase (Si) and maltase and glucoamylase (Mgam). We ablated Mgam to study its roles. The in vitro effect was a slowing of null mucosal activity to 10% of WT. Here we report in vivo effects of Mgam KO on mouse glucose metabolism. α‐Glucogenesis was assayed by digestion of IG 13C‐starch to blood 13C‐glucose. UL13C‐starch (Sigma: M+6) was predigested with amylase (Sigma) to 13C‐limit dextrin (LD), which contained 3.48 ± 0.12 g/L glucose equivalents. 13C‐LD was infused IG. D‐Glucose‐C‐d7 (Isotec: M+7) was infused IV (7.5 g/L) to measure total glucose flux. Mice were fasted overnight and tail vein and gastric cannulas inserted. Both IG snd IV were infused at 0.0001 L/h. 4 h blood was collected from submandibular sinus. Total glucose was measured by analyzer and glucose isotopomers as penta‐acetate derivatives by PCI‐GC/MS. Baseline bloods were from uninfused mice. The null has only 60% of WT α‐glucogenesis. Because of increased null gluconeogenesis, total glucose metabolism in Mgam null mice was identical to WT.
Eicosapentaenoic acid ameliorates lipid accumulation via up‐regulation of lipolytic gene expression and down‐regulation of adipogenic gene expression in 3T3‐L1 adipocytes
Authors: Lee Mak‐Soon , Kim Chong‐Tai , Cho Yong‐Jin , Kim Chul‐Jin , Kim Yangha
Published: June 2021

In this study, the lipolytic effect of eicosapentaenoic acid (EPA) was investigated in 3T3‐L1 adipocytes. In comparison with the control, intracellular lipid accumulation was significantly decreased by 24% after 24 hours following the addition of EPA. A 61% increase of glycerol release into the medium occurred with the addition of EPA in the differentiated 3T3‐L1 adipocytes. Also, free fatty acids release into the medium increased by 44.6% compared to the control. The mRNA level of carnitine palmitoyltransferase I‐a (CPTI‐a), a component of the fatty‐acid shuttle system involved in the mitochondrial oxidation of long‐chain fatty acids, was significantly increased by EPA. However, the expression of peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) and acetyl‐CoA carboxylase (ACC), which are involved in adipogenesis, was significantly down‐regulated by EPA. These results suggest that EPA may ameliorate lipid accumulation by stimulation of lipolysis accompanied with induction of lipolytic gene expression and suppression of adipogenic gene expression in 3T3‐L1 adipocytes. “This work supported by the Food Nanotechnology Development Project (2007) and KOSEF (No. M10510130005‐07N1013‐00510).”
n‐3 fatty acids on utero programming of insulin resistance, NASH and hyperlipidemia in Psammomys obesus
Authors: Grenier Emilie , Ziv Ehud , Delvin Edgard , Leduc Line , Spahis Schohraya , Lafond Julie , Levy Emile
Published: June 2021

Nonalcoholic steatohepatitis (NASH) represents the inflammatory form of liver steatosis, which can progress to fibrosis and cirrhosis. NASH is associated with features of the insulin resistance syndrome. In view of the hypothesis that under nutrition in utero enduringly affects the function and metabolism, thereby leading to coronary heart disease in later life, we attempted to examine whether n‐3 fatty acid (FA) supplementation during pregnancy may prevent metabolic disorders and NASH in the adult period. Experiments were carried out on Psammomys obesus gerbils, a model of nutritionally induced metabolic syndrome. Pregnant females were assigned to an isocaloric control diet or a diet rich in fish oil and analyses were carried out in the offspring of diabetic mothers. Our findings could document: (1) a decrease in obesity as assessed by body weight and adipose tissue mass; (2) diminished hyperlipidemia, hyperglycemia and insulin resistance; (3) reduced amounts of lipid content in the liver; (4) lessened inflammation as reflected by the levels of TNFα; and (5) raised levels of adiponectine. Further experiments disclosed a reduced gene expression of FA synthase, malic enzyme and acetyl CoA carboxylase, emphasizing attenuation in hepatic lipogenesis. Overall, our results suggest that maternal nutrition influences fetal programming, insulin resistance and diabetes in later life. Furthermore, our works emphasizes the impact of n‐3 FA on intrauterine mechanisms regulating risk factors of cardiovascular health during adulthood.
Analysis of Locus‐Specific DNA Methylation in Response to Chronic Folic Acid Supplementation and Withdrawal in Chinese Women
Authors: Brant Jason O , Zhu Jiang‐Hui , Crider Krista , Berry R J , Hao Ling , Li Zhu , Maneval David , Rasmussen Sonja , Bailey Lynn B , Yang Thomas P
Published: June 2021

