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BackgroundPhosphatase and tensin homolog (Pten) is capable of mediating microbe-induced immune responses in the gut. Thus, Pten deficiency in the intestine accelerates colitis development in Il10-/- mice. As some ambient pollutants inhibit Pten function and exposure to ambient pollutants may increase inflammatory bowel disease (IBD) incidence, it is of interest to examine how Pten inhibition could affect colitis development in genetically susceptible hosts.MethodsWith human colonic mucosa biopsies from pediatric ulcerative colitis and non-IBD control subjects, we assessed the mRNA levels of the PTEN gene and the gene involved in IL10 responses. The data from the human tissues were corroborated by treating Il10-/-, Il10rb-/-, and wild-type C57BL/6 mice with Pten-specific inhibitor VO-OHpic. We evaluated the severity of mouse colitis by investigating the tissue histology and cytokine production. The gut microbiome was investigated by analyzing the 16S ribosomal RNA gene sequence with mouse fecal samples.ResultsPTEN and IL10RB mRNA levels were reduced in the human colonic mucosa of pediatric ulcerative colitis compared with non-IBD subjects. Intracolonic treatment of the Pten inhibitor induced colitis in Il10-/- mice, characterized by reduced body weight, marked colonic damage, and increased production of inflammatory cytokines, whereas Il10rb-/- and wild-type C57BL/6 mice treated with the inhibitor did not develop colitis. Pten inhibitor treatment changed the fecal microbiome, with increased abundance of colitogenic bacteria Bacteroides and Akkermansia in Il10-/- mice.ConclusionsLoss of Pten function increases the levels of colitogenic bacteria in the gut, thereby inducing deleterious colitis in an Il10-deficient condition.

ObjectiveChronic HIV-1 infection leads to widespread inflammation and immune dysregulation. The gastrointestinal mucosa, a primary site for HIV-1 replication, is thought to play a significant role in this response. MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression, including immune activation and inflammation. Here we investigate miR expression and function in the colonic mucosa during HIV-1 infection.Design and methodsUsing miR profiling, we examined miR expression in the colonic mucosa of HIV-infected patients. These miRs were further parsed to identify those that most likely function in HIV-related inflammation. Using bioinformatics tools, we identified potential target genes which were confirmed using in-vitro functional testing.ResultsWe identified 12 miRs that were differentially expressed in the colonic mucosa of HIV-infected patients with high versus undetectable plasma viral concentrations. Of these, both miR-26a and miR-29a were downregulated in untreated HIV-1 infection, yet not in the colonic mucosa from inflammatory bowel disease. This downregulation occurs within the first hours after infection. These miRs were further shown to directly target IL-6 and STAT3, respectively, with similar changes confirmed in an ex-vivo explant infection model.ConclusionmiR-26a and miR-29a levels are decreased in the colonic mucosa during chronic HIV-1 infection, and this change may be initiated during acute infection. Both miRs de-repress the IL-6/STAT3 signaling pathway, which could contribute to increased inflammation during infection. These miRs may represent novel therapeutic targets for HIV-1-associated inflammation in the colonic mucosa.

Background & aimsPersistent activation of the inflammatory response contributes to the development of inflammatory bowel diseases, which increase the risk of colorectal cancer. We aimed to identify microRNAs that regulate inflammation during the development of ulcerative colitis (UC) and progression to colitis-associated colon cancer (CAC).MethodsWe performed a quantitative polymerase chain reaction analysis to measure microRNAs in 401 colon specimens from patients with UC, Crohn's disease, irritable bowel syndrome, sporadic colorectal cancer, or CAC, as well as subjects without these disorders (controls); levels were correlated with clinical features and disease activity of patients. Colitis was induced in mice by administration of dextran sodium sulfate (DSS), and carcinogenesis was induced by addition of azoxymethane; some mice also were given an inhibitor of microRNA214 (miR214).ResultsA high-throughput functional screen of the human microRNAome found that miR214 regulated the activity of nuclear factor-κB. Higher levels of miR214 were detected in colon tissues from patients with active UC or CAC than from patients with other disorders or controls and correlated with disease progression. Bioinformatic and genome-wide profile analyses showed that miR214 activates an inflammatory response and is amplified through a feedback loop circuit mediated by phosphatase and tensin homolog (PTEN) and PDZ and LIM domain 2 (PDLIM2). Interleukin-6 induced signal transducer and activator of transcription 3 (STAT3)-mediated transcription of miR214. A miR214 chemical inhibitor blocked this circuit and reduced the severity of DSS-induced colitis in mice, as well as the number and size of tumors that formed in mice given azoxymethane and DSS. In fresh colonic biopsy specimens from patients with active UC, the miR214 inhibitor reduced inflammation by increasing levels of PDLIM2 and PTEN.ConclusionsInterleukin-6 up-regulates STAT3-mediated transcription of miR214 in colon tissues, which reduces levels of PDLIM2 and PTEN, increases phosphorylation of AKT, and activates nuclear factor-κB. The activity of this circuit correlates with disease activity in patients with UC and progression to colorectal cancer.

