Search

Background: Microsatellite instability-high (MSI-high) tumors comprise ~15% of sporadic colorectal cancers (CRC) and are associated with elevated T cell infiltration. However, the universality of this response across T cell subtypes with distinct functions is unknown. Methods: Including 1,236 CRC tumors from three observational studies, we conducted in-situ T cell profiling using a customized 9-plex (CD3, CD4, CD8, CD45RA, CD45RO, FOXP3, KRT, MKI67, and DAPI) multispectral immunofluorescence assay. MSI status was assessed through polymerase chain reaction or immunohistochemical assays. We used multivariable ordinal logistic regression to estimate odds ratios (OR per increasing quantile) and 95% confidence intervals (CIs) for the association of MSI status with quantiles of T cell densities in either tumor epithelial or stromal tissue areas. Results: Compared to microsatellite instability low or microsatellite stable (MSI-low/MSS) tumors, MSI-high status was associated with higher density for the majority of immune subsets (twelve out of eighteen) in both epithelial and stromal tissue areas. The strongest associations were for CD3+CD8+ T cells in epithelial areas [OR (95% CI) for naive, memory, and regulatory subsets = 3.49 (2.57, 4.75); 2.82 (2.10, 3.78); 3.04 (2.24, 4.13), respectively]. Conversely, stromal area CD3+CD4+ memory T cells were inversely associated [OR (95% CI) = 0.68 (0.51, 0.91)]. Discussion: MSI-high status was strongly associated with higher densities of most T cell subsets in both epithelial and stromal tissue areas. Our investigation supports efforts to identify patients who may be more likely to respond to current immunotherapy treatments. Significance: This study helps us better understand how a clinically relevant tumor phenotype, microsatellite instability status, is related to different functioning T cell densities in colorectal tumors, which may impact future immunotherapy strategies. [ABSTRACT FROM AUTHOR]

Objectives The objective of this study was to evaluate SOX17, a transcription factor from the Sry high-mobility group–related box superfamily, as a diagnostic marker to determine site of origin using both whole-tissue sections and tissue microarrays (TMAs). Methods SOX17 immunohistochemistry was performed on gynecologic and nongynecologic tissues (N = 1004) using whole-tissue sections and both internally constructed and commercially available TMAs. SOX17 nuclear reactivity was scored as positive or negative on the whole-tissue sections and using the semiquantitative H score method on TMAs. Results Using both whole-tissue sections and TMAs, SOX17 was positive in 94% (n = 155) of endometrial tumors and 96% (n = 242) of ovarian tumors. All breast cases (n = 241) and vulvar/cervical squamous cell carcinomas (n = 150) were negative. Among 1004 tumors from 20 sites, the only organs with positive tumors were ovary, uterus, and testis. Conclusions SOX17 is a sensitive and specific marker for gynecologic origin in the tissues tested and may be a valuable adjunct to PAX8 and other commonly used markers to confirm endometrial or ovarian origin. SOX17 expression is lower in mucinous tumors, endocervical adenocarcinoma, high-grade neuroendocrine tumors, and undifferentiated/dedifferentiated endometrial carcinoma. [ABSTRACT FROM AUTHOR]

Superficial duodenal epithelial tumors were previously considered rare. Laparoscopic and endoscopic cooperative surgery for duodenal tumors (D-LECS) has been developed to achieve successful endoscopic treatment. Patients who have undergone living-donor liver transplantation (LDLT) may have severe abdominal adhesions, and immunosuppressive agents (IAs) may affect the degree of postoperative abdominal adhesions and wound healing, but their effects remain unclear. Herein, we present the first case of D-LECS for duodenal adenoma after LDLT. A 66-year-old man underwent D-LECS for a non-ampullary duodenal high-grade adenoma after LDLT with an IA. The patient's condition was uneventful 36 months after the surgery. In gastrointestinal surgery, IAs may affect the resected duodenal repair process. For duodenal neoplasms in high-risk patients, D-LECS may be better than endoscopic submucosal dissection alone. D-LECS after LDLT is a feasible and less invasive procedure. [ABSTRACT FROM AUTHOR]

Liver masses are common in children, however primary malignant neoplasms are rare, representing only 1% of all pediatric cancers. Hepatocellular neoplasms are the most common primary liver malignancies and hepatoblastoma (HB) is the most frequently diagnosed. The incidence of HB, which is increasing, is approximately of 2 cases per million in the United States, followed by hepatocellular carcinoma (HCC). Pediatric primary liver tumors of mesenchymal origin are less common, except for benign vascular tumors (hemangiomas). Malignant mesenchymal neoplasms represent approximately 10–15% of all, the most common being embryonal sarcoma and malignant rhabdoid tumor. Malignant vascular tumors are rare, but epithelioid hemangioendothelioma (EHE) and angiosarcoma can be seen in children. The development and adoption of consensus diagnostic, therapeutic and risk-stratifying approaches for pediatric patients with malignant liver tumors has been historically challenged by their rarity and by their diverse clinical and histological appearance. On-going collaborative efforts of international consortia including the Children's Oncology Group (COG) in North America, the German Society of Paediatric Oncology and Haematology (GPOH), the Societe Internationale d' Oncologie Pediatrique Liver Tumor Study Group (SIOPEL) in Europe and the Japanese Liver Tumor group (JPLT), have made significant contributions to understanding the clinical and histopathological features, as well as the underlying biology of pediatric liver tumors, in particular HB. A new classification of pediatric liver tumors drafted at the international consensus meeting held in Los Angeles, has been incorporated in the recent WHO classification and is currently used by the PHITT (Paediatric Hepatic Malignancy International Tumour Trial) and other therapeutic protocols. This manuscript provides an overview of salient diagnostic features and updates in classification and molecular characterization for the most common pediatric primary liver neoplasms. It also includes a brief overview of other less common but relevant tumors, which should be considered in the differential diagnosis. [ABSTRACT FROM AUTHOR]

