Identification and validation of a blood- based diagnostic lipidomic signature of pediatric inflammatory bowel disease.

Improved biomarkers are needed for pediatric inflammatory bowel disease. Here we identify a diagnostic lipidomic signature for pediatric inflammatory bowel disease by analyzing blood samples from a discovery cohort of incident treatment-naïve pediatric patients and validating findings in an independent inception cohort. The lipidomic signature comprising of only lactosyl ceramide (d18:1/16:0) and phosphatidylcholine (18:0p/22:6) improves the diagnostic prediction compared with high-sensitivity C-reactive protein. Adding high-sensitivity C-reactive protein to the signature does not improve its performance. In patients providing a stool sample, the diagnostic performance of the lipidomic signature and fecal calprotectin, a marker of gastrointestinal inflammation, does not substantially differ. Upon investigation in a third pediatric cohort, the findings of increased lactosyl ceramide (d18:1/16:0) and decreased phosphatidylcholine (18:0p/22:6) absolute concentrations are confirmed. Translation of the lipidomic signature into a scalable diagnostic blood test for pediatric inflammatory bowel disease has the potential to support clinical decision making.

Construct validity of the international standards to document remaining autonomic function after spinal cord injury (ISAFSCI) (1st edition).

STUDY DESIGN: Observational study. OBJECTIVES: To assess the construct validity of the International Standards to Document Remaining Autonomic Function after Spinal Cord Injury (ISAFSCI) (2012 1st Edition). SETTING: Two Canadian spinal cord injury (SCI) centers. METHODS: Data were collected between 2011-2014. Assessments included the ISAFSCI, standardized measures of autonomic function and a clinical examination. Construct validity of ISAFSCI was assessed by testing a priori hypotheses on expected ISAFSCI responses to standard measures (convergent hypotheses) and clinical variables (clinical hypotheses). RESULTS: Forty-nine participants with an average age of 45 ± 12 years were included, of which 42 (85.7%) were males, 37 (77.6%) had a neurological level of injury at or above T6, and 23 (46.9%) were assessed as having motor and sensory complete SCI. For the six General Autonomic Function component hypotheses, two hypotheses (1 clinical, 1 convergent) related to autonomic control of blood pressure and one clinical hypothesis for temperature regulation were statistically significant. In terms of the Lower Urinary Tract, Bowel and Sexual Function component of the ISAFSCI, all the hypotheses (5 convergent, 3 clinical) were statistically significant except for the hypotheses on female sexual items (2 convergent, 2 clinical), likely due to small sample size. CONCLUSION: The construct validity of ISAFSCI (2012 1st Edition) for the General Autonomic Function component was considered to be weak while it was much stronger for the Lower Urinary Tract, Bowel and Sexual Function component based on a priori hypotheses. These results can inform future psychometric studies of the ISAFSCI (2021 2nd Edition).

Inflammatory bowel disease and asthma. Results from the RHINE study.

BACKGROUND: Asthma and inflammatory bowel disease (IBD) are common inflammatory diseases. The aim of this study was to investigate the associations of IBD with asthma and respiratory symptoms. METHODS: This study is based on 13,499 participants from seven northern European countries that filled in a postal questionnaire on asthma, respiratory symptoms, IBD including ulcerative colitis and Crohn's disease and various lifestyle variables. RESULTS: There were 195 participants with IBD. The prevalence of asthma (14.5 vs 8.1%, p = 0.001), different respiratory symptoms (range 11.9-36.8% vs range 6.0-18.6%, p < 0.005), non-infectious rhinitis (52.1 vs. 41.6%, p = 0.004) and chronic rhinosinusitis (11.6 vs 6.0%, p = 0.001) were higher in subjects with IBD than in those without IBD. In multivariable regression analysis, the association between IBD and asthma was statistically significant (OR 1.95 (95% CI 1.28-2.96)) after adjusting for confounders such as sex, BMI, smoking history, educational level and physical activity. There was a significant association between asthma and ulcerative colitis (adjusted OR 2.02 (95% CI 1.27-2.19)), and asthma but not Crohn's disease (adjusted OR 1.66 (95% CI 0.69-3.95)). A significant gender interaction was found with a significant association between IBD and asthma in women but not in men ((OR 2.72 (95% CI 1.67-4.46) vs OR 0.87 (95% CI 0.35-2.19), p = 0.038). CONCLUSIONS: Patients with IBD, particularly those with ulcerative colitis and female, have a higher prevalence of asthma and respiratory symptoms. Our findings indicate that it is important to consider respiratory symptoms and disorders when examining patients with manifest or suspected IBD.

