Pouch Tissue Research Articles

A Rat Model of Pouchitis Following Proctocolectomy and Ileal Pouch-Anal Anastomosis Using Dextran Sulfate Sodium.

Ulcerative colitis (UC) is a chronic immune-mediated disease that affects the entire colon and rectum with a relapsing and remitting course, causing lifelong morbidity. When medical treatment is ineffective, especially in cases of massive gastrointestinal bleeding, perforation, toxic megacolon, or carcinogenesis, surgery becomes the last line of defense to cure UC. Total colorectal resection and ileal pouch-anal anastomosis (IPAA) offer the best chance for long-term treatment. Pouchitis is the most common and troublesome postoperative complication. In this investigation, microsurgery is employed to create an ileal pouch model in experimental rats via IPAA surgery. Subsequently, a sustained rat model of pouchitis is established by inducing inflammation of the ileal pouch with dextran sulfate sodium (DSS). The successful establishment of rat pouchitis is validated through analysis of postoperative general status, weight, food and water intake, fecal data, as well as pouch tissue pathology, immunohistochemistry, and inflammatory factor analysis. This experimental animal model of pouchitis provides a foundation for studying the pathogenesis and treatment of the condition.

MUCOSAL SINGLE-CELL PROFILING OF CROHN’S-LIKE DISEASE OF THE POUCH REVEALS COMMON PATHOGENICS AND THERAPEUTIC TARGETS

Abstract BACKGROUND & AIMS After restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC), a subset of patients develop Crohn’s-like disease of the pouch (CDP), an inflammatory condition that affects both the ileoanal pouch and extra-pouch organs including pre-pouch ileum. The cellular and molecular identities of CDP are unknown, and its diagnosis and treatment remain challenging. To define the pathophysiology of CDP, we examined mucosal cells from patients with and without CDP using single cell analyses. METHODS Endoscopic samples from the pouch and pre-pouch ileum of 50 patients with an IPAA were collected for single-cell RNA sequencing (scRNA-seq) or mass cytometry (or CyTOF). We analyzed immune and non-immune cells from pouch and ileal tissues of patients with normal pouch/ileum and CDP (n = 5 or 6) using scRNA-seq. CyTOF was performed on mucosal immune cells from independent cohorts of patients with normal pouch/ileum, CDP, and pouchitis (n = 9 or 10). Mucosal samples from patients with familial adenomatous polyposis (FAP) after pouch formation were also analyzed by CyTOF. Some scRNA-seq and CyTOF findings were independently validated using immunohistochemistry (n = 6 or 8). RESULTS We revealed distinct immune and non-immune cell clusters in normal pouch and pre-pouch ileum. Colitogenic immune cells expanded in normal UC pouch compared to normal FAP pouch. Compared to normal pouch/pre-pouch ileum, CDP tissues exhibited expanded TCR clonotypes, elevated Th17 signaling, and diminished T cell exhaustion markers. Elevated frequencies of plasma cells, inflammatory fibroblasts and monocytes were noted in CDP pouch and ileum (Fig 1; only pouch data were shown). CDP also harbored elevated Th17-inducing cytokines such as IL23, IL1B, and IL6 produced by myeloid cells. ScRNA-seq and CyTOF identified increased CD14+TREM1+ pathogenic monocytes in both pouch and pre-pouch ileum of CDP. Ligand-receptor interactions further uncovered an essential role of proinflammatory myeloid cells in the pathogenesis of CDP. In addition, pouch and pre-pouch ileum of CDP showed prominent activation of the unfolded protein response (UPR) across all major cell compartments (Fig 2), which was not present in UC, CD or pouchitis based on integrated analysis of published databases. CONCLUSIONS CDP demonstrates altered immune and non-immune cell populations/states in the pouch and pre-pouch ileum. CDP represents a distinct entity of inflammatory bowel disease with extensive UPR activation, a unique feature that may serve as novel diagnostic markers and therapeutic targets using ER stress-alleviating agents including chemical chaperones. Fig 1 CDP reshapes immune and non-immune landscapes in pouch and pre-pouch ileum. (A) UMAP visualization of all cells from the pouch and ileum of CLDP and normal controls. (B) Diversity of epithelial cells, stromal cells, myeloid cells, T cells, innate lymphoid cells (ILCs), B cells and plasma cells in CLDP vs. normal controls in the pouch estimated by scRNA-seq (n = 5 or 6). *p<0.01. Fig 2 Extensive UPR activation in CDP tissues. (A) UPR gene expression in major cell compartments in CDP and normal controls, highlighted genes including CALR, XBP1, and SERP1 were upregulated in all 5 cell compartments. (B) ER stress/UPR pathways are enriched in CDP. (C) IHC of UPR proteins XBP1 and HSPA5/BiP.

Pouchitis Is Associated with Paneth Cell Dysfunction and Ameliorated by Exogenous Lysosome in a Rat Model Undergoing Ileal Pouch Anal Anastomosis.

