Spatial cluster mapping and environmental modeling in pediatric inflammatory bowel disease.

BACKGROUND: Geographical (geospatial) clusters have been observed in inflammatory bowel disease (IBD) incidence and linked to environmental determinants of disease, but pediatric spatial patterns in North America are unknown. We hypothesized that we would identify geospatial clusters in the pediatric IBD (PIBD) population of British Columbia (BC), Canada and associate incidence with ethnicity and environmental exposures. AIM: To identify PIBD clusters and model how spatial patterns are associated with population ethnicity and environmental exposures. METHODS: One thousand one hundred eighty-three patients were included from a BC Children's Hospital clinical registry who met the criteria of diagnosis with IBD ≤ age 16.9 from 2001-2016 with a valid postal code on file. A spatial cluster detection routine was used to identify areas with similar incidence. An ecological analysis employed Poisson rate models of IBD, Crohn's disease (CD), and ulcerative colitis (UC) cases as functions of areal population ethnicity, rurality, average family size and income, average population exposure to green space, air pollution, and vitamin-D weighted ultraviolet light from the Canadian Environmental Health Research Consortium, and pesticide applications. RESULTS: Hot spots (high incidence) were identified in Metro Vancouver (IBD, CD, UC), southern Okanagan regions (IBD, CD), and Vancouver Island (CD). Cold spots (low incidence) were identified in Southeastern BC (IBD, CD, UC), Northern BC (IBD, CD), and on BC's coast (UC). No high incidence hot spots were detected in the densest urban areas. Modeling results were represented as incidence rate ratios (IRR) with 95%CI. Novel risk factors for PIBD included fine particulate matter (PM2.5) pollution (IRR = 1.294, CI = 1.113-1.507, P < 0.001) and agricultural application of petroleum oil to orchards and grapes (IRR = 1.135, CI = 1.007-1.270, P = 0.033). South Asian population (IRR = 1.020, CI = 1.011-1.028, P < 0.001) was a risk factor and Indigenous population (IRR = 0.956, CI = 0.941-0.971, P < 0.001), family size (IRR = 0.467, CI = 0.268-0.816, P = 0.007), and summer ultraviolet (IBD = 0.9993, CI = 0.9990-0.9996, P < 0.001) were protective factors as previously established. Novel risk factors for CD, as for PIBD, included: PM2.5 air pollution (IRR = 1.230, CI = 1 .056-1.435, P = 0.008) and agricultural petroleum oil (IRR = 1.159, CI = 1.002-1.326, P = 0.038). Indigenous population (IRR = 0.923, CI = 0.895-0.951, P < 0.001), as previously established, was a protective factor. For UC, rural population (UC IRR = 0.990, CI = 0.983-0.996, P = 0.004) was a protective factor and South Asian population (IRR = 1.054, CI = 1.030-1.079, P < 0.001) a risk factor as previously established. CONCLUSION: PIBD spatial clusters were identified and associated with known and novel environmental determinants. The identification of agricultural pesticides and PM2.5 air pollution needs further study to validate these observations.

Transition Readiness in Youth with Inflammatory Bowel Disease.

OBJECTIVES: To examine readiness of adolescents and young adults (AYAs) with inflammatory bowel disease (IBD) to transition to adult care. STUDY DESIGN: A cross-sectional multicenter study evaluating transition readiness in individuals with IBD 16-19 years old prospectively recruited from 8 Canadian IBD centers using the validated ON Taking Responsibility for Adolescent to Adult Care (ON TRAC) questionnaire. Secondary aims included (1) screening for depression and anxiety using the 8-item Personal Health Questionnaire Depression Scale and The Screen for Child Anxiety Related Emotional Disorders questionnaires, respectively; (2) evaluating the association between depression and anxiety with readiness and disease activity; and (3) subjectively evaluating AYA readiness based on physician and parent assessments. RESULTS: In total, 186 participants (139 adolescent, 47 young adult) were enrolled, mean age 17.4 years (SD, 0.87). ON TRAC scores determined that 26.6% of AYAs at pediatric and 40.4% at adult centers reached the threshold of readiness. On multivariable linear regression analysis age was positively (P = .001) and disease remission negatively (P = .03) associated with ON TRAC scores. No statistically significant differences were determined across centers. A significant percentage of AYAs reported moderate-to-severe depression (21.7%) and generalized anxiety (36%); however, neither were significantly associated with ON TRAC scores. Notably, physician and parental assessment of AYA readiness correlated poorly with ON TRAC scores (⍴ = 0.11, ⍴ = 0.24, respectively). CONCLUSIONS: Assessment of transition readiness in AYAs with IBD highlighted that a large proportion do not have adequate knowledge or behavior skills needed for transition to adult care. This study infers that readiness assessment tools are essential during transition to identify deficits in knowledge and behavior skills that could be specifically targeted by the youth, caregivers, and multidisciplinary team.

