Lamina Propria Research Articles

Abstract B039: T cell recruitment dynamics at different stages of primary and metastatic colorectal cancer

Abstract Colorectal cancer (CRC) is the fourth deadliest cancer worldwide, primarily due to metastasis. The current standard therapy for patients with CRC includes neoadjuvant therapies combined with surgery, often resulting in severe complications that affect patients’ lifestyles. Hence, there is a critical need to improve our understanding of the tumor microenvironment to improve the current therapeutics for metastatic CRC. We characterized T cell dynamics in response to tumors at early (1-2 weeks) versus late stages (6-7 weeks) of CRC development in different colon compartments (tumor, epithelium, lamina propria), as well as in colon-draining lymph nodes and the liver as potential metastasis sites. To achieve this, we injected by colonoscopy tumor AKP mouse organoids (mutations in APC, Kras, and P53 genes) or metastatic AKP mouse organoids in the colon walls of a novel fate-mapping strain (iSell-tomato). This model allows for the labelling of peripheral T cells that are recruited to the tissue or tumor sites. Our initial data showed an enhanced recruitment of CD4 and CD8ab T cells to the colon epithelium in early stage AKP (non-metastatic) tumor models compared to late-stage AKP and metastatic-AKP models at both stages. These recruited cells exhibited an enhanced anti-tumorigenic profile characterized by the production of IFNg and reduced frequency of FoxP3+ CD4 regulatory T cells (Tregs). In contrast, T cells recruited to early stage metastatic-AKP tumors and late-stage tumors of the colon and liver exhibited enhanced exhaustion characterized by an increased frequency of PD1+ CD4 and CD8ab T cells. These data suggest a time- and aggressiveness-dependent effect of tumors on immune cell infiltrates that, in turn, affect tumor progression and metastasis. Citation Format: Marwa A Saad, Angelina Bilate, Daniel Mucida. T cell recruitment dynamics at different stages of primary and metastatic colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B039.

A case of large-cell undifferentiated carcinoma of the bladder

Abstract Large-cell undifferentiated carcinoma of the urinary bladder is an extremely rare and aggressive neoplasm. We present a unique case of painless gross hematuria and a past surgical history of cystolithotomy. The patient underwent transurethral resection of the bladder tumor, which revealed high-grade urothelial cell carcinoma with lamina propria involvement. Subsequent radical cystoprostatectomy with orthotopic neobladder urinary diversion and pelvic lymphadenectomy was performed, and the postoperative pathologic examination indicated large-cell undifferentiated. This case report highlights the importance of accurate diagnosis and management for this rare malignancy and adds to the limited existing literature on Large-cell undifferentiated carcinoma.

Delineation of the healthy rabbit tongue by immunohistochemistry – A technical note

In the oral cavity the tongue is an important muscular organ that supports the swallowing of food and liquids. It is responsible for the sense of taste, based on the many different taste buds it contains. Research in the field of tongue diseases demands for suitable preclinical models. The healthy rabbit tongue may therefore serve as baseline and reference for the pathological situation. With this consideration, we covered the fixation and histological stainings as well as the immunohistochemical labelling of the healthy rabbit tongue. In this technical note, initial choice of the fixative is discussed, with a comparison of formalin fixation and subsequent paraffin embedding versus cryopreservation. Moreover, we delineate the effect of an antigen retrieval step for formalin fixation by several examples. Finally, we provide ECM markers collagen I, collagen III, fibronectin, α-SMA and elastin staining as well as ki67 for proliferative status and PAR-2 protein expression as a marker for inflammatory status and nociception in tongue sections, mainly from the tongue body. Technically, we found superiority of paraffin sections for collagen I, collagen III, fibronectin, ki67 and α-SMA labelling, for selected detections systems. As for ECM components, the lamina propria was very rich in collagen and fibronectin, while the muscular body of the tongue showed only collagen and fibronectin positive areas between the muscle fibers. Moreover, α-SMA was clearly expressed in the walls of arteries and veins. The inflammatory marker PAR-2 on the other hand was prominently expressed in the salivary glands and to some extent in the walls of the vessels. Particular PAR-2 expression was found in the excretory ducts of the tongue. This technical note has the aim to provide baseline images that can be used to compare the pathological state of the diseased rabbit tongue as well as for inter-species comparison, such as mouse or rat tongue. Finally, it can be used for the comparison with the human situation.

Spatial mapping of ectonucleotidase gene expression in the murine urinary bladder.