Pouchitis is a common complication of restorative proctocolectomy and ileal pouch anal anastomosis (IPAA) for ulcerative colitis (UC), significantly affecting the postoperative quality of life. Paneth cells play an important role in the maintenance of gut homeostasis. This study aimed to investigate the role of Paneth cells in the pathogenesis of pouchitis. Endoscopic biopsies from the pouch body and terminal ileum of UC patients undergoing IPAA with or without pouchitis were obtained to analyze Paneth cell function. Acute pouchitis was induced with 5% dextran sulfate sodium (DSS) for seven consecutive days in a rat model of IPAA. The Paneth cell morphology was examined by immunofluorescence and electron microscopy. The effect of exogenous lysozyme supplementation on pouchitis was also investigated. The fecal microbiota profile after DSS and lysozyme treatment was determined by 16s rRNA ITS2 sequence analysis. Abnormal mucosal lysozyme expression was observed in patients with pouchitis. The rat model of pouchitis showed increased pouch inflammation, increased CD3+ and CD45+ T cell infiltration, and decreased tight junction proteins, including ZO-1 and Occludin. There is a significant deficiency of Paneth cell-derived lysozyme granules in the rat model of pouchitis. Supplementation with exogenous lysozyme significantly ameliorated pouchitis, lowering the levels of inflammatory cytokines such as TNF-α and IL-6 in the pouch tissue. 16s rRNA analysis revealed a higher Lachnospiraceae level after lysosome treatment. Paneth cell dysfunction is prominent in patients and rat models of pouchitis and may be one of its causes. The decrease in Lachnospiraceae, a characteristic of dysbiosis in pouchitis, could be reserved by lysosome treatment. Lysozyme supplementation shows promise as a novel treatment strategy for pouchitis.

Therapeutic Effects of Hydrogel Formulations Incorporating Troxipide Nanoparticles on Oral Mucositis in Hamsters.

Medical therapies, such as the use of anti-inflammatory agents, are commonly used for the treatment of oral mucositis (OM). However, these treatments have limited efficacy in treating severe cases of OM. In this study, we aimed to develop a carbopol gel incorporating troxipide (TRO) nanoparticles and methylcellulose (TRO-NP gel) and demonstrate its efficacy in accelerating wound healing in a hamster model of OM (OM model) induced by acetic acid injection. TRO nanoparticles were prepared using bead milling. The crystalline form was determined by powder X-ray diffraction, and the particle size was measured using a NanoSight LM10 instrument. The drug release was determined using a Franz diffusion cell, and the hamsters injected with acetic acid were selected to evaluate the therapeutic effect of OM. After preparing TRO nanoparticles, we observed a mixture of crystals and amorphous TRO, and the particle size of TRO in the TRO-NP gel ranged from 50 to 280 nm. The TRO-NP gel exhibited a more uniform TRO distribution and viscosity compared to the Carbopol gel containing TRO microparticles (TRO-MP gel). However, the solubility of TRO was comparable in both TRO-MP and TRO-NP gels. The TRO-NP gel released a higher amount of TRO than that from the TRO-MP gel, with detectable release of TRO nanoparticles. TRO levels in the cheek pouches of hamsters treated with TRO-NP gel were higher than those treated with TRO-MP gel. The increased TRO levels in the cheek pouches of hamsters treated with TRO-NP gel were attenuated by treatment with 40 μM dynasore, an inhibitor of clathrin-dependent endocytosis (CME). Moreover, the therapeutic effect of the TRO-NP gel was superior to that of the TRO-MP gel in the hamster model of OM. We have designed a TRO-NP gel, and this gel showed excellent TRO delivery into the cheek pouch tissue through the CME pathway. Moreover, the TRO-NP gel treatment enhanced wound healing after acetic acid injection.

Anti-inflammatory and antinociceptive activities of Daniellia oliveri (Fabaceace) stem bark extract.

