State of the art; microbiology in health and disease. Intestinal bacterial flora in autism

Autism of the regressive variety is selected as an example of the importance of intestinal bacterial microflora in disease other than classical infection. Our studies have indicated that intestinal bacteria play a role in this disease since it responds to oral vancomycin, a drug that is not absorbed from the GI tract. Pyrosequencing studies document an abnormal gut microflora in regressive autism subjects as compared to controls. Finally, we present preliminary evidence suggesting that Desulfovibrio may play a key role in this disease.     

Gene-Network Analysis Identifies Susceptibility Genes Related to Glycobiology in Autism

The recent identification of copy-number variation in the human genome has opened up new avenues for the discovery of positional candidate genes underlying complex genetic disorders, especially in the field of psychiatric disease. One major challenge that remains is pinpointing the susceptibility genes in the multitude of disease-associated loci. This challenge may be tackled by reconstruction of functional gene-networks from the genes residing in these loci. We applied this approach to autism spectrum disorder (ASD), and identified the copy-number changes in the DNA of 105 ASD patients and 267 healthy individuals with Illumina Humanhap300 Beadchips. Subsequently, we used a human reconstructed gene-network, Prioritizer, to rank candidate genes in the segmental gains and losses in our autism cohort. This analysis highlighted several candidate genes already known to be mutated in cognitive and neuropsychiatric disorders, including RAI1, BRD1, and LARGE. In addition, the LARGE gene was part of a sub-network of seven genes functioning in glycobiology, present in seven copy-number changes specifically identified in autism patients with limited co-morbidity. Three of these seven copy-number changes were de novo in the patients. In autism patients with a complex phenotype and healthy controls no such sub-network was identified. An independent systematic analysis of 13 published autism susceptibility loci supports the involvement of genes related to glycobiology as we also identified the same or similar genes from those loci. Our findings suggest that the occurrence of genomic gains and losses of genes associated with glycobiology are important contributors to the development of ASD.     

A pilot study of the relationship between bowel habits and sleep health by actigraphy measurement and fecal flora analysis

We have previously reported that there may be a relationship between bowel habits including functional constipation (FC) and irritable bowel syndrome and sleep health. However, our previous studies were based on only subjective parameters by self-reported questionnaire. The aim of this study is to investigate the relationship between bowel habits such as FC and sleep health using objective parameters. Sleep health was assessed by actigraphy measurement and bowel habits by fecal flora analysis. The FC and control subjects, whose bowel habits were defined at Rome II, were recruited from evaluated respondents in our previous study directed at middle-aged Japanese women, ten FC and ten control subjects participating in this study. Wake after sleep onset (WASO) and WASO (%) (WASO/total sleep time multiplied by 100) in FC subjects was significantly longer and greater than those in control subjects, respectively. Average activity during sleep in FC subjects was significantly higher than that in control subjects. FC had no effect on total sleep time. Bifidobacterium is broadly accepted to be useful intestinal bacteria for human health and one of the indices showing that the intestinal environment is in a desirable condition. Bifidobacterium counts per gram of wet feces and proportion in total bacterial cell counts in FC subjects were significantly lower than those in control subjects. In conclusion, these results suggest that corresponding to low Bifidobacterium counts and proportion, sleep in FC subjects may be worse than that in control subjects. There may be a relationship between bowel habits and sleep health. Bowel habits such as FC might be a risk factor for sleep disorders.     

Paneth cells: the hub for sensing and regulating intestinal flora

The complex interplay between symbiotic bacteria and host immunity plays a key role in shaping intestinal homeostasis and maintaining host health. Paneth cells, as one of the major producers of antimicrobial peptides in the intestine under steady-state conditions, play a vital role in regulating intestinal flora. Many studies on inflammatory bowel disease (IBD)-associated genes have put Paneth cells at the center of IBD pathogenesis. In this perspective, we focus on mechanistic studies of different cellular processes in Paneth cells that are regulated by various IBD-associated susceptibility genes, and we discuss the hypothesis that Paneth cells function as the central hub for sensing and regulating intestinal flora in the maintenance of intestinal homeostasis.     

益生菌疗法——抑郁症患者的福音?

