Approaches to studying and manipulating the enteric microbiome to improve autism symptoms.

There is a growing body of scientific evidence that the health of the microbiome (the trillions of microbes that inhabit the human host) plays an important role in maintaining the health of the host and that disruptions in the microbiome may play a role in certain disease processes. An increasing number of research studies have provided evidence that the composition of the gut (enteric) microbiome (GM) in at least a subset of individuals with autism spectrum disorder (ASD) deviates from what is usually observed in typically developing individuals. There are several lines of research that suggest that specific changes in the GM could be causative or highly associated with driving core and associated ASD symptoms, pathology, and comorbidities which include gastrointestinal symptoms, although it is also a possibility that these changes, in whole or in part, could be a consequence of underlying pathophysiological features associated with ASD. However, if the GM truly plays a causative role in ASD, then the manipulation of the GM could potentially be leveraged as a therapeutic approach to improve ASD symptoms and/or comorbidities, including gastrointestinal symptoms. One approach to investigating this possibility in greater detail includes a highly controlled clinical trial in which the GM is systematically manipulated to determine its significance in individuals with ASD. To outline the important issues that would be required to design such a study, a group of clinicians, research scientists, and parents of children with ASD participated in an interdisciplinary daylong workshop as an extension of the 1st International Symposium on the Microbiome in Health and Disease with a Special Focus on Autism (www.microbiome-autism.com). The group considered several aspects of designing clinical studies, including clinical trial design, treatments that could potentially be used in a clinical trial, appropriate ASD participants for the clinical trial, behavioral and cognitive assessments, important biomarkers, safety concerns, and ethical considerations. Overall, the group not only felt that this was a promising area of research for the ASD population and a promising avenue for potential treatment but also felt that further basic and translational research was needed to clarify the clinical utility of such treatments and to elucidate possible mechanisms responsible for a clinical response, so that new treatments and approaches may be discovered and/or fostered in the future.

Relationships between feeding problems, behavioral characteristics and nutritional quality in children with ASD.

Many children with autism spectrum disorders (ASD) have co-occurring feeding problems. However, there is limited knowledge about how these feeding habits are related to other behavioral characteristics ubiqitious in ASD. In a relatively large sample of 256 children with ASD, ages 2-11, we examined the relationships between feeding and mealtime behaviors and social, communication, and cognitive levels as well repetitive and ritualistic behaviors, sensory behaviors, and externalizing and internalizing behaviors. Finally, we examined whether feeding habits were predictive of nutritional adequacy. In this sample, we found strong associations between parent reported feeding habits and (1) repetitive and ritualistic behaviors, (2) sensory features, and (3) externalizing and internalizing behavior. There was a lack of association between feeding behaviors and the social and communication deficits of ASD and cognitive levels. Increases in the degree of problematic feeding behaviors predicted decrements in nutritional adequacy.

Using allopurinol above the dose based on creatinine clearance is effective and safe in patients with chronic gout, including those with renal impairment.

OBJECTIVE: To determine the efficacy and safety of increasing the allopurinol dose above the proposed creatinine clearance-based dose in patients with gout. METHODS: Patients with gout who had been receiving a stable dose of allopurinol for ≥ 1 month were recruited. The dose of allopurinol was increased to obtain the target serum urate level of <0.36 mmoles/liter (<6 mg/dl). Patients were seen monthly until the serum urate concentration was <0.36 mmoles/liter for 3 consecutive months and then were seen every 3 months for at least 12 months. Data were analyzed using the dosage of allopurinol (mg/day) greater than the recommended dosage, as defined by the Hande criteria. RESULTS: Ninety patients were enrolled. The mean age of the patients was 58.7 years (range 27-83 years), 87.9% were male, and 81.9% were of European ancestry. Forty-five patients had a serum urate concentration of ≥ 0.36 mmoles/liter, and the dose of allopurinol was increased in these patients. Rashes developed in 3 patients, and either allopurinol was discontinued or dose escalation was ceased in these patients. Seven patients were lost to followup or developed intervening medical problems that precluded dose escalation. In 31 (88.8%) of 35 patients who completed the study, the serum urate level was <0.36 mmoles/liter at 12 months. Two of the 5 patients who had a serum urate level ≥ 0.36 mmoles/liter had undetectable levels of plasma oxypurinol, indicating noncompliance with allopurinol treatment. A significant reduction in the serum urate concentration was observed for all allopurinol doses above the recommended dose. Eighteen of 45 patients were receiving furosemide; those receiving furosemide were just as likely as those not receiving furosemide to achieve a serum urate concentration of <0.36 mmoles/liter (72% versus 88.5%; P = 0.24). Patients receiving furosemide required a higher dose of allopurinol to achieve the target serum urate concentration. No serious adverse events were observed. CONCLUSION: Increasing the dose of allopurinol above the proposed creatinine clearance-based dose led to a significant reduction in the serum urate concentration. Approximately 89% of patients achieved a serum urate concentration of <0.36 mmoles/liter. In this cohort, toxicity was not increased in patients receiving higher doses of allopurinol, including those with renal impairment.