Arthroscopic-Assisted Removal of Proximal Humerus Locking Plates With Capsular Release Significantly Improves Range of Motion and Function.

PURPOSE: To investigate the clinical outcomes following the arthroscopic removal of proximal humerus locking plates for symptomatic hardware after open reduction and internal fixation (ORIF) of proximal humerus fractures. METHODS: Patients who underwent arthroscopic removal of hardware (ROH) with capsular release due to pain and/or immobility after receiving locking plates to treat proximal humerus fractures from 2009 to 2016 were identified. Operative and clinic records were reviewed to obtain demographic information, concomitant procedures during ROH, and pre- and postoperative active shoulder range of motion. Postoperative patient-reported outcomes included the QuickDASH, PROMIS Pain Intensity, Constant, and University of California, Los Angeles shoulder rating scale. RESULTS: In total, 88 patients were included. Patients were evaluated at a minimum of 6 weeks postoperatively after ROH. Patients with pre- and postoperative active range of motion values demonstrated significant improvements in mean forward elevation (n = 69; 78.4%; 115.1° to 152.1°, P < .001), abduction (n = 29; 33.0%; 70.9° to 138.7°, P < .001), external rotation (n = 49; 55.7%; 43.7° to 58.6°, P = .012), and internal rotation (n = 45; 51.1%; 25.7° to 61.9°, P < .001). Patients also reported positive patient-reported scores, including the QuickDASH (4.1 ± 7.8), PROMIS Pain Intensity (3.5 ± 0.9), Constant (84.6 ± 10.7), and University of California, Los Angeles shoulder rating scale (33 ± 2.9), which were measured 70.6 ± 26.6 months postoperatively. There were no surgical complications, no arthroscopic cases were converted to open, but 2 reported refractures (2.3%). CONCLUSIONS: Arthroscopic-assisted removal of proximal humerus locking plates significantly improves motion and function while allowing for management of concomitant shoulder pathology and potentially avoiding open surgery complications. Given that patients undergoing this procedure frequently have multiple comorbidities, arthroscopic-assisted removal with smaller incisions may minimize risks while restoring shoulder mobility. Therefore, arthroscopic ROH for patients experiencing symptomatic hardware after ORIF is recommended. LEVEL OF EVIDENCE: Level IV, therapeutic case series.

Comparing Health Administrative and Clinical Registry Data: Trends in Incidence and Prevalence of Pediatric Inflammatory Bowel Disease in British Columbia.

PURPOSE: Canada maintains robust health administrative databases and British Columbia Children's Hospital (BCCH), as the only tertiary care pediatric hospital in British Columbia (BC), maintains a comprehensive clinical inflammatory bowel disease (IBD) registry. To evaluate the strengths and weaknesses of utilizing health administrative and clinical registry data to study the epidemiology of IBD in BC, we conducted a population-based retrospective cohort study of all children <18 years of age who were diagnosed with IBD between 1996 and 2008 in BC. METHODS: IBD cases from health administrative data were identified using a combination of IBD-coded physician encounters and hospitalizations while a separate IBD cohort was identified from the BCCH clinical registry data. Age and gender standardized incidence and prevalence rates were fitted to Poisson regression models. RESULTS: The overall incidence of pediatric IBD identified in health administrative data increased from 7.1 (95% CI 5.5-9.2) in 1996 to 10.3 (95% CI 8.2-12.7) per 100,000 children in 2008. Similarly, the incidence of the BCCH cohort increased from 4.3 (95% CI 3.0-6.0) to 9.7 (95% CI 7.6-12.1) per 100,000. Children aged 10-17 had the highest rise in incidence in both data sources; however, the administrative data identified significantly more 10-17-year-olds and significantly less 6-9-year-olds (p<0.05) compared to clinical registry data. CONCLUSION: While the application of both health administrative and clinical registry data demonstrates that the incidence of IBD is increasing in BC, we identify strengths and limitations to both and suggest that the utilization of either data source requires unique considerations that mitigate misclassification biases.

