Mesenteric Tissue Research Articles

Incidental finding of intramural splenic heterotopy in the colon mimicking subepithelial neoplasm: a case report

AimThe aim of this case report is describe an unprecedented case with histological and immunohistochemical diagnosis of splenic heterotopy in the colon using material obtained by endoscopic ultrasound-guided biopsy.BackgroundSplenic heterotopia is a benign condition characterized by the implantation of splenic tissue in areas distant from its usual anatomical site, such as the peritoneum, omentum, mesentery, liver, pancreas, and subcutaneous tissue and, more rarely, in locations such as the colon and brain. It is generally associated with a history of splenic trauma or splenectomy and typically does not cause specific symptoms.Case presentationA 35-year-old white male patient who was healthy, with no history of trauma or splenectomy, but had a family history of colorectal neoplasia underwent colonoscopy for screening. The examination revealed a large bulge in the proximal descending colon, covered by normal-appearing mucosa. Endoscopic ultrasound-guided puncture was performed with a 22 gauge fine needle biopsy, and the histopathological and immunohistochemical analysis results were consistent with a heterotopic spleen.ConclusionsThis is the first report of a primary intramural colic splenosis case with histological and immunohistochemical diagnosis of splenic heterotopia in the colon, using material obtained by endoscopic ultrasound and ultrasound-guided biopsy.

EXERCISE PRECONDITIONING IMPROVES MESENTERIC LYMPHATIC CONTRACTILITY THROUGH MAM IN RATS FOLLOWING HEMORRHAGIC SHOCK.

Restoration of mesenteric lymphatic microcirculation is crucial for alleviating severe hemorrhagic shock-induced death. Exercise preconditioning (EP) enhances adaptability and resistance to injury and disease. The mitochondria-associated endoplasmic reticulum membrane (MAM) plays a crucial role in the energy and information exchange between the two organelles. Therefore, we hypothesized that EP ameliorates mesenteric lymphatic contractility through MAM in rats following hemorrhagic shock, aiming to confirm that EP enhances resistance to hemorrhagic shock and further popularizes the idea that exercise is beneficial for health. To test this hypothesis, we observed the effects of EP for 4 weeks on survival time and mesenteric lymphatic contractility in conscious rats following hemorrhagic shock and further explored the effects of MAM agonists and inhibitors. The results showed that EP prolonged the survival time and improved the mesenteric lymphatic contractility and reactivity in vivo and in vitro in rats underwent hemorrhagic shock, ameliorated the MAM ultrastructure in lymphatic smooth muscle cells (LSMCs) and reduced the voltage-dependent anion channel 1 (VDAC1, a vital protein of MAM) and IP3R1 expressions in mesenteric lymphatic tissue. Importantly, treatment with 2-APB (IP3R1 inhibitor) or VBIT-12 (VDAC1 inhibitor) prolonged the survival time, improved mesenteric lymphatic contractility in vivo , ameliorated the MAM ultrastructure injury, and decreased the IP3R1 or VDAC1 expressions in LSMCs in rats following hemorrhagic shock. In contrast, the administration of drinking water containing CdCl 2 (IP3R1 activator) abolished the beneficial effect of EP on hemorrhagic shock. Taken together, the protective effect of EP on lymphatic contractility following hemorrhagic shock was achieved by improving MAM in LSMCs.

Effects of dietary fish to rapeseed oil ratio on steatosis symptoms in Atlantic salmon (Salmo salar L) of different sizes

Choline is recognized as an essential nutrient for Atlantic salmon at all developmental stages. However, its dietary requirement is not well defined. Choline plays a critical role in lipid transport, and the clearest deficiency sign is intestinal steatosis. The present work, aiming to find whether lipid source and fish size may affect steatosis symptoms, was one of a series of studies conducted to identify which production-related conditions may influence choline requirement. Six choline-deficient diets were formulated varying in ratios of rapeseed oil to fish oil and fed to Atlantic salmon of 1.5 and 4.5 kg. After eight weeks, somatic characteristics were observed, and the severity of intestinal steatosis was assessed by histological, biochemical, and molecular analyses. Fatty acid composition in pyloric intestine, mesenteric tissue, and liver samples was also quantified. The increasing rapeseed oil level increased lipid digestibility markedly, enhancing lipid supply to the fish. Moreover, small fish consumed more feed, and consequently had a higher lipid intake. In conclusion, the results showed that choline requirement depends on dietary lipid load, which depends on the fatty acid profile as well as the fish size.