BackgroundTwenty to 25% of the patients with inflammatory bowel disease (IBD) present the disease before the age of 18 to 20, with worse extent and severity, compared with adult-onset IBD. We sought to identify the differential expression of microRNAs in pediatric ulcerative colitis (UC) and their association with different clinical phenotypes.MethodsMicroRNA expression analysis was performed in colonic tissues derived from pediatric patients with UC and controls without IBD. MiR-4284 levels were verified by real-time quantitative polymerase chain reaction in 2 additional cohorts of pediatric patients with UC. Bioinformatics analysis was performed to predict the targets of miR-4284. In vitro experiments using luciferase reporter assays and real-time polymerase chain reaction evaluated the direct effect of miR-4284 on CXCL5 mRNA. In vivo experiments were performed in 2 mouse models of experimental colitis.ResultsA 24-microRNA signature was identified in colonic tissues derived from pediatric patients with UC. The most downregulated microRNA in the tissue of pediatric patients UC, relative to non-IBD controls, was miR-4284. In situ hybridization revealed that miR-4284 is present in colonic epithelial cells, and its levels correlate with the disease activity. Furthermore, we found that miR-4284 regulates CXCL5 mRNA expression through binding to its 3'UTR. CXCL5 had increased mRNA levels in colonic tissue from pediatric patients with UC and correlated with disease activity. Furthermore, we found an inverse correlation between miR-4284 and CXCL5 levels in the colonic pediatric UC tissues and in 2 mouse models of experimental colitis.ConclusionsOur data reveal a novel microRNA pediatric UC signature and provide evidence that miR-4284 directly regulates CXCL5 and correlates with the disease activity.

IntroductionAnimal studies have shown that stress could induce epigenetic and transcriptomic alterations essential in determining the balance between adaptive or maladaptive responses to stress. We tested the hypothesis that chronic stress in rats deregulates coding and non-coding gene expression in the spinal cord, which may underline neuroinflammation and nociceptive changes previously observed in this model.MethodsMale Wistar rats were exposed to daily stress or handled, for 10 days. At day 11, lumbar spinal segments were collected and processed for mRNA/miRNA isolation followed by expression profiling using Agilent SurePrint Rat Exon and Rat miRNA Microarray platforms. Differentially expressed gene lists were generated using the dChip program. Microarrays were analyzed using the Ingenuity Pathways Analysis (IPA) tool from Ingenuity Systems. Multiple methods were used for the analysis of miRNA-mRNA functional modules. Quantitative real time RT-PCR for Interleukin 6 signal transducer (gp130), the Signal Transducer And Activator Of Transcription 3 (STAT3), glial fibrillary acidic protein and mir-17-5p were performed to confirm levels of expression.ResultsGene network analysis revealed that stress deregulated different inflammatory (IL-6, JAK/STAT, TNF) and metabolic (PI3K/AKT) signaling pathways. MicroRNA array analysis revealed a signature of 39 deregulated microRNAs in stressed rats. MicroRNA-gene network analysis showed that microRNAs are regulators of two gene networks relevant to inflammatory processes. Specifically, our analysis of miRNA-mRNA functional modules identified miR-17-5p as an important regulator in our model. We verified miR-17-5p increased expression in stress using qPCR and in situ hybridization. In addition, we observed changes in the expression of gp130 and STAT3 (involved in intracellular signaling cascades in response to gp130 activation), both predicted targets for miR-17-5p. A modulatory role of spinal mir17-5p in the modulation of visceral sensitivity was confirmed in vivo.ConclusionUsing an integrative high throughput approach, our findings suggest a link between miR-17-5p increased expression and gp130/STAT3 activation providing new insight into the possible mechanisms mediating the effect of chronic stress on neuroinflammation in the spinal cord.