The objective of this study was to evaluate the effect of chronic folic acid supplementation and withdrawal on DNA methylation at specific genetic loci in a Chinese population. Women of reproductive age were assigned a daily folic acid supplement of 400 μg for 6 months, and whole blood samples were drawn at baseline, after 6 months of daily folic acid intake, and 3 months after folic acid supplementation was withdrawn. Blood DNA from 10 subjects (5 MTHFR CC and 5 TT) was analyzed by methylation‐specific PCR (MSP) for changes in methylation status at the promoters of 6 tumor suppressor genes: DAPK, ESR1, HIC1, MGMT, p16, and TIMP, genes known to be aberrantly silenced by hypermethylation of their promoter regions in a variety of human cancers. Overall, no widespread changes in normal DNA methylation patterns (i.e., hypermethylation) were detected for any of the genes tested in all 10 subjects in response to 6 months of folic acid supplementation and 3 months of folic acid withdrawal. To further assess potential changes in DNA methylation in response to folic acid fortification, additional tumor suppressor genes are being analyzed by MSP, and high resolution DNA methylation analyses by sodium bisulfite genomic sequencing are currently in progress. Supported by a collaborative CDC agreement.
Carbohydrate response element binding‐protein is activated by elevated glucose levels in the normal murine liver cell line NMuLi
Authors: Adams Jackson , Peterson Daniel G
Published: June 2021

Carbohydrate response element (ChRE) binding protein (ChREBP) is a transcription factor that is regulated by intracellular glucose levels and activates several lipogenic genes including acetyl CoA carboxylase (ACC). Previous studies of the ChREBP system have used both in vivo models as well as isolated primary hepatocytes. Due to the difficulties in isolation and genetic variability of primary cell culture, we propose the use of a normal murine liver cell line (NMuLi) as a convenient alternative to primary isolated hepatocytes. We show that high (25 mM) but not low (0.5 or 5 mM) glucose activates a luciferase reporter driven by 4 repeated ChREs, as well as the murine ACC promoter segment from −220 to +21. Furthermore, using small interfering RNA to knock‐down ChREBP, we show that ChREBP is required for this glucose‐induced luciferase reporter activation. Finally, using chromatin immunoprecipitation we show that ChREBP directly binds to the ChRE within the promoter region of the ACC gene in the presence of high but not low glucose. Together, these results suggest that NMuLi cells may be used as an alternative to primary isolated hepatocytes for studies of the ChREBP system.
Zinc transproter‐2 (ZnT2) is associated with mitochondria and functions as a zinc importer in mammary epithelial cells
Authors: Kelleher Shannon L , Lopez Veronica
Published: June 2021

Zinc (Zn) transport is regulated by Zn‐specific transporters. ZnT2 expression is restricted to unique tissues, several of which have unusual Zn requirements including mammary and prostate glands. Mammary and prostate glands transport an extraordinary amount of Zn for secretion into milk and seminal fluid, respectively. These tissues have likely developed unique mechanisms to regulate this transfer. The objective of this study was to further characterize the role of ZnT2 in Zn transport using cultured mammary epithelial cells (MEC). We detected a ZnT2 variant (~58 kDa) by immunoblotting, and abundance was attenuated in MECs transfected with ZnT2 siRNA. ZnT2 co‐localized with the mitochondrial membrane protein cytochrome c oxidase subunit IV using confocal microscopy. Localization was confirmed by immunoblot of purified mitochondria proteins. ZnT2 was detected in mitoplasts (mitochondria devoid of the outer membrane) indicating that ZnT2 resides in the inner mitochondria membrane. Pre‐incubation of mitoplasts with ZnT2 antibody reduced Zn uptake but not Zn export relative to mitoplasts pre‐incubated with control IgG. ZnT2, Zn uptake and Zn concentration in the mitochondria was higher in non‐secreting relative to secreting cells. To our knowledge, this is the first identification of a Zn transporter associated with mitochondria. Our data indicate that discrete ZnT2 variants reside in unique sub‐cellular compartment and suggest that mitochondria may serve as a labile Zn pool in highly specialized cells which may be redistributed in response to extrinsic or intrinsic cues. Supported by UCD intramural grants and PSU to SLK.
Intestinal tumors adapt to excess dietary iron by altering expression of lipocalin‐2 and divalent metal ion transporter
Authors: Swain James , Li Chunbiao , Leahy Patrick
Published: June 2021

