Introduction

Sarcopenia is a syndrome characterized by the progressive and generalized loss of skeletal muscle mass. Primary sarcopenia is associated with the aging process, while the secondary one is caused by several factors including inflammatory conditions, such as inflammatory bowel disease (IBD). Since there is a complex interaction between inflammation, intestinal dysbiosis and malnutrition which lead to muscle degeneration in IBD patients, it is referred to as the gut-muscle axis. In particular, the high level of cytokines and the gut dysbiosis which characterizes IBD is responsible for the activation of pathways that cause an imbalance in muscle protein synthesis. However, the involvement of molecular and microbial mechanisms of sarcopenia, at present, has not been extensively studied in IBD.

Aims & Methods

In order to identify the mechanisms that are potentially involved in the onset of sarcopenia, a DSS-induced colitis mouse model has been created. To ensure that the model developed sarcopenia, we performed physical performance assessment at rotarod. On this well-characterized model, we studied the muscle by immunohistochemistry, by fiber count per field and by Cross Sectional Area. Next, we evaluated the gut microbiota and serum LPS detection. In vitro, with the aim to create a situation of intestinal inflammation and to assess the activation of pathways of muscle atrophy, we used the immortalized murine myoblast cell line C2C12 at different time points, 30'-2h-6h. Treatments performed included pro-inflammatory cytokines (TNF-α, IFN-γ, IL-6), LPS and drugs (Infliximab, Upadacitinib, Tofacitinib) in order to assess cellular response to the most promising drugs used in the treatment of IBD.

Results

The mouse model showed a reduction in body weight and muscle performance, probably associated with quantitative and qualitative alterations in muscle fibers. In addition, increased expression of markers of muscle atrophy (myostatin and Murf-1) was found in colitic mouse muscle. Also we observed increasing expression of the pathway STAT3-pSTAT3 involved in muscle wasting and decreasing pAKT, which is associated with muscle growth. Most relevant, a condition of dysbiosis was found in the colitic model, as well as increased LPS in the serum of mice.
In vitro, Upadacitinib and Tofacitinib in the presence of inflammatory cytokines decrease the expression of muscle atrophy-associated pathways (STAT3, its active form pSTAT3, and the transcriptional factor NFkB) and increase the expression of pAKT, a protein associated with muscle synthesis. When cells were treated with LPS, NFkB expression did not decrease with drug addition, suggesting that LPS is able to activate NFkB independently of the JAK/STAT pathway.

Conclusion

These data open new scenario for the gut-muscle axis as a promising area of research for the management of sarcopenia in IBD. Therefore, we might also consider the role of microbiota and LPS in addition to inflammatory cytokines in the pathogenesis of sarcopenia in IBD, since LPS-induced signaling is only partially controlled by drugs.

Introduction

Autoimmune Atrophic Gastritis (AAG) is a chronic immune-mediated condition that mainly affects the body and fundus of the stomach. AAG is often under-diagnosed due to the slow progression of the disease and a variable spectrum of symptoms. Recent data suggests a potential role of altered gastric microbiota in the natural history of AAG, due to the potential overgrowth of bacteria linked to the development of neoplastic complications (neuroendocrine tumors- NETs and gastric cancer) (1,2). Moreover, the same data hypothesizes the use of fecal microbiota as a possible non-invasive marker to diagnose and monitor AAG. Thus, we aimed to prospectively characterize the salivary and fecal microbiota of patients with AAG to provide further insights in this field.

Aims & Methods

We collected salivary and fecal samples from consecutive patients with an established diagnosis of AAG, enrolled at the Gastroenterology Unit of Azienda Ospedale Università Padova from March to June 2023 at the time of endoscopic follow-up examination. All patients needed to be off Proton-Pump Inhibitors, antibiotics, and probiotics for at least one month before enrolment. Stool samples were collected by the patients on the day of the endoscopy, transferred in a buffer solution, and stored at -20°C. Oral samples were collected through salivary commercial kits and freezed at -20°C. Both salivary and faecal samples were analyzed by BMR Genomic (Padua, Italy) that amplified the V3-V4 hypervariable ribosomal RNA region. Sequencing of the 16S rRNA gene V3- V4 region was performed using the Illumina MiSeq. Preliminary analyses were conducted on individual fecal samples compared to an internal population of healthy subjects (provided by BMR genomics) using Qiime2 tools. Shannon–Wiener index was used to calculate α-diversity and then the microbial biodiversity index (MBI) and Dysbiosis Index (DI). Mann–Whitney tests were performed to verify statistical differences in the α-diversity across AAG patients and healthy subjects.