Investigation of antigen-presenting cells in the intestinal mesentery in normal and adhesive processes

The morphology and topography of dendritic cells in the mesentery of the small intestine, their quantity, and their presence under normal conditions and during progressive adhesive processes have been insufficiently studied, presenting an important issue in immunomorphology. This study aimed to identify and determine the functional activity of antigen-presenting cells using lectin histochemistry with peanut and lentil lectins under normal conditions and during adhesive processes. The methods employed included morphometric analysis, lectin histochemistry, and statistical analysis. For the first time, using lectin histochemistry with lentil and soy lectins, functionally active and immunologically immature antigen-presenting cells were found in the structure of lymphoid clusters of the small intestine mesentery. A correlation was established between the progression of adhesion processes and the number of antigen-presenting cells. Correlation between dendritic cells and the quantity of immunologically immature lymphocytes and B-lymphocytes was identified, enhancing the understanding of the functional mechanisms of the local arm of innate immunity. An increasing trend in immunologically immature lymphocytes and B-lymphocytes was observed alongside an increase in antigen presenting cells. The results indicated that the activation of dendritic cells in mesenteric tissues induced an increase in immunologically immature lymphocytes, from which B-lymphocytes subsequently developed, initiating a local immune response. The increased frequency of PNA+- and LCA+- antigen-presenting cells pointed to an elevated immune reactivity of lymphoid clusters. This study on the distribution of antigen-presenting cells contributes to a deeper understanding of the structure of lymphoid tissue associated with serous membranes, as well as fat-associated lymphoid aggregates, and underscores the connection between innate and adaptive immunity in the abdominal cavity

Pathological Investigation of Double–Stranded DNA Breaks and DNA Oxidation in Natural Infection with Mycobacterium avium subspecies paratuberculosis in Goats

Paratuberculosis, created by Mycobacterium avium subspecies paratuberculosis (MAP), manifests as a chronic affliction marked by persistent diarrhoea and granulomatous enteritis, pervasive in both domestic and global wild ruminants. In this investigation, DNA disruption in lesioned tissues of goat as natural infecte with MAP was pathologically assessed. Accordingly, goats manifesting symptoms suggestive to paratuberculosis, including pronounced emaciation and continual episodic diarrhoea, were subjected to an ELISA diagnostic procedure to ascertain the presence of MAP. This diagnostic approach confirmed the presence of the infectious agent in 20 patients. These patients were subsequently euthanized, and tissue samples from intestinal and regional lenf nods. It were subjected to Hematoxylin and Eosin (HE) staining for histopathological investigatıon, Ziehl Neelsen (ZN) staining to identify acid–fast mycobacteria, γ–H2AX to discern disruptions in double stranded DNA, and 8–Ohdg to detect DNA oxidation by immunohistochemical (IHC) method. Gross anatomical observation serous adipose atrophy, augmented dimensions of mesenterial lymphatic nodes, mucosal hypertrophy and non–retractable mucosal undulations. Histological assessment highlighted epithelial cellular degeneration, an abundance of epithelioid macrophages, lymphocytes, plasmocytes, infiltrating in mucosa. Acid–fast entities, discernible through ZN staining, appeared as luminescent red conglomerates in intestinal and mesenterial tissue. The immunohistochemical analyses evinced positive results for both γ–H2AX and 8–Ohdg across all sampled tissues. Intriguingly, this investigation presented the inaugural global evidence of γ–H2AX and 8–Ohdg expression in a natural MAP infection, demonstrating that this pathological agent precipitates DNA degradation and oxidation, thereby augmenting comprehension of the disease’s pathogenesis.

Nutritional Practice-Related Alteration of Mesenteric Tissue Oxygenation in Fully Enteral-Fed Very and Extremely Preterm Neonates

Nutritional Practice-Related Alteration of Mesenteric Tissue Oxygenation in Fully Enteral-Fed Very and Extremely Preterm Neonates

A Novel Ex Vivo Tumor Spheroid-Tissue Model for Investigating Microvascular Remodeling and Lymphatic Blood Vessel Plasticity.