近几年,肠道菌群与精神疾病,如孤独症谱系障碍、焦虑症、抑郁症等之间的关系越来越受到重视。本文主要介绍了肠道菌群调控精神行为的现有证据,并总结了益生菌对抑郁症干预作用的现有主要临床前及临床研究结果。虽然目前在这一领域取得了许多令人激动的进展,但仍需要更多深入的研究来促进我们理解肠道菌群与抑郁症二者之间的因果关联,并充分评估益生菌防治抑郁症的潜力。     

Increased intestinal permeability correlates with sigmoid mucosa alpha-synuclein staining and endotoxin exposure markers in early Parkinson's disease

Parkinson's disease (PD) is the second most common neurodegenerative disorder of aging. The pathological hallmark of PD is neuronal inclusions termed Lewy bodies whose main component is alpha-synuclein protein. The finding of these Lewy bodies in the intestinal enteric nerves led to the hypothesis that the intestine might be an early site of PD disease in response to an environmental toxin or pathogen. One potential mechanism for environmental toxin(s) and proinflammatory luminal products to gain access to mucosal neuronal tissue and promote oxidative stress is compromised intestinal barrier integrity. However, the role of intestinal permeability in PD has never been tested. We hypothesized that PD subjects might exhibit increased intestinal permeability to proinflammatory bacterial products in the intestine. To test our hypothesis we evaluated intestinal permeability in subjects newly diagnosed with PD and compared their values to healthy subjects. In addition, we obtained intestinal biopsies from both groups and used immunohistochemistry to assess bacterial translocation, nitrotyrosine (oxidative stress), and alpha-synuclein. We also evaluated serum markers of endotoxin exposure including LPS binding protein (LBP). Our data show that our PD subjects exhibit significantly greater intestinal permeability (gut leakiness) than controls. In addition, this intestinal hyperpermeability significantly correlated with increased intestinal mucosa staining for E. coli bacteria, nitrotyrosine, and alpha-synuclein as well as serum LBP levels in PD subjects. These data represent not only the first demonstration of abnormal intestinal permeability in PD subjects but also the first correlation of increased intestinal permeability in PD with intestinal alpha-synuclein (the hallmark of PD), as well as staining for gram negative bacteria and tissue oxidative stress. Our study may thus shed new light on PD pathogenesis as well as provide a new method for earlier diagnosis of PD and suggests potential therapeutic targets in PD subjects. TRIAL REGISTRATION: Clinicaltrials.gov NCT01155492.     

Pyrosequencing study of fecal microflora of autistic and control children

There is evidence of genetic predisposition to autism, but the percent of autistic subjects with this background is unknown. It is clear that other factors, such as environmental influences, may play a role in this disease. In the present study, we have examined the fecal microbial flora of 33 subjects with various severities of autism with gastrointestinal symptoms, 7 siblings not showing autistic symptoms (sibling controls) and eight non-sibling control subjects, using the bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP) procedure. The results provide us with information on the microflora of stools of young children and a compelling picture of unique fecal microflora of children with autism with gastrointestinal symptomatology. Differences based upon maximum observed and maximum predicted operational taxonomic units were statistically significant when comparing autistic and control subjects with p-values ranging from <0.001 to 0.009 using both parametric and non-parametric estimators. At the phylum level, Bacteroidetes and Firmicutes showed the most difference between groups of varying severities of autism. Bacteroidetes was found at high levels in the severely autistic group, while Firmicutes were more predominant in the control group. Smaller, but significant, differences also occurred in the Actinobacterium and Proteobacterium phyla. Desulfovibrio species and Bacteroides vulgatus are present in significantly higher numbers in stools of severely autistic children than in controls. If the unique microbial flora is found to be a causative or consequent factor in this type of autism, it may have implications with regard to a specific diagnostic test, its epidemiology, and for treatment and prevention.     

氢气与肠道菌群的关系研究进展

目前,氢气已被证实在多种疾病中具有显著的医学效应,然而其发挥效应的分子机制并不清楚。肠道菌群被人们看作人体的一个重要“器官”,与人类健康的关系密不可分。研究表明,人类肠道菌群中存在着大量能够进行氢气代谢的菌群,这些菌群的变化可能与多种疾病的发生发展密切相关。此外,研究还发现外源氢气干预可能通过重塑肠道菌群改善炎症性肠病、脂肪性肝病等。综述了肠道菌群的氢气代谢及其与疾病发生发展的关系以及外源氢气干预通过调节肠道菌群影响疾病进展的相关研究,希望能为致力于从肠道菌群角度研究氢气医学效应的科研工作者提供帮助。     

Do musicians with perfect pitch have more autism traits than musicians without perfect pitch? An empirical study

