Mucosal Content Research Articles

Treating Lower Urinary Tract Symptoms in Older Adults: Intravesical Options.

This article provides an overview of the diagnosis and the treatment of lower urinary tract symptoms in older adults complicated by the neurodegenerative changes in the micturition reflex and further confounded by age-related decline in hepatic and renal clearance raising the propensity of adverse drug reactions. The first-line drug treatment for lower urinary tract symptoms, orally administered antimuscarinics, fails to reach the equilibrium dissociation constant of muscarinic receptors even at their maximum plasma concentration and tends to evoke a half-maximal response at a muscarinic receptor occupancy of just 0.206% in the bladder with a minimal difference from exocrine glands, which raises the adverse drug reaction risk. On the contrary, intravesical antimuscarinics are instilled at concentrations 1000-fold higher than the oral maximum plasma concentration and the equilibrium dissociation constant erects a downhill concentration gradient that drives passive diffusion and achieves a mucosal concentration around ten-fold lower than the instilled concentration for a long-lasting occupation of muscarinic receptors in mucosa and sensory nerves. A high local concentration of antimuscarinics in the bladder triggers alternative mechanisms of action and is supposed to engage retrograde transport to nerve cell bodies for neuroplastic changes that underlie a long-lasting therapeutic effect, while an intrinsically lower systemic uptake of the intravesical route lowers the muscarinic receptor occupancy of exocrine glands to lower the adverse drug reaction relative to the oral route. Thus, the traditional pharmacokinetics and pharmacodynamics of oral treatment are upended by intravesical antimuscarinics to generate a dramatic improvement (~ 76%) noted in a meta-analysis of studies enrolling children with neurogenic lower urinary tract symptoms on the primary endpoint of maximum cystometric bladder capacity as well as the secondary endpoints of filling compliance and uninhibited detrusor contractions. The therapeutic success of intravesical multidose oxybutynin solution or oxybutynin entrapped in the polymer for sustained release in the pediatric populationbodes well for patients with lower urinary tract symptoms at the other extreme of the age spectrum. Though generally used to predict oral drug absorption, Lipinski's rule of five can also explain the ten-fold lower systemic uptake from the bladder of positively charged trospium over oxybutynin, a tertiary amine. Chemodenervation by an intradetrusor injection of onabotulinumtoxinA is merited for patients with idiopathic overactive bladder discontinuing oral treatment because of a lack of efficacy. However, age-related peripheral neurodegeneration potentiates the adverse drug reaction risk of urinary retention that motivates the quest of liquid instillation, delivering larger fraction of onabotulinumtoxinA to the mucosa as opposed to muscle by an intradetrusor injection can also probe the neurogenic and myogenic predominance of idiopathic overactive bladder. Overall, the treatment paradigm of lower urinary tract symptoms in older adults should be tailored to individual's overall health status and the risk tolerance for adverse drug reactions.

Potential of Mesalazine Therapeutic Drug Monitoring by Measuring Fecal Excretion in Patients With Ulcerative Colitis.

Therapeutic drug monitoring of mesalazine (5-ASA) in patients with ulcerative colitis is unavailable. Mucosal 5-ASA concentrations are assumed to be higher during remission, but biopsy is not practical. Therefore, we investigated the feasibility of measuring mesalazine levels in feces. To explore the potential role of fecal mesalazine measurements in therapeutic drug monitoring, we compared the dry fecal concentration and daily fecal excretion of 5-ASA and its metabolite N-acetyl-5-ASA in patients with ulcerative colitis with active and quiescent disease. Adults with ulcerative colitis on oral mesalazine and scheduled for colonoscopy were eligible for inclusion in this cross-sectional study. Stool and urine samples were collected for 48 and 24 hours, respectively, and rectal biopsies were performed. (N-acetyl-)5-ASA was measured using mass spectrometry. Biochemically active disease was defined as a fecal calprotectin level above 100 mcg/g and endoscopically active disease as any activity following the endoscopic Mayo score (≥1). Approximately 28 patients were included in the study. Daily fecal excretion of (N-acetyl-)5-ASA did not differ between patients with (n = 13) and without (n = 15) endoscopically active disease [median 572 mg/d versus 597 mg/d ( P = 0.86) for 5-ASA and 572 mg/d versus 554 mg/d ( P = 0.86) for N-acetyl-5-ASA]. The same applied to the fecal concentration [median 9.7 mcg/mg dry weight versus 10.3 ( P = 0.53) and 12.0 versus 9.9 ( P = 0.89)]. The results were comparable when the biochemical disease activity definition was used. The mucosal concentrations and urinary excretion of (N-acetyl-)5-ASA did not differentiate between quiescent and active activity. Fecal (N-acetyl-)5-ASA measurements do not correlate with disease activity, which renders it an unsuitable tool for therapeutic drug monitoring of mesalazine.

Vitamin A-Deficient Rats have Only Mild Changes in Jejunal Structure and Function

This study investigated the effect of clinical and subclinical vitamin A deficiency on intestinal structure and function in rats. Weanling male rats fed a vitamin A-deficient diet (VA-) for 40–42 or 60–63 d were compared with rats either pair-fed (PF) or with free access to the same diet supplemented with vitamin A (VA+). A reference (REF) group was fed a standard rat diet. Weight began to plateau in VA- rats after 42 d, becoming significantly different from PF rats at 60–63 d (P < 0.02). Diarrhea did not develop in any study group. VA- rats had clinical signs of vitamin A deficiency in the 60–63 d study, but not in the 40–42 d study. However, serum and liver retinol concentrations were negligible in all VA- rats. VA- rats in the 60–63 d study had significantly reduced villus height (P < 0.02), and sucrase and maltase activities (P < 0.02) compared with PF rats. There were no differences between VA- and PF rats in mucosal wet weights, protein and DNA concentrations, thymidine kinase activity and glucose transport. No differences were detected in the 40–42 d study for any variable measured. Because clinical vitamin A deficiency in rats causes only mild changes in intestinal structure and function, it is unlikely that these alterations alone are responsible for the interactions observed in epidemiological studies between vitamin A deficiency and diarrheal disease.