Biomimetic tumor microenvironment models bridge the gap between in vitro and in vivo systems and serve as a useful way to address the modeling challenge of how to recreate the cell and system complexity associated with real tissues. Our laboratory has developed an ex vivo rat mesentery culturemodel, which allows for simultaneous investigation of blood and lymphatic microvascular network remodeling in an intact tissue environment. Given that angiogenesis and lymphangiogenesis are key contributors to the progression of cancer, the objective of this study was to combine tissue and tumor spheroid culture methods to establish a novel ex vivo tumor spheroid-tissue model by verifying its use for evaluating the effects of cancer cell behavior on the local microvascular environment. H1299 or A549 tumor spheroids were formed via hanging drop culture and seeded onto rat mesenteric tissues harvested from adult male Wistar rats. Tissues with transplanted spheroids were cultured in serum-free media for 3 to 5days. PECAM, NG2, CD11b, and αSMA labeling identified endothelial cells, pericytes, immune cells, and smooth muscle cells, respectively. Time-lapse imaging confirmed cancer cell type specific migration. In addition to increasing PECAM positive capillary sprouting and LYVE-1 positive endothelial cell extensions indicative of lymphangiogenesis, tumor spheroid presence induced the formation of lymphatic/blood vessel connections and the formation of hybrid, mosaic vessels that were characterized by discontinuous LYVE-1 labeling. The results support the application of a novel tumor spheroid microenvironment model for investigating cancer cell-microvascular interactions.

Mesenteric panniculitis on 18F-FDG-PET/CT caused by G-CSF therapy mimicking recurrence of lymphoma

We present the case of a 55-year-old woman with Follicular Lymphoma, with basal 18F-FDG-PET/TC showing supra and infradiaphragmatic lymphatic, pleural and bone involvement. Treatment with Epcoritamab + Lenalidomide + Rituximab was iniciated with complete metabolic response (CR). Due to neutropenia, granulocyte colony-stimulating factor (G-CSF) was indicated on 02/22/23 for 5 days. A control PET/CT was performed on 02/23/23 with a pathological increase in metabolism in new-appearing mesenteric soft tissue lesions, suggestive of disease relapse. QT continued without changes and a PET/CT performed on 04/12/23 showed disappearance of the hypermetabolic mesenteric lesions. This suggests that it was a mesenteric panniculitis probably due to the use of G-CSF.

Stromal Vascular Fraction Derived Vasculogenesis Is Associated with the Formation of Malformed Lymphatic Vessel Structure

Tissue engineering and the development of therapies aimed at manipulating microvascular function require the ability to generate both blood and lymphatic vessels. Adipose-derived stromal vascular fraction (SVF), consisting of endothelial cells, stem cells, pericytes, smooth muscle cells, fibroblasts and immune cells, has emerged as a potential heterogeneous stem cell population for de novo vessel growth. SVF derived vasculogenesis has been reported to result in the dramatic formation of blood vessel segments that can connect with host vasculatures. Understanding how SVF can be used to therapeutically form vessels requires 1) exploration of the cell dynamics involved in the vasculogenesis process and 2) cauterization of the vessel structures. The objective of this study was to characterize SVF de novo vessel formation in an intact adult murine tissue environments. SVF was isolated from subcutaneous adipose in the inguinal region from adult C57BL/6 mice, labeled with DiI and seeded onto avascular mesentery tissues and then cultured in MEM + 10% fetal bovine serum (FBS) for up to 5 days (35 μl SVF cells/tissue; isolated from 1.8 g inguinal fats and resuspended in 200 μl MEM+FBS). Tissues were then labeled for PECAM (endothelial marker) + Lyve-1 (lymphatic marker) or NG2 (pericyte marker). By day 1, individual SVF cells started to form multi-cellular PECAM positive chord structures. Day 3 tissues displayed apparent vessel structures characterized by asterisk shaped clusters displaying a central PECAM positive “hub” and radial “spoke-like” extensions. At later time points and in regions of increased vascular coverage, the vessel structures were connected in a network pattern. In additional studies for which angiogenesis was pre-stimulated in the normally avascular mouse mesenteries, the SVF derived vessels connected to the host networks. A subset of structures during the vasculogenesis process displayed blunt ended lymphatic-like vessel structures termed “blebs.” The endothelial cell “blebs” were characterized by large, irregular diameters and a mosaic of Lyve-1 positive and negative cells. The “blebs” were also observed to connect with nearby SVF derived PECAM positive vessel segments. Our evidence of PECAM positive hub and lymphatic-like blebs provoke questions about SVF derived vessel function and suggest a previously undiscovered potential for SVF to form lymphatic vessel structures. NIH grant R21HL159501. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Peritoneal remodeling and changes in its lymphoid component in experimental modeling of adhesion disease in rats

