Gut microbiome of treatment-naïve MS patients of different ethnicities early in disease course.

Although the intestinal microbiome has been increasingly implicated in autoimmune diseases, much is unknown about its roles in Multiple Sclerosis (MS). Our aim was to compare the microbiome between treatment-naïve MS subjects early in their disease course and controls, and between Caucasian (CA), Hispanic (HA), and African American (AA) MS subjects. From fecal samples, we performed 16S rRNA V4 sequencing and analysis from 45 MS subjects (15 CA, 16 HA, 14 AA) and 44 matched healthy controls, and whole metagenomic shotgun sequencing from 24 MS subjects (all newly diagnosed, treatment-naïve, and steroid-free) and 24 controls. In all three ethnic groups, there was an increased relative abundance of the same single genus, Clostridium, compared to ethnicity-matched controls. Analysis of microbiota networks showed significant changes in the network characteristics between combined MS cohorts and controls, suggesting global differences not restricted to individual taxa. Metagenomic analysis revealed significant enrichment of individual species within Clostridia as well as particular functional pathways in the MS subjects. The increased relative abundance of Clostridia in all three early MS cohorts compared to controls provides candidate taxa for further study as biomarkers or as etiologic agents in MS.

Helicobacter pylori in children with asthmatic conditions at school age, and their mothers.

BACKGROUND: Helicobacter pylori prevalence in Western countries has been declining simultaneously with increases in childhood asthma and allergic diseases; prior studies have linked these phenomena. AIMS: To examine the association between H. pylori colonisation in children and risk of asthma and related conditions at school age. We secondly examined additional effects of maternal H. pylori status by pairing with children's status. METHODS: This study was embedded in a multi-ethnic population-based cohort in Rotterdam, The Netherlands. We measured anti-H. pylori and anti-CagA antibodies in serum of children obtained at age 6 years, and of their mothers obtained during midpregnancy. Asthma or related conditions were reported for children at age 6 years. We used multivariate logistic regression analyses among 3797 subjects. RESULTS: In children, the H. pylori positivity rate was 8.7%, and 29.2% of these were CagA-positive. A child's colonisation with a CagA-negative-H. pylori strain was associated with an increased risk of asthma (Odds ratio 2.11; 95% CI 1.23-3.60), but this differed for European (3.64; 1.97-6.73) and non-European (0.52; 0.14-1.89) children. When taking into account maternal H. pylori status, only H. pylori-positive children with an H. pylori-negative mother had increased risk of asthma (2.42; 1.11-5.27), accounting for 3.4% of the asthma risk. CONCLUSIONS: Colonisation of a European child with a CagA-negative-H. pylori strain at age 6 was associated with an increased prevalence of asthma, but there was no association for non-European children. The underlying mechanisms for the observed risk differences require further research.

Genetic risk factors for inflammatory bowel disease in a North-eastern Mexican population.

The purpose of this study was to assess the role of Helicobacter pylori and several genetic polymorphisms in relation to inflammatory bowel disease (IBD). We studied 44 unrelated patients with IBD and 75 subjects with no history of IBD as controls. Using pyrosequencing technology, we identified gene polymorphisms in IL-10, TNF-A, ILB-31, and TLR4. H. pylori status was determined by serology. Individuals homozygous for IL10-592 A or IL10-1082 A genotypes show significantly lower occurrence of IBD (P=0.03 and P<0.01, respectively). Individuals heterozygous at IL10-1082 have significantly increased occurrence of IBD, both ulcerative colitis and Crohn's disease (P<0.01). There was no difference in the prevalence of H. pylori infection between cases and controls. This study provides evidence that variation in IL10 is correlated with IBD occurrence in this Mexican population.

Effect of L-dopa on interleukin-1 beta-induced suppression of luteinizing hormone secretion in intact female rats.

