Assessment and validation of enrichment and target capture approaches to improve Mycobacterium tuberculosis WGS from direct patient samples.

Within-host Mycobacterium tuberculosis (Mtb) diversity may detect antibiotic resistance or predict tuberculosis treatment failure and is best captured through sequencing directly from sputum. Here, we compared three sample pre-processing steps for DNA decontamination and studied the yield of a new target enrichment protocol for optimal whole-genome sequencing (WGS) from direct patient samples. Mtb-positive NALC-NaOH-treated patient sputum sediments were pooled, and heat inactivated, split in replicates, and treated by either a wash, DNase I, or benzonase digestion. Levels of contaminating host DNA and target Mtb DNA were assessed by quantitative PCR (qPCR), followed by WGS with and without custom dsDNA target enrichment. The pre-treatment sample has a high host-to-target ratio of DNA (6,168 ± 1,638 host copies/ng to 212.3 ± 59.4 Mtb copies/ng) that significantly decreased with all three treatments. Benzonase treatment resulted in the highest enrichment of Mtb DNA at 100-fold compared with control (3,422 ± 2,162 host copies/ng to 11,721 ± 7,096 Mtb copies/ng). The custom dsDNA probe panel successfully enriched libraries from as little as 0.45 pg of Mtb DNA (100 genome copies). Applied to direct sputum the dsDNA target enrichment panel increased the percent of sequencing reads mapping to the Mtb target for all three pre-processing methods. Comparing the results of the benzonase sample sequenced both with and without enrichment, the percent of sequencing reads mapping to the Mtb increased to 90.95% from 1.18%. We demonstrate a low limit of detection for a new custom dsDNA Mtb target enrichment panel that has a favorable cost profile. The results also demonstrate that pre-processing to remove contaminating extracellular DNA prior to cell lysis and DNA extraction improves the host-to-Mtb DNA ratio but is not adequate to support average coverage WGS without target capture.

Dietary patterns and risk of ulcerative colitis: a case-control study.

BACKGROUND: Recent evidence indicates a role for dietary factors in the pathogenesis of ulcerative colitis (UC). The aim of the present study was to investigate the relationship between dietary patterns and UC risk. METHODS: Sixty-two newly diagnosed cases of UC and 124 healthy age and sex-matched controls were studied. Data on diet was measured using a validated country-specific food frequency questionnaire. Factor analysis was used to define major dietary patterns based on 28 food groups and nutrient content. RESULTS: After adjustment for confounding factors, subjects who were in the highest tertile of the healthy dietary pattern had a 79% lower risk of UC (odds ratio = 0.21, 95% confidence interval = 0.07-0.59, P = 0.003), whereas the consumption of an unhealthy dietary pattern was associated with a significantly increased risk of UC (odds ratio = 3.39, 95% 95% confidence interval = 1.16-9.90, P = 0.027). CONCLUSIONS: The findings of the present study suggest that dietary patterns are associated with UC risk.

The Crohn's disease-associated polymorphism in ATG16L1 (rs2241880) reduces SHIP gene expression and activity in human subjects.

Crohn's disease (CD) is a polygenic immune-mediated disease characterized by gastrointestinal inflammation. Mice deficient in the hematopoietic-restricted SH2 domain-containing inositolpolyphosphate 5'-phosphatase (SHIP) develop spontaneous CD-like ileal inflammation. Intriguingly, SHIP mRNA is not upregulated in biopsies from patients with ileal CD despite immune cell infiltration, but SHIP's role in human CD remains unknown. We analyzed SHIP mRNA expression and activity in biopsies and peripheral blood mononuclear cells (PBMCs) from control and treatment-naive subjects with ileal CD, and demonstrated that SHIP mRNA and activity were lower in hematopoietic cells in ileal biopsies and PBMCs from subjects with CD. In all tissues from our patient cohort and in PBMCs from a second healthy control cohort, subjects homozygous for the autophagy-related 16-like protein (ATG16L1) CD-associated gene variant (rs2241880), had low SHIP mRNA expression and activity. SHIP protein expression increased during autophagy and SHIP upregulation was dependent on ATG16L1 and/or autophagy, as well as the ATG16L1 CD-associated gene variant. Finally, homozygosity for the ATG16L1 risk variant and low SHIP mRNA expression is inversely related to increased (LPS+ATP)-induced IL-1ß production by PBMCs in our cohorts and was regulated by increased transcription of ILIB. These data suggest a novel mechanism by which the ATG16L1 CD-associated gene variant may predispose people to develop intestinal inflammation.