Polymorphisms within the folate pathway predict folate concentrations but are not associated with disease activity in rheumatoid arthritis patients on methotrexate.

OBJECTIVES: To determine whether genetic polymorphisms within the folate pathway are associated with red blood cell (RBC) methotrexate (MTX) polyglutamate concentrations, RBC folate concentrations and MTX efficacy/toxicity. METHODS: Disease activity in 200 rheumatoid arthritis patients on MTX was assessed by swollen and tender joint counts and Disease Activity Score 28. Genetic polymorphisms shown to have an effect on gene expression or protein function were examined. RESULTS: RBC folate concentrations were significantly associated with MTHFR 677C>T (P=0.002), MTRR 66A>G (P<0.0001), MTHFD1 1958G>A (P=0.001) and SHMT 1420C>T (P=0.012), whereas no association of these polymorphisms with disease activity was observed. None of the polymorphisms tested predicted RBC MTX polyglutamate concentrations. There were weak associations between central nervous system adverse effects and AMPD1 34C>T (P=0.04) and between gastrointestinal adverse effects and MTHFD1 1958G>A (P=0.03) and ABCC2 IVS23+56T>C (P=0.045). There was a stronger association between any adverse effect and ABCG2 914C>A (P=0.004). CONCLUSION: Although RBC folate concentrations are associated with genetic polymorphisms within the folate pathway, these variants do not predict disease activity. To accurately evaluate whether any polymorphisms are reliable predictors of MTX efficacy or toxicity, large prospective clinical trials are required. Furthermore, application of a genome-wide association strategy is likely to uncover novel predictors of MTX response.

Methotrexate polyglutamate concentrations are not associated with disease control in rheumatoid arthritis patients receiving long-term methotrexate therapy.

OBJECTIVE: There are limited data suggesting that methotrexate polyglutamate (MTXGlu) concentrations can guide MTX dosing in patients with rheumatoid arthritis (RA). The aim of this study was to define a therapeutic range of red blood cell (RBC) MTXGlu(n) concentrations (where n refers to the number of glutamate groups), including threshold values for efficacy and adverse effects in patients receiving long-term oral MTX treatment. METHODS: A cross-sectional study of 192 patients receiving oral MTX was undertaken. Disease activity was assessed by the swollen and tender joint counts, the C-reactive protein level, and the Disease Activity Score in 28 joints (DAS28). High disease activity was defined as a DAS28 of >3.2. A standardized questionnaire regarding common MTX adverse effects was completed. RESULTS: The MTX dosage was significantly higher in patients in whom the swollen joint count and DAS28 were higher. The MTXGlu(4), MTXGlu(5), MTXGlu(3-5), and MTXGlu(1-5) concentrations were significantly higher in patients with high disease activity. After correction for age, the estimated glomerular filtration rate, and the MTX dosage, the association remained significant for MTXGlu(5). RBC folate concentrations were significantly higher in the group with high disease activity. There was no association between any MTXGlu(n) concentration and adverse effects. CONCLUSION: In contrast to other studies, the results of the present study did not show a relationship between the MTXGlu(n) concentration and reduced disease activity in patients with RA who were receiving long-term MTX therapy. However, disease activity was influenced by the RBC folate level, which may be a more important factor than MTXGlu(n) concentrations for disease control. In accordance with the findings of previous studies, we were unable to show a relationship between MTXGlu(n) concentrations and adverse effects. Prospective studies will be important to determine whether there is a role for measuring MTXGlu(n) concentrations in patients receiving long-term treatment with MTX.