Introduction: Ovarian cancer is a significant global health concern for women. More than 85-90% of ovarian cancer cases originates from epithelial causes. Dysfunctional p53 is a hallmark of many cancers, including ovarian cancer. Alteration in functioning of p53 gene has been connected to the onset and spread of ovarian cancer. Aim: To analyse the Immunohistochemical (IHC) expression of p53 gene and clinicopathological features of Low-grade Serous Ovarian Cancer (LGSOC) and High-grade Serous Ovarian Cancer (HGSOC). Materials and Methods: This cohort study was conducted on 37 histopathologically confirmed cases of serous carcinomas of ovary in Department of Genetics, Maharshi Dayanand University, Rohtak, in collaboration with Department of Pathology, Pandit Bhagwat Dayal Sharma University of Health Sciences, Rohtak, Haryana, India during the period 2019 to 2023. Study was approved by the Institutional Human Ethical Research Committee (Ref no-IHEC/19/07). Parameters of study included clinical features of ovarian cancer patients, histological types, the Federation of Gynaecology and Obstetrics stage, survival of patients and prognostic factors. Analysis of p53 expression was analysed immunohistochemically. Statistical analysis was performed using GraphPad PRISM Version 8.0.1. Results: A total of 37 cases of serous carcinoma were analysed, including 26 cases of HGSOC and 11 cases of LGSOC. Comparative analysis revealed advanced-stage disease in patients HGSOC, 20 cases (76.92%) compared to 4 cases (36.36%) in LGSOC. Additionally, higher median CA125 levels were found in HGSOC patients (median 269 U/mL vs. 27 U/mL) and a higher rate of postsurgery residual disease (57.69% vs. 27.27%). Among all serous carcinoma cases, 17 cases (45.95%) showed null p53 expression, 16 cases (43.24%) showed diffuse p53 expression, and four cases (10.81%) had focal positive expression (wild type). Statistically significant differences (p-value=0.02) between the grade of serous ovarian cancer and the expression of p53 were observed. However, median Overall Survival (OS) between the two groups (39 months for HGSOC vs. 41 months for LGSOC) showed a statistically non significant difference (p-value=0.51). Log-rank statistical analysis showed a non significant association between the overall survival time of the two grades, with a Hazard Ratio of 1.384 (0.5-3.5) at 95% confidence interval with p-value=0.51. Conclusion: The significant difference in p53 IHC expression between LGSOC and HGSOC highlighted the role of p53 mutation in pathogenesis of HGSOC. However, by understanding genetic predisposition of ovarian tumour preventive steps as well as planning of early treatment can reduce the mortality rate. [ABSTRACT FROM AUTHOR]

Introduction: The bone marrow examination is an essential investigation in the diagnosis and management of many haematological disorders. The integration of all investigations, including peripheral blood analysis, bone marrow aspirate, and trephine biopsy findings, along with supplementary tests such as immunophenotyping, cytogenetic analysis, and molecular genetic studies, is crucial for arriving at a final diagnosis. Aim: To assess the presence of reticulin fibres in bone marrow biopsy sections in haematological malignancies, to evaluate the grade of BMF associated with haematological malignancies and to assess the role of angiogenesis using IHC markers in various haematological malignancies. Materials and Methods: A cross-sectional study was conducted in the Department of Pathology at the National Institute of Pathology, Safdarjung Hospital, New Delhi, India in 2009 for a duration of 18 months. Thirty-eight patients with a diagnosis of Acute Myeloid Leukaemia (AML), Acute Lymphoblastic Leukaemia (ALL), Chronic Myeloid Leukaemia (CML), and Chronic Lymphoproliferative Disorder (CLPD) were studied. Bone marrow biopsies were taken, fixed in 10% formalin, and decalcified in 10% Ethylene Diamine Tetraacetic Acid (EDTA). Routine paraffin embedding was performed, and serial sections of 4 μm were obtained on poly-L-lysine-coated slides for Immunohistochemistry (IHC) {Vimentin, Vascular Endothelial Growth Factor (VEGF), CD-34, Smooth Muscle Actin (SMA)}. The presence of reticulin fibres in the bone marrow biopsy sections was assessed using two special stains: Gomori’s Silver Impregnation and Masson’s Trichrome. Fibrosis was quantified according to the Baurmeister 0-4 grading system of Bone Marrow Fibrosis (BMF). Results: The results of the present study suggest that Endothelial to Mesenchymal Transition (EndMT) may play a role in the pathogenesis of BMF. Various grades of fibrosis were observed, with 15 cases (39.47%) in Grade 3, followed by 11 cases (28.95%) in Grade 2, 8 cases (21.05%) in Grade 1, and 4 cases (10.53%) in Grade 4. Conclusion: BMF was a significant finding even in the early stages of the majority of the lesions studied and was closely linked with angiogenesis. This study showed that angiogenesis plays an important role in the pathogenesis of haematological neoplasms and that VEGF is a prominent stimulus in the majority of these disorders. Additionally, this study suggests that EndMT has a possible role in the pathogenesis of BMF.