BACKGROUND: The cytokine, interleukin-1 beta (IL-1 beta), increases during immune stress and is known to suppress the preovulatory luteinizing hormone (LH) surge in female rats by decreasing hypothalamic norepinephrine (NE). We hypothesized that IL-1 beta could produce this effect by decreasing NE biosynthesis. METHODS: Female Sprague-Dawley rats were implanted with a push-pull cannula in the medial preoptic area (MPA) of the hypothalamus and a catheter in the jugular vein. They were treated i.p. with the vehicle or 5 microg of IL-1 beta, the NE precursor, L-dopa, or a combination of L-dopa and IL-1 beta at 1300 hours on the day of proestrus. They were subjected to push-pull perfusion and serial blood sampling. Perfusates were analyzed for NE levels and serum samples for LH. RESULTS: IL-1 beta treatment blocked the increase in NE levels in the MPA and the LH surge. Treatment with L-dopa was able to partially restore both NE and LH levels during the afternoon of proestrus. IL-1 beta treatment caused failure of ovulation and this effect was also reversed by L-dopa. CONCLUSIONS: These results suggest that IL-1 beta could decrease NE levels in the MPA to suppress reproductive functions and L-dopa can be used to counter this effect.

Antral follicle count reliably predicts number of morphologically healthy oocytes and follicles in ovaries of young adult cattle.

Methods to predict numbers of healthy oocytes in the ovaries of young adults could have important diagnostic relevance in family planning and animal agriculture. We have observed that peak antral follicle count (AFC) determined by serial ovarian ultrasonography during follicular waves is very highly reproducible within individual young adult cattle, despite 7-fold variation among animals. Herein, we tested the hypothesis that AFC is positively associated with the number of morphologically healthy oocytes and follicles in ovaries and with serum concentrations of anti-Müllerian hormone (AMH), an indirect marker for number of healthy follicles and oocytes in ovaries. In the present study, age-matched young adult cattle (12-18 mo old) were subjected to serial ultrasonography to identify animals with a consistently high (> or =25 follicles that were > or =3 mm in diameter) or low (< or =15 follicles) AFC during follicular waves. Differences in serum AMH concentrations, ovary weight, and number of morphologically healthy and atretic follicles and oocytes were determined. The phenotypic classifications of cattle based on AFC during follicular waves or AMH concentrations both predict reliably the relative number of morphologically healthy follicles and oocytes in ovaries of age-matched young adult cattle.

The association of gastric leptin with oesophageal inflammation and metaplasia.

BACKGROUND: Gastro-oesophageal reflux disease complications may reflect imbalances between protective and injurious factors. Through its effects on cell growth, leptin may influence oesophageal mucosal homeostasis. AIMS: To determine whether leptin receptors are present in the oesophagus, and whether serum or gastric leptin levels are associated with oesophageal inflammation and metaplasia. METHODS: From patients referred for upper endoscopy, biopsies were obtained from the stomach and distal oesophagus, and serum samples were collected. Patients were classified as having normal, inflamed or Barrett's oesophagus. Quantitative immunohistochemistry was performed on representative sections, and leptin levels in plasma and gastric biopsy samples were determined by specific immunoassay. RESULTS: Of 269 individuals enrolled, 105 were Helicobacter pylori-negative. Of the 88 patients with complete oesophageal biopsies, 44 were normal, 24 were inflamed and 20 were Barrett's oesophagus. Receptors for leptin were highly expressed on oesophageal epithelial cells, with similar density and staining pattern in all three conditions, and plasma and antral leptin levels did not differ significantly. Patients with Barrett's had significantly (p = 0.01) higher fundic leptin levels (median 202 (interquartile range 123-333) pg/mg) compared with normal (126 (78-221) pg/mg) or inflamed (114 (76-195) pg/mg) oesophagus. In multivariate analysis, for every twofold increase in fundic leptin, the odds of having Barrett's was 3.4 times (95% CI 1.5 to 7.6) higher compared with having a normal oesophagus. CONCLUSIONS: Leptin receptor expression on oesophageal epithelial cells provides a pathway for leptin-mediated signal transduction. Variation in gastric leptin production could contribute to differential oesophageal healing and metaplasia progression.

[Helicobacter pylori erradication and its relation to antibiotic resistance and CYP2C19 status]. / Erradicación de H. pylori y su relación con la resistencia a los antibióticos y el estatus de CYP2C19.

