Clostridioides difficile Toxin A Remodels Membranes and Mediates DNA Entry Into Cells to Activate Toll-Like Receptor 9 Signaling.

BACKGROUND & AIMS: Clostridioides difficile toxin A (TcdA) activates the innate immune response. TcdA co-purifies with DNA. Toll-like receptor 9 (TLR9) recognizes bacterial DNA to initiate inflammation. We investigated whether DNA bound to TcdA activates an inflammatory response in murine models of C difficile infection via activation of TLR9. METHODS: We performed studies with human colonocytes and monocytes and macrophages from wild-type and TLR9 knockout mice incubated with TcdA or its antagonist (ODN TTAGGG) or transduced with vectors encoding TLR9 or small-interfering RNAs. Cytokine production was measured with enzyme-linked immunosorbent assay. We studied a transduction domain of TcdA (TcdA57-80), which was predicted by machine learning to have cell-penetrating activity and confirmed by synchrotron small-angle X-ray scattering. Intestines of CD1 mice, C57BL6J mice, and mice that express a form of TLR9 that is not activated by CpG DNA were injected with TcdA, TLR9 antagonist, or both. Enterotoxicity was estimated based on loop weight to length ratios. A TLR9 antagonist was tested in mice infected with C difficile. We incubated human colon explants with an antagonist of TLR9 and measured TcdA-induced production of cytokines. RESULTS: The TcdA57-80 protein transduction domain had membrane remodeling activity that allowed TcdA to enter endosomes. TcdA-bound DNA entered human colonocytes. TLR9 was required for production of cytokines by cultured cells and in human colon explants incubated with TcdA. TLR9 was required in TcdA-induced mice intestinal secretions and in the survival of mice infected by C difficile. Even in a protease-rich environment, in which only fragments of TcdA exist, the TcdA57-80 domain organized DNA into a geometrically ordered structure that activated TLR9. CONCLUSIONS: TcdA from C difficile can bind and organize bacterial DNA to activate TLR9. TcdA and TcdA fragments remodel membranes, which allows them to access endosomes and present bacterial DNA to and activate TLR9. Rather than inactivating the ability of DNA to bind TLR9, TcdA appears to chaperone and organize DNA into an inflammatory, spatially periodic structure.

Assessing Campus Alcohol Policies: Measuring Accessibility, Clarity, and Effectiveness.

BACKGROUND: Excessive alcohol consumption poses significant hazards to health and safety on college campuses. While substantial research exists regarding effective policies for preventing alcohol-related problems in the communities surrounding campuses, on-campus alcohol policies have received far less attention. METHODS: Official campus alcohol policies (CAPs) were retrieved from the websites of the 15 member schools of the Maryland Collaborative to Reduce College Drinking and Related Problems, a voluntary statewide collaborative. CAPs were assessed for accessibility, clarity, and effectiveness. In addition to assessing whether campuses were in compliance with federal regulations for comprehensiveness of policies, a measure of likely policy effectiveness was developed through the use of 2 Delphi panels drawing on alcohol policy researchers and on-campus and community practitioners, respectively. The panels rated 35 potential policies and 13 possible sanctions; lists of policies and sanctions were compiled primarily from what was already in existence at 1 or more member schools. RESULTS: For most campuses, the CAPs could be located within 30 seconds, but tended to be spread across multiple web pages. Language used to communicate the policies tended to be complex and above the reading level of someone with a high school education. At least half of the schools had less than half of the possible policies rated most or somewhat effective by the Delphi panels. Schools were more likely to employ the most effective sanctions, but somewhat and ineffective sanctions were also not uncommon. CONCLUSIONS: CAPs are an important element in reducing negative consequences of alcohol consumption on college campuses. A higher level of research scrutiny is warranted to understand the extent to which CAPs are associated with excessive drinking, but this research describes an evidence- and expert-informed assessment approach that colleges can use to regularly analyze and update their CAPS.

Genetic impairments in folate enzymes increase dependence on dietary choline for phosphatidylcholine production at the expense of betaine synthesis.