Results

We enrolled 15 AAG patients with a mean age of ± 63 ± 11 years (9/15 females). OLGA 2 stage was reported in 10/15 patients. Sixty percent of the patients showed a MBI between 7.11 and 12.45, indicating that each sample contained phylogenetically similar species. The remaining patients have an index greater than 12.45 compared to the healthy population. More than half of the patients had dysbiosis (>0.082). The highest indices of dysbiosis were found in patients with a history of NET (n=3), gastric cancer (n=1), and previous HP- infection and OLGA 3 stage (n=1). Firmicutes, Bacteroidetes, and Proteobacteria were more frequent phyla found in the fecal samples, whereas Enterobacteriaceae and Streptococcaceae were the most common bacterial families (13/15, 87%). In particular, Klebsiella and Campylobacter pathogens genera were found in some samples. Streptococcus and Prevotella were the most abundant genera found in the salivary samples.

Conclusion

Patients with AAG were found to have a great proportion of Firmicutes and Bacteroidetes, which aligns with the literature (3,4). Moreover, a dominant of Proteobacteria, especially Enterobacteriaceae commonly associated with proinflammatory states, was found. (5) Our preliminary data showed a possible positive association between higher degrees of dysbiosis and a previous history of NET or gastric cancer.

References

1. Parsons BN, Ijaz UZ, D'Amore R, Burkitt MD, Eccles R, Lenzi L, Duckworth CA, Moore AR, Tiszlavicz L, Varro A, Hall N, Pritchard DM. Comparison of the human gastric microbiota in hypochlorhydric states arising as a result of Helicobacter pylori-induced atrophic gastritis, autoimmune atrophic gastritis and proton pump inhibitor use. PLoS Pathog. 2017 Nov 2;13(11):e1006653.

2. Conti L, Annibale B, Lahner E. Autoimmune Gastritis and Gastric Microbiota. Microorganisms. 2020 Nov 19;8(11):1827.

3. Castaño-Rodríguez N, Goh KL, Fock KM, et al. Dysbiosis of the microbiome in gastric carcinogenesis. Sci Rep. 2017;7(1):15957.

4. Conti L, Borro M, Milani C, Simmaco M, Esposito G, Canali G, Pilozzi E, Ventura M, Annibale B, Lahner E. Gastric microbiota composition in patients with corpus atrophic gastritis. Dig Liver Dis. 2021 Dec;53(12):1580-1587.

5. Rizzatti G, Lopetuso LR, Gibiino G, Binda C, Gasbarrini A. Proteobacteria: A Common Factor in Human Diseases. Biomed Res Int. 2017;2017:9351507.

Disclosure

Nothing to disclose

Introduction

A significant proportion of patients with inflammatory bowel diseases (IBD) have extraintestinal manifestations (EIM), which may be very disabling. The presence of EIM and immunomediated diseases (IMIDs) may influence the selection of the biologic agent. On the other hand, biologic agents have been associated with the onset of IMIDs.

Aims & Methods

Aim: To evaluate the impact of ustekinumab on EIMs and IMIDs in a cohort of ulcerative colitis (UC) patients in real life.
Methods: Retrospective, multicenter study including UC patients who had received the first ustekinumab dose at least 16 weeks before the inclusion. Patients were followed-up from the first ustekinumab dose to treatment discontinuation or last visit, whichever came first. The evolution of previous EIMs and IMIDs was evaluated based on clinicians’ judgment. The onset of new EIMs and IMIDs under ustekinumab treatment was recorded.

Results

376 patients have been included so far (91% had PMS >2 at ustekinumab initiation, 96% had been previously exposed to biologics, and 25% had failed to anti-TNF, vedolizumab and tofacitinib). Median follow-up was 12 months (m). A total of 122 patients (32%) had previous EIM and/or IMIDs at ustekinumab start. A total of 80 patients (21%) had at least one EIM and/or IMIDs inactive at ustekinumab start, being the most frequent ones peripheral arthropathy (5%), axial spondyloarthropathy (3%) and psoriasis (3%). Some patients worsened from these conditions: a patient (6%) with peripheral arthropathy (partial response of UC), 2 (15%) patients with axial spondyloarthropathy (one in remission and one non-responder), a patient (33%) with rheumatoid arthritis (non-responder) and a patient (20%) with uveitis (non-responder). A total of 65 patients (17%) had active EIM and/or IMIDs at ustekinumab start; the outcomes of these patients are summarized in table 1. A total of 15 patients (4%) developed EIM and/or IMIDs after ustekinumab start: 6 (1.6%) patients developed peripheral arthropathy, 2 (0.5%) patients erythema nodosum, a patient (0.3%) was diagnosed with primary sclerosing cholangitis, a patient (0.3%) was diagnosed with vasculitis, and 6 (1.6%) patients developed other conditions.