CREATINE KINASE EXPRESSION IS PROTECTIVE IN A MOUSE COLITIS MODEL

Abstract BACKGROUND We have previously identified a loss of expression of creatine pathway proteins in mucosal tissue samples from inflammatory bowel disease patients. This is important as creatine is vital to ATP buffering in energetically active cells. The goal of this study was to determine the impact of decreased creatine kinase (CK) expression on mucosal inflammation by utilizing a whole body CK Brain and CK Mitochondrial knockout mouse (CKB/MitKO) in the dextran sulfate sodium (DSS) model of acute colitis. METHODS CKB/MitKO and matched controls were administered 1.5% DSS in drinking water for 5 days and assessed by body weight loss, disease activity index, mucosal permeability, histology and inflammatory cytokine production by Mesoscale analysis. In subsequent experiments, interferon-γ (IFN-γ) was given by intraperitoneal injection during the DSS experiment. We also analyzed transcriptional differences between CKB/MitKO and controls using a transcription factor array and western blot. Finally, we evaluated the impact of CK on the regulation of IFN-γ and transcriptional regulation of IFN-γ production in immune cells by flow cytometry. RESULTS Loss of CKB/Mit did not result in detectable baseline inflammation or barrier dysfunction in CKB/MitKO mice as compared to controls. However, in the setting of the chemical irritant DSS, CKB/MitKO mice had significantly increased weight loss, disease activity, intestinal permeability, histologic inflammation and decreased colon length. While control mice treated with DSS had significant increases in mucosal IFN-γ, the CKB/MitKO mice had strikingly absent mucosal protein concentrations of IFN-γ. Although IFN-γ is commonly considered a pro-inflammatory cytokine, it has also been shown to help promote inflammatory resolution. Therefore, we proceeded to add back IFN-γ in DSS experiments and we identified significant protection from weight loss and histologic damage with the addition of IFN-γ. We went on to identify transcription factor differences between CKB/MitKO and control splenocytes that revealed a &amp;gt;40-fold increase in the hypoxia responsive factor, HIF-1a, in CKB/MitKO splenocytes. We confirmed a causative relationship between HIF-1 expression and IFN-γ by treating mouse splenocytes with a HIF stabilizer and identified that HIF stabilization results in a reduction of IFN-γ production by CD4+ and CD8+ T cells. CONCLUSIONS CK is an essential energetic regulator. Using CK knockout mice we have identified an important role for CK in regulating IFN-γ production, mediated at least in part by HIF stabilization. These findings support a novel role for CK in tissue protection by immune regulation during acute colitis. Better understanding the role of energetics and the creatine pathway in particular may provide novel therapeutic options in inflammatory bowel disease.

2160. Mucosal Antiviral Cytokine Responses Are Impaired in Children and Adolescents with Severe COVID-19

Abstract Background The factors associated with severe COVID-19 in pediatric patients remain poorly understood. We sought to determine whether mucosal innate immunity cytokines were associated with disease severity in children and adolescents with acute COVID-19. Methods Single-center, prospective study including children and adolescents &amp;lt; 21 years of age hospitalized because of symptomatic COVID-19 from 3/2020 to 1/2021, and age, sex and race matched pre-pandemic (2016-2019) healthy controls. Nasopharyngeal (NP) samples were obtained at enrollment for measurement of SARS-CoV-2 viral loads by rt-PCR and cytokine concentrations using a 92-plex inflammation/antiviral panel (Olink). Disease severity was assessed by the need for supplemental oxygen or PICU admission, and patients classified as severe and non-severe based on these two parameters. Statistical analyses were performed in R studio and Benjamini-Hochberg applied to adjust for multiple comparisons. Results Of the 75 children with acute COVID-19 (median IQR age: 3.4 [0.2-15.3] years), 28 (37%) were classified as severe COVID-19 (19 PICU; 25 supplemental oxygen) and 47 (63%) were non-severe. Children with severe COVID-19 were predominantly male and had an underlying condition more frequently than those with non-severe disease (79% vs 49% respectively; p&amp;lt; 0.01, Table 1). SARS-CoV-2 viral loads were comparable between groups, yet patients with severe COVID-19 had significantly higher concentrations of C-reactive protein (p=0.04), more frequent lymphopenia (p=0.03) and cardiac involvement (p=0.04), and received COVID-19 directed therapies more commonly (p&amp;lt; 0.001). Comparative analyses identified 24 cytokines that were significantly different between children with acute COVID-19 versus 45 healthy controls. Of those, concentrations of IFN-γ (p=0.004), CXCL10 (p=0.01), CXCL11 (p=0.02) and CCL19 (p=0.02) were significantly lower in children with severe versus those with non-severe COVID-19 (Fig 1). Table 1.Demographic and clinical characteristics in children with symptomatic COVID-19*Bolded p-values indicates statistical significance. Abbreviations: N, number. CRP, C-Reactive protein; PICU, pediatric intensive care unit.**Other including the following underlying conditions: prematurity, endocrine conditions, immunocompromising conditions, prematurity, trisomy 21 and complex care. Ł Lymphopenia defined as an ALC of &amp;lt;4,500 cells/μl in infants, and &amp;lt;1,500 cells /μl in children &amp;gt;12 months of age Continuous variables displayed as medians and 25%-75% interquartile ranges (IQR), Mann-Whitney test was used to determine differences between two groups; Kruskal-Wallis &amp; Dunn’s multiple test correction was used to determine differences between three groups (p-values*). Categorical data expressed as numbers and proportions (in parenthesis) and analyzed by Fisher’s exact test or Chi square test. Figure 1.Nasal mucosal concentrations of antiviral/regulatory cytokines in children with symptomatic COVID-19 according to disease severity Conclusion Mucosal concentrations of antiviral/regulatory cytokines were decreased in children with severe COVID-19. These findings suggest that impaired mucosal innate immune responses might favor SARS-CoV-2 disease progression and severity in children. Disclosures Amy Leber, PhD, BioFire: Advisor/Consultant|BioFire: Grant/Research Support|Biorad: Advisor/Consultant|Cepheid: Grant/Research Support Octavio Ramilo, MD, Adagio: Advisor/Consultant|Bill &amp; Melinda Gates Foundation: Grant/Research Support|Janssen: Advisor/Consultant|Janssen: Grant/Research Support|Lilly: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Grant/Research Support|NIH: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Honoraria|Sanofi: Advisor/Consultant|Sanofi: Honoraria Asuncion Mejias, MD, PhD, MsCS, AstraZeneca: Honoraria|Janssen: Advisor/Consultant|Janssen: Grant/Research Support|Merck: Advisor/Consultant|Merck: Grant/Research Support|Roche: Advisor/Consultant|Sanofi-Pasteur: Advisor/Consultant|Sanofi-Pasteur: Honoraria.

