Characterization of Adherent Bacteroidales from Intestinal Biopsies of Children and Young Adults with Inflammatory Bowel Disease

There is extensive evidence implicating the intestinal microbiota in inflammatory bowel disease [IBD], but no microbial agent has been identified as a sole causative agent. Bacteroidales are numerically dominant intestinal organisms that associate with the mucosal surface and have properties that both positively and negatively affect the host. To determine precise numbers and species of Bacteroidales adherent to the mucosal surface in IBD patients, we performed a comprehensive culture based analysis of intestinal biopsies from pediatric Crohn''s disease [CD], ulcerative colitis [UC], and control subjects. We obtained biopsies from 94 patients and used multiplex PCR or 16S rDNA sequencing of Bacteroidales isolates for species identification. Eighteen different Bacteroidales species were identified in the study group, with up to ten different species per biopsy, a number higher than demonstrated using 16S rRNA gene sequencing methods. Species diversity was decreased in IBD compared to controls and with increasingly inflamed tissue. There were significant differences in predominant Bacteroidales species between biopsies from the three groups and from inflamed and uninflamed sites. Parabacteroides distasonis significantly decreased in inflamed tissue. All 373 Bacteroidales isolates collected in this study grew with mucin as the only utilizable carbon source suggesting this is a non-pathogenic feature of this bacterial order. Bacteroides fragilis isolates with the enterotoxin gene [bft], previously associated with flares of colitis, were not found more often at inflamed colonic sites or within IBD subjects. B. fragilis isolates with the ability to synthesize the immunomodulatory polysaccharide A [PSA], previously shown to be protective in murine models of colitis, were not detected more often from healthy versus inflamed tissue.     

Regulation and localization of endogenous human tristetraprolin

Tumor necrosis factor (TNF) has been implicated in the development and pathogenicity of infectious diseases and autoimmune disorders, such as septic shock and arthritis. The zinc-finger protein tristetraprolin (TTP) has been identified as a major regulator of TNF biosynthesis. To define its intracellular location and examine its regulation of TNF, a quantitive intracellular staining assay specific for TTP was developed. We establish for the first time that in peripheral blood leukocytes, expression of endogenous TTP is confined to the cytoplasm. Baseline expression of TTP was higher in monocytes than in lymphocytes or neutrophils. After in vitro incubation with lipopolysaccharide (LPS), leukocyte TTP levels increased rapidly, peaking after approximately 2 hours. Monocytes showed the greatest response to LPS stimulation and lymphocytes the least. TTP levels were also studied in leukocytes isolated from healthy volunteers infused with a bolus dose of LPS. TTP expression and initial upregulation in response to LPS infusion were consistent with the in vitro data. Neutrophil TTP levels responded first, reaching an initial peak within 1 hour, monocyte levels peaked next at 2 hours, followed by lymphocytes at 4 hours. This response paralleled plasma TNF levels, which peaked 2 hours after infusion and were no longer detectable after 12 hours. A second rise in intracellular TTP levels, which did not parallel plasma TNF levels, was observed in all leukocyte populations, starting 12 hours after infusion. These data establish the cytoplasmic location of TTP, supporting a major role for this protein in regulating TNF production, and suggest that TTP levels are not regulated solely by TNF.     

HIV-1 does not provoke alteration of cytokine gene expression in lymphoid tissue after acute infection ex vivo

The cytokine response to invading microorganisms is critical for priming the adaptive immune response. During acute HIV infection, the response is disrupted, but the mechanism is poorly understood. We examined the cytokine response in human lymphoid tissue, acutely infected ex vivo with HIV. Lymphoid tissue was cultured either as blocks or as human lymphocyte aggregate cultures (HLAC) of tonsils and lymph nodes. This approach allowed us to examine the effects of HIV on cytokines using distinct culture techniques. In contrast to HLAC, mock-infected tissue blocks displayed a 50- to 100-fold up-regulation of mRNAs for IL-1beta, -6, and -8 in the first 6 days of culture. Parallel increases were also noted at the protein level in the supernatants. Although IL-1beta, -6, and -8 are known to synergistically enhance HIV replication, peak HIV replication (measured as p24 Ag) was similar in tissue blocks and HLAC. Surprisingly, vigorous HIV replication of CXCR4- and CCR5-tropic HIV strains did not result in characteristic mRNA profiles for IL-1beta, -2, -4, -6, -8, -10, -12, -15, IFN-gamma, TNF-alpha, TGF-beta, and beta-chemokines in tissue blocks or HLAC. The increased expression of IL-1beta, -6, and -8 in tissue blocks may approximate clinical situations with heightened immune activation; neutralization of these cytokines resulted in inhibition of HIV replication, suggesting that these cytokines may contribute to HIV replication in certain clinical settings. These results also indicate that different molecular mechanisms govern HIV replication in tissue blocks and HLAC. Prevention of effective cytokine responses may be an important mechanism that HIV uses during acute infection.     