Conclusion

In patients with active EIM at the start of ustekinumab treatment, improvement of those related to disease (UC) activity was observed. Other EIM and IMIDs not related to disease activity (axial spondyloarthropathy, uveitis, or rheumatoid arthritis) worsened. Treatment with ustekinumab does not appear to be associated with an increased risk of developing EIM or IMIDs.

Disclosure

This is an investigator-initiated study with financial support from Janssen (CNTO1275UCO4008).

Introduction

Urgency is not an IBD Patient Reported Outcome (PRO) in the STRIDE II but it has been assessed at baseline and as an outcome measure in recent pivotal RCTs with newer biologicals and targeted synthetic small molecules in both Crohn’s disease CD) and ulcerative colitis (UC). However, although it is reasonable to assume that UC patients or CD patients with rectal involvement may complain for severe urgency, urgency has been assessed even in patients with Crohn’s ileitis (iCD).

Aims & Methods

The aim of this study was to assess whether urgency is reported by iCD patients with flares or quiescent disease in a Real-World (RW) setting. This prospective, pilot, RW study included CD patients with solely L1 phenotype. L1 location was documented by ileocolonoscopy, histology and, imaging (MRE, US, or capsule endoscopy). Patients with colonic and/or rectal disease, perianal involvement and complicated ileal disease were excluded. Patients were interviewed by office visits during disease flare [HBI>5, CRP <5mg/dl, faecal calprotectin (FC)>250μg/g faecal tissue and SES-CD for terminal ileum >3 if colonoscopy was performed within a month prior to the visit) and during clinical remission (HBI<5, CRP <5mf/dl and FC <200μg/g and ileal SES-CD=0, in a recent ileocolonoscopy). The presence of and severity of urgency was recorded by a Symptom Rating Score of 0-10 (absent to very severe). The presence and severity of abdominal pain, the number and consistency of stools, nocturnal stools, blood and/or mucus in the stools, and the feeling of inadequate evacuation were sought by a structed questionnaire.

Results

During a 15-month period, 85 iCD patients [45 males, median age (range) 33 (18-65) years, median (range) disease duration 9 (0.6-25) years] were enrolled in the study. Patients were receiving a wide range of treatments, including anti-TNF agents (n=35), ustekinumab (N=25), vedolizumab (N=5), azathioprine (n=10), budesonide (n=5) and mesalazine (n=15). Fifty patients had a flare, 65 were in remissionand 30 patients were interviewed sequentially in flare and remission of disease. All patients in disease flare reported abnormal classical PROs of CD. Moderate to severe urgency [median score 5, range 4-8] was reported by 5/50 (10%) patients with disease flare, 8/65 (12%) patients with disease remission and 2/30 (6.5%) patients who were interviewed during flare and remission of disease. 3/50 patients with urgency during a flare of CD did not report urgency during disease remission. Six of the 8 patients who were interviewed only with iCD in remission and reported urgency had concurrently typical Irritable Bowel Symptoms (IBS, abdominal pain relieved by defecation, alternating diarrhoea/constipation, inadequate bowel emptying).

Conclusion

This prospective, pilot, real-world study shows that the reliability of urgency as classical PRO in patients with iCD is questioned and raises doubts whether urgency should be included in the spectrum of PROs for patients with iCD both in RCTs and in RW studies. Validation of the terminology in different ethnic languages may also pose additional barriers in patient understanding and reporting the symptom in the presence of additional IBS-type symptoms. Large studies are needed to assess if urgency is present in patients with iCD and how reliably is reported especially in multinational RCTs.

Disclosure

Dr GJ Mantzaris: Research grants: AbbVie, Genesis, Merck Sharp & Dohme, Takeda; advisor/speaker: AbbVie, Aenorasis, Dr Falk, Ferring, Hospira, Janssen, Merck Sharp & Dohme, MYLAN, Pfizer, Takeda, Vianex.
Dr A Loukou: Nothing to declare