Daniellia oliveri (Rolfe) Hutch. & Dalziel (Fabaceae) is used for the treatment of inflammatory diseases and pains (chest pain, toothache and lumbago) and rheumatism. The study investigates the anti-inflammatory and antinociceptive properties of D. oliveri and possible mechanism of antiinflammatory action. Acute toxicity of the extract was evaluated in mice using the limit test. The anti-inflammatory activity was assessed in xylene-induced paw oedema and carrageenan-induced air-pouch models at doses of 50, 100 and 200mg/kg, p.o. Volume of exudate, total protein, leukocyte counts, myeloperoxidase (MPO) and concentration of cytokines (TNF-α and IL-6) were measured in the exudate of rats in the carrageenan-induced air-pouch model. Other parameters include lipid peroxidation (LPO), nitric oxide (NO) and antioxidant indices (SOD, CAT and GSH). Histopathology of the air pouch tissue was also carried out. The antinociceptive effect was assessed using acetic acid-induced writhing, tail flick and formalin tests. Locomotor activity was done in the open field test. The extract was analysed with HPLC-DAD-UV technique. The extract showed significant anti-inflammatory effect (73.68 and 75.79%, inhibition) in xylene-induced ear oedema test at the dose of 100 and 200mg/kg, respectively. In carrageenan air pouch model, the extract significantly reduced exudate volume, protein concentration, the migration of leukocytes and MPO production in the exudate. The concentrations of cytokines TNF-α (12.25±1.80pg/mL) and IL-6 (21.12pg/mL) in the exudate at the dose of 200mg/kg were reduced compared to carrageenan alone group (48.15±4.50pg/mL; 82.62pg/mL) respectively. The extract showed significant increase in the activities of CAT and SOD and GSH concentration. The histopathological assessment of the pouch lining revealed reduction of immuno-inflammatory cell influx. Nociception was significantly inhibited by the extract in acetic acid-induced writhing model and the second phase of formalin test indicating a peripheral mechanism of action. The open field test showed that D. oliveri did not alter locomotor activity. The acute toxicity study did not cause mortality or signs of toxicity at 2000mg/kg, p.o. We identified and quantified caffeic acid, p-coumaric acid, ferulic acid, rutin, apigenin-7-glucoside, quercetin and kaempferol in the extract. The results of our study showed that the stem bark extract of D. oliveri possesses anti-inflammatory and antinociceptive activities thereby supporting its traditional use in the treatment of some inflammatory and painful disorders.

EFFECTS OF WOUND IRRIGATION SOLUTIONS ON HOST TISSUE: MURINE AIR POUCH INFECTION MODEL

Irrigation with antiseptic agents, antibiotics, and surfactants are used for treatment and prevention of infections. Despite desirable microbicidal actions, studies have demonstrated cytotoxic effects on host tissue that may impair healing. This study investigated the extent of tissue damage caused by commonly used irrigation solutions in the presence or absence of infection.Air pouches created in 60 balb/c mice were divided into two groups (n=30): infected with Staphylococcus aureus and control. One week later the infected group was subdivided into 5 subgroups (n=6) based on irrigation solutions and by day 0 (immediately) and day7 after irrigation (n=3). Solutions included Saline, Bacitracin, Clorpactin, Irrisept and Bactisure. In infected group wash fluid was collected for quantitative analysis of bacterial growth. At the specified times mice were sacrificed, pouch tissue sent for histology, and sections analyzed for inflammation, necrosis, and edema.Inflammation decreased in infected vs sterile pouches for all solutions except Bacitracin day 0 and for all solutions day 7 with significance in all except Bacitracin (p<0.05). On day 0, necrosis increased in infected vs sterile pouches in Bacitracin (p=0.006), Irrisept (p=0.18), or Bactisure (p=0.07); however, on day 7, necrosis significantly decreased in infected pouches for all solutions (p<0.05) except for Clorpactin (p=0.18). Edema decreased in infected vs sterile pouches on day 0 for all solutions with significance in saline, Irrisept, and Bacitracin (p<0.05). On day 7, infected pouches had decreased edema in saline, Bacitracin, and Bactisure (p<0.05) and increased in Irrisept (p<0.05) and Clorpactin (p=0.069) compared to sterile pouches. Bacterial culture of washouts demonstrated that Clorpactin, Irrisept and Bactisure controlled the infection, whereas saline and Bacitracin showed bacterial multiplication 3.9 × 10^7 CFU/ml and 6.7 × 10^7 CFU/ml respectively. Bacitracin wash showed significantly more bacteria growth compared to Clorpactin (p=0.024), Irrisept (p=0.025) and Bactisure (p=0.025).Tissue damage varied with irrigation solutions and the presence or absence of infection. Presence of bacteria appeared to lead to less tissue inflammation and edema. Tissue necrosis varied over time with different solutions. Surgeons must weigh risks and benefits when selecting solutions and determining when to irrigate.

EFFICACY OF SALINE WASH PLUS VANCOMYCIN/TOBRAMYCIN-DOPED PVA COMPOSITE (PVA-VAN/TOB-P) IN A MOUSE POUCH INFECTION MODEL IMPLANTED WITH 3D-PRINTED POROUS TITANIUM CYLINDERS