Age changes, inflammatory processes, surgical interventions and heterogeneous pathological effects on the physiological processes of the peritoneum lead to certain changes in the structure of the components of the peritoneum, which leads to remodeling of the tissue structures of the abdominal cavity. According to the literature, the most frequent consequence of such remodeling of tissue structures of the peritoneum is the development of an adhesion process. To date, there are no data on the study of the lymphoid component of mesentery tissues, which is the goal of further research. The purpose of the work: to study the process of remodeling of peritoneal tissue during experimental adhesion formation and the specificity of changes in its lymphoid component in rats compared to the norm. Research materials and methods: preparation, macroscopic, microscopic, histological (production of film preparations), staining of preparations with hematoxylin and eosin, mathematical (morphometric grids - counting the number of immunocompetent cells per 1000 μm2 standard area), statistical processing according to Student. Results: experimental adhesion disease is characterized by a gradual process of remodeling of the mesenteric tissues of the small intestine and, as a result, the formation of connective tissue neoplasms. The mesentery of the small intestine loses its elasticity and mobility and thickens considerably. The process of experimental adhesion formation was accompanied by dynamic changes in the number of lymphocytes. Conclusions: these structures are thin and homogeneous on the 7th day; hard, dense and granular on the 14th day; contain solid conglomerates of heterogeneous structures on the 21st day after talc injection. The number of lymphocytes in this structure gradually increases: on day 7 - by 2 % in animals of group II, on day 14 - by 30 % in animals of group III, and on day 21 - by 36 % in animals of group IV, compared to animals of the intact group

Abstract B045: In vivo ovarian cancer detection using folate receptor-α targeted iron oxide nanoparticles

Abstract Ovarian cancer is the second most common and most deadly gynecological cancer in women. Early and accurate detection is crucial in improving survival rate and quality of life of patients. Superparamagnetic iron oxide nanoparticles (SPIONs) have been used in a variety of cancer detection/imaging applications, including Magnetic Resonant Imaging (MRI), Magnetic Particle Imaging (MPI) as well as Superparamagnetic Relaxometry (SPMR) detection currently being developed in-house. SPMR is a highly sensitive detection technology that can differentiate the magnetic signature of nanoparticles bound to tumor cells from unbound nanoparticles (NP). Folate receptor alpha (FRα) is a folate transporter overexpressed in approximately 90% of ovarian cancer, which makes it a suitable molecular target to use in developing tumor imaging methods. Our earlier study using xenograft model with KB (FRα +ve) and A549 (FRα -ve) cells implanted subcutaneously on the flank region of female athymic nude mice demonstrated higher level tumor accumulation of folate-NPs in KB tumor compared to A549 tumor, detectable by SPMR and MRI. To translate such results into a potential clinical application, we recently generated two orthotopic mice ovarian cancer models by implanting KB cells surgically into ovary as well intraperitoneally (IP) into mice abdominal cavity (metastasis model). Implanted tumor cells were allowed to grow for about 2 weeks. Folate-NPs (20mg Fe/kg) were delivered via tail vein or IP injections. PEG-NPs were also delivered as controls. Mice were euthanized 24-hour post-dosing. Mesenteric tumor tissue and reproductive organs with tumors were resected and measured on SPMR. Same tissues from mice without NP injection or with PEG-NP injection were also measured. Our results demonstrated that Folate-NPs reached the tumor sites in both orthotopic models and accumulated specifically at high level detectable by SPMR ex vivo. The amount of NP accumulation in mice with orthotopically implanted tumor was 3x-10x more than those of the xenograft subcutaneous flank implanted tumor model, indicating efficient NP delivery and binding of NP with tumor cells in its natural tissues/organs. The repeat studies of these orthotopic mice models used in the SPMR experiment are currently being conducted using MRI as an alternative in vivo detection method with images taken pre- and post- dosing of the NPs. We anticipate that folate-NPs presence in the tumors will be easily observable on MRI images as it produces T2 contrast along with additional anatomical information. Favorable orthotopic mice model results presented here will lay the groundwork for IND-enabling development of these targeted NP as T2 contrast agent for early ovarian cancer detection using MRI. This type of targeted imaging technology will improve the accuracy of cancer detection in vivo without the use of radioisotopes or radiation, therefore, minimize the needs of invasive biopsies/surgery, with further potential of monitoring therapy response or recurrence. Citation Format: Marie Zhang, Yamitha Perera, Kathirvel Kandasamy, Dan Inglese. In vivo ovarian cancer detection using folate receptor-α targeted iron oxide nanoparticles [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B045.

