Chronic inflammatory bowel diseases (IBD) are associated with an abnormal immune response to intestinal bacteria. Although the pro-inflammatory mediator osteopontin (OPN) is upregulated in IBD, its role in disease pathogenesis remains unclear. The aim of this study, therefore, was to determine the role of OPN in mediating host responses to a non-invasive intestinal bacterial pathogen, using Citrobacter rodentium infection of mice as a murine model of IBD, as well as infecting mouse-derived OPN+/+ and OPN-/- fibroblasts. OPN-/-” and wild-type (WT) mice were inoculated with either C. rodentium or LB broth (as sham control). Colonic epithelial cell hyperplasia, which is the hallmark of C. rodentium infection, was reduced in infected OPN-/- mice (218±13μm, N = 15) relative to infected WT mice (295±15μm; N=8; p=0.01), and daily rectal administration of OPN to OPN-/-” mice partially restored the WT phenotype (239±21μm; N = 6; p<0.05). Colonization of mice with C. rodentium was also reduced in OPN-/- mice (3.9×104±3.2×103 vs 2.8×105±8×104; p<0.005). An increase in spleen weight and in the number of splenocytes observed in infected WT mice was absent in null mice. By contrast, there was no difference in body weight, disease activity, bacterial translocation, and overall histology score between infected WT and infected OPN-/- mice. While lack of OPN did not decrease bacterial adhesion to mouse fibroblasts (54±21 vs. 44±15 bacteria/cell; N = 3; p>0.05), rearrangements of the cytoskeleton, demonstrated by attaching and effacing lesions, were reduced in cells derived from OPN-/- mice and restored in OPN-/- cell rescued with human OPN by stable transfection and with exogenous application of OPN. Lack of OPN results in decreased pedestal formation and colonic epithelial cell hyperplasia in response to C. rodentium infection, indicating that OPN mediates signaling events that impact on the pathogenesis of disease through rearrangements of the host cell cytoskeleton. These findings demonstrate a role for OPN in mediating host responses to injury and inflammation caused by a microbial pathogen, and could thereby explain its role in the pathogenesis of IBD.
A decade ago, the Gene Expression Omnibus (GEO) database was established at the National Center for Biotechnology Information (NCBI). The original objective of GEO was to serve as a public repository for high-throughput gene expression data generated mostly by microarray technology. However, the research community quickly applied microarrays to non-gene-expression studies, including examination of genome copy number variation and genome-wide profiling of DNA-binding proteins. Because the GEO database was designed with a flexible structure, it was possible to quickly adapt the repository to store these data types. More recently, as the microarray community switches to next-generation sequencing technologies, GEO has again adapted to host these data sets. Today, GEO stores over 20 000 microarray- and sequence-based functional genomics studies, and continues to handle the majority of direct high-throughput data submissions from the research community. Multiple mechanisms are provided to help users effectively search, browse, download and visualize the data at the level of individual genes or entire studies. This paper describes recent database enhancements, including new search and data representation tools, as well as a brief review of how the community uses GEO data. GEO is freely accessible at http://www.ncbi.nlm.nih.gov/geo/ .