To determine the effect of excess dietary iron on gene expression of intestinal tumors in an animal model of human familial adenomatous polyposis, male C57BL/6J ApcMin/+ mice were fed adequate and excess amounts of iron (ferrous sulfate: 45 vs. 450 mg iron/kg AIN‐93M[M] diet). After 10 wk, mice were sacrificed and intestines removed. Tumors and adjacent non‐tumor tissue were excised and RNA isolated for micro‐array analysis (n=3/group; Mouse Genome 430A 2.0 arrays; Affymetrix) and RT‐PCR. Pathway mapping was performed to reveal gene networks (Pathway Studio 4.0, Ariadne Genomics, Inc.; GeneSpring, Agilent, Inc.; Searcher, Lucidyx). Bayesian ANOVA was used for micro‐arrays. Data show there was an approximate 3‐fold increase in the expression of lipocalin‐2 and a 9‐fold decrease in expression of the proton‐coupled divalent metal ion transporter (DMT; solute carrier family 11, member 2) with excess iron. Findings reveal that intestinal tumors adapt to excess dietary iron by increasing expression of lipocalin‐2, possibly to assist in sequestering increased cytosolic iron, and decreasing expression of DMT, thereby limiting iron absorption. Supported in part by grants IRG‐91‐022 from ACS (to JS) and P30CA43703 from NIH. Ferrous sulfate provided by Crown Technologies, Inc.
Effects of three types of inulin on dietary iron bioavailability and site of their disappearance in the digestive tract of young pigs
Authors: Yasuda Koji , Roneker Karl R. , Rice Sarah , Welch Ross M. , Miller Dennis D. , Lei Xin Gen
Published: June 2021

Inulin may be used to improve mineral nutrition in humans. We showed positive effects of Synergy 1, a mixture of short oligofructose P95 and long‐chain HP (Orafti, Tienen, Belgium) on hemoglobin repletion efficiency (HRE) and cecum as the major degradation site in young anemic pigs. The present study was conducted to compare the effect of HP and P95, relative to Synergy 1, on HRE and their degradation site in weanling pigs. Thirty‐two pigs (6‐wk old) were individually housed and fed a corn‐soybean meal based diet (BD) without supplemental inorganic iron, or the BD plus 4% of Synergy 1, HP, or P95 for 5 wk. Blood hemoglobin (Hb) concentrations were measured weekly. Daily feed intake and weekly body weight gain were recorded to calculate HRE. At wk 5, digesta samples were collected from lower jejunum, ileum, and cecum of pigs at slaughtering for inulin analysis. Blood Hb concentrations were 10% greater (P < 0.05) in the inulin‐fed pigs than those fed the BD at wk 5. Pigs fed Synergy 1 and P95 had higher (P < 0.05) and lower (P < 0.05) HRE than that of BD, respectively. No inulin was detected in the cecum digesta of any treatment group. Compared with those fed Synergy 1, pigs fed HP had higher (P < 0.05) whereas pigs fed P95 had lower (P = 0.07) inulin concentrations in lower jejunum digesta. In conclusion, benefit of supplemental inulin to dietary iron bioavailability and site of its disappearance were a function of the chain length.
Selenium is critical for the regulation of tumor suppressor and pro‐inflammatory gene expression in human colon Caco‐2 cells
Authors: Zeng Huawei , Botnen James H
Published: June 2021