Abstract Postnatal kidney growth is substantial and involves expansion in kidney tubules without growth of new nephrons, which are the functional units of the kidney. Proliferation and differentiation pathways underpinning nephron elongation are not well defined. To address this, we performed sequential characterization of mouse kidney transcriptomics at the single cell level. Single nuclear RNA sequencing (snRNA-seq) was performed on kidney tissue from male and female mice at 1, 2, 4 and 12 weeks of age using the 10x Chromium platform. Unbiased clustering was performed on 68,775 nuclei from 16 animals. 31 discrete cellular clusters were seen, which were identified through comparison of their gene expression profiles to canonical markers of kidney cell populations. High levels of proliferation were evident at early time points in some cell types, especially tubular cells, but not in other cell types, for example podocytes. Proliferation was especially evident in Proximal Tubular Cells (PTCs) which are the most abundant cell type in the adult kidney. Uniquely when compared to other kidney cell types, PTCs demonstrated sex-specific expression profiles at late, but not early, time points. Mapping of PTC differentiation pathways using techniques including trajectory and RNA Velocity analyses delineated increasing PTC specialization and sex-specific phenotype specification. Our single-cell transcriptomics data characterise cellular states observed during kidney growth. We have identified PTC differentiation pathways that lead to sex-specific tubular cell phenotypes. Tubular proliferative responses are of central importance in postnatal kidney growth and have also been linked to kidney recovery versus fibrosis following injury. Our unbiased and comprehensive dataset of tubular cell development can be used to identify candidate pathways for therapeutic targeting.

Introduction: The bone marrow examination is an essential investigation in the diagnosis and management of many haematological disorders. The integration of all investigations, including peripheral blood analysis, bone marrow aspirate, and trephine biopsy findings, along with supplementary tests such as immunophenotyping, cytogenetic analysis, and molecular genetic studies, is crucial for arriving at a final diagnosis. Aim: To assess the presence of reticulin fibres in bone marrow biopsy sections in haematological malignancies, to evaluate the grade of BMF associated with haematological malignancies and to assess the role of angiogenesis using IHC markers in various haematological malignancies. Materials and Methods: A cross-sectional study was conducted in the Department of Pathology at the National Institute of Pathology, Safdarjung Hospital, New Delhi, India in 2009 for a duration of 18 months. Thirty-eight patients with a diagnosis of Acute Myeloid Leukaemia (AML), Acute Lymphoblastic Leukaemia (ALL), Chronic Myeloid Leukaemia (CML), and Chronic Lymphoproliferative Disorder (CLPD) were studied. Bone marrow biopsies were taken, fixed in 10% formalin, and decalcified in 10% Ethylene Diamine Tetraacetic Acid (EDTA). Routine paraffin embedding was performed, and serial sections of 4 µm were obtained on poly-L-lysine-coated slides for Immunohistochemistry (IHC) {Vimentin, Vascular Endothelial Growth Factor (VEGF), CD-34, Smooth Muscle Actin (SMA)}. The presence of reticulin fibres in the bone marrow biopsy sections was assessed using two special stains: Gomori's Silver Impregnation and Masson's Trichrome. Fibrosis was quantified according to the Baurmeister 0-4 grading system of Bone Marrow Fibrosis (BMF). Results: The results of the present study suggest that Endothelial to Mesenchymal Transition (EndMT) may play a role in the pathogenesis of BMF. Various grades of fibrosis were observed, with 15 cases (39.47%) in Grade 3, followed by 11 cases (28.95%) in Grade 2, 8 cases (21.05%) in Grade 1, and 4 cases (10.53%) in Grade 4. Conclusion: BMF was a significant finding even in the early stages of the majority of the lesions studied and was closely linked with angiogenesis. This study showed that angiogenesis plays an important role in the pathogenesis of haematological neoplasms and that VEGF is a prominent stimulus in the majority of these disorders. Additionally, this study suggests that EndMT has a possible role in the pathogenesis of BMF. [ABSTRACT FROM AUTHOR]

OBJECTIVES Minimally invasive thymectomy is an accepted approach for early-stage thymic epithelial neoplasia, reducing pain and length of stay compared with open surgery. In this study, we compare robotic and video-assisted thymectomy to assess pathological resection status, overall and disease-free survival. METHODS Data were retrieved from the European Society of Thoracic Surgeons prospectively maintained thymic database. Eighty-two international centres were invited to participate in the ESTS registry. Thirty-seven centres agreed to take part. We included all patients who had undergone complete thymectomy for malignancy through a minimally invasive approach and excluded patients in whom complete data were not available. RESULTS Between October 2001 and May 2021, a total of 899 patients with thymic malignancy underwent minimal access surgical resection and were included in the study. A propensity matched analysis was conducted with interrogation of 732 patients. Median age was 55 years, and 408 (56%) patients were female. Propensity matched was performed with 1:1 matching for surgical approach (video assisted = 366, robot assisted = 366). Robot-assisted surgery conferred significantly lower odds of incomplete resection (R1; 0.203 95% CI 0.13–0.317; P  < 0.001). However, there was no difference in terms of overall and disease-free survival between the 2 techniques. CONCLUSIONS In this analysis, after adjusting for thymoma stage, the odds of incomplete surgical resection were higher in patients undergoing video-assisted surgery than robotic. However, there was no difference in overall or disease-free survival. With data maturation and increased follow-up, this would need repeat analysis and perhaps may provide more credence to the concept of a prospective randomized study to compare outcomes in thymic epithelial neoplasia by surgical approach with a standardized pathological work-up. [ABSTRACT FROM AUTHOR]

Sebaceous glands (SGs) release oils that protect our skin, but how these glands respond to injury has not been previously examined. Here, we report that SGs are largely self-renewed by dedicated stem cell pools during homeostasis. Using targeted single-cell RNA sequencing, we uncovered both direct and indirect paths by which resident SG progenitors ordinarily differentiate into sebocytes, including transit through a Krt5+PPARγ+ transitional basal cell state. Upon skin injury, however, SG progenitors depart their niche, reepithelialize the wound, and are replaced by hair-follicle-derived stem cells. Furthermore, following targeted genetic ablation of >99% of SGs from dorsal skin, these glands unexpectedly regenerate within weeks. This regenerative process is mediated by alternative stem cells originating from the hair follicle bulge, is dependent upon FGFR2 signaling, and can be accelerated by inducing hair growth. Altogether, our studies demonstrate that stem cell plasticity promotes SG durability following injury.