Purinergic signaling is important for normal bladder function, as it is thought to initiate the voiding reflex and modulate smooth muscle tone. The availability of adenine nucleotides and nucleosides (aka purines) at receptor sites of various cell types in the bladder wall is regulated by ectonucleotidases (ENTDs). ENTDs hydrolyze purines such as adenosine 5'-triphosphate (ATP) and adenosine 5'-diphosphate (ADP) with varying preference for the individual substrate. Therefore, the end effect of extracellular purines may depend significantly on the type of ENTD that is expressed in close proximity to the target cells. ENTDs likely have distinct cellular associations, but the specific locations of individual enzymes in the bladder wall are poorly understood. We used RNAscope™, an RNA in situ hybridization (ISH) technology, to visualize the distribution and measure the levels of gene expression of the main recognized ectonucleotidases in large high-resolution images of murine bladder sections. The relative gene expression of ENTDs was Entpd3 > Alpl >> Enpp1 = Entpd2 >> Enpp3 > Entpd1 (very low to no signal) in the urothelium, Entpd1 ≥ Entpd2 >> Enpp3 > Enpp1 = Alpl ≥ Nt5e (very low to no signal) in the lamina propria, and Entpd1 >> Nt5e = Entpd2 >> Enpp1 > Alpl = Enpp3 in the detrusor. These layer-specific differences might be important in compartmentalized regulation of purine availability and subsequent functions in the bladder wall and may explain reported asymmetries in purine availability in the bladder lumen and suburothelium/lamina propria spaces.

Colonic Duplication Cyst with Gastric Heterotopia Haemorrhage and Mucosal Ulceration: A Case Report

Present report relates to duplication of ascending colon with cyst formation along with ectopia of gastric glands in lamina propria of colon. Heterotopia or ectopia is defined as presence of an organ outside its normal anatomic site. Ileum is the commonest site of heterotopia in gastrointestinal tract. Rarely, heterotopia may involve ascending colon. In the present case, cyst wall also had a thickened area, measuring 1×1×0.5 cm, consisting of hypertrophic muscularis. Cyst wall showed ulceration in continuity with colonic columnar epithelium. In addition, lamina propria also showed mixed leucocytic infiltration. The lesion was finally diagnosed as duplication of ascending colon with cyst formation and heterotopia of benign gastric glands, hemorrhage and ulceration. Keywords: Large intestinal cyst with two corresponding parts, ectopia of gastric glands with ulceration and haemorrhage.

Intestinal microbiota-specific Th17 cells possess regulatory properties and suppress effector T cells via c-MAF and IL-10

Commensal microbes induce cytokine-producing effector tissue-resident CD4+ Tcells, but the function of these Tcells in mucosal homeostasis is not well understood. Here, we report that commensal-specific intestinal Th17 cells possess an anti-inflammatory phenotype marked by expression of interleukin (IL)-10 and co-inhibitory receptors. The anti-inflammatory phenotype of gut-resident commensal-specific Th17 cells was driven by the transcription factor c-MAF. IL-10-producing commensal-specific Th17 cells were heterogeneous and derived from a TCF1+ gut-resident progenitor Th17 cell population. Th17 cells acquired IL-10 expression and anti-inflammatory phenotype in the small-intestinal lamina propria. IL-10 production by CD4+ Tcells and IL-10 signaling in intestinal macrophages drove IL-10 expression by commensal-specific Th17 cells. Intestinal commensal-specific Th17 cells possessed immunoregulatory functions and curbed effector Tcell activity invitro and invivo in an IL-10-dependent and c-MAF-dependent manner. Our results suggest that tissue-resident commensal-specific Th17 cells perform regulatory functions in mucosal homeostasis.