Arthroscopic-Assisted Coracoclavicular Ligament Reconstruction: Clinical Outcomes and Return to Activity at Mean 6-Year Follow-Up.

PURPOSE: To report clinical and functional outcomes including return to preinjury activity level following arthroscopic-assisted coracoclavicular (CC) ligament reconstruction (AA-CCR) and to determine associations between return to preinjury activity level, radiographic outcomes, and patient-reported outcomes following AA-CCR. METHODS: A institutional registry review of all AA-CCR using free tendon grafts from 2007 to 2016 was performed. Clinical assessment included Single Assessment Numeric Evaluation (SANE) score and return to preinjury activity level at final follow-up. Treatment failure was defined as (1) revision acromioclavicular stabilization surgery, (2) unable to return to preinjury activity level, or (3) radiographic loss of reduction (RLOR, >25% CC distance compared with contralateral side). SANE scores, return to activity, and RLOR were compared between patients within each category of treatment failure, by grade of injury, and whether concomitant pathology was treated. RESULTS: There were 88 patients (89.8% male) with mean age of 39.6 years and minimum 2-year clinical follow-up (mean 6.1 years). Most injuries were Rockwood grade V (63.6%). Mean postoperative SANE score was 86.3 ± 17.5. Treatment failure occurred in 17.1%: 8.0% were unable to return to activity, 5.7% had RLOR, and 3.4% underwent revision surgery due to traumatic reinjury. SANE score was lower among patients who were unable to return to activity compared with those with RLOR and compared with nonfailures (P = .0002). There were no differences in revision surgery rates, return to activity, or SANE scores according to Rockwood grade or if concomitant pathology was treated. CONCLUSIONS: AA-CCR with free tendon grafts resulted in good clinical outcomes and a high rate of return to preinjury activity level. RLOR did not correlate with return to preinjury activity level. Concomitant pathology that required treatment did not adversely affect outcomes. Return to preinjury activity level may be a more clinically relevant outcome measure than radiographic maintenance of acromioclavicular joint reduction. LEVEL OF EVIDENCE: IV (Case Series).

Upper Extremity and Hip Range of Motion Changes Throughout a Season in Professional Baseball Players.

BACKGROUND: Loss of upper and lower extremity range of motion (ROM) is a significant risk factor for injuries in professional baseball players. PURPOSE/HYPOTHESIS: The purpose was to determine changes in ROM in professional baseball players over the course of a single season and their careers. We hypothesized that pitchers and position players would lose ROM, specifically total shoulder motion (total ROM [TROM]) and hip internal rotation (IR), over the course of a season and their careers. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Upper and lower extremity ROM measurements were recorded during pre-, mid-, and postseason on all professional baseball players for a single organization between 2011 and 2018. ROM measurements were compared for pitchers and position players over the course of the season and their careers. Also, ROM measurements over the pre-, mid-, and postseason were compared between pitchers and position players. RESULTS: A total of 166 professional baseball players (98 pitchers, 68 position players) were included. Pitcher hip external rotation (ER; P < .001), IR (P = .010), and TROM (P < .001) for lead and trail legs decreased over the course of the season. Pitcher shoulder ER (P = .005), TROM (P = .042), and horizontal adduction (P < .001) significantly increased over the course of the season. Position player shoulder flexion (P = .046), hip ER (P < .001, lead leg; P < .001, trail leg), and hip TROM (P = .001; P = .002) decreased over the course of the season. Position player shoulder ER (P = .031) and humeral adduction (P < .001) significantly increased over the course of the season. Over the course of pitchers' careers, there was decreased shoulder IR (P = .014), increased shoulder horizontal adduction (P < .001), and hip IR (P = .042) and hip TROM (P = .027) for the lead leg. Position players experienced loss of hip TROM (P = .010, lead leg; P = .018, trail leg) over the course of their careers. Pitchers started with and maintained more shoulder ER and gained more shoulder TROM over a season as compared with position players. CONCLUSION: Pitchers and position players saw overall decreases in hip ROM but increases in shoulder ROM over the course of the season and career.