Fecal Biomarkers of Neutrophil and Eosinophil Origin Reflect the Response to Biological Therapy and Corticosteroids in Patients With Inflammatory Bowel Disease.

INTRODUCTION: Fecal calprotectin (FC) is a noninvasive tool for examining response to biologics in inflammatory bowel disease (IBD), but its performance in relation to other novel fecal markers of various cellular origins is unknown. METHODS: We performed a prospective multicenter cohort study and included patients with active IBD who provided a fecal sample at initiation of biological therapy. Levels of FC, myeloperoxidase (MPO), human neutrophil lipocalin (HNL), and eosinophil-derived neurotoxin (EDN) were analyzed and related to clinical remission status at 3 months. Changes in levels of markers at 3 months were calculated, and the impact of concomitant use of corticosteroids at baseline was estimated. RESULTS: In patients achieving clinical remission (n = 27), a decrease in levels of FC ( P = 0.005), MPO ( P < 0.001), HNL ( P < 0.001), and EDN ( P < 0.001) was observed, whereas no significant decrease was seen in patients not achieving remission (n = 39). There was a significant difference in the change in the level of MPO ( P = 0.01) and HNL ( P = 0.02) between patients achieving clinical remission and those who did not, but changes in FC and EDN could not differentiate between these groups. Patients with concomitant systemic corticosteroids at inclusion had lower levels of HNL ( P = 0.01) and EDN ( P < 0.001) at baseline, compared with patients without corticosteroids. DISCUSSION: Fecal MPO, HNL, and EDN are all promising biomarkers for assessing the treatment outcome of biologics in patients with IBD. Fecal levels of EDN and HNL are significantly affected by corticosteroids indicating a greater sensitivity to the effects of corticosteroids compared with levels of FC and MPO.

Downregulated Mucosal Autophagy, Alpha Kinase-1 and IL-17 Signaling Pathways in Active and Quiescent Ulcerative Colitis.

Background: Improved mucosal immune profiling in active and quiescent colonic inflammatory bowel disease (IBD) is needed to develop therapeutic options for treating and preventing flares. This study therefore aimed to provide a comprehensive mucosal characterization with emphasis on immunological host response of patients with active ulcerative colitis (UC active), UC during remission (UC remission) and active colonic Crohn's disease (CD active). Methods: Colonic biopsies from 47 study subjects were collected for gene expression and pathway analyses using the NanoString host-response panel, including 776 genes and 56 immune-related pathways. Results: The majority of mucosal gene expression and signaling pathway scores were increased in active IBD (n=27) compared to healthy subjects (n=10). However, both active IBD and UC remission (n=10) demonstrated decreased gene expression and signaling pathway scores related to autophagy, alpha kinase-1 and IL-17 signaling pathways compared to healthy subjects. Further, UC remission was characterized by decreased scores of several signaling pathways linked to homeostasis along with increased mononuclear cell migration pathway score as compared to healthy subjects. No major differences in the colonic mucosal gene expression between CD active (n=7) and UC (n=20) active were observed. Conclusion: This study indicates that autophagy, alpha kinase-1 and IL-17 signaling pathways are persistently downregulated in UC irrespective of disease activity. Further, UC patients in remission present a unique mucosal environment, potentially preventing patients from reaching and sustaining true homeostasis. These findings may enable better comprehension of the remitting and relapsing pattern of colonic IBD and guide future treatment and prevention of flares.

Using the Intersectionality-Based Policy Analysis Framework to Evaluate a Policy Supporting Sexual Health and Intimacy in Long-Term Care, Assisted Living, Group Homes & Supported Housing.