Effect of Dietary Supplementation with Omega-3 Fatty Acid on the Generation of Regulatory T Lymphocytes and on Antioxidant Parameters and Markers of Oxidative Stress in the Liver Tissue of IL-10 Knockout Mice.

chronic low-grade inflammation, or inflammaging, emerges as a crucial element in the aging process and is associated with cardiovascular and neurological diseases, sarcopenia, and malnutrition. Evidence suggests that omega-3 fatty acids present a potential therapeutic agent in the prevention and treatment of inflammatory diseases, mitigating oxidative stress, and improving muscle mass, attributes that are particularly relevant in the context of aging. The objective of the present study was to evaluate the effectiveness of supplementation with omega-3 fish oil in improving the immune response and oxidative stress in knockout mice for interleukin IL-10 (IL-10-/-). female C57BL/6 wild-type (WT) and interleukin IL-10 knockout (IL-10-/-) mice were fed during 90 days with a standard diet (control groups), or they were fed/supplemented with 10% of the omega-3 polyunsaturated fatty acid diet (omega-3 groups). Muscle, liver, intestinal, and mesenteric lymph node tissue were collected for analysis. the IL-10-/-+O3 group showed greater weight gain compared to the WT+O3 (p = 0.001) group. The IL-10-/-+O3 group exhibited a higher frequency of regulatory T cells than the IL-10-/- group (p = 0.001). It was found that animals in the IL-10-/-+O3 group had lower levels of steatosis when compared to the IL-10-/- group (p = 0.017). There was even greater vitamin E activity in the WT group compared to the IL-10-/-+O3 group (p = 0.001) and WT+O3 compared to IL-10-/-+O3 (p = 0.002), and when analyzing the marker of oxidative stress, MDA, an increase in lipid peroxidation was found in the IL-10-/-+O3 group when compared to the IL-10-/- group (p = 0.03). Muscle tissue histology showed decreased muscle fibers in the IL-10-/-+O3, IL-10-/-, and WT+O3 groups. the findings show a decrease in inflammation, an increase in oxidative stress markers, and a decrease in antioxidant markers in the IL-10-/-+O3 group, suggesting that supplementation with omega-3 fish oil might be a potential intervention for inflammaging that characterizes the aging process and age-related diseases.

A181 EXPLORING THE ROLE OF KLEBSIELLA VARIICOLA AND KLEBSIELLA PNEUMONIAE IN PEDIATRIC ULCERATIVE COLITIS PATHOGENESIS

Abstract Background Recent studies indicated that Klebsiella (K) pneumoniae (isolated from the stool of IBD patients) and K. variicola (isolated from the mesenteric tissue of Crohn disease patients) have the potential to induce inflammation in epithelial and preadipocyte cells, exacerbating colitis in murine models. Aims We isolated these strains from pediatric ulcerative colitis (UC) patients' appendix (relevant to UC pathogenesis given the protective effects of appendectomy) and other non-inflamed colon sections. We hypothesized that these isolates are invasive and induce inflammation in UC. These strains have not been previously studied in UC. Methods K. variicola and K. pneumoniae were isolated from two pediatric UC patients' appendices and other non-inflamed colon sections and identified using 16S DNA Sanger sequencing. Virulence of the two strains was determined by infecting Caco2 cell lines and performing adhesion and invasion assays, quantifying biofilm formation, and assessing barrier functions using transepithelial electrical resistance (TEER) measurement. Importantly, we monitored the production of pro-inflammatory (e.g., IL-8, TNF-α) and anti-inflammatory (IL-10) cytokines using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Results K. variicola and K. pneumoniae exhibited high levels of biofilm formation compared to adherent-invasive Escherichia coli (AIEC), which are commonly isolated from IBD patients. Furthermore, the Klebsiella strains adhered to epithelial cells within 2-3 hours post infection. TEER experiments showed compromised barrier integrity after 6 hours and overnight infection. Except for K. pneumoniae isolated from the ascending colon, the tested strains exhibited a significant increase in IL-8 expression. Similarly, K. variicola from the peri-appendicular region demonstrated elevated TNF-α gene expression. Conclusions K. variicola and K. pneumoniae isolated from UC patients have the potential to form biofilms, disrupt barrier integrity, and trigger inflammatory responses. These findings unravel a potential role for pathogenic Klebsiella strains in driving UC pathogenesis. Importantly, these data shed light on the role of appendix-associated bacteria in the development of UC. Future work includes using comparative genomics to map the virulence determinants of these bacteria. Funding Agencies CIHRWomen and Children's Health Research Institute (WCHRI)

P020 Effects of Anti-TNF Treatment on Browning of Mesenteric Adipose Tissue with Crohn's Disease