Vasoactive intestinal peptide prevents PKCε-induced intestinal epithelial barrier disruption during EPEC infection.

We previously showed that vasoactive intestinal peptide (VIP) protects against bacterial pathogen-induced epithelial barrier disruption and colitis, although the mechanisms remain poorly defined. The aim of the current study was to identify cellular pathways of VIP-mediated protection with use of pharmacological inhibitors during enteropathogenic Escherichia coli (EPEC) infection of Caco-2 cell monolayers and during Citrobacter rodentium-induced colitis. EPEC-induced epithelial barrier disruption involved the PKC pathway but was independent of functional cAMP, Rho, and NF-κB pathways. VIP mediated its protective effects by inhibiting EPEC-induced PKC activity and increasing expression of the junctional protein claudin-4. Short-term treatment with TPA, which is known to activate PKC, was inhibited by VIP pretreatment, while PKC degradation via long-term treatment with TPA mimicked the protective actions of VIP. Immunostaining for specific PKC isotypes showed upregulated expression of PKCθ and PKCε during EPEC infection. Treatment with specific inhibitors revealed a critical role for PKCε in EPEC-induced barrier disruption. Furthermore, activation of PKCε and loss of barrier integrity correlated with claudin-4 degradation. In contrast, inhibition of PKCε by VIP pretreatment or the PKCε inhibitor maintained membrane-bound claudin-4 levels, along with barrier function. Finally, in vivo treatment with the PKCε inhibitor protected mice from C. rodentium-induced colitis. In conclusion, EPEC infection increases intracellular PKCε levels, leading to decreased claudin-4 levels and compromising epithelial barrier integrity. VIP inhibits PKCε activation, thereby attenuating EPEC-induced barrier disruption.

Infections by Renibacterium salmoninarum and Nanophyetus salmincola Chapin are associated with reduced growth of juvenile Chinook salmon, Oncorhynchus tshawytscha (Walbaum), in the Northeast Pacific Ocean.

We examined 1454 juvenile Chinook salmon, Oncorhynchus tshawytscha (Walbaum), captured in nearshore waters off the coasts of Washington and Oregon (USA) from 1999 to 2004 for infection by Renibacterium salmoninarum, Nanophyetus salmincola Chapin and skin metacercariae. The prevalence and intensities for each of these infections were established for both yearling and subyearling Chinook salmon. Two metrics of salmon growth, weight residuals and plasma levels of insulin-like growth factor-1, were determined for salmon infected with these pathogens/parasites, both individually and in combination, with uninfected fish used for comparison. Yearling Chinook salmon infected with R. salmoninarum had significantly reduced weight residuals. Chinook salmon infected with skin metacercariae alone did not have significantly reduced growth metrics. Dual infections were not associated with significantly more severe effects on the growth metrics than single infections; the number of triple infections was very low and precluded statistical comparison. Overall, these data suggest that infections by these organisms can be associated with reduced juvenile Chinook salmon growth. Because growth in the first year at sea has been linked to survival for some stocks of Chinook salmon, the infections may therefore play a role in regulating these populations in the Northeast Pacific Ocean.

Repeat treatment of acute hereditary angioedema attacks with open-label icatibant in the FAST-1 trial.