Although single nucleotide polymorphisms (SNPs) in folate-mediated pathways predict susceptibility to choline deficiency during severe choline deprivation, it is unknown if effects persist at recommended intakes. Thus, we used stable isotope liquid chromatography-mass spectrometry (LC-MS) methodology to examine the impact of candidate SNPs on choline metabolism in a long-term, randomized, controlled feeding trial among pregnant, lactating, and nonpregnant (NP) women consuming 480 or 930 mg/d choline (22% as choline-d9, with d9 indicating a deuterated trimethyl amine group) and meeting folate-intake recommendations. Variants impairing folate metabolism, methylenetetrahydrofolate reductase (MTHFR) rs1801133, methionine synthase (MTR) rs1805087 [wild-type (WT)], MTR reductase (MTRR) rs1801394, and methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase (MTHFD1) rs2236225, influenced choline dynamics, frequently through interactions with reproductive state and choline intake, with fewer genotypic alterations observed among pregnant women. Women with these variants partitioned more dietary choline toward phosphatidylcholine (PC) biosynthesis via the cytidine diphosphate (CDP)-choline pathway at the expense of betaine synthesis even when use of betaine as a methyl donor was increased. Choline intakes of 930 mg/d restored partitioning of dietary choline between betaine and CDP-PC among NP (MTHFR rs1801133 and MTR rs1805087 WT) and lactating (MTHFD1 rs2236225) women with risk genotypes. Overall, our findings indicate that loss-of-function variants in folate-metabolizing enzymes strain cellular PC production, possibly via impaired folate-dependent phosphatidylethanolamine-N-methyltransferase (PEMT)-PC synthesis, and suggest that women with these risk genotypes may benefit from choline intakes exceeding current recommendations.-Ganz, A. B., Shields, K., Fomin, V. G., Lopez, Y. S., Mohan, S., Lovesky, J., Chuang, J. C., Ganti, A., Carrier, B., Yan, J., Taeswuan, S., Cohen, V. V., Swersky, C. C., Stover, J. A., Vitiello, G. A., Malysheva, O. V., Mudrak, E., Caudill, M. A. Genetic impairments in folate enzymes increase dependence on dietary choline for phosphatidylcholine production at the expense of betaine synthesis.

Genetic Variation in Choline-Metabolizing Enzymes Alters Choline Metabolism in Young Women Consuming Choline Intakes Meeting Current Recommendations.

Single nucleotide polymorphisms (SNPs) in choline metabolizing genes are associated with disease risk and greater susceptibility to organ dysfunction under conditions of dietary choline restriction. However, the underlying metabolic signatures of these variants are not well characterized and it is unknown whether genotypic differences persist at recommended choline intakes. Thus, we sought to determine if common genetic risk factors alter choline dynamics in pregnant, lactating, and non-pregnant women consuming choline intakes meeting and exceeding current recommendations. Women (n = 75) consumed 480 or 930 mg choline/day (22% as a metabolic tracer, choline-d9) for 10-12 weeks in a controlled feeding study. Genotyping was performed for eight variant SNPs and genetic differences in metabolic flux and partitioning of plasma choline metabolites were evaluated using stable isotope methodology. CHKA rs10791957, CHDH rs9001, CHDH rs12676, PEMT rs4646343, PEMT rs7946, FMO3 rs2266782, SLC44A1 rs7873937, and SLC44A1 rs3199966 altered the use of choline as a methyl donor; CHDH rs9001 and BHMT rs3733890 altered the partitioning of dietary choline between betaine and phosphatidylcholine synthesis via the cytidine diphosphate (CDP)-choline pathway; and CHKA rs10791957, CHDH rs12676, PEMT rs4646343, PEMT rs7946 and SLC44A1 rs7873937 altered the distribution of dietary choline between the CDP-choline and phosphatidylethanolamine N-methyltransferase (PEMT) denovo pathway. Such metabolic differences may contribute to disease pathogenesis and prognosis over the long-term.

Development and Validation of Digital Enzyme-Linked Immunosorbent Assays for Ultrasensitive Detection and Quantification of Clostridium difficile Toxins in Stool.

The currently available diagnostics for Clostridium difficile infection (CDI) have major limitations. Despite mounting evidence that toxin detection is paramount for diagnosis, conventional toxin immunoassays are insufficiently sensitive and cytotoxicity assays too complex; assays that detect toxigenic organisms (toxigenic culture [TC] and nucleic acid amplification testing [NAAT]) are confounded by asymptomatic colonization by toxigenic C. difficile. We developed ultrasensitive digital enzyme-linked immunosorbent assays (ELISAs) for toxins A and B using single-molecule array technology and validated the assays using (i) culture filtrates from a panel of clinical C. difficile isolates and (ii) 149 adult stool specimens already tested routinely by NAAT. The digital ELISAs detected toxins A and B in stool with limits of detection of 0.45 and 1.5 pg/ml, respectively, quantified toxins across a 4-log range, and detected toxins from all clinical strains studied. Using specimens that were negative by cytotoxicity assay/TC/NAAT, clinical cutoffs were set at 29.4 pg/ml (toxin A) and 23.3 pg/ml (toxin B); the resulting clinical specificities were 96% and 98%, respectively. The toxin B digital ELISA was 100% sensitive versus cytotoxicity assay. Twenty-five percent and 22% of the samples positive by NAAT and TC, respectively, were negative by the toxin B digital ELISA, consistent with the presence of organism but minimal or no toxin. The mean toxin levels by digital ELISA were 1.5- to 1.7-fold higher in five patients with CDI-attributable severe outcomes, versus 68 patients without, but this difference was not statistically significant. Ultrasensitive digital ELISAs for the detection and quantification of toxins A and B in stool can provide a rapid and simple tool for the diagnosis of CDI with both high analytical sensitivity and high clinical specificity.