Poor Correlation of Oral Swabs with Esophageal Eosinophil Counts.

Eosinophilic esophagitis (EoE) is a chronic condition that requires repeated endoscopies/biopsies to track the disease and treatment response. This invasive procedure involves risk to the patient and has significant costs. We studied whether the detection of specific proteins (cytokines and eosinophil degranulation products) from oral swabs could serve as a minimally invasive test for EoE. Swabs of the oral cavity (buccal and oropharyngeal) were obtained prior to endoscopy/biopsies in patients with EoE, possible EoE, and non-EoE patients in addition to obtaining additional esophageal biopsy tissue. ELISAs measuring the levels of cytokines IL-5, IL-8, IL-13, and eosinophil degranulation products including major basic protein (MBP), eosinophil derived neurotoxin (EDN), and eosinophil peroxidase (EPO) were performed on the samples. Comparisons were made to peak esophageal eosinophil counts. Tolerability of the swabs was evaluated. 43 patients, 4-17 years old, participated in the study. Swabs were well tolerated and all showed measurable protein. 26 patients had EoE [14 active (> 15 eosinophils/high power field), 12 non-active], 17 patients did not have EoE. Results obtained from oral swabs showed poor correlation with those from esophageal tissue. Only measurement of eosinophil degranulation products EDN and EPO from esophageal tissues showed strong correlations with eosinophil counts. In this study, the levels of cytokines and eosinophil degranulation products detected from oral swabs did not correlate with esophageal eosinophilia, and their detection would be insufficient to displace endoscopy/biopsies.

Milk Fat Globule Membrane Supplementation in Formula Modulates the Neonatal Gut Microbiome and Normalizes Intestinal Development.

Breast milk has many beneficial properties and unusual characteristics including a unique fat component, termed milk fat globule membrane (MFGM). While breast milk yields important developmental benefits, there are situations where it is unavailable resulting in a need for formula feeding. Most formulas do not contain MFGM, but derive their lipids from vegetable sources, which differ greatly in size and composition. Here we tested the effects of MFGM supplementation on intestinal development and the microbiome as well as its potential to protect against Clostridium difficile induced colitis. The pup-in-a-cup model was used to deliver either control or MFGM supplemented formula to rats from 5 to 15 days of age; with mother's milk (MM) reared animals used as controls. While CTL formula yielded significant deficits in intestinal development as compared to MM littermates, addition of MFGM to formula restored intestinal growth, Paneth and goblet cell numbers, and tight junction protein patterns to that of MM pups. Moreover, the gut microbiota of MFGM and MM pups displayed greater similarities than CTL, and proved protective against C. difficile toxin induced inflammation. Our study thus demonstrates that addition of MFGM to formula promotes development of the intestinal epithelium and microbiome and protects against inflammation.

Investigation of Host and Pathogen Contributions to Infectious Colitis Using the Citrobacter rodentium Mouse Model of Infection.

Citrobacter rodentium is used as a model organism to study enteric bacterial infections in mice. Infection occurs via the oral-fecal route and results in the pathogen forming attaching and effacing lesions on infected epithelial cells. Moreover, infection leads to a subsequent host-mediated form of colitis. C. rodentium infection is thus an excellent model to study infectious colitis in vivo, while the ability to genetically manipulate C. rodentium virulence genes provides the opportunity to develop clear insights into the pathogenesis of this and related infectious microbes. This chapter outlines the basic techniques involved in setting up a C. rodentium infection in mice and several different methodologies to assess the severity of the infection.