BackgroundMicrosatellite instability-high (MSI-high) tumors comprise ~15% of sporadic colorectal cancers (CRC) and are associated with elevated T cell infiltration. However, the universality of this response across T cell subtypes with distinct functions is unknown.MethodsIncluding 1,236 CRC tumors from three observational studies, we conducted in-situ T cell profiling using a customized 9-plex (CD3, CD4, CD8, CD45RA, CD45RO, FOXP3, KRT, MKI67, and DAPI) multispectral immunofluorescence assay. MSI status was assessed through polymerase chain reaction or immunohistochemical assays. We used multivariable ordinal logistic regression to estimate odds ratios (OR per increasing quantile) and 95% confidence intervals (CIs) for the association of MSI status with quantiles of T cell densities in either tumor epithelial or stromal tissue areas.ResultsCompared to microsatellite instability low or microsatellite stable (MSI-low/MSS) tumors, MSI-high status was associated with higher density for the majority of immune subsets (twelve out of eighteen) in both epithelial and stromal tissue areas. The strongest associations were for CD3+CD8+ T cells in epithelial areas [OR (95% CI) for naive, memory, and regulatory subsets = 3.49 (2.57, 4.75); 2.82 (2.10, 3.78); 3.04 (2.24, 4.13), respectively]. Conversely, stromal area CD3+CD4+ memory T cells were inversely associated [OR (95% CI) = 0.68 (0.51, 0.91)].DiscussionMSI-high status was strongly associated with higher densities of most T cell subsets in both epithelial and stromal tissue areas. Our investigation supports efforts to identify patients who may be more likely to respond to current immunotherapy treatments.SignificanceThis study helps us better understand how a clinically relevant tumor phenotype, microsatellite instability status, is related to different functioning T cell densities in colorectal tumors, which may impact future immunotherapy strategies.

The prostone analog, lubiprostone, is approved to manage constipation-predominant irritable bowel syndrome. Lubiprostone also protects intestinal mucosal barrier function in animal models of colitis. The aim of this study was to determine if lubiprostone improves barrier properties in isolated colonic biopsies from Crohn's disease (CD) and ulcerative colitis (UC) patients. Sigmoid colon biopsies from healthy subjects, CD and UC patients in remission, and CD patients with active disease were mounted in Ussing chambers. Tissues were treated with lubiprostone or vehicle to determine the effects on transepithelial electrical resistance (TER), FITC-dextran 4kD (FD4) permeability, and electrogenic ion transport responses to forskolin and carbachol. Localization of the tight junction protein, occludin, was determined by immunofluorescence. Lubiprostone significantly increased ion transport across control, CD and UC remission biopsies but not active CD. Lubiprostone selectively improved TER in both CD remission and active disease biopsies but not in control or UC biopsies. The improved TER was associated with increased membrane localization of occludin. Lubiprostone selectively improved barrier properties of biopsies from CD patients vs. UC and independent of an ion transport response. These data indicate that lubiprostone has potential efficacy in improving mucosal integrity in Crohn's disease.

Simple Summary: High levels of the transcription factor Grainyhead-like protein 2 (GRHL2) contribute to worse outcomes for patients with breast cancer tumors that express estrogen receptor (ER). Using multiple methods to increase GRHL2 expression in breast cancer cells, we found that high GRHL2 promotes both epithelial and mesenchymal phenotypes. This is indicative of an intermediate state between epithelial and mesenchymal cells, which is associated with metastasis. We also observed that elevated GRHL2 stimulates properties characteristic of cellular plasticity, including a decrease in cellular proliferation with a reciprocal increase in dormancy and stem cell markers. Elevated levels of GRHL2 broadly changed its control of gene expression at the level of DNA and shifted nearby transcription factor binding sites from those associated with development to those associated with disease progression. These results provide a possible explanation for how elevated GRHL2 levels could contribute to poorer prognosis in ER-positive breast cancer. Estrogen receptor (ER)-positive breast cancer is characterized by late recurrences following initial treatment. The epithelial cell fate transcription factor Grainyhead-like protein 2 (GRHL2) is overexpressed in ER-positive breast cancers and is linked to poorer prognosis as compared to ER-negative breast cancers. To understand how GRHL2 contributes to progression, GRHL2 was overexpressed in ER-positive cells. We demonstrated that elevated GRHL2 imparts plasticity with stem cell- and dormancy-associated traits. RNA sequencing and immunocytochemistry revealed that high GRHL2 not only strengthens the epithelial identity but supports a hybrid epithelial to mesenchymal transition (EMT). Proliferation and tumor studies exhibited a decrease in growth and an upregulation of dormancy markers, such as NR2F1 and CDKN1B. Mammosphere assays and flow cytometry revealed enrichment of stem cell markers CD44 and ALDH1, and increased self-renewal capacity. Cistrome analyses revealed a change in transcription factor motifs near GRHL2 sites from developmental factors to those associated with disease progression. Together, these data support the idea that the plasticity and properties induced by elevated GRHL2 may provide a selective advantage to explain the association between GRHL2 and breast cancer progression. [ABSTRACT FROM AUTHOR]