The efficacy of saline irrigation for the treatment of periprosthetic infection (PJI) is limited in the presence of infected implants. This study evaluated the efficacy of vancomycin/tobramycin-doped polyvinyl alcohol (PVA)/ceramic composites (PVA-VAN/TOB-P) after saline irrigation in a mouse pouch infection model.3D printed porous titanium (Ti) cylinders (400, 700 and 100 µm in pore size) were implanted into mice pouches, then inoculated with S. aureus at the amounts of 1X103 CFU and 1X106 CFU per pouch, respectively. Mice were randomized into 4 groups (n=6 for each group): (1) no bacteria; (2) bacteria without saline wash; 3) saline wash only, and (4) saline wash+PVA-VAN/TOB-P. After seven days, pouches were washed out alone or with additional injection of 0.2 ml of PVA-VAN/TOB-P. Mice were sacrificed 14 days after pouch wash. Bacteria cultures of collected Ti cylinders and washout fluid and histology of pouch tissues were performed.The low-grade infection (1X103 CFU) was more significant in 400 µm Ti cylinders than that in Ti cylinders with larger pore sizes (700 and 1000 µm (p<0.05). A similar pattern of high-grade infection (1X106 CFU) was observed (p<0.05). For the end wash, the bacteria burden (0.49±0.02) in saline wash group was completely eradicated by the addition of PVA-VAN/TOB-P (0.005±0.001, p<0.05).We noticed that 400 µm Ti cylinders have the highest risk of implant infection. Our data supported that the effect of saline irrigation was very limited in the presence of contaminated porous Ti cylinders. PVA-VAN/TOB-P was biodegradable, biocompatible, and was effective in eradicating bacteria retention after saline irrigation in a mouse model of low grade and high-grade infection. We believe that PVA-VAN/TOB-P represents an alternative to reduce the risk of PJI by providing a sustained local delivery of antibiotics.

Prolactin and the evolution of male pregnancy

Prolactin (PRL) is a multifunctional hormone of broad physiological importance, and is involved in many aspects of fish reproduction, including the regulation of live birth (viviparity) and both male and female parental care. Previous research suggests that PRL also plays an important reproductive role in syngnathid fishes (seahorses, pipefish and seadragons), a group with a highly derived reproductive strategy, male pregnancy - how the PRL axis has come to be co-opted for male pregnancy remains unclear. We investigated the molecular evolution and expression of the genes for prolactin and its receptor (PRLR) in an evolutionarily diverse sampling of syngnathid fishes to explore how the co-option of PRL for male pregnancy has impacted its evolution, and to clarify whether the PRL axis is also involved in regulating reproductive function in species with more rudimentary forms of male pregnancy. In contrast to the majority of teleost fishes, all syngnathid fishes tested carry single copies of PRL and PRLR that cluster genetically within the PRL1 and PRLRa lineages of teleosts, respectively. PRL1 gene expression in seahorses and pipefish is restricted to the pituitary, while PRLRa is expressed in all tissues, including the brood pouch of species with both rudimentary and complex brooding structures. Pituitary PRL1 expression remains stable throughout pregnancy, but PRLRa expression is specifically upregulated in the male brood pouch during pregnancy, consistent with the higher affinity of pouch tissues for PRL hormone during embryonic incubation. Finally, immunohistochemistry of brood pouch tissues reveals that both PRL1 protein and PRLRa and Na+/K+ ATPase-positive cells line the inner pouch epithelium, suggesting that pituitary-derived PRL1 may be involved in brood pouch osmoregulation during pregnancy. Our data provide a unique molecular perspective on the evolution and expression of prolactin and its receptor during male pregnancy, and provide the foundation for further manipulative experiments exploring the role of PRL in this unique form of reproduction.

Hedgehog regulation of epithelial cell state and morphogenesis in the larynx.

The larynx enables speech while regulating swallowing and respiration. Larynx function hinges on the laryngeal epithelium which originates as part of the anterior foregut and undergoes extensive remodeling to separate from the esophagus and form vocal folds that interface with the adjacent trachea. Here we find that sonic hedgehog (SHH) is essential for epithelial integrity in the mouse larynx as well as the anterior foregut. During larynx-esophageal separation, low Shh expression marks specific domains of actively remodeling epithelium that undergo an epithelial-to-mesenchymal transition (EMT) characterized by the induction of N-Cadherin and movement of cells out of the epithelial layer. Consistent with a role for SHH signaling in regulating this process, Shh mutants undergo an abnormal EMT throughout the anterior foregut and larynx, marked by a cadherin switch, movement out of the epithelial layer and cell death. Unexpectedly, Shh mutant epithelial cells are replaced by a new population of FOXA2-negative cells that likely derive from adjacent pouch tissues and form a rudimentary epithelium. These findings have important implications for interpreting the etiology of HH-dependent birth defects within the foregut. We propose that SHH signaling has a default role in maintaining epithelial identity throughout the anterior foregut and that regionalized reductions in SHH trigger epithelial remodeling.

Mark Coventry Award: Efficacy of Saline Wash Plus Antibiotics Doped Polyvinyl Alcohol (PVA) Composite (PVA-VAN/TOB-P) in a Mouse Pouch Infection Model.