Background & aimsAltered levels and functions of microRNAs (miRs) have been associated with inflammatory bowel diseases (IBDs), although little is known about their roles in pediatric IBD. We investigated whether colonic mucosal miRs are altered in children with ulcerative colitis (UC).MethodsWe used a library of 316 miRs to identify those that regulate phosphorylation of signal transducer and activator of transcription 3 (STAT3) in NCM460 human colonocytes incubated with interleukin-6. Levels of miR-124 were measured by real-time polymerase chain reaction analysis of colon biopsies from pediatric and adult patients with UC and patients without IBD (controls), and of HCT-116 colonocytes incubated with 5-aza-2'-deoxycytidine (5-AZA). Methylation of the MIR124 promoter was measured by quantitative methylation-specific polymerase chain reaction.ResultsLevels of phosphorylated STAT3 and the genes it regulates (encoding vascular endothelial growth factor (VEGF), BCL2, BCLXL, and matrix metallopeptidase 9 [MMP9]) were increased in pediatric patients with UC compared with control tissues. Overexpression of miR-124, let-7, miR-125, miR-26, or miR-101 reduced STAT3 phosphorylation by ≥ 75% in NCM460 cells; miR-124 had the greatest effect. miR-124 was down-regulated specifically in colon tissues from pediatric patients with UC and directly targeted STAT3 messenger RNA (mRNA). Levels of miR-124 were decreased, whereas levels of STAT3 phosphorylation increased in colon tissues from pediatric patients with active UC compared with those with inactive disease. In addition, levels of miR-124 and STAT3 were inversely correlated in mice with experimental colitis. Down-regulation of miR-124 in tissues from children with UC was attributed to hypermethylation of its promoter region. Incubation of HCT-116 colonocytes with 5-AZA up-regulated miR-124 and reduced levels of STAT3 mRNA.ConclusionsmiR-124 appears to regulate the expression of STAT3. Reduced levels of miR-124 in colon tissues of children with active UC appear to increase expression and activity of STAT3, which could promote inflammation and the pathogenesis of UC in children.

The effect of point mutations and deletions in the carboxy-terminal region of apolipo-protein (apoA-1) on the biogenesis of high-density lipoproteins (HDL) was investigated by using adenovirus-mediated gene transfer in apoA-I-deficient mice. The findings suggest that deletions that eliminate the 220-231 region of apoA-I prevent the synthesis of [alpha]-HDL but allow the synthesis of preB-HDL particles in vivo.

The recombinant adenoviruses expressing apolipoproteins are generated to explain the etiology and find a mode of therapy of genetically determined low levels of high-density lipoprotein (HDL). The study establishes that apoA-I[Leu141Arg.sub.Pisn] and apoA-I[Leu159Arg.sub.FIN] inhibit an early step in the biogenesis of HDL.

Glomeruli number and size are important for determining the pathogenesis of glomerular disease, chronic kidney disease, and hypertension. Moreover, renal injury can occur in specific cortical layers and alter glomerular spatial distribution. In this study, we present a comprehensive structural analysis of glomeruli in a model of Adriamycin (doxorubicin) nephropathy. Glomeruli are imaged (micro-CT at 10 × 10 × 10 μm³) in kidney specimens from C57Bl/6 mouse cohorts: control treated with saline (n = 9) and Adriamycin treated with 20 mg/kg Adriamycin (n = 7). Several indices were examined, including glomerular number, glomerular volume, glomerular volume heterogeneity, and spatial density at each glomerulus and in each cortical layer (superficial, midcortical, and juxtamedullary). In the Adriamycin-treated animals, glomerular number decreased significantly in the left kidney [control: 8,298 ± 221, Adriamycin: 6,781 ± 630 (mean ± SE)] and right kidney (control: 7,317 ± 367, Adriamycin: 5,522 ± 508), and glomerular volume heterogeneity increased significantly in the left kidney (control: 0.642 ± 0.015, Adriamycin: 0.786 ± 0.018) and right kidney (control: 0.739 ± 0.016, Adriamycin: 0.937 ± 0.023). Glomerular spatial density was not affected. Glomerular volume heterogeneity increased significantly in the superficial and midcortical layers of the Adriamycin cohort. Adriamycin did not affect glomerular volume or density metrics in the juxtamedullary region, suggesting a compensatory mechanism of juxtamedullary glomeruli to injury in the outer cortical layers. Left/right asymmetry was observed in kidney size and various glomeruli metrics. The methods presented here can be used to evaluate renal disease models with subtle changes in glomerular endowment locally or across the entire kidney, and they provide an imaging tool to investigate diverse interventions and therapeutic drugs. [ABSTRACT FROM AUTHOR]