Hereditary angioedema (HAE) is characterized by potentially life-threatening recurrent episodes of oedema. The open-label extension (OLE) phase of the For Angioedema Subcutaneous Treatment (FAST)-1 trial (NCT00097695) evaluated the efficacy and safety of repeated icatibant exposure in adults with multiple HAE attacks. Following completion of the randomized, controlled phase, patients could receive open-label icatibant (30 mg subcutaneously) for subsequent attacks. The primary end-point was time to onset of primary symptom relief, as assessed by visual analogue scale (VAS). Descriptive statistics were reported for cutaneous/abdominal attacks 1-10 treated in the OLE phase and individual laryngeal attacks. Post-hoc analyses were conducted in patients with ≥ 5 attacks across the controlled and OLE phases. Safety was evaluated throughout. During the OLE phase, 72 patients received icatibant for 340 attacks. For cutaneous/abdominal attacks 1-10, the median time to onset of primary symptom relief was 1·0-2·0 h. For laryngeal attacks 1-12, patient-assessed median time to initial symptom improvement was 0·3-1·2 h. Post-hoc analyses showed the time to onset of symptom relief based on composite VAS was consistent across repeated treatments with icatibant. One injection of icatibant was sufficient to treat 88·2% of attacks; rescue medication was required in 5·3% of attacks. No icatibant-related serious adverse events were reported. Icatibant provided consistent efficacy and was well tolerated for repeated treatment of HAE attacks.

Growth and condition of juvenile coho salmon Oncorhynchus kisutch relate positively to species richness of trophically transmitted parasites.

The aims of this study were first, to test the hypothesis that metrics of fish growth and condition relate positively to parasite species richness (S(R)) in a salmonid host; second, to identify whether S(R) differs as a function of host origin; third, to identify whether acquisition of parasites through marine v. freshwater trophic interactions was related to growth and condition of juvenile salmonids. To evaluate these questions, species diversity of trophically transmitted parasites in juvenile coho salmon Oncorhynchus kisutch collected off the coast of the Oregon and Washington states, U.S.A. in June 2002 and 2004 were analysed. Fish infected with three or more parasite species scored highest in metrics of growth and condition. Fish originating from the Columbia River basin had lower S(R) than those from the Oregon coast, Washington coast and Puget Sound, WA. Parasites obtained through freshwater or marine trophic interactions were equally important in the relationship between S(R) and ocean growth and condition of juvenile O. kisutch salmon.

Risk factors for and timing of presumptive recurrent TB.

Understanding factors associated with increased risk for tuberculosis (TB) recurrence is essential in lowering the TB burden. We aimed to quantify the burden, risk factors, and timing of TB presumptive recurrence.We analyzed test results from 2013 to 2017 in the South African National Health Laboratory Service's database. We defined a person's TB episode to start with their first positive TB test. In the absence of treatment outcome data, we assumed the episode concluded 6 months later for rifampicin-susceptible TB (RS-TB) and 18 months later for rifampicin-resistant TB (RR-TB), provided that at least one negative smear or culture test was recorded within this period. We defined a presumptive recurrent TB episode to start with a positive TB test after the completion of a prior episode. We calculated recurrence measures stratified by various demographics and RR-TB status.Of 574,316 people with RS-TB, 4.7% experienced at least one presumptive recurrent TB episode. Higher local TB notification rates, HIV coinfection, and males experienced higher recurrence rates. Most (89.4%) of the first RS-TB recurrences occurred within a year of the initial episode.Our findings of when and among whom recurrent TB is more likely to occur can be used to assist early interventions and inform impact on patient care..

Strategies for isoniazid preventive therapy in HIV-positive patients who consume alcohol.

WHO guidance to defer isoniazid preventive therapy (IPT) among those with regular alcohol use because of hepatotoxicity concerns may exclude many people living with HIV (PLWH) at high TB risk in these settings.To evaluate hepatotoxicity during TB preventive therapy (TPT) in PLWH who report alcohol use in Uganda over 10 years.We developed a Markov model of latent TB infection, isoniazid preventive therapy (IPT - a type of TPT), and TB disease using data from the Alcohol Drinkers' Exposure to Preventive Therapy for TB (ADEPTT) study. We modeled several treatment scenarios, including no IPT, IPT with liver enzyme monitoring (AST/ALT) during treatment, and IPT with pre-screening using the tuberculin skin test (TST).The no IPT scenario had 230 TB deaths/100,000 population over 10 years, which is more than that seen in any IPT scenario. IPT, even with no monitoring, was preferred over no IPT when population TB disease incidence was >50 in 100,000.For PLWH who report alcohol use in high TB burden settings, IPT should be offered, ideally with regular AST/ALT monitoring. However, even if regular monitoring is not possible, IPT is still preferable to no IPT in almost every modeled scenario..