Strain level and comprehensive microbiome analysis in inflammatory bowel disease via multi-technology meta-analysis identifies key bacterial influencers of disease.

ObjectiveInflammatory bowel disease (IBD) is a heterogenous disease in which the microbiome has been shown to play an important role. However, the precise homeostatic or pathological functions played by bacteria remain unclear. Most published studies report taxa-disease associations based on single-technology analysis of a single cohort, potentially biasing results to one clinical protocol, cohort, and molecular analysis technology. To begin to address this key question, precise identification of the bacteria implicated in IBD across cohorts is necessary.MethodsWe sought to take advantage of the numerous and diverse studies characterizing the microbiome in IBD to develop a multi-technology meta-analysis (MTMA) as a platform for aggregation of independently generated datasets, irrespective of DNA-profiling technique, in order to uncover the consistent microbial modulators of disease. We report the largest strain-level survey of IBD, integrating microbiome profiles from 3,407 samples from 21 datasets spanning 15 cohorts, three of which are presented for the first time in the current study, characterized using three DNA-profiling technologies, mapping all nucleotide data against known, culturable strain reference data.ResultsWe identify several novel IBD associations with culturable strains that have so far remained elusive, including two genome-sequenced but uncharacterized Lachnospiraceae strains consistently decreased in both the gut luminal and mucosal contents of patients with IBD, and demonstrate that these strains are correlated with inflammation-related pathways that are known mechanisms targeted for treatment. Furthermore, comparative MTMA at the species versus strain level reveals that not all significant strain associations resulted in a corresponding species-level significance and conversely significant species associations are not always re-captured at the strain level.ConclusionWe propose MTMA for uncovering experimentally testable strain-disease associations that, as demonstrated here, are beneficial in discovering mechanisms underpinning microbiome impact on disease or novel targets for therapeutic interventions.

Effects of dietary Bopu powder supplementation on intestinal development and microbiota in broiler chickens.

This study aimed to investigate the effect of dietary supplementation with Bopu powder on intestinal development and bacterial community composition in broiler chickens. A total of 486 1-day-old arbor acres broilers were fed a basal diet (CON group), a basal diet supplemented with 50 mg/kg aureomycin (AB group), or a basal diet supplemented with 40 mg/kg Bopu powder (BP group). The results showed that the BP group had significantly lower serum tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and diamine oxidase concentrations and had significantly higher serum IL-10 concentrations than CON group (p < 0.05). Groups AB and BP had a significantly higher weight per unit length of the small intestine and villus height than the CON group (p < 0.05), and BP group had a significantly higher ratio of villus height to crypt depth than groups CON and AB (p < 0.05). Compared to the CON group, dietary Bopu powder or aureomycin supplementation significantly increased transforming growth factor-α concentration and mRNA expressions of zonula occludens-1 (ZO-1) and occludin, and decreased intestinal mucosal concentrations of TNF-α, IL-6, IL-10, caspase-3, and caspase-8 and mRNA expressions of nuclear factor-kappa-B and Bax/Bcl-2 ratio in the intestinal mucosa (p < 0.05). Meanwhile, BP group had significantly higher ZO-1, secretory immunoglobulin A, interferon-γ concentrations, and mRNA expressions of glucose transporter type-2 and sirtuin-1, and significantly lower IL-1β concentration than groups CON and AB in intestinal mucosa (p < 0.05). Dietary Bopu powder supplementation significantly increased the concentration of trefoil factor family member and mRNA expressions of superoxide dismutase-1 and bcl-2 associated X, and significantly reduced casepase-9 concentration and myeloid differentiation primary response-88 expression in the intestinal mucosa of broiler chickens relative to CON group (p < 0.05). Moreover, results of high-throughput sequencing showed that broilers in the BP group had microbial community structure distinct from that in CON group, and the addition of Bopu powder increased the abundances of Faecalibacterium and Colidextribacter (p < 0.05). Therefore, our study suggests a synergic response of intestinal development and microbiota to the Bopu powder, and provides a theoretical basis as a potential substitute for antibiotics.

New Approach to Addison Disease: Oral Manifestations Due to Endocrine Dysfunction and Comorbidity Burden.