Perfect pitch, also known as absolute pitch (AP), refers to the rare ability to identify or produce a musical tone correctly without the benefit of an external reference. AP is often considered to reflect musical giftedness, but it has also been associated with certain disabilities due to increased prevalence of AP in individuals with sensory and developmental disorders. Here, we determine whether individual autistic traits are present in people with AP. We quantified subclinical levels of autism traits using the Autism-Spectrum Quotient (AQ) in three matched groups of subjects: 16 musicians with AP (APs), 18 musicians without AP (non-APs), and 16 non-musicians. In addition, we measured AP ability by a pitch identification test with sine wave tones and piano tones. We found a significantly higher degree of autism traits in APs than in non-APs and non-musicians, and autism scores were significantly correlated with pitch identification scores (r = .46, p = .003). However, our results showed that APs did not differ from non-APs on diagnostically crucial social and communicative domain scores and their total AQ scores were well below clinical thresholds for autism. Group differences emerged on the imagination and attention switching subscales of the AQ. Thus, whilst these findings do link AP with autism, they also show that AP ability is most strongly associated with personality traits that vary widely within the normal population.     

First isolation of Desulfovibrio from the human vaginal flora

Four Desulfovibrio species, including 2 subtypes of 1 species, namely, Desulfovibrio piger, Desulfovibrio desulfuricans MB subtype and Essex 6 subtype, Desulfovibrio fairfieldensis, and Desulfovibrio vulgaris, have been isolated from the human oral and intestinal flora, but not previously from the vaginal flora. They are opportunistic pathogens and have been considered as possible environmental and etiologic agents involved in ulcerative colitis and chronic periodontitis. We isolated Desulfovibrio intestinalis from vaginal specimens of four Japanese women; a species which has not been previously isolated from humans. The vaginal isolates were highly resistant to cefoxitin, piperacillin, and piperacillin-tazobactam but were susceptible to the other antimicrobial agents tested. Our findings suggested that vaginal Desulfovibrio species may be involved in gynecological or obstetric pathology, and provides additional information of the medical relevance on human Desulfovibrio species.     

Ultrastructure of cyclic changes in the murine uterus,cervix, and vagina

The regional and cyclic changes in the murine genital epithelium were studied by transmission and scanning electron microscopy to provide a morphological standard to serve as a basis for investigation of host-parasite relationships in genital infections. Thus, we examined not only mucosal epithelial cell changes, but also surface mucus, normal flora and inflammatory cells. Ultrastructurally, at proestrus/estrus, we found uterine and most cervical epithelial cells covered with microvilli overlaid with mucus-like secretions and evidence of internal secretory activity. There was little normal flora anywhere in the tract. At early metestrus, we found squamous cervicovaginal epithelial cells with low discontinuous microrugae, extensive normal flora and many neutrophils beginning to migrate through the epithelium. The flora and neutrophils could explain the relative lack of susceptibility to infection at that time. At diestrus the appearance of a newly regenerated epithelium and lack of normal flora suggested that initiation of infection could occur at this stage; however, the presence of large numbers of neutrophils ready to phagocytize invading bacteria indicated a deterrent to infection. This study of cyclic changes in flora, mucus, neutrophils and epithelial cells provided ultrastructural evidence to support an earlier hypothesis that the greatest susceptibility to gonococcal infection in mice occurred at proestrus/estrus.     

Novel antibody assessment method for microbial compositional alteration in the oral cavity

Recently, it has been demonstrated that dysbiosis, an alteration in commensal microflora composition, is intimately involved in the onset of a variety of diseases. It is becoming increasingly evident that the composition of commensal microflora in the oral cavity is closely connected to oral diseases, such as periodontal disease, and systemic diseases, such as inflammatory bowel disease. Next-generation sequencing techniques are used as a method to examine changes in bacterial flora, but additional analytical methods to assess bacterial flora are needed to understand bacterial activity in more detail. In addition, the oral environment is unique because of the role of secretory antibodies contained in saliva in the formation of bacterial flora. The present study aimed to develop a new method for evaluating the compositional change of microbiota using flow cytometry (FCM) with specific antibodies against the bacterial surface antigen, as well as salivary antibodies. Using specific antibodies against Streptococcus mutans, a causative agent of dental caries, and human IgA, bacterial samples from human saliva were analyzed via FCM. The results showed that different profiles could be obtained depending on the oral hygiene status of the subjects. These results suggest that changes in the amount and type of antibodies that bind to oral bacteria may be an indicator for evaluating abnormalities in the oral flora. Therefore, the protocol established in this report could be applied as an evaluation method for alterations in the oral microbiota.     