Russell body gastritis: a case series and review of literature

Abstract Introduction/Objective Russell body gastritis is considered a rare finding characterized by Russell body-containing plasma cells in the lamina propria. Patients typically present with nonspecific findings. In this study, clinical and histopathological features of 3 cases with incidental findings of Russell body gastritis (RBG).<quillbot-extension- portal></quillbot-extension-portal> Methods/Case Report The Epic SlicerDicer tool was used to identify institutional cases between April 2022 to April 2023 using the keyword “Russell Body”. Using the PubMed database, we searched for articles published between January 2003 and April 2023, with the keyword "Russell body gastritis". Studies that described novel RBG cases were included.<quillbot-extension- portal></quillbot-extension-portal> Results (if a Case Study enter NA) The three cases were female with a mean age of 47 (42-51). Two patients were presented with chronic dyspepsia and one patient was being evaluated for right upper quadrant pain. The endoscopic finding for one patient was significant for multifocal erythematous gastric mucosa and the other two patients had unremarkable findings. Biopsies from the three patients revealed active gastritis in 2 cases and inactive chronic gastritis in 1 case. A dense accumulation of plasma cells with Mott cells and Russell bodies were identified in the lamina propria. Helicobacter pylori was identified on H. pylori stain in one case. Upon further investigation, CD138 highlights the plasma cells and kappa and lambda ISH demonstrated polyclonal plasma cells and cytokeratin AE1:AE3 was negative for a carcinoma A review of 64 previously reported cases of RBG shows that the average age of presentation is 63. Half of the cases were associated with Helicobacter pylori infection. Other associated conditions include multiple myeloma (3%) and HIV (6%). There have been 12 cases with monoclonal plasma cells with 10 cases of kappa-type monoclonality (including one case of duodenitis) and 1 case of lambda-type monoclonality.<quillbot-extension-portal></quillbot- extension-portal> Conclusion Most cases of RBG have been linked with helicobacter pylori. However, the underlying etiology is not well understood. Differentials include plasmacytoma, MALToma, and signet ring carcinoma. The clinical significance and its long-term effect remain unknown. Our report is the largest review of RBG. With its wide range of clinical presentations, additional cases are to define this entity better.<quillbot-extension-portal></quillbot-extension-portal>

Inhibition of microRNA-34c reduces detrusor ROCK2 expression and urinary bladder inflammation in experimental cystitis

Interstitial cystitis (IC), also called painful bladder syndrome (PBS), is 2 to 5 times more common in women than in men, yet its cause and pathogenesis remain unclear. In our study using the cyclophosphamide (CYP)-induced mouse model of cystitis, histological evaluation of the urinary bladder (UB) lamina propria (LP) showed immune cell infiltrations, indicating moderate to severe inflammation. In this study, we noticed a differential expression of a subset of microRNAs (miRs) in the UB cells (UBs) of CYP-induced cystitis as compared to the control. UB inflammatory scores and inflammatory signaling were also elevated in CYP-induced cystitis as compared to control. We identified eight UBs miRs that exhibited altered expression after CYP induction and are predicted to have a role in inflammation and smooth muscle function (miRs-34c-5p, −34b-3p, −212-3p, −449a-5p, −21a-3p, −376b-3p, −376b-5p and − 409-5p). Further analysis using ELISA for inflammatory markers and real-time PCR (RT-PCR) for differentially enriched miRs identified miR-34c as a potential target for the suppression of UB inflammation in cystitis. Blocking miR-34c by antagomir ex vivo reduced STAT3, TGF-β1, and VEGF expression in the UBs, which was induced during cystitis as compared to control. Interestingly, miR-34c inhibition also downregulated ROCK2 but elevated ROCK1 expression in bladder and detrusor cells. Thus, the present study shows that targeting miR-34c can mitigate the STAT3, TGF-β, and VEGF, inflammatory signaling in UB, and suppress ROCK2 expression in UBs to effectively suppress the inflammatory response in cystitis. This study highlights miR-34c as a potential biomarker and/or serves as the basis for new therapies for the treatment of cystitis.

Microbiota-Specific T Cells Contribute to Graft-Versus-Host Disease after Allogeneic Stem Cell Transplantation