To explain the etiology and find a mode of therapy of genetically determined low levels of high-density lipoprotein (HDL), we have generated recombinant adenoviruses expressing apolipoprotein A-I (apoA-I)(Leu141Arg)Pisa and apoA-I(Leu159Arg)FIN and studied their properties in vitro and in vivo. Both mutants were secreted efficiently from cells but had diminished capacity to activate lecithin/cholesterol acyltransferase (LCAT) in vitro. Adenovirus-mediated gene transfer of either of the two mutants in apoA- I-deficient (apoA-I-1- ) mice resulted in greatly decreased total plasma cholesterol, apoA-I, and HDL cholesterol levels. The treatment also decreased the cholesteryl ester to total cholesterol ratio (CE/TC), caused accumulation of preβ1-HDL and small size α4-HDL particles, and generated only few spherical HDL particles, as compared to mice expressing wild-type (WT) apoA-I. Simultaneous treatment of the mice with adenoviruses expressing either of the two mutants and human LCAT normalized the plasma apoA-I, HDL cholesterol levels, and the CE/TC ratio, restored normal preβ- and α-HDL subpopulations, and generated spherical HDL. The study establishes that apoA-I(Leu141Arg)Pisa and apoA-I(Leu159Arg)FIN inhibit an early step in the biogenesis of HDL due to inefficient esterification of the cholesterol of the preβ1-HDL particles by the endogenous LCAT. Both defects can be corrected by treatment with LCAT. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) are remarkably versatile signaling systems that are activated by a large number of different agonists on the outside of the cell. However, the inside surface of the receptors that couple to G proteins has not yet been effectively modulated for activity or treatment of diseases. Pepducins are cell-penetrating lipopeptides that have enabled chemical and physical access to the intracellular face of GPCRs. The structure of a third intracellular (i3) loop agonist, pepducin, based on protease-activated receptor-1 (PAR1) was solved by NMR and found to closely resemble the i3 loop structure predicted for the intact receptor in the on-state. Mechanistic studies revealed that the pepducin directly interacts with the intracellular H8 helix region of PAR1 and allosterically activates the receptor through the adjacent (D/N)PXXYYY motif through a dimer-like mechanism. The i3 pepducin enhances PAR1/Gα subunit interactions and induces a conformational change in fluorescently labeled PAR1 in a very similar manner to that induced by thrombin. As pepducins can potentially be made to target any GPCR, these data provide insight into the identification of allosteric modulators to this major drug target class. [ABSTRACT FROM AUTHOR]

Objectives: The aim of this study is to investigate the prevalence of bla TEM and nim genes that encode resistance to β-lactams and nitroimidazoles, respectively, in the oral cavity of systemically healthy Greek subjects., Materials and Methodology: After screening 720 potentially eligible subjects, 154 subjects were recruited for the study, including 50 periodontally healthy patients, 52 cases of gingivitis and 52 cases of chronic periodontitis. The clinical parameters were assessed with an automated probe. Various samples were collected from the tongue, first molars and pockets >6mm, and analysed by polymerase chain reaction-amplification of the bla TEM and nim genes, using primers and conditions previously described in the literature., Results: There was a high rate of detection of bla TEM in plaque and tongue samples alike in all periodontal conditions (37% of plaque and 60% of tongue samples, and 71% of participants). The bla TEM gene was detected more frequently in the tongue samples of the periodontally healthy (56%) and chronic periodontitis (62%) groups compared to the plaque samples from the same groups (36% and 29%, respectively; z-test with Bonferroni corrections-tests, P<0.05). The nim gene was not detected in any of the 343 samples analysed., Conclusion: The oral cavity of Greek subjects often harbours bla TEM but not nim genes, and therefore the antimicrobial activity of β-lactams might be compromised.

G protein-coupled receptors (GPCR) are a superfamily of receptors that are vital in a wide array of physiological processes. Modulation of GPCR signaling has been an intensive area of therapeutic study, mainly due to the diverse pathophysiological significance of GPCRs. Pepducins are cell-penetrating lipidated peptides designed to target the intracellular loops of the GPCR of interest. Pepducins can function as agonists or antagonists of their cognate receptor, making them highly useful compounds for the study of GPCR signaling. Pepducins have been used to control platelet-dependent hemostasis and thrombosis, tumor growth, invasion, and angiogenesis, as well as to improve sepsis outcomes in mice. Pepducins have been successfully designed against a wide variety of GPCRs including the protease-activated receptors (PAR1, 2, 4), the chemokine receptors (CXCR1, 2, 4), the sphingosine-1-phosphate receptor (S1P3), the adrenergic receptor (ADRA1B), and have the potential to help reveal the functions of intractable GPCRs. Pharmacokinetic, pharmacodynamic, and biodistribution studies have showed that pepducins are widely distributed throughout the body except the brain and possess appropriate drug-like properties for use in vivo. Here, we discuss the delivery, pharmacology, and biodistribution of pepducins, as well as the effects of pepducins in models of inflammation, cardiovascular disease, cancer, and angiogenesis.