Prolonged antibiotic treatment induces a diabetogenic intestinal microbiome that accelerates diabetes in NOD mice.

Accumulating evidence supports that the intestinal microbiome is involved in Type 1 diabetes (T1D) pathogenesis through the gut-pancreas nexus. Our aim was to determine whether the intestinal microbiota in the non-obese diabetic (NOD) mouse model played a role in T1D through the gut. To examine the effect of the intestinal microbiota on T1D onset, we manipulated gut microbes by: (1) the fecal transplantation between non-obese diabetic (NOD) and resistant (NOR) mice and (2) the oral antibiotic and probiotic treatment of NOD mice. We monitored diabetes onset, quantified CD4+T cells in the Peyer's patches, profiled the microbiome and measured fecal short-chain fatty acids (SCFA). The gut microbiota from NOD mice harbored more pathobionts and fewer beneficial microbes in comparison with NOR mice. Fecal transplantation of NOD microbes induced insulitis in NOR hosts suggesting that the NOD microbiome is diabetogenic. Moreover, antibiotic exposure accelerated diabetes onset in NOD mice accompanied by increased T-helper type 1 (Th1) and reduced Th17 cells in the intestinal lymphoid tissues. The diabetogenic microbiome was characterized by a metagenome altered in several metabolic gene clusters. Furthermore, diabetes susceptibility correlated with reduced fecal SCFAs. In an attempt to correct the diabetogenic microbiome, we administered VLS#3 probiotics to NOD mice but found that VSL#3 colonized the intestine poorly and did not delay diabetes. We conclude that NOD mice harbor gut microbes that induce diabetes and that their diabetogenic microbiome can be amplified early in life through antibiotic exposure. Protective microbes like VSL#3 are insufficient to overcome the effects of a diabetogenic microbiome.

Characterization of gait in late onset Pompe disease.

The skeletal muscle manifestations of late-onset Pompe disease (LOPD) cause significant gait impairment. However, the specific temporal and spatial characteristics of abnormal gait in LOPD have not been objectively analyzed or described in the literature. This pilot study evaluated the gait of 22 individuals with LOPD using the GAITRite® temporospatial gait analysis system. The gait parameters were compared to normal reference values, and correlations were made with standard measures of disease progression. The LOPD population demonstrated significant abnormalities in temporospatial parameters of gait including a trend towards decreased velocity and cadence, a prolonged stance phase, prolonged time in double limb support, shorter step and stride length, and a wider base of support. Precise descriptions and analyses of gait abnormalities have much potential in increasing our understanding of LOPD, especially in regards to how its natural history may be modified by the use of enzyme replacement therapy (ERT) and other interventions. Gait analysis may provide a sensitive early marker of the onset of clinical symptoms and signs, offer an additional objective measure of disease progression and the impact of intervention, and serve as a potentially important clinical endpoint. The additional data from comprehensive gait analysis may personalize and optimize physical therapy management, and the clarification of specific gait patterns in neuromuscular diseases could be of clinical benefit in the ranking of a differential diagnosis.

Goblet Cell Derived RELM-ß Recruits CD4+ T Cells during Infectious Colitis to Promote Protective Intestinal Epithelial Cell Proliferation.