Galectin-1 (Gal-1), a member of the human lectin family, has garnered attention for its association with aggressive behavior in human tumors, prompting research into the development of targeted drugs. This study aims to assess the staining pattern and prognostic significance of Gal-1 immunohistochemical expression in a homogeneous cohort of Western patients with gastric cancer (GC). A total of 149 cases were included and tissue microarrays were constructed. Stromal Gal-1 expression was observed to some extent in most tumors, displaying a cytoplasmic pattern. Cases with stromal Gal-1 overexpression showed significantly more necrosis, lymphovascular invasion, advanced pTNM stages, recurrences, and cancer-related deaths. Epithelial Gal-1 expression was present in 63.8% of the cases, primarily exhibiting a cytoplasmic pattern, and its overexpression was significantly associated with lymphovascular invasion, peritumoral lymphocytic infiltration, and tumor-related death. Kaplan/Meier curves for cancer-specific survival (CSS) revealed a significantly worse prognosis for patients with tumors exhibiting stromal or epithelial Gal-1 overexpression. Furthermore, stromal Gal-1 expression stratified stage III patients into distinct prognostic subgroups. In a multivariable analysis, increased stromal Gal-1 expression emerged as an independent prognostic factor for CSS. These findings underscore the prognostic relevance of Gal-1 and suggest its potential as a target for drug development in Western patients with GC. [ABSTRACT FROM AUTHOR]

Ovarian cancer (OC) is the fourth most common cancer of women in sub‐Saharan Africa (SSA), although few data have been published on population‐level survival. We estimate ovarian cancer survival in SSA by human development index and histological subtype, using data from seven population‐based cancer registries in six countries: Kenya (Nairobi and Eldoret), Mauritius, Uganda (Kampala), Cote d'Ivoire (Abidjan), Ethiopia (Addis Ababa) and South Africa (Eastern Cape). A total of 644 cases diagnosed during 2008–2014 were included, with 77% being of epithelial subtypes (range 47% [Abidjan]—80% [Mauritius]). The overall observed survival in the study cohort was 73.4% (95% CI: 69.8, 77.0) at 1 year, 54.4% (95% CI: 50.4, 58.7) at 3 years and 45.0% (95% CI: 41.0, 49.4) at 5 years. Relative survival at Year 1 ranged from 44.4% in Kampala to 86.3% in Mauritius, with a mean for the seven series of 67.4%. Relative survival was highest in Mauritius at 72.2% and lowest in Kampala, Uganda at 19.5%, with a mean of 47.8%. There was no difference in survival by age at diagnosis. Patients from high and medium HDI countries had significantly better survival than those from low HDI countries. Women with cancers of epithelial cell origin had much lower survival compared to women with other histological subtypes (p =.02). Adjusted for the young age of the African patients with ovarian cancer (44% aged <50) survival is much lower than in USA or Europe, and underlines the need for improvements in the access to diagnosis and treatment of OC in SSA. [ABSTRACT FROM AUTHOR]

Mixed neuroendocrine–nonneuroendocrine epithelial neoplasms are rare malignant neoplasms that may occur in the bladder with highly aggressive behavior. Because of its worse prognosis, when compared to the pure urothelial carcinoma without the neuroendocrine component, the bladder mixed neuroendocrine–nonneuroendocrine epithelial neoplasm may be considered a distinct clinicopathologic entity. We present a case of mixed neuroendocrine–nonneuroendocrine epithelial neoplasm occurring in the urinary bladder of an elderly female with a personal history of chronic kidney disease, drug-resistant urinary tract infections, and neurogenic bladder. Her presenting symptoms included complaints of abdominal pain, urinary urgency, oliguria, dysuria, and occasional hematuria. Recognition of the clinicopathologic features of these rare aggressive neoplasms is important for accurate early diagnosis, necessitating appropriate therapeutic management. [ABSTRACT FROM AUTHOR]

Clear cell renal cell carcinoma (ccRCC) is a malignant tumor of kidney epithelial cells, one of the most common tumors in the world. Transforming growth factor beta (TGFβ)1 is a crucial factor that induces epithelial–mesenchymal transition (EMT) in cancer cells. microRNA-141-3p (miR-141-3p) is a microRNA that is considered a tumor suppressor. However, the role and mechanism of miR-141-3p in TGFβ1-induced ccRCC cells are not fully understood. This study investigated the roles of miR-141-3p and its target gene in regulating EMT in ccRCC development. 786-0 and Caki-1cells were treated with TGFβ1 to induce EMT. The levels of miR-141-3p and TGFβ2 were determined by quantitative real-time polymerase chain reaction and Western blotting. The progression of EMT was evaluated by E-cadherin detection by immunofluorescence, and E-cadherin, N-cadherin, and vimentin detection by Western blotting. Furthermore, migration and invasion capacities were assessed using a Transwell system. The direct binding of miR-141-3p with the target gene TGFβ2 was confirmed by dual luciferase reporter gene assay. Results indicated that TGFβ1 treatment decreased the protein abundance of E-cadherin while increasing the protein expression of N-cadherin and vimentin, indicating TGFβ1-induced EMT was constructed successfully. Moreover, TGFβ1 treatment repressed the expression of miR-141-3p. miR-141-3p mimics reversed the effect of TGFβ1 on the migration, invasion, and expression of E-cadherin, N-cadherin, and vimentin. The miR-141-3p directly binds with the 3′ untranslated region of TGFβ2 mRNA and suppresses its expression. Furthermore, TGFβ2 overexpression abrogated the above changes regulated by miR-141-3p mimics. Taken together, miR-141-3p inhibited TGFβ1-induced EMT by suppressing the migration and invasion of ccRCC cells via directly targeting TGFβ2 gene expression. [ABSTRACT FROM AUTHOR]