Smoked drug use in patients with TB is associated with higher bacterial burden.

BACKGROUND: Smoking of illicit drugs may lead to more rapid TB disease progression or late treatment presentation, yet research on this topic is scant. We examined the association between smoked drug use and bacterial burden among patients newly initiated on drug-susceptible TB (DS-TB) therapy.METHODS: Data from 303 participants initiating DS-TB treatment in the Western Cape Province, South Africa, were analyzed. Smoked drug use was defined as self-reported or biologically verified methamphetamine, methaqualone and/or cannabis use. Proportional hazard and logistic regression models (adjusted for age, sex, HIV status and tobacco use) examined associations between smoked drug use and mycobacterial time to culture positivity (TTP), acid-fast bacilli sputum smear positivity and lung cavitation.RESULTS: People who smoked drugs (PWSD) comprised 54.8% (n = 166) of the cohort. TTP was faster for PWSD (hazard ratio 1.48, 95% CI 1.10-1.97; P = 0.008). Smear positivity was higher among PWSD (OR 2.28, 95% CI 1.22-4.34; P = 0.011). Smoked drug use (OR 1.08, 95% CI 0.62-1.87; P = 0.799) was not associated with increased cavitation.CONCLUSIONS: PWSD had a higher bacterial burden at diagnosis than those who do not smoke drugs. Screening for TB among PWSD in the community may facilitate earlier linkage to TB treatment and reduce community transmission.

Moderating effects of childhood maltreatment on associations between social information processing and adult aggression.

BACKGROUND: Associations between early life maltreatment, social information processing (SIP) and aggression in childhood and adolescence have been widely documented. Few studies have examined the importance of childhood maltreatment independent of SIP in the etiology of adult aggression. Furthermore, moderating effects of childhood maltreatment on the SIP-aggression links have not been explored. METHOD: Hierarchical, multi-level models were fitted to data from n=2752 twins aged 20-55 years from the PennTwins Cohort. Adult aggression was assessed with the Life History of Aggression questionnaire. Childhood maltreatment was measured using the Childhood Trauma Questionnaire. Two aspects of SIP were examined: hostile attribution biases (HAB); negative emotional responses (NER). RESULTS: Childhood maltreatment was positively correlated with adult aggression, independently of HAB and NER. In addition, childhood maltreatment moderated the relationships between both aspects of SIP and adult aggression. Specifically, the relationship between NER and aggression was stronger among individuals with higher levels of childhood maltreatment and NER was not associated with aggression for adults who experienced low levels of childhood maltreatment. Moderating effects of childhood maltreatment on the NER-aggression link were supported for total childhood maltreatment, emotional neglect and emotional abuse. In contrast, HAB was more strongly associated with adult aggression at lower levels of emotional abuse and physical neglect. CONCLUSIONS: The current study provides insight into the mechanisms by which early life experiences influence adult aggression. Our findings suggest that childhood maltreatment may not only lead to increased levels of aggression in adulthood but may also modify the associations between SIP and adult aggression.

The Phenotypic Spectrum of New-onset IBD in Canadian Children of South Asian Ethnicity: A Prospective Multi-Centre Comparative Study.