Enterohemorrhagic Escherichia coli and related food and waterborne pathogens pose significant threats to human health. These attaching/effacing microbes infect the apical surface of intestinal epithelial cells (IEC), causing severe diarrheal disease. Colonizing the intestinal luminal surface helps segregate these microbes from most host inflammatory responses. Based on studies using Citrobacter rodentium, a related mouse pathogen, we speculate that hosts rely on immune-mediated changes in IEC, including goblet cells to defend against these pathogens. These changes include a CD4+ T cell-dependent increase in IEC proliferation to replace infected IEC, as well as altered production of the goblet cell-derived mucin Muc2. Another goblet cell mediator, REsistin-Like Molecule (RELM)-ß is strongly induced within goblet cells during C. rodentium infection, and was detected in the stool as well as serum. Despite its dramatic induction, RELM-ß's role in host defense is unclear. Thus, wildtype and RELM-ß gene deficient mice (Retnlb-/-) were orally infected with C. rodentium. While their C. rodentium burdens were only modestly elevated, infected Retnlb-/- mice suffered increased mortality and mucosal ulceration due to deep pathogen penetration of colonic crypts. Immunostaining for Ki67 and BrDU revealed Retnlb-/- mice were significantly impaired in infection-induced IEC hyper-proliferation. Interestingly, exposure to RELM-ß did not directly increase IEC proliferation, rather RELM-ß acted as a CD4+ T cell chemoattractant. Correspondingly, Retnlb-/- mice showed impaired CD4+ T cell recruitment to their infected colons, along with reduced production of interleukin (IL)-22, a multifunctional cytokine that directly increased IEC proliferation. Enema delivery of RELM-ß to Retnlb-/- mice restored CD4+ T cell recruitment, concurrently increasing IL-22 levels and IEC proliferation, while reducing mucosal pathology. These findings demonstrate that RELM-ß and goblet cells play an unexpected, yet critical role in recruiting CD4+ T cells to the colon to protect against an enteric pathogen, in part via the induction of increased IEC proliferation.

CD4+ T cells drive goblet cell depletion during Citrobacter rodentium infection.

Both idiopathic and infectious forms of colitis disrupt normal intestinal epithelial cell (IEC) proliferation and differentiation, although the mechanisms involved remain unclear. Recently, we demonstrated that infection by the attaching and effacing murine pathogen Citrobacter rodentium leads to a significant reduction in colonic goblet cell numbers (goblet cell depletion). This pathology depends on T and/or B cells, as Rag1(-/-) mice do not suffer this depletion during infection, instead suffering high mortality rates. To address the immune mechanisms involved, we reconstituted Rag(-/-) mice with either CD4(+) or CD8(+) T cells. Both T cell subsets increased Rag1(-/-) mouse survival during infection, with mice that received CD8(+) T cells developing colonic ulcers but not goblet cell depletion. In contrast, mice that received CD4(+) T cells showed goblet cell depletion in concert with exaggerated IEC proliferation. To define the possible involvement of T cell-derived cytokines, we infected gamma interferon receptor gene knockout (IFN-γR(-/-)) mice and wild-type mice given interleukin 17A (IL-17A) neutralizing antibodies and found that IFN-γ signaling was required for both goblet cell depletion and increased IEC proliferation. Immunostaining revealed that C. rodentium cells preferentially localized to nonhyperplastic crypts containing numerous goblet cells, whereas hyperplastic, goblet cell-depleted crypts appeared protected from infection. To address whether goblet cell depletion benefits the C. rodentium-infected host, we increased goblet cell numbers using the γ-secretase inhibitor dibenzazepine (DBZ), which resulted in greatly increased pathogen burdens and mortality rates. These results demonstrate that goblet cell depletion reflects host immunomodulation of IEC homeostasis and reflects a novel host defense mechanism against mucosal-adherent pathogens.

SIGIRR, a negative regulator of TLR/IL-1R signalling promotes Microbiota dependent resistance to colonization by enteric bacterial pathogens.