Abstract Background Crohn's disease (CD) is characterized by the expansion of mesenteric fat, known as creeping fat (CrF), to the inflamed segments of the intestine. Our group observed that CrF showed a conversion to beige tissue, a phenomenon known as "Browning", characterized by an elevated gene expression of thermogenin (UCP-1), the gene characteristic of browning1. A recent study also showed that when browning was induced in mice in a TNBS colitis model, mesenteric hypertrophy improved and inflammation in adipose tissue was reduced2. The aim of this study is to assess whether the administration of anti-TNF biologic treatment (infliximab) in patients with CD is related to the increase in adipose beige tissue in the CrF. Methods Immunohistochemistry staining of UCP-1 was conducted on CrF samples from CD patients receiving anti-TNF treatment (≥6 months) and anti-TNF-naïve patients. Additionally, we analysed the gene expression of beige adipose markers in CrF from CD patients treated with anti-TNF drugs, comparing them to those untreated. UCP-1 gene expression was also studied in adipose-stem cells isolated from CrF. Ongoing experiments involve treating adipose stem cells with infliximab (10ug/ml) for 24 hours to assess the potential conversion of white to beige adipose tissue. Results The study revealed a significant increase in UCP-1 staining in CrF samples among patients receiving anti-TNF treatment compared to anti-TNF-naïve CD patients (Fig 1.A). This trend was consistently observed in mesenteric CrF tissue. Beige markers TMEM26 and CPTB1 exhibited significant increases in anti-TNF-treated CrF, and other beige tissue markers showed a notable trend (Fig 1.B). Adipose stem cells displayed a significant increase in UCP-1 gene expression in anti-TNF-treated CrF compared to untreated patients (Fig 1.C). Exciting preliminary data suggest that infliximab treatment increases UCP-1 and the anti-inflammatory cytokine IL-4 gene expression in adipose-stem cells from active CD patients (Fig 1.D). This novel data implies a direct relationship between anti-TNF treatment and heightened browning in white adipose tissue of CD patients. Conclusion This study reveals a notable increase in UCP-1 gene expression in adipose tissue CrF of CD patients undergoing anti-TNF treatment. The findings suggest that anti-TNF therapy may trigger the transformation of white adipose tissue into the "beige" type, potentially mitigating inflammation. Browning CrF presents a promising therapeutic avenue for CD management, warranting further investigation. Ref: 1. D. Monfort-Ferré et al. J Crohn Colitis., 2022;16(10):1571–83, 2. Zuo L et al. J Crohn Colitis. 2023 21;17(8):1179-1192.

P1238 Exploring the Role of Klebsiella variicola and Klebsiella pneumoniae in Pediatric Ulcerative Colitis Pathogenesis

Abstract Background Recent studies indicated that Klebsiella (K) pneumoniae (isolated from the stool of IBD patients) and K. variicola (isolated from the mesenteric tissue of Crohn disease patients) have the potential to induce inflammation in epithelial and preadipocyte cells, exacerbating colitis in murine models. We isolated these strains from pediatric ulcerative colitis (UC) patients' appendix (relevant to UC pathogenesis given the protective effects of appendectomy) and other non-inflamed colon sections. We hypothesized that these isolates are invasive and induce inflammation in UC. These strains have not been previously studied in UC. Methods K. variicola and K. pneumoniae were isolated from two pediatric UC patients' appendices and other non-inflamed colon sections and identified using 16S DNA Sanger sequencing. Virulence of the two strains was determined by infecting Caco2 cell lines and performing adhesion and invasion assays, quantifying biofilm formation, and assessing barrier functions using transepithelial electrical resistance (TEER) measurement. Importantly, we monitored the production of pro-inflammatory (e.g., IL-8, TNF-α) and anti-inflammatory (IL-10) cytokines using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Results K. variicola and K. pneumoniae exhibited high levels of biofilm formation compared to adherent-invasive Escherichia coli (AIEC), which are commonly isolated from IBD patients. Furthermore, the Klebsiella strains adhered to epithelial cells within 2-3 hours post infection. TEER experiments showed compromised barrier integrity after 6 hours and overnight infection. Except for K. pneumoniae isolated from the ascending colon, the tested strains exhibited a significant increase in IL-8 expression. Similarly, K. variicola from the peri-appendicular region demonstrated elevated TNF-α gene expression. Conclusion K. variicola and K. pneumoniae isolated from UC patients have the potential to form biofilms, disrupt barrier integrity, and trigger inflammatory responses. These findings unravel a potential role for pathogenic Klebsiella strains in driving UC pathogenesis. Importantly, these data shed light on the role of appendix-associated bacteria in the development of UC. Future work includes using comparative genomics to map the virulence determinants of these bacteria.