The essential role of Se in growth of most mammalian cells is well recognized but certain cancer cells appear to have acquired a survival advantage under conditions of Se‐deficiency. We generated Se‐deficient colon Caco‐2 cells by gradually reducing serum in the media because serum contains a trace amount of Se, which is sufficient to maintain the expression of cellular selenoproteins such as glutathione peroxidase (GPx). The GPx activity of Se‐deficient Caco‐2 cells was 10.8 mU/mg protein compared to 133.6∼146.3 mU/mg protein in Caco‐2 cells supplemented with 500 nmol/L selenite, Se‐ (methyl)selenocysteine or selenomethionine (three tested Se chemical forms) after 7‐ day culture in serum free media. Interestingly, there were not detectable differences on cell growth, cell cycle progression between Se‐deficient and Se‐supplemented cells. However, cancer signal pathway‐specific array assay and the real time PCR analysis demonstrated that Se increased the expression of humoral defense gene (A2M) and tumor suppressor‐related genes (IGFBP3, HHIP) but decreased pro‐inflammatory gene (CXC L9, HSPB2) expression at cellular nutritional Se doses, even though colon Caco‐2 cells were resistant to Se deprivation.
Medicinal Plants from Indian Subcontinent Decrease Quorum Sensing Dependent Virulence in Pseudomonas aeruginosa
Authors: Vattem Dhiraj , Huerta Veronica , Mihalik Kevin , Crixell Sylvia
Published: June 2021

Pseudomonas aeruginosa (PAO1), an opportunistic pathogen in immune compromised patients is under the regulation of the LasR‐RhlR system for quorum sensing (QS) mediated development of virulence. Medicinal plants from Indian subcontinent used in traditional medicine are well known for their antimicrobial effects on a variety of human pathogens. However, few studies have investigated its QS related antivirulent activities. Our objective was to screen for the QS inhibitory properties of 16 high quality ayurvedic medicinal plants derived from Indian sub‐continent, understand their mechanism of action and investigate their effect on the expression of QS regulated virulence factors in PAO1. QS inhibition of sub‐lethal concentrations (SLC) of plant extracts was measured in violacein producing Chromobacterium violaceum bioassay model. Effect of these extracts on PAO1 virulent factors pyocyanin, elastase, and total protease were quantified by standard protocols. Results indicated that all extracts reduced violacein production significantly. The results from modified screening assay suggested the primary mechanism of QSI was a combination of (1) reduction in AI formation and (2) inhibition of AI activity. The extracts reduced pyocyanin synthesis, inhibited the activity of elastase and other proteolytic enzymes.
DNA Repair in Human Lymphocytes by Sinigrin ‐ a Possible Mechanism of Action: Comet –FLARE Assay.
Authors: Tope Avinash M , Rogers Phyllis I
Published: June 2021

Chronic low level exposure to environmental pollutants such as synthetic pesticides has been implicated in many health conditions such as oxidative stress, leading to DNA damage, a cause behind many serious diseases such as cancer. The objective of the present study was to investigate the possible mode of action of Sinigrin (10–30μM) a bio‐active compound found in vegetables such as broccoli, cabbage, Brussels sprouts, in the DNA repair process, using Single Cell Gel Electrophoresis (Comet Assay) ‐ Fragment Length Analysis using Repair Enzymes (FLARE) assay. Human lymphocytes were exposed to various concentrations of Sinigrin (10–30 μM) along with (a) a mixture of most widely used pesticides and (b) 100 mM of H2O2 incubated simultaneously at 37° C, at 5% CO2 for 3 hours and DNA repair was evaluated using formamidopyrimidine FLARE‐Comet Assay. The efficiency of Sinigrin in protecting DNA damage was found at and above 20 μM, suggesting a possible role in the functioning of DNA glycosylase involved in DNA repair process.
The plasma concentration‐time profile of beta‐alanine using a controlled‐release formulation (Carnosyn®)
Authors: Harris Roger C , Jones Glenys , Wise John A
Published: June 2021

Carnosine (β‐Alanylhistidine) synthesis in muscle cells is limited by the availability of β‐Alanine, but can be increased 80% or more when the diet is supplemented with β‐Ala. This is of interest to athletes since increased levels of muscle Carnosine have been shown to improve exercise performance. Above 10mg/kg bwt, however, β‐Ala in solution or rapidly‐dissolving capsules causes symptoms of paraesthesia, despite the amount being less than that available from normal‐size portions of meat.

PURPOSE

To investigate β‐Ala absorption using a controlled‐release formulation.

METHOD

Three males and 3 females were given 1600mg β‐Ala dissolved in water (C), 2 × 800mg β‐Ala controlled release tablets (T2) and 4 tablets (T4), on separate occasions. Controlled release tablets were Carnosyn™ supplied by Collegiate Sports Nutrition. Plasma from 100μl capillary blood samples was collected over 6h, initially every 0.25h, and analysed for β‐Ala by HPLC.