Gene Expression Profiling of Lymphoblasts from Autistic and Nonaffected Sib Pairs: Altered Pathways in Neuronal Development and Steroid Biosynthesis

Despite the identification of numerous autism susceptibility genes, the pathobiology of autism remains unknown. The present “case-control” study takes a global approach to understanding the molecular basis of autism spectrum disorders based upon large-scale gene expression profiling. DNA microarray analyses were conducted on lymphoblastoid cell lines from over 20 sib pairs in which one sibling had a diagnosis of autism and the other was not affected in order to identify biochemical and signaling pathways which are differentially regulated in cells from autistic and nonautistic siblings. Bioinformatics and gene ontological analyses of the data implicate genes which are involved in nervous system development, inflammation, and cytoskeletal organization, in addition to genes which may be relevant to gastrointestinal or other physiological symptoms often associated with autism. Moreover, the data further suggests that these processes may be modulated by cholesterol/steroid metabolism, especially at the level of androgenic hormones. Elevation of male hormones, in turn, has been suggested as a possible factor influencing susceptibility to autism, which affects ∼4 times as many males as females. Preliminary metabolic profiling of steroid hormones in lymphoblastoid cell lines from several pairs of siblings reveals higher levels of testosterone in the autistic sibling, which is consistent with the increased expression of two genes involved in the steroidogenesis pathway. Global gene expression profiling of cultured cells from ASD probands thus serves as a window to underlying metabolic and signaling deficits that may be relevant to the pathobiology of autism.     

肠道菌群与抑郁症相关性的研究进展

正常人体内的肠道菌群数量可达100万亿,可参与人体的多项生理活动,包括营养物质的吸收与代谢、免疫系统的发育与成熟、抵挡外来病原体的入侵等,对人类的健康有着至关重要的作用。近年来发现肠道中的定植微生物与抑郁症、自闭症、焦虑症和帕金森病等一系列的神经精神疾病密切相关。最新研究表明,肠道菌群是通过神经、体液、代谢和免疫多种途径双向调节肠道和中枢神经系统的。目前,随着医学技术和医学理论的的提高,抑郁症与肠道菌群间的关系受到极大地重视。本文从肠道菌群对抑郁症的影响机制以及益生菌对抑郁症的改善作用两方面来综述肠道菌群与抑郁症相关性的最新进展。     

High-throughput sequencing of mGluR signaling pathway genes reveals enrichment of rare variants in autism

Identification of common molecular pathways affected by genetic variation in autism is important for understanding disease pathogenesis and devising effective therapies. Here, we test the hypothesis that rare genetic variation in the metabotropic glutamate-receptor (mGluR) signaling pathway contributes to autism susceptibility. Single-nucleotide variants in genes encoding components of the mGluR signaling pathway were identified by high-throughput multiplex sequencing of pooled samples from 290 non-syndromic autism cases and 300 ethnically matched controls on two independent next-generation platforms. This analysis revealed significant enrichment of rare functional variants in the mGluR pathway in autism cases. Higher burdens of rare, potentially deleterious variants were identified in autism cases for three pathway genes previously implicated in syndromic autism spectrum disorder, TSC1, TSC2, and SHANK3, suggesting that genetic variation in these genes also contributes to risk for non-syndromic autism. In addition, our analysis identified HOMER1, which encodes a postsynaptic density-localized scaffolding protein that interacts with Shank3 to regulate mGluR activity, as a novel autism-risk gene. Rare, potentially deleterious HOMER1 variants identified uniquely in the autism population affected functionally important protein regions or regulatory sequences and co-segregated closely with autism among children of affected families. We also identified rare ASD-associated coding variants predicted to have damaging effects on components of the Ras/MAPK cascade. Collectively, these findings suggest that altered signaling downstream of mGluRs contributes to the pathogenesis of non-syndromic autism.     

Association between the g.296596G > A genetic variant of RELN gene and susceptibility to autism in a Chinese Han population

Autism is a childhood neuro-developmental disorder, and Reelin (RELN) is an important candidate gene for influencing autism. This study aimed at investigating the influence of genetic variants of the RELN gene on autism susceptibility. In this study, 205 autism patients and 210 healthy controls were recruited and the genetic variants of the RELN gene were genotyped by the created restriction site-polymerase chain reaction (CRS-PCR) method. The influence of genetic variants on autism susceptibility was analyzed by association analysis, and the g.296596G > A genetic variant in exon10 of the RELN gene was detected. The frequencies of allele/genotype in autistic patients were significantly different from those in healthy controls, and a statistically significant association was detected between this genetic variant and autism susceptibility. Our data lead to the inference that the g.296596G > A genetic variant in the RELN gene has a potential influence on autism susceptibility in the Chinese Han population.     