CT energy spectral parameters of creeping fat in Crohn's disease and correlation with inflammatory activity.

Creeping fat is a kind of unique abnormal mesenteric tissue at the sites of diseased bowel of Crohn's disease. By using dual-energy CT enterography, this study aimed to evaluate the feasibility of spectral parameters in the quantitative analysis of mesenteric adipose tissue or creeping fat. In this study, patients with known or suspected Crohn's disease who underwent dual-energy CT enterography from March 1, 2019, to March 31, 2021, were enrolled. Among them, 40 patients with surgery and pathology-proven creeping fat were selected as the creeping fat Crohn's disease group, and 40 normal patients were selected as the control group. The quantitative spectral parameters including the slope of the Hounsfield unit curve, normalised fat-water concentration, normalised fat-iodine concentration, and normalised fat volume fraction at the enteric phases were obtained. Mann-Whitney U test, Kruskal-Wallis H test, and receiver operating characteristic curve analysis were applied to compare quantitative parameters among various groups. A significant difference was observed in the slope of the Hounsfield unit curve, normalised fat-water concentration, normalised fat-iodine concentration, and normalised fat volume fraction between mesenteric adipose tissue and creeping fat with Crohn's disease at the enteric phase (all p < 0.001). The slope of the Hounsfield unit curve of creeping fat at the enteric phase had a better capability to distinguish inactive and active Crohn's disease (AUC = 0.93, p < 0.001). Dual-energy CT enterography with quantitative spectral parameters is a potentially novel noninvasive tool for evaluating creeping fat in Crohn's disease. Energy spectral parameters of creeping fat in Crohn's disease are significantly different from normal mesenteric adipose tissues and are correlated with inflammatory activity. • Dual-energy CT enterography allows quantitatively assessing creeping fat with spectral parameters. • The creeping fat has distinct spectral parameters to normal mesenteric adipose. • The spectral parameters accurately differentiate active and inactive Crohn's disease.

Pediatrik İnflamatuar Bağırsak Hastalıklarının Tanısında ve Ayrımında Manyetik Rezonans Enterografinin Rolü

Background/Aims: Inflammatory bowel diseases (IBD) are of two types: Crohn’s disease (CD) and ulcerative colitis (UC). The differential diagnosis of these two diseases is important because their treatment strategies are different. The present study aimed to evaluate the Magnetic Resonance Enterography (MRE) findings of pediatric patients who were clinically diagnosed with CD or UC. Material and Method: The patients under the age of 18, who underwent MRE examination, were retrospectively screened. Fifty five of those patients, who were clinically diagnosed with CD or UC, were included in the study. Bowel wall thickening, mesenteric fat tissue changes and complications were evaluated, and the findings were recorded. The sensitivity and specificity of MRE in the diagnosis of CD and UC in IBD patients were calculated. Results: Three of the patients clinically diagnosed with CD (7.3%) and six patients who had UC (42.9%) had involvement from the rectum to the proximal colonic segments (continuous lesions). Of those diagnosed with CD, 11 (26.8%) had skip lesions. In patients with IBD, the sensitivity of MRE for the diagnosis of CD was 56.1% and the specifity was 100%. The sensitivity of MRE for the diagnosis of UC was 42.9% and the specificity was 90.2%. Conclusion: MRE is helpful to identify the signs and complications of inflammatory bowel diseases. Although MRE has low sensitivity to differentiate between CD or UC in pediatric patients, it may help with its high specificity rates.

STELLATE GANGLION BLOCK REVERSES PHSML-INDUCED VASCULAR HYPOREACTIVITY THROUGH INHIBITING AUTOPHAGY-MEDIATED PHENOTYPIC TRANSFORMATION OF VSMCs.