Sexuality is an integral part of being human throughout life. This does not change when moving into long-term care (LTC). However, the sexual health of persons living in LTC is often overlooked. This paper presents an analysis of the recently released health organizational policy: Supporting Sexual Health and Intimacy in Long-Term Care, Assisted Living, Group Homes & Supported Housing. The Intersectionality-Based Policy Analysis Framework is used to outline the policy problem, examine how this policy was developed, and evaluate its potential to address the problem. Key findings are that both the development process and the policy constructs align with principles of intersectionality, such as equity, reflexivity, and diverse knowledges. In conclusion, this analysis suggests this policy is feasible, equitable and could effectively address sexual health for persons living in LTC, while leading to an improved workplace for staff. We recommend that this policy be more widely adopted across Canada.

AGPAT1 as a Novel Colonic Biomarker for Discriminating Between Ulcerative Colitis With and Without Primary Sclerosing Cholangitis.

INTRODUCTION: Ulcerative colitis (UC) associated with primary sclerosing cholangitis (PSC-UC) is considered a unique inflammatory bowel disease (IBD) entity. PSC diagnosis in an IBD individual entails a significantly higher risk of gastrointestinal cancer; however, biomarkers for identifying patients with UC at risk for PSC are lacking. We, therefore, performed a thorough PSC-UC biomarker study, starting from archived colonic tissue. METHODS: Proteins were extracted out of formalin-fixed paraffin-embedded proximal colon samples from PSC-UC (n = 9), UC (n = 7), and healthy controls (n = 7). Patients with IBD were in clinical and histological remission, and all patients with UC had a history of pancolitis. Samples were processed by the multienzyme digestion FASP and subsequently analyzed by liquid chromatography-tandem mass spectrometry. Candidate proteins were replicated in an independent cohort (n: PSC-UC = 16 and UC = 21) and further validated by immunohistochemistry. RESULTS: In the discovery step, 7,279 unique proteins were detected. The top 5 most differentiating proteins (PSC-UC vs UC) based on linear regression analysis were selected for replication. Of these, 1-acetylglycerol-3-phosphate O-acyltransferase 1 (AGPAT1) was verified as higher in PSC-UC than UC (P = 0.009) in the replication cohort. A difference on the group level was also confirmed by immunohistochemistry, showing more intense AGPAT1 staining in patients with PSC-UC compared with UC. DISCUSSION: We present AGPAT1 as a potential colonic biomarker for differentiating PSC-UC from UC. Our findings have possible implication for future PSC-IBD diagnostics and surveillance.

Elevated F-EDN correlates with mucosal eosinophil degranulation in patients with IBS-A possible association with microbiota?

Eosinophils have been linked to functional dyspepsia; however, less is known about their role in irritable bowel syndrome (IBS). This study tested the hypothesis of alterations in levels of fecal eosinophil-derived neurotoxin (F-EDN) and eosinophil density and degranulation within the colonic mucosa of IBS patients compared with healthy controls (HC). Colonic biopsies were collected from 37 IBS patients and 20 HC and analyzed for eosinophil numbers and local degranulation of eosinophil cationic protein (ECP) by histologic procedures. Fecal samples were collected for F-EDN and microbiota analysis. Differentiated 15HL-60 cells were used in vitro to investigate the direct effect of live bacteria on eosinophil activation measured by a colorimetric assay with o-phenylenediamine (OPD) substrate. We observed a higher number of eosinophils and increased extracellular ECP in the mucosa of IBS patients compared with HC. Moreover, F-EDN levels in IBS samples were elevated compared with HC and positively correlated to extracellular ECP. Metagenomic analysis showed significant correlations between bacterial composition and eosinophil measurements in both HC and IBS patients. In vitro experiments revealed an increased degranulation of 15HL-60 after stimulation with Salmonella typhimurium, Salmonella enterica, and Yersinia enterocolitica. To conclude, we could demonstrate alterations related to eosinophils in IBS, and, for the first time, a positive correlation between F-EDN levels and degranulated eosinophils in the colonic mucosa of IBS patients. Together our results suggest that eosinophils play a role in the pathophysiology of IBS and the mechanisms might be linked to an altered microbiota.

Localized IgG4-related disease manifested on the tongue: a case report.