This review highlights oral anomalies with major clinical impact in Addison disease (AD), including dental health and dermatologic features, through a dual perspective: pigmentation issues and AD comorbidities with oral manifestations. Affecting 92% of AD patients, cutaneomucosal hyperpigmentation is synchronous with or precedes general manifestations by up to a decade, underlying melanocytic infiltration of the basal epidermal layer; melanophages in the superficial dermis; and, rarely, acanthosis, perivascular lymphocytic infiltrate, and hyperkeratosis. Intraoral pigmentation might be the only sign of AD; thus, early recognition is mandatory, and biopsy is helpful in selected cases. The buccal area is the most affected location; other sites are palatine arches, lips, gums, and tongue. Pigmented oral lesions are patchy or diffuse; mostly asymptomatic; and occasionally accompanied by pain, itchiness, and burn-like lesions. Pigmented lingual patches are isolated or multiple, located on dorsal and lateral areas; fungiform pigmented papillae are also reported in AD individuals. Dermoscopy examination is particularly indicated for fungal etiology; yet, it is not routinely performed. AD’s comorbidity burden includes the cluster of autoimmune polyglandular syndrome (APS) type 1 underlying AIRE gene malfunction. Chronic cutaneomucosal candidiasis (CMC), including oral CMC, represents the first sign of APS1 in 70–80% of cases, displaying autoantibodies against interleukin (IL)-17A, IL-17F ± IL-22, and probably a high mucosal concentration of interferon (IFN)-γ. CMC is prone to systemic candidiasis, representing a procarcinogenic status due to Th17 cell anomalies. In APS1, the first cause of mortality is infections (24%), followed by oral and esophageal cancers (15%). Autoimmune hypoparathyroidism (HyP) is the earliest endocrine element in APS1; a combination of CMC by the age of 5 years and dental enamel hypoplasia (the most frequent dental complication of pediatric HyP) by the age of 15 is an indication for HyP assessment. Children with HyP might experience short dental roots, enamel opacities, hypodontia, and eruption dysfunctions. Copresence of APS-related type 1 diabetes mellitus (DM) enhances the risk of CMC, as well as periodontal disease (PD). Anemia-related mucosal pallor is related to DM, hypothyroidism, hypogonadism, corresponding gastroenterological diseases (Crohn’s disease also presents oral ulceration (OU), mucogingivitis, and a 2–3 times higher risk of PD; Biermer anemia might cause hyperpigmentation by itself), and rheumatologic diseases (lupus induces OU, honeycomb plaques, keratotic plaques, angular cheilitis, buccal petechial lesions, and PD). In more than half of the patients, associated vitiligo involves depigmentation of oral mucosa at different levels (palatal, gingival, alveolar, buccal mucosa, and lips). Celiac disease may manifest xerostomia, dry lips, OU, sialadenitis, recurrent aphthous stomatitis and dental enamel defects in children, a higher prevalence of caries and dentin sensitivity, and gingival bleeding. Oral pigmented lesions might provide a useful index of suspicion for AD in apparently healthy individuals, and thus an adrenocorticotropic hormone (ACTH) stimulation is useful. The spectrum of autoimmune AD comorbidities massively complicates the overall picture of oral manifestations.

Mitochondria-targeted hydrogen sulfide donors versus acute oxidative gastric mucosal injury.

Hydrogen sulfide (H2S) as a gaseous molecule prevents gastrointestinal (GI)-tract against various injuries. This study aimed to evaluate for the first time the detailed molecular mechanism of mitochondria-targeting H2S-prodrugs, AP39 and RT01 in gastroprotection against ischemia/reperfusion (I/R)-induced lesions. Wistar rats exposed to I/R were pretreated i.g. with vehicle, AP39 (0.004-2mg/kg), RT01 (0.1mg/kg), or with AP219 (0.1mg/kg) as structural control without ability to release H2S. AP39 was also administered with mTOR1 inhibitor, rapamycin (1mg/kg i.g.). Gastric damage area was assessed micro-/macroscopically, gastric blood flow (GBF) by laser flowmetry, mRNA level of HIF-1α, GPx, SOD1, SOD2, annexin-A1, SOCS3, IL-1RA, IL-1β, IL-1R1, IL-1R2, TNFR2, iNOS by real-time PCR. Gastric mucosal and/or serum content of IL-1β, IL-4, IL-5, IL-10, G-CSF, M-CSF, VEGFA, GRO, RANTES, MIP-1α, MCP1, TNF-α, TIMP1, FABP3, GST-α, STAT3/5 and phosphorylation of mTOR, NF-κB, ERK, Akt was evaluated by microbeads-fluorescent assay. Mitochondrial complexes activities were measured biochemically. RNA damage was assessed as 8-OHG by ELISA. AP39 and RT01 reduced micro-/macroscopic gastric I/R-injury increasing GBF. AP39-gastroprotection was accompanied by maintained activity of mitochondrial complexes, prevented RNA oxidation and enhanced mRNA/protein expression of SOCS3, IL-1RA, annexin-A1, GST-α, HIF-1α. Rapamycin reversed AP-39-gastroprotection. AP39-gastroprotection was followed by decreased NF-κB, ERK, IL-1β and enhanced Akt and mTOR proteins phosphorylation. AP39-prevented gastric mucosal damage caused by I/R-injury, partly by mitochondrial complex activity maintenance. AP39-mediated attenuation of gastric mucosal oxidation, hypoxia and inflammation involved mTOR1 and Akt pathways activity and modulation of HIF-1α, GST-α, SOCS3, IL1RA and TIMP1 molecular interplay.

Zinc Oxide Nanoparticle Improves the Intestinal Function of Intrauterine Growth Retardation Finishing Pigs via Regulating Intestinal Morphology, Inflammation, Antioxidant Status and Autophagy.