Background: Allogeneic T-cell expansion is the primary determinant of graft-versus-host disease (GVHD), and current dogma dictates that this is driven by histocompatibility antigen disparities between donor and recipient. This paradigm represents a fixed genetic system within which donor T cells interact with recipient-derived peptide-MHC complexes. However, the current model does not account for potential cognate interactions between donor T cells and microbe-derived antigen presented by the recipient. Prior studies in healthy patients have demonstrated the existence of circulating αβT cells targeting common bacterial species in the gastrointestinal (GI) tract. Whether microbe-specific T cells derived from the donor graft impact GVHD outcomes has not been studied. We tested the hypothesis that the recipient GI microbiota serve as a source of cognate antigen, contributing to clonotypic donor T-cell expansion and induction of GVHD. Methods: Functional studies utilized allogeneic stem cell transplant models in murine systems including both matched (Marilyn Tg to B6 male) and mismatched (B6 to B6D2F1) transplants as well as inducible MHCII knockout mice. Microbiota-specific transgenic T cells (MSTCs) targeting commensal flagellin (CBir1) and vendor-specific microbiome (segmented filamentous bacteria - SFB) in the context of I-A(b) were incorporated as part of the donor graft. Mechanistic studies utilized flow cytometry, cytokine stimulation and profiling, single-cell RNA sequencing, gene set enrichment analysis, NFAT-GFP T-hybridoma reporters, live-cell imaging (xCelligence RTCA eSight), and tissue analysis with RNA in-situ hybridization (ISH). Results: MSTCs lethally augmented GVHD when given as part of the donor graft in respectively colonized recipients. Taconic B6 male recipients (SFB-bearing) developed lethal GVHD when both donor Marilyn (Allo) Tg T cells and SFB T cells were introduced, but not when either transgenic T cell was introduced alone ( Figure A, p<0.0001). In contrast, Jackson B6 recipients (SFB-absent) receiving both Allo and SFB T cells did not develop lethal GVHD ( Figure A, p<0.0001), confirming functional relevance of microbe-specificity among the donor T cell pool. Lethal GVHD was also seen with addition of CBir1 T cells to the donor graft but not with addition of bulk T cells (log-rank p<0.0001). Similar results were seen in a mismatched transplant system. To determine how MSTCs promoted lethality in the context of an allogeneic (allo) response, we performed scRNA sequencing of T cells harvested in the recipient spleen at day 6 post-transplant (prior to mortality onset). Post-transplant MSTCs harbored a naïve phenotype (Sell high/CD44 low/CD69 low) when given as the primary source of donor T cells but shifted to an early activated phenotype (Sell int/CD44 low/CD69 high/TCF7 low) when Allo T cells were concurrently introduced (ANOVA q<0.01). MSTC activation was confirmed with augmented expression of IFNγ upon PMA/ionomycin stimulation observed in allo vs. syngeneic response (Wilcoxon rank-sum, p< 0.01). Introduction of Allo T cells increased the abundance of MSTCs in the mesenteric lymph nodes (day 5, 6, Wilcoxon, p<0.01). Ablation of MHCII on the GI epithelial surface using Villin-CreERT2 I-Ab flox recipients abrogated microbe-specific T cell activation in co-culture with recipient GI intraepithelial cells harvested at day 6 post-transplant vs. non-ablated control (8, 12, 24-hr NFAT-GFP intensity, p<0.001). RNA ISH probes targeting the CDR3 region of anti-microbial CBir1 T cells demonstrated an increase in MSTC infiltration at day 6 in the recipient GI lamina propria during an allo response ( Figure B) compared to a syngeneic response (# of MSTCs/terminal ileum cross section at day 6, Wilcoxon p<0.01). Conclusions: We confirm that clonotypic T cell expansion following allogeneic transplantation is not only driven by donor-recipient genetics, as reflected by major and minor histocompatibility matching, but also by differences in microbiota composition that can serve as a source of antigenic stimulation, providing a feed-forward mechanism to propagate GVHD. Furthermore, in the context of an alloreactive T cell response that damages gut integrity, microbial antigens can be presented by GI epithelia, allowing the gut to be concurrently targeted by microbiota specific T cells whose function is additionally enhanced by the alloreactive response.

Macrophage polarization toward M1 phenotype in T cell transfer colitis model

BackgroundT cell transfer colitis model is often used to study the CD4+ T cell functions in the intestine. However, the specific roles of macrophages in colitis remain unclear. In this study, we aimed to evaluate the phenotype and functions of macrophages in the colonic lamina propria (LP) in a colitis model.MethodsColitis was induced in scid mice via the adaptive transfer of CD4+CD45RBhi T cells. Then, flow cytometry was used to determine the number of macrophages in the colonic LP and expression of cytokines in macrophages at the onset of colitis. Moreover, M1/M2 macrophage markers were detected in the colonic LP during colitis development using high-dimensional single-cell data and gating-based analyses. Expression levels of M1 markers in macrophages isolated from the colonic LP were measured using quantitative reverse transcription-polymerase chain reaction. Additionally, macrophages were co-cultured with T cells isolated from the colon to assess colitogenic T cell activation.ResultsInfiltration of macrophages into the colon increased with the development of colitis in the T cell transfer colitis model. M1/M2 macrophage markers were observed in this model, as observed in the colon of patients with inflammatory bowel disease (IBD). Moreover, number of M1 macrophages increased, whereas that of M2 macrophages decreased in the colonic LP during colitis development. M1 macrophages were identified as the main source of inflammatory cytokine production, and colitogenic T cells were activated via interactions with these macrophages.ConclusionsOur findings revealed that macrophages polarized toward the M1 phenotype in LP during colitis development in the T cell transfer colitis model. Therefore, the colitis model is suitable for the evaluation of the efficacy of macrophage-targeted drugs in human IBD treatment. Furthermore, this model can be used to elucidate the in vivo functions of macrophages in the colon of patients with IBD.

Effect of acupuncture on the expression of neuropeptides and related inflammatory factors in rats with diarrhea-predominant irritable bowel syndrome.