Upregulation of Ras/Raf/ERK1/2 signaling and ERK5 in the brain of autistic subjects

Autism is a neurodevelopmental disorder characterized by impairments in social interaction, verbal communication and repetitive behaviors. A number of studies have shown that the Ras/Raf/ERK1/2 (extracellular signal-regulated kinase) signaling pathway plays important roles in the genesis of neural progenitors, learning and memory. Ras/Raf/ERK1/2 and ERK5 have also been shown to have death-promoting apoptotic roles in neural cells. Recent studies have shown a possible association between neural cell death and autism. In addition, two recent studies reported that a deletion of a locus on chromosome 16, which included the mitogen-activated protein kinase 3 (MAPK3) gene that encodes ERK1, is associated with autism. Most recently, our laboratory detected that Ras/Raf/ERK1/2 signaling activities were significantly enhanced in the brain of BTBR mice that model autism, as they exhibit many autism-like behaviors. We thus hypothesized that Ras/Raf/ERK1/2 signaling and ERK5 could be abnormally regulated in the brain of autistic subjects. In this study, we show that the expression of Ras protein was significantly elevated in the frontal cortex of autistic subjects. C-Raf phosphorylation was increased in the frontal cortex, while both C-Raf and A-Raf activities were enhanced in the cerebellum of autistic subjects. We also detected that both the protein expression and activities of ERK1/2 were significantly upregulated in the frontal cortex of autistic subjects, but not in the cerebellum. Furthermore, we showed that ERK5 protein expression is upregulated in both frontal cortex and cerebellum of autistic subjects. These results suggest that the upregulation of Ras/Raf/ERK1/2 signaling and ERK5 activities mainly found in the frontal cortex of autistic subjects may be critically involved in the pathogenesis of autism.     

Reduced activity of protein kinase C in the frontal cortex of subjects with regressive autism: relationship with developmental abnormalities

Autism is a neurodevelopmental disorder with unknown etiology. In some cases, typically developing children regress into clinical symptoms of autism, a condition known as regressive autism. Protein kinases are essential for G-protein-coupled receptor-mediated signal transduction, and are involved in neuronal functions, gene expression, memory, and cell differentiation. Recently, we reported decreased activity of protein kinase A (PKA) in the frontal cortex of subjects with regressive autism. In the present study, we analyzed the activity of protein kinase C (PKC) in the cerebellum and different regions of cerebral cortex from subjects with regressive autism, autistic subjects without clinical history of regression, and age-matched control subjects. In the frontal cortex of subjects with regressive autism, PKC activity was significantly decreased by 57.1% as compared to age-matched control subjects (p = 0.0085), and by 65.8% as compared to non-regressed autistic subjects (p = 0.0048). PKC activity was unaffected in the temporal, parietal and occipital cortices, and in the cerebellum in both autism groups, i.e., regressive and non-regressed autism as compared to control subjects. These results suggest brain region-specific alteration of PKC activity in the frontal cortex of subjects with regressive autism. Further studies showed a negative correlation between PKC activity and restrictive, repetitive and stereotyped pattern of behavior (r= -0.084, p = 0.0363) in autistic individuals, suggesting involvement of PKC in behavioral abnormalities in autism. These findings suggest that regression in autism may be attributed, in part, to alterations in G-protein-coupled receptor-mediated signal transduction involving PKA and PKC in the frontal cortex.     

Type 2 Diabetes and Three Calpain-10 Gene Polymorphisms in Samoans: No Evidence of Association

Although genomewide scans have identified several potential chromosomal susceptibility regions in several human populations, finding a causative gene for type 2 diabetes has remained elusive. Others have reported a novel gene, calpain-10 (CAPN10), located in a previously identified region on chromosome 2q37.3, as a putative susceptibility gene for type 2 diabetes. Three single-nucleotide polymorphisms (SNPs) (UCSNP43, UCSNP19, and UCSNP63) were shown to be involved in increased risk of the disease among Mexican Americans. We have tested the association of these three SNPs with type 2 diabetes among the Samoans of Polynesia, who have a very high prevalence of the disease. In the U.S. territory of American Samoa, prevalence is 25% and 15% in men and women, respectively, whereas, in the independent nation of Samoa, prevalence is 3% and 5% in men and women, respectively. In our study sample, which consisted of 172 unrelated affected case subjects and 96 control subjects, we failed to detect any association between case subjects and control subjects in allele frequencies, haplotype frequencies, or haplotype combinations of UCSNP43, -19, and -63. Also, our data showed no evidence of linkage, among 201 affected sib pairs, in the region of chromosome 2 that contains these SNPs. Three plausible scenarios could explain these observations. (1) CAPN10 is a susceptibility gene only in particular ethnic groups; (2) our study lacks power to detect the effects of CAPN10 polymorphisms (but our sample size is comparable to that of earlier reports); or (3) the underlying biological mechanism is too complex and requires further research.