BACKGROUND: Canadian-born children of South Asian [SA] ethnicity develop inflammatory bowel disease [IBD] at similar rates to those among Caucasian children. We evaluated the variation in phenotypic spectrum of IBD in SA and Caucasian children in a national paediatric inception cohort of new-onset IBD. METHODS: Patients aged <17 years, enrolled in a Canadian nationwide inception cohort study, were included. Baseline demographic and IBD phenotypic features were compared between SA and Caucasian children. Longitudinal outcomes through 18 months of follow-up were compared matched by propensity scores. RESULTS: Of 1156 children enrolled over 2014 to 2019, 623 were Caucasian [98% and 88% parents Canadian born] and 114 SA [79% Canadian born, 87% parents SA born]. Fewer SAs have a first-degree relative with IBD, 6% vs 19% in Caucasians, p = 0.002. SAs present at a younger age, median age 11.4 years (interquartile range [IQR] 9.2-14.3) vs 13 years [IQR 10.9-15 years], p = 0.03 and more commonly with a UC/IBD-U [ulcerative colitis/IBD-unclassified] subtype [ratio of UC/IBD-U to CD 1.2:1 vs 1:1.8 for Caucasians, p <0.001]. Additionally, a greater proportion of SA CD patients present with colonic-only disease [colonic-only CD/UC/IBD-U in SAs 67% vs 57% for Caucasians, p = 0.001], and among those with CD, colonic CD in SAs 31% vs 23% in Caucasians, p = 0.20]. Perianal fistulising disease was also numerically more common in SAs (14 [27%] vs 64 [18%], p = 0.06]. Adjusting for differences in phenotypic presentation, anti-tumour necrosis factor [TNF] exposure, and time to initiation was similar, and two-thirds of children, whether anti-TNF exposed or naïve, were in corticosteroid-free clinical remission at 18 months irrespective of ethnicity. CONCLUSIONS: The phenotypic spectrum of new-onset IBD in SA children differs from that of Caucasian children, but treatment and clinical course are similar within phenotypic subgroups.

CF102 an A3 adenosine receptor agonist mediates anti-tumor and anti-inflammatory effects in the liver.

The Gi protein-associated A(3) adenosine receptor (A(3) AR) is a member of the adenosine receptor family. Selective agonists at the A(3) AR, such as CF101 and CF102 were found to induce anti-inflammatory and anti-cancer effects. In this study, we examined the differential effect of CF102 in pathological conditions of the liver. The anti-inflammatory protective effect of CF101 was tested in a model of liver inflammation induced by Concanavalin A (Con. A) and the anti-cancer effect of CF102 was examined in vitro and in a xenograft animal model utilizing Hep-3B hepatocellular carcinoma (HCC) cells. The mechanism of action was explored by following the expression levels of key signaling proteins in the inflamed and tumor liver tissues, utilizing Western blot (WB) analysis. In the liver inflammation model, CF102 (100 µg/kg) markedly reduced the secretion of serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase in comparison to the vehicle-treated group. Mechanistically, CF102 treatment decreased the expression level of phosphorylated glycogen synthase kinase-3ß, NF-κB, and TNF-α and prevented apoptosis in the liver. This was demonstrated by decreased expression levels of Fas receptor (FasR) and of the pro-apoptotic proteins Bax and Bad in liver tissues. In addition, CF102-induced apoptosis of Hep-3B cells both in vitro and in vivo via de-regulation of the PI3K-NF-κB signaling pathway, resulting in up-regulation of pro-apoptotic proteins. Taken together, CF102 acts as a protective agent in liver inflammation and inhibits HCC tumor growth. These results suggest that CF102 through its differential effect is a potential drug candidate to treat various pathological liver conditions.

PTEN and PIK3CA gene copy numbers and poor outcomes in non-small cell lung cancer patients with gefitinib therapy.

METHODS: Fluorescent in situ hybridisation analyses of PTEN, PIK3CA, EGFR and CEN7 were performed on tumour specimens from patients treated on the expanded access gefitinib trial. Progression-free survival (PFS) and overall survival (OS) were correlated with outcomes in all patients and EGFR wild-type patients. RESULTS: Progression-free survival (hazard ratio=2.54, P<0.001) and OS (hazard ratio=4.04, P<0.001) were significantly shorter in patients whose tumours had all of the following molecular patterns: CEN7 <4 copies per cell, PTEN loss (<2 copies in at least 20% of cells), and PIK3CA gain (>2 copies in at least 40% of cells) both in all and EGFR wild-type only patients. CONCLUSION: The combination of low CEN7 copy number, PTEN loss, and PI3KCA gain may be useful for identifying NSCLC patients unlikely to benefit from treatment with EGFR (TKIs), specifically in wild-type EGFR cases.

New insights into membrane dynamics from the analysis of cell surface interactions by physical methods.

The physical, chemical and mechanical properties of the cell surface can be probed using a variety of microscopy-based techniques. The movements of membrane components are currently being characterized, and recent experiments have begun to define the structural origins of these modes of transport at a molecular level. However, explicit relationships between new knowledge of membrane structure and complex, linked functions, such as signal transduction and adhesion, remain elusive.