This study was to investigate effects of zinc oxide nanoparticle (Nano-ZnO) on growth, immunity, intestinal morphology and function of intrauterine growth retardation (IUGR) finishing pigs. Six normal birth weight (NBW) and 12 IUGR male piglets were obtained and weaned at 21 d. NBW-weaned piglets fed basal diets (NBW group), IUGR-weaned piglets allocated to two groups fed basal diets (IUGR group) and basal diets further supplemented 600 mg Zn/kg from Nano-ZnO (IUGR+Zn group), respectively. All pigs were slaughtered at 163 d. Results showed: (1) IUGR pigs showed no difference in body weight at 77d and 163d (P > 0.05), while had increased villus height (VH) and villus surface area in jejunum (P < 0.05) and enhanced interleukin-6, TNF-α and NF-κB mRNA expression (P < 0.05) as compared to NBW group; Compared with IUGR group, dietary Nano-ZnO did not affect the body weight (P > 0.05), but increased VH to crypt depth ratio and IgA concentration (P < 0.05) and decreased TNF-α and NF-κB mRNA expression in jejunum (P < 0.05). (2) IUGR increased the number of swollen mitochondria and autolysosomes, and protein expressions of sequestosome-1 (P62) and microtubule-associated protein light chain 3 B/A (LC3B/A) in jejunum as compared to NBW group (P < 0.05); Compared with IUGR group, Nano-ZnO decreased the number of swollen mitochondria and autolysosomes, and P62 and LC3B/A protein expression (P < 0.05). (3) IUGR increased mucosal contents of malondialdehyde and protein carbonyl (PC) and Keap1 protein expression (P < 0.05) as compared to NBW group; Compared with IUGR group, dietary Nano-ZnO increased activities of total antioxidant capacity, catalase, glutathione peroxidase, and glutathione content (P < 0.05), and enhanced nuclear respiratory factor 2 (Nrf2), glutamate-cysteine ligase modifier subunit and glutathione peroxidase 1 mRNA expression, and increased total and nuclear Nrf2 protein expression (P < 0.05), and decreased malondialdehyde and PC content, and Keap1 protein expression (P < 0.05) in jejunum. Results suggested that IUGR pigs showed postnatal catch-up growth and improved intestinal morphology, and dietary Nano-ZnO may further improve intestinal morphology, reduce inflammation, decrease autophagy and alleviate oxidative stress via Nrf2/Keap1 pathway in jejunum of IUGR pigs.

OBESTATIN ACCELERATES THE HEALING OF GINGIVAL ULCERS IN SALIVARY INTACT AND SIALOADENECTOMIZED RATS

<h3>Objectives</h3> There are numerous strategies for the prevention or treatment of oral mucositis. However, their effectiveness is limited and does not correspond to expectations. Recent studies have shown that obestatin exhibits protective effects and accelerates the healing of gastrointestinal mucosa. The aim of the present study was to examine the influence of obestatin administration on oral ulcers in rats. <h3>Study Design</h3> Studies were performed on salivary-intact and sialoadenectomized rats. Gingival ulcers were induced by the use of acetic acid. Rats were treated intraperitoneally twice a day with saline or obestatin (4, 8, or 16 nmol/kg/dose) for 6 days. The study determined the oral mucosa morphology, cell proliferation, mucosal blood flow, and mucosal pro-inflammatory IL-1β level. <h3>Results</h3> In animals with intact salivary glands without induction of oral ulcers, treatment with obestatin was without any effect. Obestatin administration in rats with gingival ulcersbut with intact salivary glands significantly increased healing rate of these ulcers. Obestatin given at a dose of 8 or 16 nmol/kg/dose caused the strongest and similar therapeutic effects. This result was associated with a significant increase in blood flow and cell proliferation in gingival mucosa, as well as a significant decrease in IL-1β level. Sialoadenectomy caused a reduction in cell proliferation and an increase in IL-1β level. Administration of obestatin reversed this effect. In sialoadenectomized rats, the healing rate of ulcers was reduced. As in rats without induction of ulcers, this effect was associated with a reduction in blood flow and cell proliferation in oral mucosa, as well as an increase in mucosal IL-1β concentration. In these rats, obestatin reversed the effect of sialoadenectomy and doses of 8 or 16 nmol/kg increased the healing rate of oral ulcers above a value observed in control rats with intact salivary glands. <h3>Conclusions</h3> Obestatin accelerates the healing of gingival ulcers in both sialoadenectomized and non-sialoadenectomized rats. This therapeutic effect is well correlated with an increase in blood flow and cell proliferation in oral mucosa, as well as decrease of pro-inflammatory IL-1β level. Obestatin is potentially useful candidate for the prevention and treatment of oral mucositis.

Dietary Dracocephalum kotschyi essential oil improved growth, haematology, immunity and resistance to Aeromonas hydrophila in rainbow trout ( Oncorhynchus mykiss )

In the present study, the effect of Dracocephalum kotschyi essential oil (DKE) was investigated on growth, haematology, immune and antioxidant defence system and resistance of rainbow trout juveniles to bacterial infection (Aeromonas hydrophila). For this purpose, the fish were fed a diet containing different concentrations of DKE including 0 (control), 0.2, 0.25 and 0.3 mg/kg diet in three replicates for 60 days. After feeding trial, the fish were challenged with a pathogenic dose of A. hydrophila. Based on the results, immune components in plasma (alternative complement activity [ACH50], IgM content, lysozyme activity, total protein and total albumin) and mucus (protease activity, IgM content and lysozyme activity) significantly elevated in fish fed diet containing 0.2 and 0.25 mg DKE/kg diet compared with other groups (p < 0.01). The plasma bactericidal activity increased in all DKE-supplemented fish. Supplementation of fish with 0.3 mg DKE/kg diet depressed the mucosal lysozyme activity and IgM content. The liver antioxidant enzymes, glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD) showed increased activity in response to 0.25 and 0.3 mg DKE/kg diet compared with other experimental diets (p < 0.01). Dietary DKE changed the haematology of the fish. The haematocrit, red blood cell count (RBC), white blood cell (WBC), haemoglobin content and mean erythrocyte cell haemoglobin content (MCHC) increased in response to DKE. In contrast, the mean corpuscular haemoglobin (MCH) decreased in fish fed 0.2 and 0.25 mg DKE/kg diet compared with other diets (p < 0.01). The supplementation of fish with 0.2 and 0.25 mg DKE/kg diet decreased the expression of cytokine-related genes, TNF-α and IL-8. In contrast, the DKE up-regulated the expression of the immune-related genes, C3 and lysozyme. DKE at concentration of 0.3 mg/kg diet depressed the C3 and lysozyme gene expressions. The DKE supplementation decreased the mortality rate of the fish after bacterial challenge compared with non-supplemented ones (p < 0.01). In conclusion, the findings of this study revealed the immune-stimulating effects of DKE at optimized dietary concentrations. In addition, DKE decreased the mortality induced by A. hydrophila infection.