Transfusion-associated Anaplasma phagocytophilum infection in a pregnant patient with thalassemia trait: a case report.

BACKGROUND: Human granulocytic anaplasmosis (HGA) is an acute nonspecific febrile illness caused by the bacterium Anaplasma phagocytophilum. Although usually transmitted via tick bite, HGA may rarely also be acquired through transfusion. HGA during pregnancy may pose significant gestational risks due to altered maternal immune status and the potential for perinatal transmission. CASE REPORT: A pregnant 34-year-old Massachusetts woman with ß-thalassemia trait was diagnosed at 32 weeks of gestation with transfusion-associated HGA (TAHGA) after receiving nine leukoreduced red blood cell transfusions. She was successfully treated with rifampin therapy and gave birth to a healthy child who tested negative for HGA after delivery. An implicated blood donor was subsequently identified through physician collaboration with the regional American Red Cross and Massachusetts Department of Public Health. DISCUSSION: This is the 11th reported case of HGA in pregnancy and is at least the sixth known case in which leukoreduction did not prevent TAHGA. As seen in this case, nonspecific symptomatology of variable onset can impede diagnosis and treatment. This may increase risk of poor outcomes in maternal HGA patients. Cases of TAHGA, although currently uncommon, may increase as the incidence of HGA in certain parts of the country increases. CONCLUSION: Heightened cross-institutional awareness of the potential risk of TAHGA is warranted. Clinicians need to consider transfusion-associated infections when fever occurs in a transfusion recipient. This case provides additional evidence that leukoreduction does not obviate risk of A. phagocytophilum contamination of donated blood components.

Recurrent Clostridium difficile infection: From colonization to cure.

Clostridium difficile infection (CDI) is increasingly prevalent, dangerous and challenging to prevent and manage. Despite intense national and international attention the incidence of primary and of recurrent CDI (PCDI and RCDI, respectively) have risen rapidly throughout the past decade. Of major concern is the increase in cases of RCDI resulting in substantial morbidity, morality and economic burden. RCDI management remains challenging as there is no uniformly effective therapy, no firm consensus on optimal treatment, and reliable data regarding RCDI-specific treatment options is scant. Novel therapeutic strategies are critically needed to rapidly, accurately, and effectively identify and treat patients with, or at-risk for, RCDI. In this review we consider the factors implicated in the epidemiology, pathogenesis and clinical presentation of RCDI, evaluate current management options for RCDI and explore novel and emerging therapies.

Reduced MTHFD1 activity in male mice perturbs folate- and choline-dependent one-carbon metabolism as well as transsulfuration.

Impaired utilization of folate is caused by insufficient dietary intake and/or genetic variation and has been shown to prompt changes in related pathways, including choline and methionine metabolism. These pathways have been shown to be sensitive to variation within the Mthfd1 gene, which codes for a folate-metabolizing enzyme responsible for generating 1-carbon (1-C)-substituted folate derivatives. The Mthfd1(gt/+) mouse serves as a potential model of human Mthfd1 loss-of-function genetic variants that impair MTHFD1 function. This study investigated the effects of the Mthfd1(gt/+) genotype and folate intake on markers of choline, folate, methionine, and transsulfuration metabolism. Male Mthfd1(gt/+) and Mthfd1(+/+) mice were randomly assigned at weaning (3 wk of age) to either a control (2 mg/kg folic acid) or folate-deficient (0 mg/kg folic acid) diet for 5 wk. Mice were killed at 8 wk of age following 12 h of food deprivation; blood and liver samples were analyzed for choline, methionine, and transsulfuration biomarkers. Independent of folate intake, mice with the Mthfd1(gt/+) genotype had higher hepatic concentrations of choline (P = 0.005), betaine (P = 0.013), and dimethylglycine (P = 0.004) and lower hepatic concentrations of glycerophosphocholine (P = 0.002) relative to Mthfd1(+/+) mice. Mthfd1(gt/+) mice also had higher plasma concentrations of homocysteine (P = 0.0016) and cysteine (P < 0.001) as well as lower plasma concentrations of methionine (P = 0.0003) and cystathionine (P = 0.011). The metabolic alterations observed in Mthfd1(gt/+) mice indicate perturbed choline and folate-dependent 1-C metabolism and support the future use of Mthfd1(gt/+) mice as a tool to investigate the impact of impaired 1-C metabolism on disease outcomes.