C1 esterase inhibitor concentrate in 1085 Hereditary Angioedema attacks--final results of the I.M.P.A.C.T.2 study.

BACKGROUND: The placebo-controlled study International Multicentre Prospective Angioedema C1-INH Trial 1 (I.M.P.A.C.T.1) demonstrated that 20 U/kg C1 esterase inhibitor (C1-INH) concentrate (Berinert®; CSL Behring, Marburg, Germany) is effective in treating acute abdominal and facial Hereditary Angioedema (HAE) attacks. METHODS: I.M.P.A.C.T.2 was an open-label extension study of I.M.P.A.C.T.1 to evaluate the safety and efficacy of long-term treatment with 20 U/kg C1-INH for successive HAE attacks at any body location. Efficacy outcomes included patient-reported time to onset of symptom relief (primary) and time to complete resolution of all symptoms (secondary), analysed on a per-patient and per-attack basis. Safety assessments included adverse events, vital signs, viral safety and anti-C1-INH antibodies. RESULTS: During a median study duration of 24 months, 1085 attacks were treated in 57 patients (10-53 years of age). In the per-patient analysis, the median time to onset of symptom relief was 0.46 h and was similar for all types of attacks (0.39-0.48 h); the median time to complete resolution of symptoms was 15.5 h (shortest for laryngeal attacks: 5.8 h; 12.8-26.6 h for abdominal, peripheral and facial attacks). Demographic factors, type of HAE, intensity of attacks, time to treatment, use of androgens and presence of anti-C1-INH antibodies had no clinically relevant effect on the efficacy outcomes. There were no treatment-related safety concerns. No inhibitory anti-C1-INH antibodies were detected in any patient. CONCLUSIONS: A single dose of 20 U/kg C1-INH concentrate is safe and provides reliable efficacy in the long-term treatment of successive HAE attacks at any body location.

Direct, high-resolution measurement of furrow stiffening during division of adherent cells.

It is unclear whether cell division is driven by cortical relaxation outside the equatorial region or cortical contractility within the developing furrow alone. To approach this question, a technique is required that can monitor spatially-resolved changes in cortical stiffness with good time resolution. We employed atomic force microscopy (AFM), in force-mapping mode, to track dynamic changes in the stiffness of the cortex of adherent cultured cells along a single scan-line during M phase, from metaphase to cytokinesis. Video microscopy, which we used to correlate the AFM data with mitotic events identified by light microscopy, indicated that the AFM force-mapping technique does not perturb dividing cells. Here we show that cortical stiffening occurs over the equatorial region about 160 seconds before any furrow appears, and that this stiffening markedly increases as the furrow starts. By contrast, polar relaxation of cells does not seem to be an obligatory event for cell division to occur.

The impact of alcohol use on tuberculosis treatment outcomes: a systematic review and meta-analysis.

Alcohol use is associated with increased risk of developing tuberculosis (TB) disease, yet the impact of alcohol use on TB treatment outcomes has not been summarized. We aimed to quantitatively review evidence of the relationship between alcohol use and poor TB treatment outcomes. We conducted a systematic review of PubMed, EMBASE, and Web of Science (January 1980-May 2018). We categorized studies as having a high- or low-quality alcohol use definition and examined poor treatment outcomes individually and as two aggregated definitions (i.e., including or excluding loss to follow-up [LTFU]). We analyzed drug-susceptible (DS-) and multidrug-resistant (MDR-) TB studies separately. Our systematic review yielded 111 studies reporting alcohol use as a predictor of DS- and MDR-TB treatment outcomes. Alcohol use was associated with increased odds of poor treatment outcomes (i.e., death, treatment failure, and LTFU) in DS (OR 1.99, 95% CI 1.57-2.51) and MDR-TB studies (OR 2.00, 95% CI 1.73-2.32). This association persisted for aggregated poor treatment outcomes excluding LTFU, each individual poor outcome, and across sub-group and sensitivity analyses. Only 19% of studies used high-quality alcohol definitions. Alcohol use significantly increased the risk of poor treatment outcomes in both DS- and MDR-TB patients. This study highlights the need for improved assessment of alcohol use in TB outcomes research and potentially modified treatment guidelines for TB patients who consume alcohol.