OBJECTIVE: to assess the efficacy of rabeprazole (RPZ), amoxicillin (Am), and clarithromycin (Cla) (7 vs. 14 days) in the eradication of H. pylori, and to determine the effect of strain-specific antibiotic resistance and host CYP2C19 status. MATERIAL AND METHODS: first, we determined the CYP2C19 status of 100 healthy subjects to establish a sample size for the clinical trial. Then, 59 H. pylori-infected patients were randomized to receive RPZ (20 mg daily) plus Cla (500 mg b.d.) and Am (1,000 mg b.d.) for 7 vs. 14 days. The MIC for Am and Cla were determined using the agar dilution method. The CYP2C19 genotype was determined by the PCR-RFLP method. RESULTS: In the per-protocol analysis (PP) eradication rates were 89.7 and 72% for the 7- and 14-day groups (p = 0.159). In the intention to-treat analysis (ITT) eradication rates were 86.7 and 62.1% in the 7- and 14-day groups, respectively (p = 0.06). None of the strains was resistant to Am, and 4 strains were resistant to Cla: 3 (11.1%) in the 14-day group and 1 (4%) in the 7-day group. Neither strain-specific antibiotic resistance nor host CYP2C19 status influenced eradication rates. CONCLUSIONS: both 7- and 14-day therapies were effective for H. pylori eradication. Strain resistance and CYP2C19 status do not seem to influence eradication rates in the studied population.

Caspase-12 compensates for lack of caspase-2 and caspase-3 in female germ cells.

Previously, we analyzed mice lacking either caspase-2 or caspase-3 and documented a role for caspase-2 in developmental and chemotherapy-induced apoptosis of oocytes. Those data also revealed dispensability of caspase-3, although we found this caspase critical for ovarian granulosa cell death. Because of the mutual interdependence of germ cells and granulosa cells, herein we generated caspase-2 and -3 double-mutant (DKO) mice to evaluate how these two caspases functionally relate to each other in orchestrating oocyte apoptosis. No difference was observed in the rate of spontaneous oocyte apoptosis between DKO and wildtype (WT) females. In contrast, the oocytes from DKO females were more susceptible to apoptosis induced by DNA damaging agents, compared with oocytes from WT females. This increased sensitivity to death of DKO oocytes appears to be a specific response to DNA damage, and it was associated with a compensatory upregulation of caspase-12. Interestingly, DKO oocytes were more resistant to apoptosis induced by methotrexate (MTX) than WT oocytes. These results revealed that in female germ cells, insults that directly interfere with their metabolic status (e.g. MTX) require caspase-2 and caspase-3 as obligatory executioners of the ensuing cell death cascade. However, when DNA damage is involved, and in the absence of caspase-2 and -3, caspase-12 becomes upregulated and mediates apoptosis in oocytes.

Genetic variance modifies apoptosis susceptibility in mature oocytes via alterations in DNA repair capacity and mitochondrial ultrastructure.

Although the identification of specific genes that regulate apoptosis has been a topic of intense study, little is known of the role that background genetic variance plays in modulating cell death. Using germ cells from inbred mouse strains, we found that apoptosis in mature (metaphase II) oocytes is affected by genetic background through at least two different mechanisms. The first, manifested in AKR/J mice, results in genomic instability. This is reflected by numerous DNA double-strand breaks in freshly isolated oocytes, causing a high apoptosis susceptibility and impaired embryonic development following fertilization. Microinjection of Rad51 reduces DNA damage, suppresses apoptosis and improves embryonic development. The second, manifested in FVB mice, results in dramatic dimorphisms in mitochondrial ultrastructure. This is correlated with cytochrome c release and a high apoptosis susceptibility, the latter of which is suppressed by pyruvate treatment, Smac/DIABLO deficiency, or microinjection of 'normal' mitochondria. Therefore, background genetic variance can profoundly affect apoptosis in female germ cells by disrupting both genomic DNA and mitochondrial integrity.

Helicobacter pylori and oesophageal and gastric cancers in a prospective study in China.