Clostridium difficile toxins induce VEGF-A and vascular permeability to promote disease pathogenesis.

Clostridium difficile infection (CDI) is mediated by two major exotoxins, toxin A (TcdA) and toxin B (TcdB), that damage the colonic epithelial barrier and induce inflammatory responses. The function of the colonic vascular barrier during CDI has been relatively understudied. Here we report increased colonic vascular permeability in CDI mice and elevated vascular endothelial growth factor A (VEGF-A), which was induced in vivo by infection with TcdA- and/or TcdB-producing C. difficile strains but not with a TcdA-TcdB- isogenic mutant. TcdA or TcdB also induced the expression of VEGF-A in human colonic mucosal biopsies. Hypoxia-inducible factor signalling appeared to mediate toxin-induced VEGF production in colonocytes, which can further stimulate human intestinal microvascular endothelial cells. Both neutralization of VEGF-A and inhibition of its signalling pathway attenuated CDI in vivo. Compared to healthy controls, CDI patients had significantly higher serum VEGF-A that subsequently decreased after treatment. Our findings indicate critical roles for toxin-induced VEGF-A and colonic vascular permeability in CDI pathogenesis and may also point to the pathophysiological significance of the gut vascular barrier in response to virulence factors of enteric pathogens. As an alternative to pathogen-targeted therapy, this study may enable new host-directed therapeutic approaches for severe, refractory CDI.

A Multi-Center Prospective Derivation and Validation of a Clinical Prediction Tool for Severe Clostridium difficile Infection.

BACKGROUND AND AIMS: Prediction of severe clinical outcomes in Clostridium difficile infection (CDI) is important to inform management decisions for optimum patient care. Currently, treatment recommendations for CDI vary based on disease severity but validated methods to predict severe disease are lacking. The aim of the study was to derive and validate a clinical prediction tool for severe outcomes in CDI. METHODS: A cohort totaling 638 patients with CDI was prospectively studied at three tertiary care clinical sites (Boston, Dublin and Houston). The clinical prediction rule (CPR) was developed by multivariate logistic regression analysis using the Boston cohort and the performance of this model was then evaluated in the combined Houston and Dublin cohorts. RESULTS: The CPR included the following three binary variables: age ≥ 65 years, peak serum creatinine ≥ 2 mg/dL and peak peripheral blood leukocyte count of ≥ 20,000 cells/µL. The Clostridium difficile severity score (CDSS) correctly classified 76.5% (95% CI: 70.87-81.31) and 72.5% (95% CI: 67.52-76.91) of patients in the derivation and validation cohorts, respectively. In the validation cohort, CDSS scores of 0, 1, 2 or 3 were associated with severe clinical outcomes of CDI in 4.7%, 13.8%, 33.3% and 40.0% of cases respectively. CONCLUSIONS: We prospectively derived and validated a clinical prediction rule for severe CDI that is simple, reliable and accurate and can be used to identify high-risk patients most likely to benefit from measures to prevent complications of CDI.

Choline intakes exceeding recommendations during human lactation improve breast milk choline content by increasing PEMT pathway metabolites.

Demand for the vital nutrient choline is high during lactation; however, few studies have examined choline metabolism and requirements in this reproductive state. The present study sought to discern the effects of lactation and varied choline intake on maternal biomarkers of choline metabolism and breast milk choline content. Lactating (n=28) and control (n=21) women were randomized to 480 or 930 mg choline/day for 10-12 weeks as part of a controlled feeding study. During the last 4-6 weeks, 20% of the total choline intake was provided as an isotopically labeled choline tracer (methyl-d9-choline). Blood, urine and breast milk samples were collected for choline metabolite quantification, enrichment measurements, and gene expression analysis of choline metabolic genes. Lactating (vs. control) women exhibited higher (P < .001) plasma choline concentrations but lower (P ≤ .002) urinary excretion of choline metabolites, decreased use of choline as a methyl donor (e.g., lower enrichment of d6-dimethylglycine, P ≤ .08) and lower (P ≤ .02) leukocyte expression of most choline-metabolizing genes. A higher choline intake during lactation differentially influenced breast milk d9- vs. d3-choline metabolite enrichment. Increases (P ≤ .03) were detected among the d3-metabolites, which are generated endogenously via the hepatic phosphatidylethanolamine N-methyltransferase (PEMT), but not among the d9-metabolites generated from intact exogenous choline. These data suggest that lactation induces metabolic adaptations that increase the supply of intact choline to the mammary epithelium, and that extra maternal choline enhances breast milk choline content by increasing supply of PEMT-derived choline metabolites. This trial was registered at clinicaltrials.gov as NCT01127022.