The efficacy of saline irrigation for treatment of periprosthetic infection (PJI) is limited by the presence of contaminated medical devices. This study evaluated treatment efficacy of locally placed polyvinyl alcohol (PVA)/bioceramic composite doped with vancomycin (PVA-VAN-P) or vancomycin and tobramycin (PVA-VAN/TOB-P) after saline irrigation in a mouse pouch infection model. Sutures were implanted into air pouches of BALB/cJ mice, then inoculated with Staphylococcus aureus. Mice were randomized into 6 groups (n= 6 each): (1) no bacteria; (2) bacteria without saline wash; (3) saline wash only; (4) saline wash+ PVA-P; (5) saline wash+ PVA-VAN-P, and (6) saline wash+ PVA-VAN/TOB-P. After 7 days, pouches were washed with saline alone or with additional injection of 0.2 mL of the composites. Sacrifice occurred 14 days after the washout. Histology was performed on the pouch tissues and bacteria cultures on the washout fluid. Bacterial culture (optical density) showed that infection persisted after saline irrigation (0.10±0.14) but was effectively eradicated by the addition of PVA-VAN-P (0.05±0.09) and PVA-VAN/TOB-P (0.002±0.003, P < .05). These effects were confirmed by histology. Importantly, no residues of the PVA-P were detected in either the pouch washouts or pouch tissues. PJI is common and problematic, and few innovations have changed clinical practice and/or outcome. Our data confirmed that the effect of saline irrigation was very limited in the presence of contaminated sutures. PVA-VAN/TOB-P was biodegradable, biocompatible, and effective in eradicating bacterial retention after saline irrigation. Application of PVA-VAN/TOB-P after saline irrigation could be an option for treatment of PJI and should be evaluated in future PJI animal models.

Human outgrowth knee fibroblasts from patients undergoing total knee arthroplasty exhibit a unique gene expression profile and undergo myofibroblastogenesis upon TGFβ1 stimulation.

Arthrofibrosis is characterized by excessive extracellular matrix (ECM) deposition that results in restricted joint motion after total knee arthroplasties (TKAs). Currently, treatment options are limited. Therefore, an in vitro model of knee-related myofibroblastogenesis is valuable to facilitate investigation of the arthrofibrotic process, diagnostic and therapeutic options. In this study, we obtained intraoperative posterior capsule (PC), quadriceps tendon (QT), and suprapatellar pouch (SP) tissues from the knees of four patients undergoing primary TKAs for osteoarthritis. From these tissues, we isolated primary cells by the outgrowth method and subsequently characterized these cells in the absence and presence of the pro-myofibroblastic cytokine, transforming growth factor beta 1 (TGFβ1). Light microscopy of knee outgrowth cells revealed spindle-shaped cells, and immunofluorescence (IF) analysis demonstrated staining for the fibroblast-specific markers TE-7 and vimentin (VIM). These knee outgrowth fibroblasts differentiated readily into myofibroblasts as reflected by enhanced α-smooth muscle actin (ACTA2) mRNA and protein expression and increased mRNA expression of collagen type 1 (COL1A1) and type 3 (COL3A1) with collagenous matrix deposition in the presence of TGFβ1. Outgrowth knee fibroblasts were more sensitive to TGFβ1-mediated myofibroblastogenesis than adipose-derived mesenchymal stromal/stem cells (MSCs). While outgrowth knee fibroblasts isolated from three anatomical regions in four patients exhibited similar gene expression, these cells are distinct from other fibroblastic cell types (i.e., Dupuytren's fibroblasts) as revealed by RNA-sequencing. In conclusion, our study provides an in vitro myofibroblastic model of outgrowth knee fibroblasts derived from patients undergoing primary TKA that can be utilized to study myofibroblastogenesis and assess therapeutic strategies for arthrofibrosis.

Oral cancer preventive potential of Polydatin: A nanoencapsulation approach

Over the period, several medicinal plants are recognized as attractive sources of anti-cancer molecules. About 35,000 plant organisms have been tested for possible anticancer activities by the National Cancer Center (NCI). Among them, reproducible anticancer behavior has been shown by around 3,000 plant organisms. Based on this information, we aimed to investigate the cancer preventive efficiency of Polydatin encapsulated PLGA nanoparticles (POL-PLGA-NPs) against 7,12 dimethylbenz (a) anthracene (DMBA) induced buccal pouch carcinogenesis by modulating the antioxidants process in male Syrian hamsters. Oral cancer was initiated on left side buccal pouches by applying DMBA (0.5%), (a known potent chemical carcinogen) on alternative days in a week (thrice a week) for a period of 14 weeks to provoke oral cancer. Sequences of oral squamous cell carcinoma are noted to be manifested exhibiting the formation of incidence, size, mass and development of tumor presented as preneoplastic and neoplastic lesions in hamsters oral buccal pouch tissues. At the same time, oral administration of POL-PLGA-NPs (30mg/kg.b.wt.) considerably increased glutathione peroxidase (GPx) and glutathione (GSH) levels in the buccal tissue thereby decreasing lipid peroxidation activity which resulted in neoplasms incidence in buccal tissue of DMBA painted animals. Thus, POL-PLGA-NPs activity seemed to be a potent cancer preventive agent.