Clinical Case Summary

A 45-year-old woman was founded with a raised serum alpha-fetoprotein (AFP) level of 89 ng/ml ( normal range < 8ug/L ) when taking a routine physical examination. During the following year, the serum AFP concentration level was gradually rising, up to 6353 ng/ml. There was no significant loss of weight or loss of appetite, and she denied having any feeling of bloating or abdominal pain.
Although the serum AFP concentration level was abnormally high, the cancer antigen (CA)125, cancer antigen (CA)199, carcinoembryonic antigen (CEA), PIVKA-II, AFP-L3, β-HCG levels were all within normal limits at each check. There was no abnormal findings in the hepatobiliary systems except the modest increased bilirubin (within 2 times). Hepatitis A, hepatitis C, hepatitis E antibody screen was negative. The result of HBV markers were: HBsAg(-), HBsAb(+), HBeAb(-), HBeAb(-), HBcAb(+), While HBV DNA quantification was less than 5.00E+2 IU/ml. Moreover, there was no significant abnormality concerning autoimmune liver disease. During the one-year follow up, several imaging examinations were performed,including abdominal ultrasonography, enhanced computer tomography (CT) , magnetic resonance imaging (MRI) and PET-CT. However, there was no evidence of liver lesion or tumour on all of them, even the PET scan.To check for hepatoid adenocarcinoma, gastroscopy, colonscopy, enhanced chest CT enhanced abdomen CT, and thyroid ultrasound were performed. Nevertheless, no suspicious lesion was found.
One year later, a 2.8×2.6 cm mass was detected in the left lobe of liver in a surveillance ultrasound scan. She underwent laparoscopic hepatocarcinoma resection, and the mass was confirmed moderately differentiated hepatocellular carcinoma by pathology. After surgery, AFP levels took 3 months to normalize.
From this case, we found that a gradual rise elevation in AFP were positive predictors for tumor development, especially hepatocellular carcinoma.

Introduction

Inflammatory bowel disease (IBD), a chronic inflammatory disease of the gastrointestinal tract, is caused by various factors, including genetic factors and environmental factors, as well as dysregulated immune responses. LMT503, a novel organic small-molecule compound, has the potential to suppress pro-inflammatory cells and induce anti-inflammatory cells by modulating their cell metabolisms. We evaluated the anti-inflammatory effects of LMT503 in a murine adoptive T cell transfer-induced colitis model.

Aims & Methods

This study was conducted to evaluate the anti-inflammatory effect of LMT503 in the murine chronic colitis model. Naïve CD4⁺CD25^-CD45RB^high CD62L^high T cells that were isolated from wild-type mice by flow cytometry based-method and transferred to Rag1 knock-out mice by intraperitoneal injection to induce murine adoptive T cell transfer-induced colitis. LMT503 was orally administrated (50mg/kg, 100mg/kg) for 2 weeks starting at 5 weeks after naïve T cell transfer. The disease activity index (DAI) was checked daily, and histopathological score, macroscopic injury score, and colon length were evaluated upon sacrifice. Macrophages and T cells from the spleen were analyzed by flow cytometry. Gene expression in colon tissue was measured by qRT-PCR and cytokine profiles in plasma was measured by cytometric bead array. Myeloperoxidase (MPO) activity was measured using the MPO activity kit.

Results

Oral administration of LMT503 ameliorated adoptive T cell transfer-induced colitis in mice. The DAI, histopathological score, and macroscopic injury score were significantly decreased after the administration of LMT503 in a dose-dependent manner. The LMT503-treated group showed significantly increased mucin production in the colon. Th1, Th17 cells, and M1 macrophages in the spleen were suppressed, and M2 macrophages were induced by the administration of LMT503. MPO activity and gene expressions of pro-inflammatory cytokines, such as IL-6, IL-1β, and TNF-α, in colon tissue were suppressed by the administration of LMT503. And the level of inflammatory cytokines, such as IL-2, TNF-α, and IFN-γ, in plasma were significantly suppressed by the administration of LMT503. Especially, LMT503 treatment significantly increased the gene expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Ppargc1a), a master regulator of mitochondrial biogenesis, suggesting that LMT503 modulates cellular metabolism towards an anti-inflammatory metabolism.

Conclusion

Our data suggest that the anti-inflammatory effect of LMT503 ameliorates murine adoptive T cell transfer-induced colitis by modulating cellular metabolism and immune cell modulation. Therefore, LMT503 may be a novel therapeutic drug that opens new avenues for the treatment of IBD.

References

1. Erben U, Loddenkemper C, Doerfel K, et al. A guide to histomorphological evaluation of intestinal inflammation in mouse models. Int J Clin Exp Pathol. 2014;7(8):4557-4576.
2. Ostanin DV, Bao J, Koboziev I, et al. T cell transfer model of chronic colitis: concepts, considerations, and tricks of the trade. Am J Physiol Gastrointest Liver Physiol. 2009;296(2):G135-G146.
3. Ghoreschi K, Jesson MI, Li X, et al. Modulation of innate and adaptive immune responses by tofacitinib (CP-690,550). J Immunol. 2011;186(7):4234-4243.
4. Burke JR, Cheng L, Gillooly KM, et al. Autoimmune pathways in mice and humans are blocked by pharmacological stabilization of the TYK2 pseudokinase domain. Sci Transl Med. 2019;11(502):eaaw1736.
5. Kiesler P, Fuss IJ, Strober W. Experimental Models of Inflammatory Bowel Diseases. Cell Mol Gastroenterol Hepatol. 2015;1(2):154-170.