RESULTS

Subjects reported significant paraesthesia with C, no symptoms with T2 and zero/trace symptoms with T4. Peak β‐Ala (P) with C was 219±78μM with return to ~10uM at 3h. Increases in P were greatly attenuated with T2 (126±36μM) and T4 (177±123μM) but greatly extended (>50μM at 3h). Areas under the plasma concentration curve were C: 286±48; T2: 309±154; T4: 518±220 μM.h. with C or T2 vs T4: P<0.05; C vs T2: P>0.05.

CONCLUSION

: T2 was bioequivalent to C but did not cause paraesthesia.
Anaplastic oligodendroglial tumor metastatic to the bone marrow: Molecular heterogeneity of glial neoplasms and the clinical importance of minor clones
Authors: Mohila Carrie A. , Simmons Nathan E. , Fadul Camilo E. , Meyer Louise P. , Hartford Alan C. , Levy Norman B. , Callahan Katherine D. , Sullivan Erin K. , Rhodes Andrea M. , Mohandas T. K. , Robirds Diane , Branson Julie , Perry Arie , Rhodes C. Harker
Published: June 2021

Anaplastic oligodendrogliomas are primary brain tumors that rarely metastasize to extracranial sites. We present a case of a 37 year old man who presented at age 34 with a cystic, ring‐enhancing left parietal tumor. Partial resection revealed a synaptophysin‐positive anaplastic oligodendroglioma with a Ki‐67 labeling index of 17%; fluorescence in situ hybridization (FISH) revealed relative deletions of both chromosomes 1p and 19q. The patient subsequently received temozolomide (TMZ), radiotherapy (RT) plus TMZ, and post‐RT TMZ with continued progression of the tumor. Re‐resection 2 years later showed recurrent anaplastic oligodendroglioma with a Ki‐67 labeling index of 50% and he was started on procarbazine, lumustine, vincristine (PCV). Nine months later a third resection revealed anaplastic mixed oligo‐astrocytoma with a Ki‐67 index of 89% and loss of heterozygosity (LOH) 1p/19q. He was started on irinotecan and Avastin. Six months later he presented with night sweats and lower back pain. Bone marrow biopsy revealed a GFAP‐positive metastatic glioma histologically similar to the intracranial tumor; FISH studies revealed normal dosages of both 1p and 19q. These findings suggest that a minor clone in the primary brain tumor did not have LOH 1p and/or 19q and that this clone metastasized to the bone marrow.
Chondroblastic Osteosarcoma Arising in a Recurrent Glioblastoma
Authors: Hagen Matthew Carlisle , Horner Terry G. , Hattab Eyas M.
Published: June 2021

Gliosarcoma is a rare, highly malignant, primary glial neoplasm accounting for less than 2% of glioblastomas. Gliosarcomas are thought to arise de novo, occasionally undergoing metaplastic transformation. The metaplastic elements, usually in the form of cartilage and bone, are typically well‐differentiated with only exceptional malignant examples. We report one such case in a 45‐year‐old man who initially presented with dysphasia. A left temporal mass was discovered by head CT and a subtotal resection was performed. Pathologic examination revealed a glioblastoma. The patient received shaped external beam radiation, was placed on chemotherapy, and followed with serial MRIs. At 3 months abnormal enhancement adjacent to the resection cavity led to stereotactic radiotherapy. At 15 months there was increased enhancement in the wall of the resection cavity, though no mass lesions were seen. At 18 months a heterogeneously enhancing mass was seen in the left temporal region measuring 4.2 cm, which grew to 6.6 cm in just over 2 weeks. The patient underwent a second resection. The tumor was composed of three components: a classic glioblastoma, a sarcomatous component resembling a fibrosarcoma or malignant fibrous histiocytoma, and a malignant cartilaginous and osteosarcomatous component reminiscent of a chondroblastic osteosarcoma. This case illustrates an exceedingly rare occurrence of malignant cartilaginous and osseous elements in glioblastoma/gliosarcoma. It is unique in that the sarcomatous elements were only represented in the recurrence, which occurred over a relatively short period of time (18 months).
Expression and phosphorylation status of β‐arrestin‐1 in normal, reactive, and neoplastic human brain
Authors: Bourne T David , Papin Jason A , Locke Corinne N , Glass George F , Mandell James W
Published: June 2021