Water extract of Artemisia scoparia Waldst. & Kitam suppresses LPS-induced cytokine production and NLRP3 inflammasome activation in macrophages and alleviates carrageenan-induced acute inflammation in mice

Ethnopharmacological relevanceArtemisia scoparia Waldst. & Kitam (A. scoparia) is a perennial herbal plant that is widely used as a folk remedy in Asian countries. Several studies have demonstrated that A. scoparia has various physiological effects, including anti-inflammation, anti-hypertension, anti-obesity, anti-hepatotoxicity, and anti-oxidant effects.Aim of the study: The objective of the present study was to examine the anti-inflammatory effects of water extract of A. scoparia (WAS). Materials and methodsMurine bone marrow-derived macrophages (BMDMs), human monocyte THP-1 and murine fibroblast 3T3-L1 cells were used for the in vitro experiments. Cell viability and cytokine production were determined by the MTT assay and ELISA, respectively. RT-PCR was performed to determine iNOS gene expression and the Griess reaction was used to measure nitrite levels. iNOS protein expression, activation of NF-κB and MAPKs, and cleavage of caspase-1 and IL-1β were determined by Western blot analysis. A carrageenan-induced mouse model of acute inflammation was used in the in vivo experiments. ResultsPretreatment with WAS concentration-dependently suppressed gene expression and IL-6, TNF-α, CXCL1 and iNOS protein levels in BMDMs stimulated with LPS. In addition, pretreatment with WAS inhibited LPS-induced production of IL-6 and TNF-α in THP-1 cells and CXCL1 in 3T3-L1. Furthermore, LPS induced phosphorylation of p65 in BMDMs, and this induction was dramatically suppressed by WAS pretreatment. We further investigated whether WAS regulates activation of the NLRP3 inflammasome, which is known to be essential for IL-1β processing. WAS inhibited the production of IL-1β, but not IL-6, in response to adenosine triphosphate (ATP) and monosodium uric acid (MSU) crystals in LPS-primed BMDMs. Cleavage of caspase-1 and IL-1β was also reduced by WAS. We finally evaluated the in vivo anti-inflammatory effects of WAS in a mouse model of carrageenan-induced acute inflammation. Subcutaneous administration of WAS reduced production of the inflammatory cytokines IL-6, TNF-α, CXCL1, and IL-1β. Recruitment of immune cells, mostly neutrophils, was also reduced by administration of WAS. Infiltration of inflammatory cells and edema in the submucosa of air pouch tissues were markedly improved in the WAS-treated groups. ConclusionsOur results indicate that WAS possesses potent anti-inflammatory properties. These findings suggest that A. scoparia is a candidate functional food targeting several inflammatory diseases.

Effect of Topical 2% Eucalyptus Extract on 5-FU-Induced Oral Mucositis in Male Golden Hamsters.

Oral mucositis is a common inflammatory complication among patients with cancer. This study evaluated the histopathological, stereological, and antioxidant markers of 2% eucalyptus extract in induced oral mucositis in male golden hamsters. In this animal study, oral mucositis was induced in 30 male golden hamsters by 5-FU (60 mg/kg) on days 0, 5, and 10 of the study. The cheek pouch was scratched with a sterile needle once daily on days 3 and 4. On days 14-17, 2% eucalyptus hydroalcoholic extract gel and Calendula officinalis extract gel groups were treated and then compared with a non-treated control group. The histopathological and stereological scores and the pouch content of malondialdehyde, as well as the activities of glutathione and myeloperoxidase in the pouch tissue, were evaluated. Histopathologic scores of oral mucositis were lower in the eucalyptus gel group than those of the calendula and control groups (p<0.05). Also, a lower malondialdehyde level and higher myeloperoxidase and glutathione activities were detected in the eucalyptus group in comparison to the calendula and control groups (p<0.001). The thickness of the mucosa and submucosa increased in the eucalyptus group. The numerical density of the fibroblast and the volume density of the collagen significantly increased in the eucalyptus group. In conclusion, the use of eucalyptus hydroalcoholic extract may be associated with reduced intensity of oral mucositis, diminished concentration of malondialdehyde, increased activity of myeloperoxidase and glutathione, increased volume of mucosa and submucosa, increased fibroblast and collagen in the induced oral mucositis in golden hamsters undergoing 5-FU consumption.

PAX1 is essential for development and function of the human thymus.