In a cohort of 29,584 residents of Linxian, China, followed from 1985 to 2001, we conducted a case-cohort study of the magnitude of the association of Helicobacter pylori seropositivity with cancer risk in a random sample of 300 oesophageal squamous cell carcinomas, 600 gastric cardia adenocarcinomas, all 363 diagnosed gastric non-cardia adenocarcinomas, and a random sample of the entire cohort (N=1050). Baseline serum was evaluated for IgG antibodies to whole-cell and CagA H. pylori antigens by enzyme-linked immunosorbent assay. Risks of both gastric cardia and non-cardia cancers were increased in individuals exposed to H. pylori (Hazard ratios (HRs) and 95% confidence intervals=1.64; 1.26-2.14, and 1.60; 1.15-2.21, respectively), whereas risk of oesophageal squamous cell cancer was not affected (1.17; 0.88-1.57). For both cardia and non-cardia cancers, HRs were higher in younger individuals. With longer time between serum collection to cancer diagnosis, associations became stronger for cardia cancers but weaker for non-cardia cancers. CagA positivity did not modify these associations. The associations between H. pylori exposure and gastric cardia and non-cardia adenocarcinoma development were equally strong, in contrast to Western countries, perhaps due to the absence of Barrett's oesophagus and oesophageal adenocarcinomas in Linxian, making all cardia tumours of gastric origin, rather than a mixture of gastric and oesophageal malignancies.

Molecular requirements for doxorubicin-mediated death in murine oocytes.

We previously published evidence that oocytes exposed to doxorubicin (DXR), a widely used chemotherapeutic agent, rapidly undergo morphological and biochemical changes via discrete effector signaling pathways consistent with the occurrence of apoptosis. In this report, we elucidated the molecular requirements for actions of this drug in oocytes. Our results indicate that within 1 h of exposure DXR causes rapid DNA damage, and commits the oocyte to cytoplasmic fragmentation by the fourth hour, followed by delayed oocyte activation and execution of cytoplasmic fragmentation. Inhibitors that interfere with oocyte activation consistently rescue cytoplasmic fragmentation, but fail to suppress DNA damage. There was evidence of depletion of Bax, Caspase-2, MA-3 and Bcl-x transcripts, suggesting that modulations by DXR caused recruitment of these maternal transcripts into the translation process. Furthermore, sphingolipids such as sphingosine-1-phosphate and ceramide modulate DXR actions by, respectively, altering its intracellular trafficking, or by sustaining the drug's contact with DNA.

Association of interleukin-1B and interleukin-1RN polymorphisms with gastric cancer in a high-risk population of Costa Rica.

Several risk factors have been associated with gastric cancer, among them Helicobacter pylori infection. This bacterium yields inflammation, the degree of which depends on the bacterial strain and the severity of the host response. The inflammatory response involves a complex cytokine network. Recently, polymorphisms of the genes coding for interleukin-1beta (IL-1B), interleukin-1Ra (ILRN) and interleukin-10 have been associated with an increased risk of gastric cancer. In order to determine the association of the IL-1B, IL-1RN and IL-10 polymorphisms with gastric cancer in a high-risk Costa Rican population, we analysed purified DNA of 58 gastric cancer patients, 99 controls and 41 patients classified as group I or II, according to the Japanese classification. Genotyping was carried out by PCR, PCR-RFLP and pyrosequencing analysis. We did not find any association of the IL-1B-31, IL-1B-511 and IL-10 polymorphisms with the risk for developing gastric cancer in the studied population. Carriers of the IL-1B+3954T/- had an increased risk for developing gastric cancer (OR 3.7; 95%CI: 1.34-10.2). Also we found an increased risk for developing gastric cancer for allele 2 heterozygotes of the IL-1RN (OR 2.94; 95%CI: 1.09-7.93). This is the first time that IL-1B+3954 has been associated with gastric cancer. This is one of the first studies trying to describe the role played by IL-1B, IL-1RN and IL-10 genetic polymorphisms in gastric cancer in one of the highest risk American countries. Further investigation on American countries is needed.

High frequency of gastric colonization with multiple Helicobacter pylori strains in Venezuelan subjects.