To observe the effect of acupuncture on the expressions of neuropeptides and related inflammatory factors in rats with diarrhea-predominant irritable bowel syndrome(IBS-D), so as to explore the mechanism of acupuncture in the treatment of IBS-D. Male Wistar rats were randomly divided into blank group, model group, medication group, and acupuncture group, with 6 rats in each group. Except for the blank group, the other groups were subjected to 14-day "acetic acid enema + restraint stress" to establish the IBS-D rat model. After successful modeling, the medication group received gavage of pinaverium bromide(15 mg/kg) once a day, and the acupuncture group received acupuncture at "Baihui"(GV20) and bilateral "Tianshu"(ST25), "Shangjuxu"(ST37), "Zusanli"(ST36), and "Taichong"(LR3) for 20 min every day, both groups were treated continuously for 14 days. The general state of the rats in each group was observed, and the body weight of the rats was measured. The open-field experiment was conducted to measure the horizontal and vertical movements, and the number of fecal pellets of rats. The histopathological morphology of hypothalamus and colon of rats was observed by HE staining. Toluidine blue staining was used to observe and count the mast cells(MCs) in the colon tissue of rats. ELISA was used to detect the serum contents of tumor necrosis factor-α(TNF-α) and interleukin(IL)-10. Real-time fluorescence quantitative PCR was performed to detect the mRNA expressions of calcitonin gene-related peptide(CGRP) in the hypothalamus and colon tissue. Western blot was used to detect the expressions of corticotropin-releasing factor(CRF) in the hypothalamus and colon tissue. HE staining showed that there was inflammatory cell infiltration in the lamina propria of colon in the model group, and it was reduced in the other groups. Compared with the blank group, the model group showed significantly decreased body weight, decreased walking distance and upright times in open field experiment, decreased serum IL-10 contents(P<0.05, P<0.01), increased fecal pellet number (P<0.01), increased MC number in the colon tissue, serum TNF-α contents, and CGRP mRNA expressions and CRF expressions in the hypothalamus and colon tissue(P<0.01). Compared with the model group, both medication and acupuncture groups showed significantly increased body weight, walking distance and upright times in the open-field experiment, and serum IL-10 contents(P<0.01, P<0.05), significantly decreased fecal pellet number (P<0.05), significantly decreased MC number in the colon tissue, serum TNF-α contents, and CGRP mRNA expressions in the hypothalamus and colon tissue(P<0.01)；at the same time, the acupuncture group showed significantly decreased CRF expressions in the hypothalamus and colon tissue(P<0.01, P<0.05). There was no significant difference in the above indicators between the medication group and the acupuncture group. Acupuncture can improve the general and emotional state, inflammatory response, and neuropeptide expression in rats with IBS-D, and alleviate the symptoms of IBS-D, which may be related to the regulation of neuropeptides and inflammatory factors levels.

Gut–Brain Axis, Microbiota and Probiotics—Current Knowledge on Their Role in Irritable Bowel Syndrome: A Review

Irritable bowel syndrome (IBS) is a common digestive disorder with a significant impact on both individuals and society in terms of quality of life and healthcare costs. A growing body of research has identified various communication pathways between the microbiota and the brain in relation to motility disorders, with the gut–brain axis being key to the pathogenesis of IBS. Multiple factors contribute to the pathogenetic pathways in IBS, including immune mechanisms, psychosocial factors, increased oxidative stress and pro-inflammatory cytokine release, as well as genetic and hormonal factors. Increased permeability of the normal intestinal barrier allows bacterial products to access the lamina propria, providing a mechanism for perpetuating chronic inflammation and characteristic symptoms. The microbiota influences inflammatory processes in IBS by altering the balance between pro-inflammatory factors and host defence. Probiotics modulate the pathophysiological mechanisms involved in IBS by influencing the composition of the microbiota and improving intestinal motility disorders, visceral hypersensitivity, immune function of the intestinal epithelium, metabolic processes in the intestinal lumen, dysfunction of the microbiota-GBA, and are recognised as effective and safe in IBS therapy. Our study aimed to provide a comprehensive overview of the relationship between the gut–brain axis, microbiota, and IBS, based on current information.

Effect of Testosterone on the Thyroarytenoid Muscle and Lamina Propria of Female Rat Vocal Folds.