Systemic and Mucosal Concentrations of Nine Cytokines Among Individuals with Neisseria gonorrhoeae infection in Nairobi Kenya.

Introduction: The human-restricted sexually transmitted Neisseria gonorrhoeae (NG) has been shown to modulate the immune response against it and consequently the cytokines produced. The levels of cytokines in NG infection in the African population have not been well described. We aimed to quantify the systemic and mucosal cytokines in NG infection. Methods: This was a comparative cross-sectional study. Levels of nine cytokines (IL-1β, IL-2, IL-4, 1L-6, 1L-10, 1L-12p70, IL-17A, TNF-α and INF-γ) were measured from plasma and genital samples (urethral swabs in men and cervicovaginal lavage in women) from 61 Neisseria gonorrhoeae infected individuals seeking treatment for sexually transmitted infections (STIs) at Casino Health Centre in Nairobi, Kenya. A comparative group of 61 NG-uninfected individuals, seeking treatment at the same facility but with laboratory-confirmed negative Neisseria gonorrhoeae, Chlamydia trachomatis (CT), Mycoplasma genitalium (MG) and Trichomonas vaginalis(TV) was also included. The Mann-Whitney U test was used to compare the cytokine levels between NG-infected and uninfected individuals. Data was analyzed using STATA ver. 15.1. Results: Overall, systemic IL-6, TNF-α and IL-10 were elevated while genital IL-10 and TNF-α were lower in NG positive participants. On subgroup analysis by sex, the levels of genital IL-1β and IL-6 and systemic IL-6 were elevated in NG-infected men. None of the genital cytokines were elevated in NG-infected women, while all systemic cytokines, except INF-γ, were elevated in NG-infected women. Conclusions: Neisseria gonorrhoeae induced the production of different cytokines in men and women, with men having a pro-inflammatory genital response. These differences should be taken into consideration during development of various interventions e.g. vaccine development.

Oral mucosal Cryoglobulin [ICD-10-CM Diagnostic Code D89.9 Cryoglobulin], Oral Mucosal Herd Immunity and the Immune Herd Plots among Periodontal Disease Patients

Forty five patients were diagnosed by specialist dentist of the team as periodontal disease patients. Basically they were found to be chronic periodontitis and chronic gingivitis. The study male and female patient groups [25 males and 20 females] were subjected to the detection of oral mucosal cryoglobulin and tempts to use cryoglobulin as a marker for oral mucosal herd immunity among these patients. Oral mucosal cyoprotein solutions were separated and characterized as oral mucosal cryoglobulin [ICD-10-CM Diagnostic code, D89.9 Cryglobulin]. Mucosal cryocrit percentages were ranged from one to seven percent. Oral mucosal cryoglobulin concentrations were ranged between 0.13 up to 2 mg/dl. In chronic periodontitis patients and 0.15 up to1.96 mg/dl.in chronic gingivitis patients. The individuals Mucosal herd immunity constitutes three basic immune fractions; low, moderate and high cryoglobulin responders. The immune herd plot types were matched as Gaussian distribution plot types. The findings of oral mucosal cryoglobulin among these patients in clinically indicative concentrations for the potential role may be played in the pathogenesis of chronic gingivitis and chronic periodontitis as it may cause precipitates in the local blood vessels of gum and periodotium.

P463 Serum and mucosal Serpin E1 concentration correlates with endoscopic activity in inflammatory bowel disease - potential new activity marker

Abstract Background Inflammatory bowel disease (IBD) is characterized by an unregulated immune response generating unbalance cytokine homeostasis. Analysis of patient-specific cytokine profiles may open new therapeutic targets or identify biomarkers that distinguish responders from non-responders before initiating therapy. Therefore, the aim of the present study was to determine cytokine profile of IBD patients and identify cytokines with predictive potential. Methods Blood and biopsy samples were obtained from 79 clinically active IBD patients and 28 controls without IBD. Inclusion criteria included change of therapy or initiation of new treatment for IBD patients. Organoid culture (OC) was generated from 5 clinically active IBD patients and 5 controls. Cytokine Array was used to analyse cytokine expression patterns. Total protein and mRNA were isolated from biopsies and from OCs. Protein levels were measured by ELISA and gene expression by qRT-PCR in serum, mucosa and in OCs. Results Pro-inflammatory cytokines were not detected in control samples, whereas in IBD biopsies the cytokine profiles enabled the discrimination between inflamed or non-inflamed areas. The mucosal expression of Serpin E1 was detected in all inflamed biopsy samples, whereas it was below the detection limit in healthy subjects. In responders Serpin E1 gene expressions were significantly (p&amp;lt;0.001) lower than in non-responders. On the other hand, we were not able to detect significant changes between responders and non-responders in gene expression of TNF-α, IL-1β, IL-6 and TGF-β. In responders, mucosal Serpin E1 concentration significantly decreased (p&amp;lt;0,05) after the initiation of therapy compared to non-responders. Serum and mucosal Serpin E1 concentrations were significantly higher in patients with endoscopically and clinically active disease compared to inactive (endoscopic: p&amp;lt;0.0001, clinical: p&amp;lt;0.05). Correlation analysis revealed that serum Serpin E1 level correlates with disease activity (p&amp;lt;0.01). In OCs the level and expression of Serpin E1 was lower in healthy organoids compared to OCs generated from inflamed samples. Conclusion Our results suggest that serum and mucosal Serpin E1 expression reflects endoscopic activity in IBD, which could be used to follow-up disease activity and possibly therapeutic response. Organoids mimicked the expression of Serpin E1 in the original biopsies suggesting that OCs can be used to study the effect of Serpin E1 inhibition.