Metformin and sulfonylureas in relation to cancer risk in type II diabetes patients: a meta-analysis using primary data of published studies.

INTRODUCTION: Accumulating evidence suggests that patients with type 2 diabetes mellitus (T2DM) and hyperinsulinemia are at increased risk for developing malignancies. It remains to be fully elucidated whether use of metformin, an insulin sensitizer, and/or sulfonylureas, insulin secretagogues, affect cancer incidence in subjects with T2DM. MATERIAL & METHODS: We performed a meta-analysis using PubMed, of randomized control trials (RCTs), cohorts, and case-control studies published through July 2012 that assess effects of metformin and/or sulfonylurea sulfonylureas on cancer risk at any site, in subjects with T2DM. Fixed and random effects meta-analysis models were used, and the effect size was summarized as relative risk (RR) for RCTs/cohorts and as odds ratio (OR) for the case-control studies. RESULTS: Analysis of 24 metformin studies in subjects with T2DM showed that metformin use is associated with reduced risk for the development of cancer, in both cohort (RR=0.70 [95% CI=0.67-0.73]) and case-control studies (OR=0.90 [95% CI=0.84-0.98]), but this finding was not supported by RCTs (RR=1.01[95% CI=0.81-1.26]). Data from 18 sulfonylurea studies in subjects with T2DM showed that sulfonylurea use is associated with an increase in all-cancer risk, in cohort studies (RR=1.55 [95% CI=1.48 -1.63]), though data from RCTs (RR=1.17 [95% CI=0.95-1.45]) and case-control studies (OR=1.02 [95% CI=0.93-1.13]) failed to demonstrate a statistically significant effect. CONCLUSIONS: This analysis using pooled primary data demonstrates that metformin use reduces, while sulfonylurea use may be associated with an increased cancer risk in subjects with T2DM. These findings need to be confirmed in large-scale RCTs before they are translated into clinical practice.

Leptin at the intersection of neuroendocrinology and metabolism: current evidence and therapeutic perspectives.

Since its discovery as an adipocyte-secreted hormone, leptin has been found to impact food intake, energy homeostasis, and metabolism through its effects on the central nervous system and peripheral organs. Recent research indicates that leptin may also be involved in cognition, immune function, and bone metabolism. These findings place leptin at the intersection of neuroendocrinology and metabolism, and possibly immune function, and render it an appealing therapeutic target for several niche areas of unmet clinical need. Current evidence regarding classic and emerging roles of leptin as well as the pros and cons of its potential clinical use are summarized herein.

Living the good life? Mortality and hospital utilization patterns in the Old Order Amish.

Lifespan increases observed in the United States and elsewhere throughout the developed world, have been attributed in part to improvements in medical care access and technology and to healthier lifestyles. To differentiate the relative contributions of these two factors, we have compared lifespan in the Old Order Amish (OOA), a population with historically low use of medical care, with that of Caucasian participants from the Framingham Heart Study (FHS), focusing on individuals who have reached at least age 30 years.Analyses were based on 2,108 OOA individuals from the Lancaster County, PA community born between 1890 and 1921 and 5,079 FHS participants born approximately the same time. Vital status was ascertained on 96.9% of the OOA cohort through 2011 and through systematic follow-up of the FHS cohort. The lifespan part of the study included an enlargement of the Anabaptist Genealogy Database to 539,822 individuals, which will be of use in other studies of the Amish. Mortality comparisons revealed that OOA men experienced better longevity (p<0.001) and OOA women comparable longevity than their FHS counterparts.We further documented all OOA hospital discharges in Lancaster County, PA during 2002-2004 and compared OOA discharge rates to Caucasian national rates obtained from the National Hospital Discharge Survey for the same time period. Both OOA men and women experienced markedly lower rates of hospital discharges than their non-Amish counterparts, despite the increased lifespan.We speculate that lifestyle factors may predispose the OOA to greater longevity and perhaps to lesser hospital use. Identifying these factors, which might include behaviors such as lesser tobacco use, greater physical activity, and/or enhanced community assimilation, and assessing their transferability to non-Amish communities may produce significant gains to the public health.