To analyze the effects of androgen therapy on the thyroarytenoid (TA) muscle, expression of androgen receptors (ARs) and hyaluronic acid (HA) concentration in the vocal folds (VFs) of adult female rats. Twenty-one adult female Wistar rats were divided into experimental and control groups. The experimental group received weekly intramuscular injections of nandrolone decanoate for 9 weeks. Following euthanasia and dissection of the VFs, histomorphometric analysis of the TA muscle, immunohistochemical evaluation of ARs, and measurement of HA concentration using the ELISA-like fluorimetric method were performed. The experimental group exhibited a significantly larger mean fiber cross-sectional area in the TA muscle compared to the control group (434.3 ± 68.6 μm2 versus 305.7 ± 110.1 μm2 ; p = 0.029), indicating muscle hypertrophy. There was no significant difference in the number of muscle fibers. The experimental group showed higher expression of ARs in the lamina propria (62.0% ± 30.3% versus 22.0% ± 22.8%; p = 0.046) and in the TA muscle (45.0% ± 22.6% versus 18.3% ± 9.8%; p = 0.024). There was no significant difference in the concentration of HA. Exposure of adult female rats to androgen therapy resulted in hypertrophy of the TA muscle and increased expression of ARs in the VFs. The TA muscle seems to be the primary target of testosterone action in the VF, and the up-regulation of ARs might contribute to the persistent deepening of the voice. Not applicable Laryngoscope, 2023.

Gasdermin D is the only Gasdermin that provides protection against acute Salmonella gut infection in mice.

Gasdermins (GSDMs) share a common functional domain structure and are best known for their capacity to form membrane pores. These pores are hallmarks of a specific form of cell death called pyroptosis and mediate the secretion of pro-inflammatory cytokines such as interleukin 1β (IL1β) and interleukin 18 (IL18). Thereby, Gasdermins have been implicated in various immune responses against cancer and infectious diseases such as acute Salmonella Typhimurium (S.Tm) gut infection. However, to date, we lack a comprehensive functional assessment of the different Gasdermins (GSDMA-E) during S.Tm infection in vivo. Here, we used epithelium-specific ablation, bone marrow chimeras, and mouse lines lacking individual Gasdermins, combinations of Gasdermins or even all Gasdermins (GSDMA1-3C1-4DE) at once and performed littermate-controlled oral S.Tm infections in streptomycin-pretreated mice to investigate the impact of all murine Gasdermins. While GSDMA, C, and E appear dispensable, we show that GSDMD i) restricts S.Tm loads in the gut tissue and systemic organs, ii) controls gut inflammation kinetics, and iii) prevents epithelium disruption by 72 h of the infection. Full protection requires GSDMD expression by both bone-marrow-derived lamina propria cells and intestinal epithelial cells (IECs). In vivo experiments as well as 3D-, 2D-, and chimeric enteroid infections further show that infected IEC extrusion proceeds also without GSDMD, but that GSDMD controls the permeabilization and morphology of the extruding IECs, affects extrusion kinetics, and promotes overall mucosal barrier capacity. As such, this work identifies a unique multipronged role of GSDMD among the Gasdermins for mucosal tissue defense against a common enteric pathogen.

Depth-Resolved Attenuation Mapping of the Vaginal Wall under Prolapse and after Laser Treatment Using Cross-Polarization Optical Coherence Tomography: A Pilot Study.

Vaginal wall prolapse is the most common type of pelvic organ prolapse and is mainly associated with collagen bundle changes in the lamina propria. Neodymium (Nd:YAG) laser treatment was used as an innovative, minimally invasive and non-ablative procedure for the treatment of early-stage vaginal wall prolapse. The purpose of this pilot study was to assess connective tissue changes in the vaginal wall under prolapse without treatment and after Nd:YAG laser treatment using cross-polarization optical coherence tomography (CP OCT) with depth-resolved attenuation mapping. A total of 26 freshly excised samples of vaginal wall from 26 patients with age norm (n = 8), stage I-II prolapses without treatment (n = 8) and stage I-II prolapse 1-2 months after Nd:YAG laser treatment (n = 10) were assessed. As a result, for the first time, depth-resolved attenuation maps of the vaginal wall in the B-scan projection in the co- and cross-polarization channels were constructed. Two parameters within the lamina propria were target calculated: the median value and the percentages of high (≥4 mm-1) and low (<4 mm-1) attenuation coefficient values. A significant (p < 0.0001) decrease in the parameters in the case of vaginal wall prolapse compared to the age norm was identified. After laser treatment, a significant (p < 0.0001) increase in the parameters compared to the normal level was also observed. Notably, in the cross-channel, both parameters showed a greater difference between the groups than in the co-channel. Therefore, using the cross-channel achieved more reliable differentiation between the groups. To conclude, attenuation coefficient maps allow visualization and quantification of changes in the condition of the connective tissue of the vaginal wall. In the future, CP OCT could be used for in vivo detection of early-stage vaginal wall prolapse and for monitoring the effectiveness of treatment.