Posthemorrhagic shock mesenteric lymph (PHSML) return-contributed excessive autophagy of vascular smooth muscle cells (VSMCs) is involved in vascular hyporeactivity, which is inhibited by stellate ganglion block (SGB) treatment. The contractile phenotype of VSMCs transforms into a synthetic phenotype after stimulation with excessive autophagy. Therefore, we hypothesized that SGB ameliorates PHSML-induced vascular hyporeactivity by inhibiting autophagy-mediated phenotypic transformation of VSMCs. To substantiate this hypothesis, a hemorrhagic shock model in conscious rats was used to observe the effects of SGB intervention or intravenous infusion of the autophagy inhibitor 3-methyladenine (3-MA) on intestinal blood flow and the expression of autophagy- and phenotype-defining proteins in mesenteric secondary artery tissues. We also investigated the effects of intraperitoneal administration of PHSML intravenous infusion and the autophagy agonist rapamycin (RAPA) on the beneficial effect of SGB. The results showed that hemorrhagic shock decreased intestinal blood flow and enhanced the expression of LC3 II/I, Beclin 1, and matrix metalloproteinase 2, which were reversed by SGB or 3-MA treatment. In contrast, RAPA and PHSML administration abolished the beneficial effects of SGB. Furthermore, the effects of PHSML or PHSML obtained from rats treated with SGB (PHSML-SGB) on cellular contractility, autophagy, and VSMC phenotype were explored. Meanwhile, the effects of 3-MA on PHSML and RAPA on PHSML-SGB were observed. The results showed that PHSML, but not PHSML-SGB, incubation decreased VSMC contractility and induced autophagy activation and phenotype transformation. Importantly, 3-MA administration reversed the adverse effects of PHSML, and RAPA treatment attenuated the effects of PHSML-SGB incubation on VSMCs. Taken together, the protective effect of SGB on vascular reactivity is achieved by inhibiting excessive autophagy-mediated phenotypic transformation of VSMCs to maintain their contractile phenotype.

Comparison of the efficiency of different etiological assays for detection of Schistosoma japonicum infections in wild mice

To compare the efficiency of multiple etiological techniques for detection of Schistosoma japonicum infections in wild mice, so as to provide technical supports to assessment of schistosomiasis transmission risk. Wild mice were captured with baited traps at night in Oncomelania hupensis snail-infested settings in schistosomiasis-endemic foci of Anhui Province from October to November, 2022. S. japonicum infections were detected in wild mice using microscopy of mouse liver tissues, microscopy of mouse mesenteric tissues, microscopy of mouse liver tissue homogenates, miracidial hatching test of mouse liver tissue homogenates, Kato-Katz technique and miracidial hatching test of mouse stool samples alone and in combinations. Identification of S. japonicum eggs or miracidia by any of these six assays was defined as an infection. The sensitivity of six assays alone or in combinations was compared for detection of S. japonicum infections in wild mice. A total of 1 703 wild mice were captured, with 366 wild mice detected positive for S. japonicum (21.49%). There were significant differences in the prevalence of S. japonicum infections in wild mice by six assays (Q = 529.33, P < 0.001) and in the sensitivity of six assays for detection of S. japonicum infections in wild mice (χ2 = 527.78, P < 0.001). In addition, the combination of microscopy of mouse liver tissues and mesenteric tissues, combination of microscopy of mouse liver tissues and liver tissue homogenates and combination of microscopy of mouse liver tissues, microscopy of mesenteric tissues, microscopy of liver tissue homogenates and Kato-Katz technique showed 86.61%, 87.16% and 97.27% sensitivities for detection of S. japonicum infections in wild mice, respectively. Diverse etiological assays show various efficiencies for detection of S. japonicum infections in wild mice. Combination of microscopy of mouse liver tissues and microscopy of mesenteric tissues, and combination of microscopy of mouse liver tissues and microscopy of liver tissue homogenates are potential approaches for field detection of S. japonicum infections in wild mice.

Time-Lapse Observation of Cell Dynamics During Angiogenesis Using the Rat Mesentery Culture Model.

The ability to track cells and their interactions with other cells during physiological processes offers a powerful tool for scientific discovery. An ex vivo model that enables real-time investigation of cell migration during angiogenesis in adult microvascular networks would enable observation of endothelial cell dynamics during capillary sprouting. Angiogenesis is defined as the growth of new blood vessels from existing ones and involves multiple cell types including endothelial cells, pericytes, and interstitial cells. The incorporation of these cell types in a physiologically relevant environment, however, represents a challenge for biomimetic model development. Recently, our laboratory has developed the rat mesentery culture model, which enables investigation of angiogenesis in an intact tissue. The objective of this chapter is to detail a protocol for tracking cellular dynamics during angiogenesis using the rat mesentery tissue culture model. The method involves harvesting mesentery tissues from adult SD-EGFP rats, culturing them in MEM+10% fetal bovine serum, and imaging network regions over the time course of angiogenesis. In example applications, time-lapse comparison of microvascular networks in cultured tissues confirmed dramatic increases in GFP-positive capillary sprouting and GFP-positive segment density. Additionally, tracking of individual capillary sprout extensions revealed their ability to "jump" by disconnecting from one vessel segment and reconnecting to another segment in the network. GFP-positive sprouts were also capable of undergoing subsequent regression. The representative results support the use of the rat mesentery culture model for identifying and tracking cellular dynamics during angiogenesis in intact microvascular networks.