DOP15 Fluorescent labelled vedolizumab for real-time visualization and quantification of local drug distribution and pharmacodynamics in Inflammatory Bowel Diseases during endoscopy

Abstract Background Vedolizumab is a monoclonal antibody which blocks integrin α4β7 inhibiting trafficking of T-lymphocytes into the gut. Unfortunately, up to 60% of vedolizumab patients experience non-response. The mechanism of action of vedolizumab is not elucidated, predictors of response are unknown and data for local drug distribution in the gut are lacking. In this clinical trial, we investigated the feasibility of assessing local distribution of fluorescently labelled vedolizumab in the gut mucosa of inflammatory bowel diseases (IBD) patients to finally enable prediction of therapy response in individual patients. Methods Vedolizumab (Entyvio, Takeda Pharma) was labelled to IRDye 800CW under cGMP conditions to yield clinical grade vedolizumab-800CW. In this dose-escalation trial, vedolizumab naïve IBD patients and IBD patients treated with vedolizumab for at least 14 weeks were included. Patients received an intravenous dose of fluorescently labelled vedolizumab of either 0 mg, 4.5 mg, 15 mg or 15 mg + 75 mg unlabelled vedolizumab 3 days prior colonoscopy. In vivo fluorescence imaging was assessed by fibre-based wide-field fluorescence molecular endoscopy (FME) and quantified by spectroscopy in healthy, mildly inflamed and severely inflamed tissue. All assessed tissue was biopsied for ex vivo examination of the fluorescent signal, fluorescence microscopy and spectroscopy. Results Up to submission 34 patients completed tracer injection and FME. An interim analysis was performed after 20 patients (5 in each dose group), which showed in severely inflamed tissue an 8 fold higher fluorescent signal in the 15 mg dose group (0.049 Q*μfa,x [mm-1]) compared to the control group (0.006 μfa,x [mm-1]) (p&amp;lt;0.05). Furthermore, the fluorescent signal within the 15 mg dose group was also 2.5 fold higher compared to healthy tissue (0.019 Q*μfa,x [mm-1]) (p&amp;lt;0.05).The addition of unlabelled vedolizumab gave similar results to the 15 mg group (p&amp;gt;0.99), suggesting that the drug target was still not saturated. The optimal dosage group of 15 mg was expanded up to 18 IBD patients, amongst them 6 IBD patients after 14 weeks of treatment regimen. Fluorescence microscopy showed clustering of fluorescent signals especially in inflamed mucosa. Additional experiments to detect vedolizumab target cells are ongoing. Conclusion In vivo visualization of fluorescent vedolizumab revealed a clear dose-dependent correlation between mucosal drug concentrations and the severity of mucosal inflammation. Fluorescence molecular endoscopy is a promising novel tool to get insight in drug distribution in IBD, detect target cells, assess target engagement and possibly predict therapy response in individual patients.

Saudi honey alleviates indomethacin-induced gastric ulcer via improving antioxidant and anti-inflammatory responses in male albino rats

Recent years have reported a rise in the occurrence of gastric ulceration especially among young children and adults. This study investigated the mechanism by which two types of Saudi honey: AlnahalAljawal honey (Wadi) or Bin Ghaithan honey (Talh) exerted their antiulcer potential in indomethacin-induced gastric ulceration. Four cohorts of rats were used: Group 1; Healthy controls, Group 2; Ulcerative animals, Group 3; Ulcerative + Wadi honey treatment, Group 4; Ulcerative + Talh honey treatment. We profiled the levels of different indicators of oxidative stress including the activities of gastric mucosal glutathione superoxide dismutase (SOD), catalase (CAT), peroxidase (GPx), reduced glutathione (GSH), and lipid peroxidation (measured as malondialdehyde; MDA). CRP content, IL-10, and plasma tumor necrosis factor-α were also evaluated. The stomach was visually examined for macroscopic lesions and using light microscope for histopathological changes in the glandular mucosa.Wadi or Talh honey significantly reduced the ulcer indices, and essentially protected the glandular mucosa from lesions. Wadi or Talh honey also significantly reduced the gastric mucosal concentrations of GPx, SOD and GSH. In addition, the administration of Wadi or Talh honey decreased gastric mucosal plasma TNF-α and MDA, CRP content, and IL-10 levels. In conclusion, Wadi or Talh honey possibly exerted their antiulcer potential via restoring the homeostasis and stabilizing the enzymatic (SOD and GPx) and non-enzymatic (GSH) antioxidants as well as reducing the levels of inflammatory cytokines (TNF-α, CRP content, IL-10 and, NF-κB activity), and inhibiting the lipid peroxidation in the gastric mucosa. Consequently, Wadi or Talh honey may be of beneficial therapy for patients diagnosed with gastric ulceration. Clinical studies need to be conducted to further support these findings.