Impaired intestinal immunity and microbial diversity in common carp exposed to cadmium

Waterborne cadmium (Cd) accumulates in the fish intestine and causes irreversible toxicity by disrupting intestinal immunity and microbial diversity. To explore the toxicity of environmentally available high Cd concentration on intestinal immunity and microbial diversity of fish, we selected the widely used bioindicator model species, Common carp (Cyprinus carpio). Literature review and Cd pollution data supported sequential doses of 0.2, 0.4, 0.8, 1.6, 3.2, and 6.4 mg/L Cd for 30 days. Based on intestinal tissue Cd accumulation, previous studies, and environmentally available Cd data, 0.4 and 1.6 mg/L Cd were selected for further studies. Intestinal Cd bioaccumulation increased significantly to ~100 times in fish exposed to 1.6 mg/L Cd. We observed villous atrophy, increased goblet cells with mucus production, muscularis erosion, and thickened lamina propria due to intense inflammatory cell infiltration in the intestine at this Cd concentration. Cd-induced immunosuppression occurred with increased lysozyme, alkaline phosphate (AKP), and acid phosphate (ACP). High levels of catalase (CAT), total antioxidant capacity (T-AOC), malondialdehyde (MDA), and hydrogen peroxide (H2O2) suggested induced oxidative stress and poor metabolism by α-amylase and lipase suppression for Cd toxicity. Proteobacteria (41.2 %), Firmicutes (21.8 %), and Bacteroidetes (17.5 %) were the dominant bacterial phyla in the common carp intestine. Additionally, potential pathogenic Cyanobacteria increased in Cd-treated fish. The decrease of beneficiary bacteria like Aeromonas, and Cetobacterium indicated Cd toxicity. Overall, these findings indicate harmful consequences of high Cd concentration in the intestinal homeostasis and health status of fish.

Single-Cell Transcriptional Analysis of Lamina Propria Lymphocytes in the Jejunum Reveals Innate Lymphoid Cell-like Cells in Pigs.

Pigs are the most suitable model to study various therapeutic strategies and drugs for human beings, although knowledge about cell type-specific transcriptomes and heterogeneity is poorly available. Through single-cell RNA sequencing and flow cytometry analysis of the types in the jejunum of pigs, we found that innate lymphoid cells (ILCs) existed in the lamina propria lymphocytes (LPLs) of the jejunum. Then, through flow sorting of live/dead-lineage (Lin)-CD45+ cells and single-cell RNA sequencing, we found that ILCs in the porcine jejunum were mainly ILC3s, with a small number of NK cells, ILC1s, and ILC2s. ILCs coexpressed IL-7Rα, ID2, and other genes and differentially expressed RORC, GATA3, and other genes but did not express the CD3 gene. ILC3s can be divided into four subgroups, and genes such as CXCL8, CXCL2, IL-22, IL-17, and NCR2 are differentially expressed. To further detect and identify ILC3s, we verified the classification of ILCs in the porcine jejunum subgroup and the expression of related hallmark genes at the protein level by flow cytometry. For systematically characterizing ILCs in the porcine intestines, we combined our pig ILC dataset with publicly available human and mice ILC data and identified that the human and pig ILCs shared more common features than did those mouse ILCs in gene signatures and cell states. Our results showed in detail for the first time (to our knowledge) the gene expression of porcine jejunal ILCs, the subtype classification of ILCs, and the markers of various ILCs, which provide a basis for an in-depth exploration of porcine intestinal mucosal immunity.

Single cell transcriptome analyses reveal the roles of B cells in fructose-induced hypertension.

While the immune system plays a crucial role in the development of hypertension, the specific contributions of distinct immune cell populations remain incompletely understood. The emergence of single-cell RNA-sequencing (scRNA-seq) technology enables us to analyze the transcriptomes of individual immune cells and to assess the significance of each immune cell type in hypertension development. We aimed to investigate the hypothesis that B cells play a crucial role in the development of fructose-induced hypertension. Eight-week-old Dahl salt-sensitive (SS) male rats were divided into two groups and given either tap water (TW) or a 20% fructose solution (HFS) for 4 weeks. Systolic blood pressure was measured using the tail-cuff method. ScRNA-seq analysis was performed on lamina propria cells (LPs) and peripheral blood mononuclear cells (PBMCs) obtained from SS rats subjected to either TW or HFS. The HFS treatment induced hypertension in the SS rats. The analysis revealed 27 clusters in LPs and 28 clusters in PBMCs, allowing for the identification and characterization of various immune cell types within each cluster. Specifically, B cells and follicular helper T (Tfh) cells were prominent in LPs, while B cells and M1 macrophages dominated PBMCs in the HFS group. Moreover, the HFS treatment triggered an increase in the number of B cells in both LPs and PBMCs, accompanied by activation of the interferon pathway. The significant involvement of B cells in intestinal and PBMC responses indicates their pivotal contribution to the development of hypertension. This finding suggests that targeting B cells could be a potential strategy to mitigate high blood pressure in fructose-induced hypertension. Moreover, the simultaneous increase in follicular B cells and Tfh cells in LPs, along with the upregulation of interferon pathway genes in B cells, underscores a potential autoimmune factor contributing to the pathogenesis of fructose-induced hypertension in the intestine.