Synaptic concentration of type-I adenylyl cyclase in cerebellar neurons

Specific subcellular targeting and spatial arrangement of signaling molecules are important for efficient signal transduction. The neuro-specific type-I adenylyl cyclase (AC1) is stimulated by Ca2+, and plays an essential role in neurodevelopment and neuroplasticity. We generated hemagglutinin (HA)-tagged AC1 to study its subcellular localization in cultured neurons. The HA-tagged AC1 has similar enzymatic activity and regulatory properties to that of non-tagged protein. HA-AC1 targeted to both apical and basolateral domains in the epithelial Madin-Darby canine kidney (MDCK) cells, and it was found in both axons and dendrites in cultured hippocampal neurons as well as in cerebellar granule neurons. Interestingly, AC1 showed a distinct punctate form of immunostaining in MDCK cells and transfected neurons, suggesting it targets to specific subcellular domains. By immunostaining with different synaptic markers, we found that AC1 puncta were located at the excitatory synapses in cerebellar granule neurons. Our data provide a possible cellular mechanism for the physiological role of AC1 in neuroplasticity.     

A Genome-Wide Association Study of Neuroticism in a Population-Based Sample

Neuroticism is a moderately heritable personality trait considered to be a risk factor for developing major depression, anxiety disorders and dementia. We performed a genome-wide association study in 2,235 participants drawn from a population-based study of neuroticism, making this the largest association study for neuroticism to date. Neuroticism was measured by the Eysenck Personality Questionnaire. After Quality Control, we analysed 430,000 autosomal SNPs together with an additional 1.2 million SNPs imputed with high quality from the Hap Map CEU samples. We found a very small effect of population stratification, corrected using one principal component, and some cryptic kinship that required no correction. NKAIN2 showed suggestive evidence of association with neuroticism as a main effect (p<10⁻⁶) and GPC6 showed suggestive evidence for interaction with age (p≈10⁻⁷). We found support for one previously-reported association (PDE4D), but failed to replicate other recent reports. These results suggest common SNP variation does not strongly influence neuroticism. Our study was powered to detect almost all SNPs explaining at least 2% of heritability, and so our results effectively exclude the existence of loci having a major effect on neuroticism.     

The Tomato spotted wilt virus cell‐to‐cell movement protein (NSM) triggers a hypersensitive response in Sw‐5‐containing resistant tomato lines and in Nicotiana benthamiana transformed with the functional Sw‐5b resistance gene copy

Although the Sw‐5 gene cluster has been cloned, and Sw‐5b has been identified as the functional gene copy that confers resistance to Tomato spotted wilt virus (TSWV), its avirulence (Avr) determinant has not been identified to date. Nicotiana tabacum ‘SR1‘ plants transformed with a copy of the Sw‐5b gene are immune without producing a clear visual response on challenge with TSWV, whereas it is shown here that N. benthamiana transformed with Sw‐5b gives a rapid and conspicuous hypersensitive response (HR). Using these plants, from all structural and non‐structural TSWV proteins tested, the TSWV cell‐to‐cell movement protein (NS_M) was confirmed as the Avr determinant using a Potato virus X (PVX) replicon or a non‐replicative pEAQ‐HT expression vector system. HR was induced in Sw‐5b‐transgenic N. benthamiana as well as in resistant near‐isogenic tomato lines after agroinfiltration with a functional cell‐to‐cell movement protein (NS_M) from a resistance‐inducing (RI) TSWV strain (BR‐01), but not with NS_M from a Sw‐5 resistance‐breaking (RB) strain (GRAU). This is the first biological demonstration that Sw‐5‐mediated resistance is triggered by the TSWV NS_M cell‐to‐cell movement protein.     

Acute exercise reduces insulin resistance‐induced TRB3 expression and amelioration of the hepatic production of glucose in the liver of diabetic mice

TRB3 (a mammalian homolog of Drosophila) is emerging as an important player in the regulation of insulin signaling. TRB3 can directly bind to Ser/Thr protein kinase Akt, the major downstream kinase of insulin signaling. Conversely, physical exercise has been linked to improved glucose homeostasis and enhanced insulin sensitivity; however, the molecular mechanisms by which exercise improves glucose homeostasis, particularly in the hepatic tissue, are only partially known. Here, we demonstrate that acute exercise reduces fasting glucose in two models diabetic mice. Western blot analysis showed that 8 h after a swimming protocol, TRB3 expression was reduced in the hepatic tissue from diet‐induced obesity (Swiss) and leptin‐deficient (ob/ob) mice, when compared with respective control groups at rest. In parallel, there was an increase in insulin responsiveness in the canonical insulin‐signaling pathway in hepatic tissue from DIO and ob/ob mice after exercise. In addition, the PEPCK expression was reduced in the liver after the exercise protocol, suggesting that acute exercise diminished hepatic glucose production through insulin‐signaling restoration. Thus, these results provide new insights into the mechanism by which physical activity improves glucose homeostasis in type 2 diabetes. J. Cell. Physiol. 221: 92–97, 2009. © 2009 Wiley‐Liss, Inc