Phenotypic Variation in Paediatric Inflammatory Bowel Disease by Age: A Multicentre Prospective Inception Cohort Study of the Canadian Children IBD Network.

BACKGROUND AND AIMS: Incidence of paediatric inflammatory bowel disease [IBD] in Canada is among the highest worldwide, and age of onset may be decreasing. In a multicentre nationwide inception cohort study, we examined variation in phenotype of IBD throughout the paediatric age spectrum. METHODS: Children aged ≥2 years [y] and <17y [A1 age at diagnosis], with new onset IBD, were systematically evaluated at sites of the Canadian Children IBD Network. Prospectively recorded phenotypic data were compared between age groups. RESULTS: Among 1092 children (70% Caucasian; 64% Crohn's disease [CD], 36% ulcerative colitis/inflammatory bowel disease unclassified [UC/IBD-U]; median age 13 y, interquartile range [IQR] 11-15 y), 210 [19%] were diagnosed before the age of age 10 y [Paris A1a] and 43 [4%] before age 6 y (very-early-onset [VEO-IBD]). CD was less common in younger children [42%, 56%, 66%, respectively, of VEO-IBD, A1a; A1b]. Colon-only IBD [UC/IBDU or CD-colon] was present in 81% of VEO-IBD and 65% of A1a; ileal disease increased progressively, reaching plateau at age 10 y. CD location was ileocolonic [L3] in 53% overall. Ileitis [L1] increased with age [6% of VEO-IBD; 13% of A1a; 21% of A1b], as did stricturing/penetrating CD [4% of A1a; 11% of A1b]. At all ages UC was extensive [E3/E4] in >85%, and disease activity moderate to severe according to Physician's Global Assessment [PGA] and weighted Paediatric Crohn's Disease Activity Index/Paediatric Ulcerative Colitis Activity Index [wPCDAI/PUCAI] in >70%. Heights were modestly reduced in CD [mean height z score -0.30 ± 1.23], but normal in UC/IBD-U. CONCLUSIONS: Paris classification of age at diagnosis is supported by age-related increases in ileal disease until age 10 years. Other phenotypic features, including severity, are similar across all ages. Linear growth is less impaired in CD than in historical cohorts, reflecting earlier diagnosis.

Vasoactive intestinal peptide ameliorates intestinal barrier disruption associated with Citrobacter rodentium-induced colitis.

Attaching and effacing bacterial pathogens attach to the apical surface of epithelial cells and disrupt epithelial barrier function, increasing permeability and allowing luminal contents access to the underlying milieu. Previous in vitro studies demonstrated that the neuropeptide vasoactive intestinal peptide (VIP) regulates epithelial paracellular permeability, and the high concentrations and close proximity of VIP-containing nerve fibers to intestinal epithelial cells would support such a function in vivo. The aim of this study was to examine whether VIP treatment modulated Citrobacter rodentium-induced disruption of intestinal barrier integrity and to identify potential mechanisms of action. Administration of VIP had no effect on bacterial attachment although histopathological scoring demonstrated a VIP-induced amelioration of colitis-induced epithelial damage compared with controls. VIP treatment prevented the infection-induced increase in mannitol flux a measure of paracellular permeability, resulting in levels similar to control mice, and immunohistochemical studies demonstrated that VIP prevented the translocation of tight junction proteins: zonula occludens-1, occludin, and claudin-3. Enteropathogenic Escherichia coli (EPEC) infection of Caco-2 monolayers confirmed a protective role for VIP on epithelial barrier function. VIP prevented EPEC-induced increase in long myosin light chain kinase (MLCK) expression and myosin light chain phosphorylation (p-MLC). Furthermore, MLCK inhibition significantly attenuated bacterial-induced epithelial damage both in vivo and in vitro. In conclusion, our results indicate that VIP protects the colonic epithelial barrier by minimizing bacterial-induced redistribution of tight junction proteins in part through actions on MLCK and MLC phosphorylation.