Immunoglobulin G4-related disease (IgG4-RD) is an immune-mediated fibroinflammatory condition that can affect multiple organs. IgG4-RD may show a variety of initial symptoms. In the oral mucosa, lesions present as inflammatory fibrosis with a large number of IgG4-positive plasma cells. Evaluating treatment is a well-known problem in IgG4-RD due to the absence of an established assessment system. There are difficulties in defining the severity of the disease, which is why treatment is primarily based on its clinical manifestations. We present a case report of localized IgG4-RD with ulcerative and proliferative manifestations on the tongue, which clinically mimicked oral squamous cell carcinoma. A tumor-like lesion on the tongue can indicate something else other than the malignant or reactive changes commonly found in the oral mucosa. Multiple differential diagnoses of these atypical oral lesions, including localized IgG4-RD, should be considered.

Genetic and shared environmental risk factors do not lead to eosinophil activation in healthy twins of IBD patients.

OBJECTIVE: To examine the role of eosinophils in the pre-diagnostic phase of inflammatory bowel disease (IBD), we studied the influence of genetic and shared environmental risk factors in a twin cohort of IBD. MATERIAL AND METHODS: We analysed eosinophil derived neurotoxin (EDN) and eosinophil cationic protein (ECP) in faecal samples from twin pairs with Crohn's disease (n = 37) or ulcerative colitis (n = 21) and from external healthy controls (n = 44). Eosinophils stained with eosinophil peroxidase (EPO) were quantified in rectal biopsies. Ratios with 95% confidence intervals were calculated. RESULTS: Twins with Crohn' disease displayed higher levels of EDN (Ratio = 2.98, 1.65-5.37) and ECP (Ratio 1.83, 1.24-2.70) than their healthy siblings. Levels did not differ between healthy twin-siblings and external controls (EDN, Ratio = 1.52, 0.79-2.94 and ECP, Ratio = 0.93, 0.56-1.54). Higher levels of EDN (Ratio = 2.43, 1.13-5.24) and ECP (Ratio = 1.53, 0.92-2.53) were observed among twins with ulcerative colitis vs their healthy siblings. Levels did not differ between healthy twin-siblings and external controls (EDN, Ratio = 1.08, 0.51-2.25 and ECP, Ratio = 1.29, 0.74-2.26). Using intra-class correlation coefficient (ICC), we found no agreement in levels of EDN or ECP in discordant pairs, except for ECP in monozygotic Crohn's disease pairs (ICC = 0.63). In contrast, agreement was observed in monozygotic pairs concordant for Crohn's disease (EDN, ICC = 0.67 and ECP, ICC = 0.66). The number of eosinophils in rectum was increased in twins with ulcerative colitis vs their healthy sibling (Ratio = 2.22, 1.50-3.27). CONCLUSIONS: Activation of eosinophils in IBD seems to be a consequence of inflammation rather than an effect of genetic and shared environmental risk factors alone.

Tissue factor in ulcerative colitis, with and without concomitant primary sclerosing cholangitis.

Background: Ulcerative colitis (UC) in patients with the severe disease primary sclerosing cholangitis (PSC) constitutes a distinct clinical phenotype (PSC-UC) with a high incidence of colorectal cancer. Today, PSC-UC diagnosis is built on clinical observations only. Tissue factor (TF) has a potential use in UC diagnostics, and also in colorectal cancer prognostication. Here we evaluate TF expression in an inflammatory bowel disease (IBD) cohort, with special focus on differences between UC and PSC-UC patients.Materials and methods: Colonic biopsies from UC (n = 23), PSC (n = 24), and healthy controls (n = 11) were stained for TF by immunohistochemistry. Mononuclear cell contribution to TF expression was verified using flow cytometry.Results: TF was distributed at three distinct colonic locations: in subepithelial pericryptal sheath cells, in mononuclear cells, and in the intestinal stroma. In contrast to UC-where inflammation was accompanied with TF up-regulation-PSC-UC activity remained low during inflammation. Stromal TF positivity was found exclusively in ongoing inflammation.Conclusion: Our study provides additional support for a divergent pathogenesis in PSC-UC, with an inflammatory environment that differs from classical UC. Stromal TF emerges as a new marker of colonic inflammation.

Prognostic significance of faecal eosinophil granule proteins in inflammatory bowel disease.