OBJECTIVES Robotic thymectomy has been suggested and considered technically feasible for thymic tumours. However, because of small-sample series and the lack of data on long-term results, controversies still exist on surgical and oncological results with this approach. We performed a large national multicentre study sought to evaluate the early and long-term outcomes after robot-assisted thoracoscopic thymectomy in thymic epithelial tumours. METHODS All patients with thymic epithelial tumours operated through a robotic thoracoscopic approach between 2002 and 2022 from 15 Italian centres were enrolled. Demographic characteristics, clinical, intraoperative, postoperative, pathological and follow-up data were retrospectively collected and reviewed. RESULTS There were 669 patients (307 men and 362 women), 312 (46.6%) of whom had associated myasthenia gravis. Complete thymectomy was performed in 657 (98%) cases and in 57 (8.5%) patients resection of other structures was necessary, with a R0 resection in all but 9 patients (98.6%). Twenty-three patients (3.4%) needed open conversion, but no perioperative mortality occurred. Fifty-one patients (7.7%) had postoperative complications. The median diameter of tumour resected was 4 cm (interquartile range 3–5.5 cm), and Masaoka stage was stage I in 39.8% of patients, stage II in 56.1%, stage III in 3.5% and stage IV in 0.6%. Thymoma was observed in 90.2% of patients while thymic carcinoma occurred in 2.8% of cases. At the end of the follow-up, only 2 patients died for tumour-related causes. Five- and ten-year recurrence rates were 7.4% and 8.3%, respectively. CONCLUSIONS Through the largest collection of robotic thymectomy for thymic epithelial tumours we demonstrated that robot-enhanced thoracoscopic thymectomy is a technically sound and safe procedure with a low complication rate and optimal oncological outcomes. [ABSTRACT FROM AUTHOR]

Differentiated primary human bronchial epithelial cell (dpHBEC) cultures grown under air-liquid interface (ALI) conditions exhibit key features of the human respiratory tract and are thus critical for respiratory research as well as efficacy and toxicity testing of inhaled substances (e.g., consumer products, industrial chemicals, and pharmaceuticals). Many inhalable substances (e.g., particles, aerosols, hydrophobic substances, reactive substances) have physiochemical properties that challenge their evaluation under ALI conditions in vitro. Evaluation of the effects of these methodologically challenging chemicals (MCCs) in vitro is typically conducted by "liquid application," involving the direct application of a solution containing the test substance to the apical, airexposed surface of dpHBEC-ALI cultures. We report that the application of liquid to the apical surface of a dpHBEC-ALI co-culture model results in significant reprogramming of the dpHBEC transcriptome and biological pathway activity, alternative regulation of cellular signaling pathways, increased secretion of proinflammatory cytokines and growth factors, and decreased epithelial barrier integrity. Given the prevalence of liquid application in the delivery of test substances to ALI systems, understanding its effects provides critical infrastructure for the use of in vitro systems in respiratory research as well as in the safety and efficacy testing of inhalable substances. [ABSTRACT FROM AUTHOR]

Many metastatic cancers with poor prognoses correlate to downregulated CD82, but exceptions exist. Understanding the context of this correlation is essential to CD82 as a prognostic biomarker and therapeutic target. Oral squamous cell carcinoma (OSCC) constitutes over 90% of oral cancer. We aimed to uncover the function and mechanism of CD82 in OSCC. We investigated CD82 in human OSCC cell lines, tissues, and healthy controls using the CRISPR-Cas9 gene knockout, transcriptomics, proteomics, etc. CD82 expression is elevated in CAL 27 cells. Knockout CD82 altered over 300 genes and proteins and inhibited cell migration. Furthermore, CD82 expression correlates with S100 proteins in CAL 27, CD82KO, SCC-25, and S-G cells and some OSCC tissues. The 37–50 kDa CD82 protein in CAL 27 cells is upregulated, glycosylated, and truncated. CD82 correlates with S100 proteins and may regulate their expression and cell migration. The truncated CD82 explains the invasive metastasis and poor outcome of the CAL 27 donor. OSCC with upregulated truncated CD82 and S100A7 may represent a distinct subtype with a poor prognosis. Differing alternatives from wild-type CD82 may elucidate the contradictory functions and pave the way for CD82 as a prognostic biomarker and therapeutic target. [ABSTRACT FROM AUTHOR]