Enteric bacterial pathogens such as enterohemorrhagic E. coli (EHEC) and Salmonella Typhimurium target the intestinal epithelial cells (IEC) lining the mammalian gastrointestinal tract. Despite expressing innate Toll-like receptors (TLRs), IEC are innately hypo-responsive to most bacterial products. This is thought to prevent maladaptive inflammatory responses against commensal bacteria, but it also limits antimicrobial responses by IEC to invading bacterial pathogens, potentially increasing host susceptibility to infection. One reason for the innate hypo-responsiveness of IEC is their expression of Single Ig IL-1 Related Receptor (SIGIRR), a negative regulator of interleukin (IL)-1 and TLR signaling. To address whether SIGIRR expression and the innate hypo-responsiveness of IEC impacts on enteric host defense, Sigirr deficient (-/-) mice were infected with the EHEC related pathogen Citrobacter rodentium. Sigirr -/- mice responded with accelerated IEC proliferation and strong pro-inflammatory and antimicrobial responses but surprisingly, Sigirr -/- mice proved dramatically more susceptible to infection than wildtype mice. Through haematopoietic transplantation studies, it was determined that SIGIRR expression by non-haematopoietic cells (putative IEC) regulated these responses. Moreover, the exaggerated responses were found to be primarily dependent on IL-1R signaling. Whilst exploring the basis for their susceptibility, Sigirr -/- mice were found to be unusually susceptible to intestinal Salmonella Typhimurium colonization, developing enterocolitis without the typical requirement for antibiotic based removal of competing commensal microbes. Strikingly, the exaggerated antimicrobial responses seen in Sigirr -/- mice were found to cause a rapid and dramatic loss of commensal microbes from the infected intestine. This depletion appears to reduce the ability of the microbiota to compete for space and nutrients (colonization resistance) with the invading pathogens, leaving the intestine highly susceptible to pathogen colonization. Thus, SIGIRR expression by IEC reflects a strategy that sacrifices maximal innate responsiveness by IEC in order to promote commensal microbe based colonization resistance against bacterial pathogens.

The Citrobacter rodentium mouse model: studying pathogen and host contributions to infectious colitis.

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection. The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

Attaching and effacing bacterial effector NleC suppresses epithelial inflammatory responses by inhibiting NF-κB and p38 mitogen-activated protein kinase activation.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli are noninvasive attaching and effacing (A/E) bacterial pathogens that cause intestinal inflammation and severe diarrheal disease. These pathogens utilize a type III secretion system to deliver effector proteins into host epithelial cells, modulating diverse cellular functions, including the release of the chemokine interleukin-8 (IL-8). While studies have implicated the effectors NleE (non-locus of enterocyte effacement [LEE]-encoded effector E) and NleH1 in suppressing IL-8 release, by preventing NF-κB nuclear translocation, the impact of these effectors only partially replicates the immunosuppressive actions of wild-type EPEC, suggesting another effector or effectors are involved. Testing an array of EPEC mutants, we identified the non-LEE-encoded effector C (NleC) as also suppressing IL-8 release. Infection by ΔnleC EPEC led to exaggerated IL-8 release from infected Caco-2 and HT-29 epithelial cells. NleC localized to EPEC-induced pedestals, with signaling studies revealing NleC inhibits both NF-κB and p38 mitogen-activated protein kinase (MAPK) activation. Using Citrobacter rodentium, a mouse-adapted A/E bacterium, we found that ΔnleC and wild-type C. rodentium-infected mice carried similar pathogen burdens, yet ΔnleC strain infection led to worsened colitis. Similarly, infection with ΔnleC C. rodentium in a cecal loop model induced significantly greater chemokine responses than infection with wild-type bacteria. These studies thus advance our understanding of how A/E pathogens subvert host inflammatory responses.

Secreted protein acidic and rich in cysteine-induced cellular senescence in colorectal cancers in response to irinotecan is mediated by P53.

Cellular senescence is another mechanism that can be exploited to achieve better chemosensitivity and greater tumor regression. Unlike apoptosis, cellular senescence can be induced at much lower concentrations of chemotherapy that are better tolerated by patients. We previously revealed that secreted protein acidic and rich in cysteine (SPARC), a matricellular protein, may function as a modulator of chemotherapy sensitivity by enhancing apoptosis. Here, we examine the effects of SPARC on cellular senescence in the presence of chemotherapy. Cellular senescence is induced only in sensitive colorectal cancer (CRC) cells with low concentrations of irinotecan (CPT-11). However, CPT-11-resistant cells exposed to endogenous or exogenous SPARC can also be triggered into cellular senescence. This induction is associated with higher levels of p16(INK4A) and phosphorylated p53. Knock down of p16(INK4A) reduces drug-induced senescence in all cells, but knock down and overexpression of p53 modulates senescence only in cells exposed to SPARC. Furthermore, treatment of mice with SPARC and CPT-11 leads to significantly increased cellular senescence and tumor regression. The chemosensitizing effects of SPARC in CRCs are, therefore, probably mediated in part by activating cellular senescence.