Multiple Helicobacter pylori strains may colonize an individual host. Using enzyme-linked immunosorbent assay and line probe assay (LiPA) techniques, we analyzed the prevalence of mixed H. pylori colonization in 127 subjects from Venezuela, a country of high H. pylori prevalence, from three regions representing different population groups: the Andes (Merida), where Caucasian mestizos predominate, a major city near the coast (Caracas), where Amerindian-Caucasian-African mestizos predominate, and an Amazonian community (Puerto Ayacucho), where Amerindians predominate and mestizos reflect Amerindian and Caucasian ancestry. Among 121 H. pylori-positive persons, the prevalence of cagA-positive strains varied from 50% (Merida) to 86% (Puerto Ayacucho) by LiPA. Rates of mixed colonization also varied, as assessed by LiPA of the vacA s (mean, 49%) and m (mean, 26%) regions. In total, 55% of the individuals had genotypic evidence of mixed colonization. vacA s1c, a marker of Amerindian (East Asian) origin, was present in all three populations, especially from Puerto Ayacucho (86%). These results demonstrate the high prevalence of mixed colonization and indicate that the H. pylori East Asian vacA genotype has survived in all three populations tested.

Characterisation of Helicobacter pylori isolates from the north-eastern region of Mexico.

The vacA and cagA genotypes of 50 Helicobacter pylori isolates from patients in the north-eastern region of Mexico were characterised by PCR, and the correlation between genotypes and different clinical outcomes was investigated. Strains of H. pylori that are vacA s1/m1 and cagA positive have previously been associated with more severe clinical outcomes, and some studies have shown differences in the vacA and cagA genotypes in different geographical regions. The six possible combinations of the vacA signal (s) and middle (m) regions were identified in this population, and the most frequent genotype was s2/m2. Thirty-two (64%) isolates were identified as cagA-positive. The s region was not amplified from seven of the cagA-positive isolates, and the m region was not amplified from one cagA-negative isolate, indicating that additional subfamilies of s and m genotypes may exist. The s1/m1 genotype was associated with cagA-positive strains (p < 0.05). No association was found between the vacA and cagA genotypes and clinical outcomes.

Antibiotic susceptibility patterns of Helicobacter pylori strains isolated from northeastern Mexico.

There are reports of increased antibiotic resistance rates in Helicobacter pylori strains around the world. The aim of this study was to determine the susceptibility patterns in H. pylori strains isolated in Monterrey, Mexico. We studied 62 strains isolated from the same number of symptomatic adult patients. Metronidazole (Mtz), clarithromycin (Cla), amoxicillin (Amx) and tetracycline (Tet) were tested by the E-test method. We observed that 37.1% of the strains were resistant to Mtz (MIC > or = 8 mg/L), and 8.1% to Cla (MIC > or = 8 mg/L), but we did not observe resistance to Amx (MIC > or = 2 mg/L) or Tet (MIC > or = 4 mg/L). In northeastern Mexico, the percentage of resistant strains was similar to that observed in developed countries. These results confirm that it is necessary to evaluate the susceptibility patterns of H. pylori strains by geographic area.

Evidence that cagA(+) Helicobacter pylori strains are disappearing more rapidly than cagA(-) strains.

BACKGROUND AND AIMS: The prevalence of Helicobacter pylori colonisation in populations in developed country has been declining, as shown by community based serological surveys of adults in Vammala, Finland in 1973 and 1994. In this study, we determined whether the proportion of subjects colonised by cagA(+) or cagA(-) H pylori strains has changed as the overall prevalence of H pylori(+) has declined. METHODS: We examined 911 sera from Vammala's study for antibodies to the CagA antigen of H pylori using a truncated CagA protein as the antigen in an ELISA and we examined the trend in acquisition and carriage of cagA(+) strains. RESULTS: As expected, the prevalence of carriage of both cagA(+) and cagA(-) strains fell between 1973 and 1994 (p<0.001). However, the prevalence of cagA(+) strains among those <45 years declined (34% to 8%) significantly (p<0.001) more than for cagA(-) strains (12% to 6%). Of 221 subjects with paired serum samples, 12 (5.4%) changed H pylori status; the estimated seroconversion and reversion rates were 0.4% and 0.13% per year, respectively. Except for the few individuals who changed serostatus, absolute antibody levels to H pylori antigens, including CagA, changed little over the 21 year period. CONCLUSIONS: The decline in CagA seroprevalence predominantly reflects declining acquisition of cag(+) strains in younger subjects. In addition, these data confirm that H pylori acquisition chiefly occurs during childhood but continues to occur during adulthood, albeit at low rates, in developed countries.

Helicobacter pylori prevalence and CagA status among children in two counties of China with high and low risks of gastric cancer.