We investigated the molecular and cellular basis of severe combined immunodeficiency (SCID) in six patients with otofaciocervical syndrome type 2 who failed to attain T cell reconstitution after allogeneic hematopoietic stem cell transplantation, despite successful engraftment in three of them. We identified rare biallelic PAX1 rare variants in all patients. We demonstrated that these mutant PAX1 proteins have an altered conformation and flexibility of the paired box domain and reduced transcriptional activity. We generated patient-derived induced pluripotent stem cells and differentiated them into thymic epithelial progenitor cells and found that they have an altered transcriptional profile, including for genes involved in the development of the thymus and other tissues derived from pharyngeal pouches. These results identify biallelic, loss-of-function PAX1 mutations as the cause of a syndromic form of SCID due to altered thymus development.

Evaluation of the Effect of a Topical Gel Form of Pistacia Atlantica and Trachyspermum Ammi on Induced Oral Mucositis in Male Golden Hamsters by Bio-Marker Indices and Stereological Assessment.

Statement of the Problem: Oral mucositis (OM) is a common inflammatory complication consequent to chemotherapy or radiotherapy in cancer patients that affects their quality of life. Therefore, finding an effective treatment for OM is always imperative. Purpose:The purpose of this experimental study was to measure the stereological, anti-inflammatory and antioxidant markers of hydro-alcoholic extract of Pistacia Atlantica (P. atlantica) leaves, Trachyspermum Ammi (T. ammi) fruits and their mixture on induced OM in hamsters. Materials and Method:In Experimental study, OM was generated by the method employed by Tanideh et al., in 90 hamsters, by 5-FU (5-fluorouracil 65 mg/kg, IP). All the animals were divided into 5 groups: control, topical gel base, hydro-alcoholic extract 3% of P. atlantica, hydro-alcoholic extract 1% of T. ammi, and a mixture of the extracts of the two plants. On days 13, 15, and 17, the cheek pouch samples were harvested. Stereological scores and the levels of malondialdehyde (MDA), Superoxide dismutase (SOD), myeloperoxidase (MPO), and interleukin-1B (IL-1B) in the pouch tissue were measured. Results:Lower levels of MPO, MDA, and IL-1B and an increase of SOD level were discovered in the mixture-treated group in comparison with other groups. As an aspect of MDA, no significant differences were detected between the topical gel form of P. atlantica and T. ammi groups on days 13 (p= 0.648), 15 (p= 0.981) and 17 (p= 0.540). No variations of MPO were detected among P. atlantica and T. ammi groups on days 13 (p= 0.159) and 15 (p= 0.694); however, the MPO activity of the T. ammi group was significantly lower than the P. atlantica group on day 17 (5.57±0.56 vs. 6.48±0.35 U/ml, p< .001). The volume density of the epithelium increased in the mixture- treated group (p< 0.05). Conclusion:The results of this study indicated that the mixture of P. atlantica leaves and T. Ammi fruits might be effective in the treatment of OM.

Impact of Whole Plant Extract of Pergularia daemia on Glycoproteins in Dimethylbenz(A)Anthracene Induced Hamster Buccal Pouch Carcinogenesis

Background: Oral squamous cell carcinoma is a major component of a diverse group of neoplasms often referred to as ‘head and neck cancer’. Frequent smoking and/or alcohol consumption are two major risk factors for oral cancer. Objectives: The present study was aimed to investigate the protective role of Pergularia daemia ethyl acetate and methanolic extracts (PDEAE and PDME, respectively) on glycoproteins in dimethylbenz(a) anthracene (DMBA) induced hamster buccal pouch carcinogenesis. Materials and Methods: Male golden Syrian hamsters were used and divided into six groups. Group 2 carried 0.5% 7,12-DMBA painting on left buccal pouch. Groups 3 and 4 were treated with DMBA and 300 mg/kg bwt of PDEAE and PDME by intragastric administration. Remaining groups served as untreated control. All the experiments were performed within 14 weeks. Results: Body weight loss and 100% tumor incidence were observed treated hamsters with DMBA alone, whereas administration of PDEAE and PDME in animals with oral cancer caused significant alterations in body weight and tumor incidence. Further, plasma and buccal pouch tissue glycoprotein levels were increased and erythrocyte glycoprotein levels were depleted in DMBA treated hamsters. The levels were significantly reversed in hamsters treated with PDEAE and PDME at 300 mg/kg bwt. Conclusion: PDEAE and PDME produce a significant protective effect against DMBA induced oral cancer by altering glycoproteins levels.

Increase in Processing Factors Is Involved in Skewed MicroRNA Expression in Patients with Ulcerative Colitis Who Develop Small Intestine Inflammation after Pouch Surgery.