204. Mucosal Cytokine Profiles in Children with COVID-19

BackgroundThe mechanisms associated with COVID-19 in children are not well understood. We sought to define the differences in nasopharyngeal (NP) cytokine profiles according to clinical presentation in children with COVID-19. MethodsSingle-center, prospective study in 137 children and adolescents < 21 years of age hospitalized with COVID-19, and 35 age, sex and race matched pre-pandemic (2016-2019) healthy controls. Children with COVID-19 were categorized according to their clinical presentation in: COVID-19-symptomatic; COVID-19-screening, and multisystem inflammatory syndrome (MIS-C). NP swabs were obtained within 24 hours of admission to measure SARS-CoV-2 loads by rt-PCR, and a 92-cytokine panel. Unsupervised and supervised analysis adjusted for multiple comparisons were performed.ResultsFrom 3/2020 to 1/2021, we enrolled 76 COVID-19-symptomatic children (3.5 [0.2-15.75] years); 45 COVID-19-screening (11.1 [4.2-16.1] years), and 16 MIS-C (11.2 [5.9-14.6] years). Median NP SARS-CoV-2 loads were higher in COVID-19-symptomatic versus screening and MIS-C (6.8 vs 3.5 vs 2.82 log10 copies/mL; p< 0.001). Statistical group comparisons identified 15 cytokines that consistently differed between groups and were clustered in three functional categories: (1) antiviral/regulatory, (2) pro-inflammatory/chemotactic, and (3) a combination of (1) and (2); (Fig 1). All 15 cytokines were higher in COVID-19-symptomatic versus controls (p< 0.05). Similarly, and except for TNF, CCL3, CCL4 and CCL23, which were comparable in COVID-19-symptomatic and screening patients, the remaining cytokines were higher in symptomatic children (p< 0.05). PDL-1 (p=0.01) and CCL3 (p=0.03) were the only cytokines significantly decreased in children with MIS-C versus symptomatic COVID-19 children. The 15 cytokines identified by multiple comparisons were correlated using Person’s in R software. Red reflects a positive correlation and blue a negative correlation with the intensity of the color indicating the strength of the association.ConclusionChildren with symptomatic COVID-19 demonstrated higher viral loads and greater mucosal cytokines concentrations than those identified via screening, whereas in MIS-C concentrations of regulatory cytokines were decreased. Simultaneous evaluation of viral loads and mucosal immune responses using non-invasive sampling could aid with the stratification of children and adolescents with COVID-19 in the clinical setting. DisclosuresOctavio Ramilo, MD, Adagio (Consultant)Bill & Melinda Gates Foundation (Grant/Research Support)Janssen (Grant/Research Support)Lilly (Consultant)Merck (Consultant, Grant/Research Support)NIH (Grant/Research Support)Pfizer (Consultant)SANOFI (Board Member) Asuncion Mejias, MD, PhD, MsCS, Janssen (Grant/Research Support, Advisor or Review Panel member)Merck (Grant/Research Support, Advisor or Review Panel member)Roche (Advisor or Review Panel member)Sanofi (Advisor or Review Panel member).

1193. Mucosal Antibody Responses to Respiratory Syncytial Virus (RSV) in Infants and Young Children

Abstract Background The burden associated with RSV infection is substantial. Although RSV initially infects the upper respiratory tract, there is limited information of the mucosal concentrations of antibodies (Abs) directed to RSV specific proteins and whether patient’s age and disease severity influence production of mucosal Abs. Methods From 2017 to2019 we enrolled previously healthy children &amp;lt; 2 years of age hospitalized with RSV infection and obtained acute and convalescent (day; D30) nasopharyngeal (NP) samples to measure preF and postF specific IgG and IgA Abs by ELISA. Demographic and clinical data were collected and analyzed according to Abs responses. Results We enrolled 77 children (median [IQR] age: 2.8 [1.5-5.2] months; 49 % females) within the first 24 hours of hospitalization. Of those 25 (33%) patients required PICU care. A significant increase in convalescent IgG preF Abs titers was detected in 62 (81%) children, while IgA preF titers significantly increased in all but one child on D30. The magnitude of the increase was 56-fold higher for preF IgA versus preF IgG (p&amp;lt; 0.0001). PostF IgG and IgA titers were also increased on D30 but at significant lower levels. Infants &amp;lt; 3 months of age compared with those &amp;gt;3-24 months had significantly higher baseline preF and postF IgG Abs titers (p &amp;lt; 0.001) but not IgA antibodies. D30 preF and post F IgG titers were higher in children &amp;gt; 6 months of age (p &amp;lt; 0.0001) but only preF titers fold change significantly correlated with age (r=0.4, p&amp;lt; 0.0001). These correlations were not identified with IgA preF antibodies. There were no statistical differences in antibody titers at baseline and on D30 according to breastfeeding, and disease severity as defined by the need for PICU care. Conclusion Children hospitalized with RSV infection demonstrated significantly increased titers of mucosal preF and post F IgG and IgA specific Abs in convalescent samples. Baseline IgG Abs where higher in younger infants, which likely reflects maternally acquired antibodies. Age significantly correlated with Abs production, suggesting a more robust immune response in older children. Disclosures Mark E. Peeples, PhD, Janssen (Research Grant or Support)Pfizer (Research Grant or Support) Octavio Ramilo, MD, Adagio (Consultant)Bill &amp; Melinda Gates Foundation (Grant/Research Support)Janssen (Grant/Research Support)Lilly (Consultant)Merck (Consultant, Grant/Research Support)NIH (Grant/Research Support)Pfizer (Consultant)SANOFI (Board Member) Asuncion Mejias, MD, PhD, MsCS, Janssen (Grant/Research Support, Advisor or Review Panel member)Merck (Grant/Research Support, Advisor or Review Panel member)Roche (Advisor or Review Panel member)Sanofi (Advisor or Review Panel member)

Higher mucosal antibody concentrations in women with genital tract inflammation

Inflammatory cytokines augment humoral responses by stimulating antibody production and inducing class-switching. In women, genital inflammation (GI) significantly modifies HIV risk. However, the impact of GI on mucosal antibodies remains undefined. We investigated the impact of GI, pre-HIV infection, on antibody isotypes and IgG subclasses in the female genital tract. Immunoglobulin (Ig) isotypes, IgG subclasses and 48 cytokines were measured prior to HIV infection in cervicovaginal lavages (CVL) from 66 HIV seroconverters (cases) and 66 matched HIV-uninfected women (controls) enrolled in the CAPRISA 004 and 008 1% tenofovir gel trials. Pre-HIV infection, cases had significantly higher genital IgM (4.13; IQR, 4.04–4.19) compared to controls (4.06; IQR, 3.90–4.20; p = 0.042). More than one-quarter of cases (27%) had GI compared to just over one-tenth (12%) in controls. Significantly higher IgG1, IgG3, IgG4 and IgM (all p < 0.05) were found in women stratified for GI compared to women without. Adjusted linear mixed models showed several pro-inflammatory, chemotactic, growth factors, and adaptive cytokines significantly correlated with higher titers of IgM, IgA and IgG subclasses (p < 0.05). The strong and significant positive correlations between mucosal antibodies and markers of GI suggest that GI may impact mucosal antibody profiles. These findings require further investigation to establish a plausible biological link between the local inflammatory milieu and its consequence on these genital antibodies.