ABSTRACT Lactiplantibacillus plantarum ATCC 202195 has probiotic properties that may be beneficial in early infancy. Since the effects of probiotics are often strain and dose specific, tools are needed to identify and enumerate L. plantarum ATCC 202195 with high sensitivity and specificity. Herein, a probe-based real-time quantitative PCR (qPCR) assay was developed to detect and enumerate L. plantarum ATCC 202195. The probe sequence contained a single nucleotide polymorphism within gene FEE41_08190 that was unique to L. plantarum ATCC 202195 at the time of the assay’s design. The linear dynamic range of the assay spanned five orders of magnitude with an R 2 that did not deviate from linearity and an amplification efficiency that ranged from 97% to 110% across six independent plates. At 95% confidence, the limit of detection in the assay was estimated at roughly 15 cells. No amplification was observed for reactions containing gDNA from seven other Lactobacillus species. This is the first report of a qPCR assay that detects and quantifies L. plantarum ATCC 202195. The assay is intended to be used as a resource for future clinical trials and commercial manufacturing settings. IMPORTANCE When administered for seven consecutive days shortly after birth, the probiotic bacterium Lactiplantibacillus plantarum ATCC 202195 plus fructooligosaccharide (FOS) was reported to reduce sepsis and lower respiratory tract infection events during early infancy in a randomized trial in India. Since probiotic effects are often strain specific, strain-level detection and quantification by routine molecular methods enables the monitoring of safety outcomes, such as probiotic-associated bacteremia, and allows for the quality of probiotic interventions to be assessed and monitored (i.e., verify strain identity and enumerate). Despite the potential clinical applications of L. plantarum ATCC 202195, an assay to detect and quantify this strain has not previously been described. Herein, we report the design of primer and probe sequences to detect L. plantarum ATCC 202195 and the development and optimization of a real-time PCR assay to detect and quantify the strain with high specificity and high sensitivity.
ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 intimately attaches to intestinal epithelial monolayers and produces attaching and effacing (A/E) lesions. In addition, EHEC infection causes disruptions of intercellular tight junctions, leading to clinical sequelae that include acute diarrhea, hemorrhagic colitis, and the hemolytic-uremic syndrome. Current therapy remains supportive since antibiotic therapy increases the risk of systemic complications. This study focused on the potential therapeutic effect of an alternative form of therapy, probiotic Lactobacillus rhamnosus strain GG, to attenuate EHEC-induced changes in paracellular permeability in polarized MDCK-I and T84 epithelial cell monolayers. Changes in epithelial cell morphology, electrical resistance, dextran permeability, and distribution and expression of claudin-1 and ZO-1 were assessed using phase-contrast, immunofluorescence, and transmission electron microscopy and macromolecular flux. This study demonstrated that pretreatment of polarized MDCK-I and T84 cells with the probiotic L. rhamnosus GG reduced morphological changes and diminished the number of A/E lesions induced in response to EHEC O157:H7 infection. With probiotic pretreatment there was corresponding attenuation of the EHEC-induced drop in electrical resistance and the increase in barrier permeability assays. In addition, L. rhamnosus GG protected epithelial monolayers against EHEC-induced redistribution of the claudin-1 and ZO-1 tight junction proteins. In contrast to the effects seen with the live probiotic, heat-inactivated L. rhamnosus GG had no effect on EHEC binding and A/E lesion formation or on disruption of the barrier function. Collectively, these findings provide in vitro evidence that treatment with the probiotic L. rhamnosus strain GG could prove to be an effective management treatment for preventing injury of the epithelial cell barrier induced by A/E bacterial enteropathogens.
The goals of therapy in chronic inflammatory bowel diseases in pediatrics include decreasing mucosa! inflammation, the restoration of optimal growth and pubertal development, and the return to a normal, age-appropriate lifestyle. The latter is best served by minimizing restrictions; for example, school attendance, extracurricular activities, travel and the child's interactions with both siblings and peers. In most instances dietary restrictions are also inappropriate and may prove detrimental. Children and adolescents are not simply 'little adults'. Teenagers dread being perceived as different from their peers. They are especially bothered therefore by cosmetic side effects associated with corticosteroid therapy and by impaired growth and pubertal development. Although not discussed openly, many adolescents are also concerned about their subsequent ability to have children and the potential for future development of neoplasms. The great majority of affected children adapt and function very well. Psychosocial factors, although clearly not the cause of inflammatory bowel diseases, are operative in many subsets of children with less than optimal function in daily activities. Children at particular risk are those with premorbid low self-esteem, dysfunction in family dynamics, or severe growth failure. Intervention and support provided through psychiatric and medical social work consultations and by the involvement of self-help groups are often key determinants for restoration of a normal lifestyle. Information about the underlying chronic disease, when provided at a level that is appropriate for age and cognitive function, can decrease anxiety and fears by reducing uncertainty and promoting a feeling of control.
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