Histopathological features of glandular atrophy of the lamina propria of the gastric mucosa during its occurrence and development

ObjectiveTo explore the histopathological features of glandular atrophy of the lamina propria of gastric mucosa during its occurrence and development.MethodWe performed detailed histological observation and immunohistochemical examination on the endoscopic biopsy and ESD endoscopic resection specimens of 896 patients with glandular atrophy of the lamina propria of gastric mucosa. The EnVision two-step method was used for immunohistochemical staining, and the slices were incubated with primary antibody CK7, CK20, villin, CDX2, MUC5AC, MUC6, p53 and ki-67. Hematoxylin staining was performed and observed under the microscope and statistically analyzed.ResultsIn the initial stage of glandular atrophy of the lamina propria, the proliferation area of the deep gastric pits, and the isthmus and neck of the gastric glands are characterized by roughly normal structure of the glandular structure, increased mesenchyme, and widened space between glands. Subsequently, the gland becomes smaller in volume and less in number, especially at the base, in the gastric glandular part of the gastric unit. The disease at this stage has higher incidence, and occurs more often in the elderly who account for 64.0% (573/896) of our study group. The disease in this stage may exhibit some lesions that are physiologic (age-related degeneration) while others are pathological. Therefore, this condition is called simple glandular atrophy of the lamina propria of the gastric mucosa. When the gastric mucosal epithelium is subjected to infection or repeated infections, chemical stimuli, immune factors, and genetic factors, it can lead to the proliferation and transformation of stem cells in the proliferation area of the deep gastric pits, and the isthmus and neck of the gastric glands, forming single ducts, multiple ducts, or a proliferation of patchy cells. Then, atypical hyperplasia (intraepithelial neoplasia) presents, finally leading to gastric adenocarcinoma.ConclusionUnderstanding the histopathological characteristics of glandular atrophy of the lamina propria of gastric mucosa is of great significance in controlling the occurrence and development of gastric cancer.

Prolonged administration of total glucosides of paeony improves intestinal immune imbalance and epithelial barrier damage in collagen-induced arthritis rats based on metabolomics-network pharmacology integrated analysis.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovial inflammation and joint damage with complex pathological mechanisms. In recent years, many studies have shown that the dysregulation of intestinal mucosal immunity and the damage of the epithelial barrier are closely related to the occurrence of RA. Total glucosides of paeony (TGP) have been used clinically for the treatment of RA in China for decades, while the pharmacological mechanism is still uncertain. The purpose of this study was to investigate the regulatory effect and mechanism of TGP on intestinal immunity and epithelial barrier in RA model rats. The results showed that TGP alleviated immune hyperfunction by regulating the ratio of CD3+, CD4+ and CD8+ in different lymphocyte synthesis sites of the small intestine, including Peyer's patches (PPs), intraepithelial lymphocytes (IELs), and lamina propria lymphocytes (LPLs). Specially, TGP first exhibited immunomodulatory effects on sites close to the intestinal lumen (IELs and LPLs), and then on PPs far away from the intestinal lumen as the administration time prolonged. Meanwhile, TGP restores the intestinal epithelial barrier by upregulating the ratio of villi height (V)/crypt depth (C) and expression of tight junction proteins (ZO-1, occludin). Finally, the integrated analysis of metabolomics-network pharmacology was also used to explore the possible regulation mechanism of TGP on the intestinal tract. Metabolomics analysis revealed that TGP reversed the intestinal metabolic profile disturbance in CIA rats, and identified 32 biomarkers and 163 corresponding targets; network pharmacology analysis identified 111 potential targets for TGP to treat RA. By intersecting the results of the two, three key targets such as ADA, PNP and TYR were determined. Pharmacological verification experiments showed that the levels of ADA and PNP in the small intestine of CIA rats were significantly increased, while TGP significantly decreased their ADA and PNP levels. In conclusion, purine metabolism may play an important role in the process of TGP improving RA-induced intestinal immune imbalance and impaired epithelial barrier.