β‐arrestins act as signal terminators and scaffold proteins for G protein‐coupled receptor‐mediated signal transduction. This study characterizes the distribution, cellular localization, and phosphorylation state of β‐arrestin‐1 (BAR1) in normal, reactive, and neoplastic human brain tissue. Fixed brain tissues from temporal lobectomy, autopsy, and glioblastoma (GBM) specimens were analyzed by immunohistochemistry using rabbit Mabs directed against BAR1 and phospho (Ser412)‐BAR1. BAR1 and p‐BAR1 immunoreactivity was identified in several neuronal cell populations and in oligodendroglia, pineocytes, ependymal cells, and subependymal glia. Areas of reactive gliosis showed both BAR1 and p‐BAR1 expression, while a majority of GBM specimens showed only BAR1 expression. BAR1 expression was prominent in most neuronal populations, showing predominantly cytoplasmic with occasional nuclear localization. Although undetectable in quiescent astrocytes, reactive astroglia exhibited BAR1 expression and phosphorylation. In contrast, although BAR1 is consistently expressed in most glioblastomas, it appears to be maintained in a dephosphorylated state by unknown mechanisms. Since dephosphorylated BAR1 is responsible for engaging Src and ERK/MAP kinase activation, this may have implications for aberrant signaling in GBM. Supported by Coulter Foundation Translational Research Grant (JWM and JAP).
Leptomeningeal sarcoma 16 years after cranial radiation in a child with acute lymphoblastic leukemia that spread to the central nervous system (CNS)
Authors: Llena Josefina , Weidenheim Karen , Radel Eva , Goodrich James , Small Trudy
Published: June 2021

With constant ongoing improvement in radiotherapy, tumors occuring after cranial radiation in children with acute lymphoblastic leukemia are increasingly rare. The latest review article is by Rimm I J et al in Cancer 59:1506–1508, 1987. Their Table 1 listed 22 cases; 20 gliomas, 1 melanocytoma and 1 meningeal fibrosarcoma. Average radiation dose was 2400 cGy, age range was 2 to 19 years and latency period range was 3 to 11 years. The meningeal fibrosarcoma was described by Tiberin P et al in J. Neurosurg 1984; 61:772–776. It was a frontal lobe tumor in an 8 year old girl irradiated at age 2. Our patient was 3 1/2 years old at diagnosis. He was treated with a regimen including 2400 cGy cranial radiation. 10 months after completing treatment, his tumor relapsed in the bone marrow and CNS. He received intensive chemotherapy including intrathecal and intraventricular chemotherapy for 9 months. About 3 years later, while in complete remission, he received an unmodified bone marrow transplant from his HLA‐identical sister after total body irradiation of 1375 cGy. At age 20, he presented with headache and seizures. Neuroradiology showed a 1 cm enhancing mass in the left frontal opercular region. A circumscribed leptomeningeal gray‐tan firm tumor located under the central sulcus was completely resected. The tumor was an undifferentiated high grade sarcoma with epithelioid and myxoid features.
Expression and localization of connexin 43 in glioblastoma
Authors: Gould Peter Vincent , Cottin Sylvine Carrondo , Caruso Manuel , Cantin Léo
Published: June 2021

The bystander effect is essential to the success of the Herpes simplex virus thymidine kinase (TK)/gancyclovir (GCV) strategy for cancer therapy. It consists of the transfer of phosphorylated GCV from TK+ cells to neighbouring TK− cells via gap junctions made up of connexin molecules. Down‐regulation and/or perinuclear localization of connexins should decrease the efficacy of TK/GCV treatment. The status of Cx43, the connexin expressed in astrocytes, in glioblastoma is unclear. In this study we have evaluated Cx43 expression, localization and functionality in glioblastoma cell lines, biopsies and primary cell cultures.

In three glioblastoma cell lines, Cx43 was the major connexin subtype, and accumulated mainly in a perinuclear location. Nevertheless, gap junction permeability was preserved and a strong bystander effect was seen. Cx43 expression was observed in 77% (57/74) of glioblastoma biopsies and seven of eight primary cell cultures derived from surgical resection. Four of seven cultures showed cytoplasmic localization of Cx43 with preservation of gap junction permeability. These results suggest that glioblastoma is a suitable candidate for the TK/GCV approach. Supported by the CTECM/FRSQ/CIHR.