ABSTRACT Candida albicans causes millions of mucosal infections in humans annually. Hyphal overgrowth on mucosal surfaces is frequently associated with tissue damage caused by candidalysin, a secreted peptide toxin that destabilizes the plasma membrane of host cells thereby promoting disease and immunopathology. Candidalysin was first identified in C. albicans strain SC5314, but recent investigations have revealed candidalysin “variants” of differing amino acid sequence in isolates of C. albicans, and the related species C. dubliniensis, and C tropicalis, suggesting that sequence variation among candidalysins may be widespread in natural populations of these Candida species. Here, we analyzed ECE1 gene sequences from 182 C. albicans isolates, 10 C. dubliniensis isolates, and 78 C. tropicalis isolates and identified 10, 3, and 2 candidalysin variants in these species, respectively. Application of candidalysin variants to epithelial cells revealed differences in the ability to cause cellular damage, changes in metabolic activity, calcium influx, MAPK signalling, and cytokine secretion, while biophysical analyses indicated that variants exhibited differences in their ability to interact with and permeabilize a membrane. This study identifies candidalysin variants with differences in biological activity that are present in medically relevant Candida species.IMPORTANCEFungal infections are a significant burden to health. Candidalysin is a toxin produced by Candida albicans that damages host tissues, facilitating infection. Previously, we demonstrated that candidalysins exist in the related species C. dubliniensis and C. tropicalis, thereby identifying these molecules as a toxin family. Recent genomic analyses have highlighted the presence of a small number of candidalysin “variant” toxins, which have different amino acid sequences to those originally identified. Here, we screened genome sequences of isolates of C. albicans, C. dubliniensis, and C. tropicalis and identified candidalysin variants in all three species. When applied to epithelial cells, candidalysin variants differed in their ability to cause damage, activate intracellular signaling pathways, and induce innate immune responses, while biophysical analysis revealed differences in the ability of candidalysin variants to interact with lipid bilayers. These findings suggest that intraspecies variation in candidalysin amino acid sequence may influence fungal pathogenicity.

Granular cell tumors are rare soft tissue neoplasms derived from Schwann cells and are characterized by their infiltrative, non-encapsulated nests and sheets of polygonal cells with fine eosinophilic cytoplasmic granules on histology. Herin, we report a case of a 10-year-old Saudi female who presented to the hospital with multiple asymptomatic skin lesions, the largest located on the right shoulder and left foot. Preoperative workup revealed the absence of liver metastasis, and the patient underwent complete surgical excision successfully. Histopathology revealed ill-defined proliferation of large bland cells with prominent eosinophilic granular cytoplasm and mild epithelial hyperplasia consistent with granular cell tumors. Granular cell tumors are a rare entity that represent only 0.5% of all soft tissue tumors. They have characteristic histological features and can present with both malignant and being features. Due to the rarity of this disease, further research is needed to enhance our understanding and improve recognition in future practice. [ABSTRACT FROM AUTHOR]

Purpose Leukoplakia is a macroscopic morphological term for thick white or grey mucosal patches that can represent various histologic diagnostic entities ranging from hyperplasia to malignancy. Aim was the study morphology of the superficial mucosa and microvascular network of the vocal cords in patients with suspected glottic squamous cell carcinoma (SCC) using contact endoscopy (CE). Material and Methods Seventy-nine patients (21 female, 58 male), with a mean age of 57.5 years ± 7.12 (range, 32–73 years), were prospectively enrolled and evaluated. Of these patients, 58 had leukoplakia (Group A/41 males and 17 females, with a mean age of 53.7 years ± 6.65), and 21 (Group B/ 17males and 4 females/ with a mean age of 60.5 years ± 6.04) had malignant lesions (pT1, n = 6; p T2, n = 8; pT3, n = 8; Group B), as proven by the results of the histological examination. Further, 79 non-smokers (control group—group C) were studied. CE imaging findings were classified into five types (I to V) based on the features of the mucosal intra-epithelial capillary loops. CE findings were correlated to the histologic findings. A separate analysis involving smoking status was done. Results The CE-based intraepithelial papillary capillary loop classification score was strongly correlated with the histological findings. Age was strongly associated with both malignancy and bilateral involvement. Smoking habits didn't significantly differ between patients with unilateral and bilateral SCC. Conclusions CE imaging of the vocal cord mucosal capillaries may be useful for the early detection of glottic SCC and pre-cancerous lesions. [ABSTRACT FROM AUTHOR]

Solid pseudopapillary epithelial neoplasm (SPEN) of the pancreas is a rare tumor of low malignant potential that occurs most often in young females. Imaging and histopathology are necessary to confirm the diagnosis as most have no symptoms. Lack of access to these technologies in sub-Saharan Africa contributes to the difficulty in making an early and accurate diagnosis, and hence, impedes treatment. We present two cases of SPEN of the pancreas in young female patients at a rural, teaching hospital in Cameroon. The diagnosis was made only with histopathology. Computed tomography scan with intravenous contrast was essential to planning a safe surgical resection. Both patients had complete surgical resection with good results. [ABSTRACT FROM AUTHOR]

Epithelial-mesenchymal transition (EMT) and its reverse mechanism, mesenchymal-epithelial transition (MET), are evolutionarily conserved mechanisms initially identified in studies of early metazoan development. EMT may even have been established in choanoflagellates, the closest unicellular relative of Metazoa. These crucial morphological transitions operate during body plan formation and subsequently in organogenesis. These findings have prompted an increasing number of investigators in biomedicine to assess the importance of such mechanisms that drive epithelial cell plasticity in multiple diseases associated with congenital disabilities and fibrosis, and, most importantly, in the progression of carcinoma. EMT and MET also play crucial roles in regenerative medicine, notably by contributing epigenetic changes in somatic cells to initiate reprogramming into stem cells and their subsequent differentiation into distinct lineages. [ABSTRACT FROM AUTHOR]