Background: Non-invasive markers for predicting relapse would be a useful tool for the management of patients with inflammatory bowel disease. Eosinophil granulocytes and their granule proteins eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) have previously been shown to reflect disease activity in Crohn's disease and ulcerative colitis.Aim: To examine the capacity of faecal ECP and EDN to predict relapse in ulcerative colitis and Crohn's disease, and to compare these proteins with faecal calprotectin.Methods: Patients with Crohn's disease (n = 49) and ulcerative colitis (n = 55) were followed prospectively until relapse or end of the two-year study period. Faecal samples were obtained every third month. The predictive value of ECP and EDN was assessed in Cox regression models.Results: In ulcerative colitis, a doubled EDN or ECP concentration was associated with a 31% and 27% increased risk of relapse, respectively. EDN levels were increased both at relapse and three months prior. By contrast, in Crohn's disease, the concentration of EDN was higher among patients in remission than in those who relapsed. Correlations between faecal calprotectin, ECP and EDN were observed in both diseases.Conclusions: We demonstrate that the risk of relapse in ulcerative colitis can be predicted by consecutively measuring faecal EDN every third month, and suggest EDN as a complementary faecal marker to calprotectin to predict future relapse in ulcerative colitis. Our finding of higher EDN in Crohn's disease-patients staying in remission than in those who relapsed indicates different functions of the protein in ulcerative colitis and Crohn's disease.

High Serum sCD40 and a Distinct Colonic T Cell Profile in Ulcerative Colitis Associated With Primary Sclerosing Cholangitis.

BACKGROUND AND AIMS: There is a strong association between primary sclerosing cholangitis [PSC] and ulcerative colitis [UC], but the immunological link between the two diseases is obscure. We compared serum cytokine profiles of patients with PSC-UC and UC, and investigated a number of selected cytokines in colonic biopsy samples. We also assessed the presence and activation of T cells in peripheral blood and colonic mucosa. METHODS: Serum samples from 22 patients with PSC-UC, 28 patients with UC, and 19 controls were analysed by a proximity extension assay including 92 inflammatory cytokines. Biopsies from caecum, sigmoid colon, and rectum were collected from the same patients. Quantitative analysis for IFN-γ, IL-2, IL-4, IL-5, IL-13, IL-17A/ E/F, IL-21, IL-22, IL-23, and IL-27 was carried out on tissue homogenates. T cell phenotype was evaluated by flow cytometry. RESULTS: By multivariate analysis we identified a cluster of serum cytokines with higher levels in PSC-UC, and sCD40 in particular was strongly associated with this patient group. In contrast, colonic cytokines were only modestly increased in PSC-UC, whereas several Th1-, Th2-, and Th17-associated cytokines were increased in UC. Patients with PSC-UC had increased colonic levels of CXCR3-positive CD8+ T cells but fewer CD25-positive CD4+ T cells. An increased CRTH2/CXCR3-quote indicated a predominance of Th-2 type CD4+ T cells in UC patients. CONCLUSIONS: Our study reveals different cytokine profiles and T cell profiles in PSC-UC and UC, with higher systemic levels of cytokines in PSC-UC, and a more pronounced colonic inflammation in UC. Serum sCD40 could potentially be investigated as a marker for PSC in UC.

Towards improved control of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is characterized by activation of both the innate and adaptive immune system in genetically susceptible individuals, resulting in chronic intestinal inflammation. The triggers that initiate and perpetuate this continuous inflammation are the subject of much speculation and research, although the central role of the intestinal microbiota is recognized, and is even a target for treatment in some circumstances. The mainstay of modern IBD treatment is suppression of the immune response towards as yet unspecified antigens, and conventional therapy includes corticosteroids, 5-aminosalicylic acid (5-ASA), thiopurines and methotrexate. Reducing activity of specific mediators has proven efficacious, including adhesion molecules, such as the gut-homing integrin α4 ß7 expressed on the surface of circulating immune cells, and cytokines, such as tumour necrosis factor α (TNF-α). This has been achieved using biologic agents including monoclonal antibodies. Recent discoveries in immunology and neuroscience have revealed that signals in the peripheral nervous system regulate inflammation, including levels of TNF-α. The understanding of the mechanisms of the neuro-immune communication involved in inflammation control in the gut is evolving, but is as yet incomplete. Clinical studies using implanted vagus nerve stimulators for treatment of IBD show encouraging results. Accordingly, the neural reflex control of inflammation is emerging as a potential therapeutic target in treatment of IBD. Here, we review current therapeutic options and neural reflex control of gut immunity in the context of intestinal inflammation.