BACKGROUND: Studies in adult populations in selected countries with widely varying rates of gastric cancer have shown a weak correlation between gastric cancer mortality rates and the prevalence of CagA+ strains of H. pylori. However, only limited data are available in ethnically homogenous populations with varying rates in the same region. METHODS; We compared the prevalence of H. pylori in general and of CagA+ strains in particular among children in Shandong Province, China in areas at high (Linqu County) and low risk (Cangshan County) of gastric cancer. H. pylori status among children aged 3 to 12 years was determined by 13C-UBT, and CagA status was determined by enzyme-linked immunosorbent assay (ELISA). Because of the difficulty in obtaining blood from young children aged 3 to 4 years and from some children aged 5 years, CagA status was determined among part of children 5 years old and children 6 to 12 years old. RESULTS; Among 98 children aged 3 to 12 years in Linqu, 68 (69.4%) was H. pylori-positive, as compared with 29 (28.7%) among 101 children in Cangshan. Among children positive for 13C-UBT, the proportion of the CagA+ strains were identified was 46 (88.5%) of 52 in Linqu and 13 (81.3%) of 16 in Cangshan, respectively. CONCLUSIONS: The prevalence of H. pylori was nearly three times higher among children in Linqu than in Cangshan, which may contribute to the large differential in gastric cancer rates for two neighboring populations in Shandong Province.

Caspase-2 deficiency prevents programmed germ cell death resulting from cytokine insufficiency but not meiotic defects caused by loss of ataxia telangiectasia-mutated (Atm) gene function.

It is well established that programmed cell death claims up to two-thirds of the oocytes produced during gametogenesis in the developing fetal ovaries. However, the mechanisms underlying prenatal germ cell loss in females remain poorly understood. Herein we report that caspase-11 null female mice are born with a reduced number of oocyte-containing primordial follicles. This phenotype is likely due to failed cytokine processing known to occur in caspase-11 mutants since neonatal female mice lacking both interleukin (IL)-1alpha and IL-1beta also exhibit a reduced endowment of primordial follicles. In addition, germ cell death in wild-type fetal ovaries cultured ex vivo is suppressed by either cytokine, likely via ligand activation of type 1 IL-1 receptors expressed in fetal germ cells. Normal oocyte endowment can be restored in caspase-11 null female mice by simultaneous inactivation of the gene encoding the cell death executioner enzyme, caspase-2. However, caspase-2 deficiency cannot overcome gametogenic failure resulting from meiotic recombination defects in ataxia telangiectasia-mutated (Atm) null female mice. Thus, genetically distinct mechanisms exist for developmental deletion of oocytes via programmed cell death, one of which probably functions as a meiotic quality-control checkpoint that cannot be overridden.

Aromatic hydrocarbon receptor-driven Bax gene expression is required for premature ovarian failure caused by biohazardous environmental chemicals.

Polycyclic aromatic hydrocarbons (PAHs) are toxic chemicals released into the environment by fossil fuel combustion. Moreover, a primary route of human exposure to PAHs is tobacco smoke. Oocyte destruction and ovarian failure occur in PAH-treated mice, and cigarette smoking causes early menopause in women. In many cells, PAHs activate the aromatic hydrocarbon receptor (Ahr), a member of the Per-Arnt-Sim family of transcription factors. The Ahr is also activated by dioxin, one of the most intensively studied environmental contaminants. Here we show that an exposure of mice to PAHs induces the expression of Bax in oocytes, followed by apoptosis. Ovarian damage caused by PAHs is prevented by Ahr or Bax inactivation. Oocytes microinjected with a Bax promoter-reporter construct show Ahr-dependent transcriptional activation after PAH, but not dioxin, treatment, consistent with findings that dioxin is not cytotoxic to oocytes. This difference in the action of PAHs versus dioxin is conveyed by a single base pair flanking each Ahr response element in the Bax promoter. Oocytes in human ovarian biopsies grafted into immunodeficient mice also accumulate Bax and undergo apoptosis after PAH exposure in vivo. Thus, Ahr-driven Bax transcription is a novel and evolutionarily conserved cell-death signaling pathway responsible for environmental toxicant-induced ovarian failure.