A large-scale increase in microRNA (miRNA) expression was observed in patients with ulcerative colitis who underwent pouch surgery and developed inflammation of the pouch (pouchitis). In this study, we assessed miRNA expression in these patients and investigated how regulation of its expression changes in the setting of pouchitis. Autologous samples that included mucosal biopsies, peripheral blood cells, and plasma were collected from the patients. Candidate primary and mature miRNA expressions were analyzed by quantitative polymerase chain reaction. A human intestinal epithelial cell line was used to test DICER activity, and the expression of key miRNA processing factors was analyzed by Western blot. miRNA-424 and its potential target serotonin reuptake transporter (SERT) expressions were examined by quantitative reverse transcription polymerase chain reaction and Western blot in human pouch tissues and in a human intestinal epithelial cell line stimulated with inflammatory cytokines TNF-α, IL-1β, and INF-γ. Candidate miRNA expression and protein expression of DICER-1, EXPORTIN-5, and AGO-2 were increased in association with pouch inflammation. Similarly, inflammatory cytokines increased protein expression of DICER-1, EXPORTIN-5, and AGO-2 and DICER activity in the epithelial cell line. The miRNA-424 expression increased whereas SERT expression decreased in the patients' mucosa. Similarly, incubation of the epithelial cell line with inflammatory cytokines resulted in increased miRNA-424 and decreased SERT mRNA and protein expression. The miRNA expression and processing are augmented in the inflamed intestinal mucosa of patients with pouchitis. These alterations are accompanied by increased expression of proteins involved in miRNA processing, suggesting that pouch inflammation contributes to miRNA processing and expression.

Nitrosative stress uncovers potent β2-adrenergic receptor-linked vasodilation further enhanced by blockade of clathrin endosome formation.

This study investigated the effect of sodium nitroprusside (SNP) preexposure on vasodilation via the β-adrenergic receptor (BAR) system. SNP was used as a nitrosative/oxidative proinflammatory insult. Small arterioles were visualized by intravital microscopy in the hamster cheek pouch tissue (isoflurane, n = 45). Control dilation to isoproterenol (EC50: 10-7 mol/l) became biphasic as a function of concentration after 2 min of exposure to SNP (10-4 M), with increased potency at picomolar dilation uncovered and decreased efficacy at the micromolar dilation. Control dilation to curcumin was likewise altered after SNP, but only the increased potency at a low dose was uncovered, whereas micromolar dilation was eliminated. The picomolar dilations were blocked by the potent BAR-2 inverse agonist carazolol (10-9 mol/l). Dynamin inhibition with dynasore mimicked this effect, suggesting that SNP preexposure prevented BAR agonist internalization. Using HeLa cells transfected with BAR-2 tagged with monomeric red fluorescent protein, exposure to 10-8-10-6 mol/l curcumin resulted in internalization and colocalization of BAR-2 and curcumin (FRET) that was prevented by oxidative stress (10-3 mol/l CoCl2), supporting that stress prevented internalization of the BAR agonist with the micromolar agonist. This study presents novel data supporting that distinct pools of BARs are differentially available after inflammatory insult. NEW & NOTEWORTHY Preexposure to an oxidative/nitrosative proinflammatory insult provides a "protective preconditioning" against future oxidative damage. We examined immediate vasoactive and molecular consequences of a brief preexposure via β-adrenergic receptor signaling in small arterioles. Blocked receptor internalization with elevated reactive oxygen levels coincides with a significant and unexpected vasodilation to β-adrenergic agonists at picomolar doses.

Biochemical, hematological, and pathological related healing effects of Elaeagnus angustifolia hydroalcoholic extract in 5-fluorouracil-induced oral mucositis in male golden hamster.

Oral mucositis (OM) is one of the cancer chemotherapy-related side effects which can affect the quality of life of affected patients. This study was designed to investigate the healing effect of Elaeagnus angustifolia in 5-flurouracil (5-FU)-induced OM in golden hamster. Fifty-six adult male golden hamsters received three intraperitoneal injections of 5-FU at a dose of 60mg/kg on days 0, 5, and 10. The cheek pouch mucosa was scratched superficially under local anesthesia. Then, two horizontal scratches were made across the everted cheek pouch on days 3 and 4. All treatments were started on day 12 for equal number of animals in control group with no treatments, gel base group that was treated with carboxy methyl cellulose as gel base which used in preparation of the topical gel, topical gel group that used gel containing 10% hydroalcoholic extract of E. angustifolia (HEEA) topically, and dietary group which was treated with 300mg/kg HEEA. At 2 and 5days after treatment, blood and pouch tissue sampling were done and analyzed for blood composition, tissue malondialdehyde (MDA) level, and myeloperoxidase (MPO) and superoxide dismutase (SOD) activities plus histopathological evaluations. Both topically and orally HEEA-treated groups showed a significant relief in OM compared to the control and base gel groups. However, the systemic form had higher efficiency in some parts especially decreasing the MPO (0.27±0.17 vs. 0.56±0.17IU/L) and increasing SOD (6.46±0.15 vs. 5.36±0.18IU/L) activities in pouch tissue in comparison to topical form mostly at 5days after treatment. It seems that hydroalcoholic extract of E. angustifolia can be used as an appropriate drug choice for the treatment of oral mucositis based on its healing stimulatory and anti-inflammatory properties.