Downregulated eosinophil activity in ulcerative colitis with concomitant primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is a chronic bile duct inflammation strongly connected to ulcerative colitis (UC). PSC is associated with an increased risk of colon cancer, but the link between the intestinal and the bile duct inflammation is still unknown. Also, the involvement of intestinal immune cells in the pathogenesis of PSC remains to be determined. The eosinophil granulocyte is one of the immune cells implicated in the inflammatory process of ulcerative colitis. This study was performed to determine how the accumulation and activation of intestinal eosinophils may differ between UC with and without concomitant PSC, and how this may be influenced by the cytokine/chemokine profile of the intestinal compartment. Eosinophils from peripheral blood and multiple parts of the colon were analyzed by flow cytometry. The intestinal level of inflammatory mediators was assessed using a multiplex proximity extension assay and a quantitative immunoassay. We found that colonic eosinophils were more abundant in both UC and PSC-UC compared with controls, but that their expression of activation markers was significantly increased in UC only. The colonic level of pro-inflammatory cytokines was increased in active UC but not in PSC-UC. In conclusion, we show for the first time that eosinophil activation phenotype discriminates between UC and PSC-UC, and that this may depend on the local cytokine profile of the colonic mucosa.

Inter-Rater Reliability of the International Standards to Document Remaining Autonomic Function after Spinal Cord Injury.

The autonomic nervous system can be profoundly affected after spinal cord injury (SCI). Despite its importance to quality of life, autonomic function is rarely systematically assessed in the clinical setting. The International Standards to Document Remaining Autonomic Function after Spinal Cord Injury (ISAFSCI) is an assessment designed to determine which autonomic functions are intact, impaired, or lost after SCI. The psychometric properties of the ISAFSCI have not yet been reported. The objective of this study was to describe the inter-rater reliability of the ISAFSCI. Participants with chronic traumatic SCI (greater than 1 year) able to remain on the same medications for the study period and communicate clearly with the assessor were recruited for the study. A standard protocol minimized variation between the sites. During the first assessment, neurologic examination (ISNCSCI) was performed and ISAFSCI completed. After 10-14 days, the ISAFSCI was repeated. Inter-rater reliability was calculated using percentage agreement, kappa, and weighted kappa statistics. Participants (n = 48) had an average age of 45 ± 12 years. Forty-one (85.4%) were male, 38 (79.2%) had a SCI at or above the T6 level, 24 (50.0%) had a complete SCI. Inter-rater reliability within the general autonomic component was moderate with kappa values ranging 0.41-0.6 (p < 0.05). Within the Lower Urinary Tract, Bowel, and Sexual Function component, agreement was good-strong with weighted kappa values 0.62-0.88 (p < 0.05). Given the results, we conclude that the ISAFSCI can be considered to have at least moderate and up to strong inter-rater reliability, especially in the bladder, bowel, and sexual function component of the assessment.

A Multidisciplinary Approach to Sexual and Fertility Rehabilitation: The Sexual Rehabilitation Framework.

Many studies have identified improvement in sexual function as a priority for persons with spinal cord injury (SCI). Due to the various secondary sensory, motor, and autonomic consequences following SCI and due to the complexity of sexuality per se, this area can be overwhelming to many health care professionals. The literature indicate that sexual and fertility rehabilitation must be addressed in a biopsychosocial manner and include various disciplines. The multidisciplinary utilization of a Sexual Rehabilitation Framework (SRF) allows the medical and/or psychosocial factors that impede or improve sexual and reproductive function to be examined. The SRF is a user-friendly and simplified way to proactively address the major biopsychosocial areas of sexuality and to create a plan of action for the person with SCI. It is an adjunct tool to the full sexual history, and it encourages all disciplines involved in SCI rehabilitation to address the issue of sexual function in the same manner as they would other activities of daily living. Eight areas are included in the SRF: sexual drive/interest, sexual functioning, fertility and contraception, factors associated with the condition, motor and sensory influences, bladder and bowel influences, sexual self-view and self-esteem, and partnership issues. The use of the SRF is encouraged in both inpatient and outpatient settings. Multidisciplinary or interdisciplinary team work is encouraged in sexual and fertility rehabilitation to move clinicians toward providing proactive and comprehensive care for individuals with SCI or other chronic disabilities.