Determinants of red blood cell methotrexate polyglutamate concentrations in rheumatoid arthritis patients receiving long-term methotrexate treatment.

OBJECTIVE: Methotrexate (MTX) is the most commonly used disease-modifying antirheumatic drug (DMARD) in the management of rheumatoid arthritis (RA). MTX is transported into cells, where additional glutamate moieties are added and it is retained as MTX polyglutamates (MTXGlu [referred to as a group as MTXGlun]). There is large interpatient variability in MTXGlun concentrations. This study was undertaken to determine nongenetic factors that influence red blood cell (RBC) MTXGlun concentrations in patients receiving long-term stable low-dose oral MTX. METHODS: One hundred ninety-two patients receiving long-term oral MTX for the treatment of RA were recruited. Trough MTXGlun concentrations were measured by high-performance liquid chromatography. Univariate analysis was performed to determine variables influencing MTXGlun concentrations. Backward stepwise multivariate regression analysis was done to determine variables that affect individual MTXGlun concentrations; variables with P values of <0.1 in the univariate analysis for any MTXGlun were included. RESULTS: Univariate analysis revealed that increased age, lower estimated glomerular filtration rate (GFR), higher MTX dosage, longer duration of MTX treatment, and use of prednisone were associated with significantly higher MTXGlun concentrations. Smokers had significantly lower concentrations of MTXGlu3, MTXGlu3-5, and MTXGlu1-5. Sex, rheumatoid factor and anti-cyclic citrullinated peptide status, RBC folate level, and body mass index had no significant effect on MTXGlun levels. Concomitant use of other DMARDs was associated with lower MTXGlu2 levels, and treatment with nonsteroidal antiinflammatory drugs was associated with lower MTXGlu3 and MTXGlu1-5 concentrations. Multivariate regression analysis revealed that age, MTX dosage, and estimated GFR were the major determinants of MTXGlun concentrations. CONCLUSION: Large interpatient variability in MTXGlun concentrations can be explained, at least in part, by a combination of factors, particularly age, MTX dosage, and renal function. There are complex interactions between smoking, RBC folate levels, and concentrations of MTXGlun.

Folate transport gene inactivation in mice increases sensitivity to colon carcinogenesis.

Low dietary folate intake is associated with an increased risk for colon cancer; however, relevant genetic animal models are lacking. We therefore investigated the effect of targeted ablation of two folate transport genes, folate binding protein 1 (Folbp1) and reduced folate carrier 1 (RFC1), on folate homeostasis to elucidate the molecular mechanisms of folate action on colonocyte cell proliferation, gene expression, and colon carcinogenesis. Targeted deletion of Folbp1 (Folbp1(+/-) and Folbp1(-/-)) significantly reduced (P < 0.05) colonic Folbp1 mRNA, colonic mucosa, and plasma folate concentration. In contrast, subtle changes in folate homeostasis resulted from targeted deletion of RFC1 (RFC1(+/-)). These animals had reduced (P < 0.05) colonic RFC1 mRNA and exhibited a 2-fold reduction in the plasma S-adenosylmethionine/S-adenosylhomocysteine. Folbp1(+/-) and Folbp1(-/-) mice had larger crypts expressed as greater (P < 0.05) numbers of cells per crypt column relative to Folbp1(+/+) mice. Colonic cell proliferation was increased in RFC1(+/-) mice relative to RFC1(+/+) mice. Microarray analysis of colonic mucosa showed distinct changes in gene expression specific to Folbp1 or RFC1 ablation. The effect of folate transporter gene ablation on colon carcinogenesis was evaluated 8 and 38 weeks post-azoxymethane injection in wild-type and heterozygous mice. Relative to RFC1(+/+) mice, RFC1(+/-) mice developed increased (P < 0.05) numbers of aberrant crypt foci at 8 weeks. At 38 weeks, RFC1(+/-) mice developed local inflammatory lesions with or without epithelial dysplasia as well as adenocarcinomas, which were larger relative to RFC1(+/+) mice. In contrast, Folbp1(+/-) mice developed 4-fold (P < 0.05) more lesions relative to Folbp1(+/+) mice. In conclusion, Folbp1 and RFC1 genetically modified mice exhibit distinct changes in colonocyte phenotype and therefore have utility as models to examine the role of folate homeostasis in colon cancer development.

Developmental consequences of in utero sodium arsenate exposure in mice with folate transport deficiencies.

Previous studies have demonstrated that mice lacking a functional folate binding protein 2 gene (Folbp2-/-) were significantly more sensitive to in utero arsenic exposure than were the wild-type mice similarly exposed. When these mice were fed a folate-deficient diet, the embryotoxic effect of arsenate was further exacerbated. Contrary to expectations, studies on 24-h urinary speciation of sodium arsenate did not demonstrate any significant difference in arsenic biotransformation between Folbp2-/- and Folbp2+/+ mice. To better understand the influence of folate pathway genes on arsenic embryotoxicity, the present investigation utilized transgenic mice with disrupted folate binding protein 1 (Folbp1) and reduced folate carrier (RFC) genes. Because complete inactivation of Folbp1 and RFC genes results in embryonic lethality, we used heterozygous animals. Overall, no RFC genotype-related differences in embryonic susceptibility to arsenic exposure were observed. Embryonic lethality and neural tube defect (NTD) frequency in Folbp1 mice was dose-dependent and differed from the RFC mice; however, no genotype-related differences were observed. The RFC heterozygotes tended to have higher plasma levels of S-adenosylhomocysteine (SAH) than did the wild-type controls, although this effect was not robust. It is concluded that genetic modifications at the Folbp1 and RFC loci confers no particular sensitivity to arsenic toxicity compared to wild-type controls, thus disproving the working hypothesis that decreased methylating capacity of the genetically modified mice would put them at increased risk for arsenic-induced reproductive toxicity.

Multiple subphases of DNA repair and poly (ADP-ribose) synthesis in Chinese hamster ovary (CHO-K1) cells.

The two types of DNA synthesis as well as poly(ADP-ribose) biosynthesis were measured simultaneously in synchronized intact populations of CHO cells throughout the duration of S phase. Naturally occurring DNA fragmentation was detected by random primed oligonucleotide synthesis (ROPS assay). Fractions of synchronous cell populations were obtained by counterflow centrifugal elutriation. By gradually increasing the resolution of centrifugal elutriation multiple non-overlapping repair and replication peaks were obtained. The elutriation profile of DNA repair peaks corresponded to the DNA fragmentation pattern measured by ROPS assay. The number and position of poly(ADP-ribose) peaks during S phase resembled those seen in the DNA replication profile. Our results indicate that PAR synthesis is coupled to DNA replication serving the purpose of genomic stability.

DNA methylation in Folbp1 knockout mice supplemented with folic acid during gestation.

Periconceptional folic acid supplementation has been shown to prevent up to 70% of neural tube and other birth defects in humans; however, the mechanism is still unknown. In this study, we tested whether defective intracellular folate transport, as achieved by inactivation of the murine folate-binding protein 1 (Folbp1), affects global DNA methylation in the liver and brain from gestational day (GD) 15 embryos. Complete Folbp1 inactivation is embryolethal but can be reversed by maternal folinic acid (FA) supplementation, and thus we also tested the effect of FA supplementation on DNA methylation in Folbp1 fetuses. Overall, the extent of global DNA methylation seems to be similar across all genotypes in unsupplemented control Folbp1 mice; however, explicit conclusions regarding Folbp1(-/-) fetuses were not possible because only a single living unsupplemented fetus was viable at GD 15. FA supplementation induced global DNA hypomethylation across all genotypes. FA-induced hypomethylation is most likely due to its ability to inhibit the enzyme glycine hydroxymethyltransferase, thereby inhibiting the homocysteine remethylation cycle necessary to regenerate S-adenosylmethionine, the methyl donor for DNA methyltransferases. Our hypothesis was that due to defective folate transport in Folbp1(-/-) embryos and fetuses, DNA would be hypomethylated, thereby altering the temporal expression of critical genes necessary for normal embryonic development. However, these results suggest that an extended examination of changes in DNA methylation prior to GD 15 is required to unequivocally prove or disprove the hypothesis.