Forkhead box protein 3 (FOXP3), traditionally recognized as a specific transcription factor for regulatory T cells (Tregs), has also been identified in various tumor epithelial cells (named as cancer-FOXP3, c-FOXP3). However, the natural state and functional role of FOXP3 positive tumor epithelial cells remain unknown. Monoclonal cells expressing varying levels of c-FOXP3 were isolated from established PANC-1 cells using limited dilution. Whole transcriptome sequencing and weighted gene co-expression network analysis (WGCNA) were conducted on these subsets, followed by in vitro and in vivo functional investigations. In addition, we identified c-FOXP3+E-cadherin- epithelial cells in human pancreatic cancer tissues after radical resection by immunofluorescence co-staining. We also investigated the connection between c-FOXP3+E-cadherin- epithelial cells and their clinicopathological features. Our study uncovered a distinct subset of c-FOXP3+ tumor epithelial cells characterized by reduced E-cadherin expression. C-FOXP3+E-cadherin- cells displayed significant proliferation potential and pro-angiogenic effect through the expression of chemokines, including C-X-C motif ligand 1 (CXCL1), C-X-C motif ligand 5 (CXCL5), and C-X-C motif ligand 8 (CXCL8). Notably, higher counts of c-FOXP3+E-Cadherin- cells correlated with poorer prognosis, lower tumor differentiation, lymph node metastasis, and vascular invasion in pancreatic ductal adenocarcinoma (PDAC). In conclusion, this work revealed the stable expression of FOXP3 in tumor epithelial cells, marking a distinct subset. C-FOXP3+E-cadherin- epithelial cells exhibit active proliferation and promote angiogenesis in a vascular endothelial growth factor A (VEGFA) independent manner. These findings provide novel insights into PDAC prognosis and therapeutic avenues. NEW & NOTEWORTHY: In this study, we revealed a novel c-FOXP3+ tumor epithelial cell subset marked by diminished E-cadherin and stable FOXP3 expression. These subpopulations not only show robust proliferation and drive angiogenesis via CXCL1, CXCL5, and CXCL8, bypassing VEGFA pathways, but their heightened presence also correlates with adverse PDAC outcomes. By challenging traditional epithelial cell definitions and extending lymphocyte markers to these cells, our findings present innovative targets for PDAC treatment and enrich our understanding of cell biology. [ABSTRACT FROM AUTHOR]

Galectin-1 (Gal-1), a member of the human lectin family, has garnered attention for its association with aggressive behavior in human tumors, prompting research into the development of targeted drugs. This study aims to assess the staining pattern and prognostic significance of Gal-1 immunohistochemical expression in a homogeneous cohort of Western patients with gastric cancer (GC). A total of 149 cases were included and tissue microarrays were constructed. Stromal Gal-1 expression was observed to some extent in most tumors, displaying a cytoplasmic pattern. Cases with stromal Gal-1 overexpression showed significantly more necrosis, lymphovascular invasion, advanced pTNM stages, recurrences, and cancer-related deaths. Epithelial Gal-1 expression was present in 63.8% of the cases, primarily exhibiting a cytoplasmic pattern, and its overexpression was significantly associated with lymphovascular invasion, peritumoral lymphocytic infiltration, and tumor-related death. Kaplan/Meier curves for cancer-specific survival (CSS) revealed a significantly worse prognosis for patients with tumors exhibiting stromal or epithelial Gal-1 overexpression. Furthermore, stromal Gal-1 expression stratified stage III patients into distinct prognostic subgroups. In a multivariable analysis, increased stromal Gal-1 expression emerged as an independent prognostic factor for CSS. These findings underscore the prognostic relevance of Gal-1 and suggest its potential as a target for drug development in Western patients with GC.

Differentiated primary human bronchial epithelial cell (dpHBEC) cultures grown under air-liquid interface (ALI) conditions exhibit key features of the human respiratory tract and are thus critical for respiratory research as well as efficacy and toxicity testing of inhaled substances (e.g., consumer products, industrial chemicals, and pharmaceuticals). Many inhalable substances (e.g., particles, aerosols, hydrophobic substances, reactive substances) have physiochemical properties that challenge their evaluation under ALI conditions in vitro. Evaluation of the effects of these methodologically challenging chemicals (MCCs) in vitro is typically conducted by “liquid application,” involving the direct application of a solution containing the test substance to the apical, air-exposed surface of dpHBEC-ALI cultures. We report that the application of liquid to the apical surface of a dpHBEC-ALI co-culture model results in significant reprogramming of the dpHBEC transcriptome and biological pathway activity, alternative regulation of cellular signaling pathways, increased secretion of pro-inflammatory cytokines and growth factors, and decreased epithelial barrier integrity. Given the prevalence of liquid application in the delivery of test substances to ALI systems, understanding its effects provides critical infrastructure for the use of in vitro systems in respiratory research as well as in the safety and efficacy testing of inhalable substances.

Objective: Cancers are currently adjudged as a hindrance to achieving a desirable life expectancy in most countries. The high mortality rate from ovarian cancer is a result of the pathology not having specific symptoms, late presentation, and lack of proper screening that results in its diagnosis in the advanced stages. The study aimed to determine the prevalence, clinical presentation, and outcome after the management of patients with ovarian cancer seen in the tertiary institution. Methodology: It was a retrospective study. Relevant information was extracted from the case files of the patients with histologically confirmed ovarian cancer. The data obtained were analysed using Statistical Package for Social Sciences (SPSS) statistical software, version 23. Results: Thirty-two cases of ovarian cancer were admitted during the period under review. This constituted 21.0% of gynaecological malignancies managed in the hospital during the study. The mean age of the ovarian cancer patients was 50.94±12.01 years with 56.25% of the patients being postmenopausal. The majority of the patients (62.4%) have 3 or more parous experiences with 53.1% of the patients presenting with advanced disease. Epithelial ovarian cancer was the most common histological variant. The treatment modality was surgery and chemotherapy. However, 65.7% of patients defaulted from initiating or completing chemotherapy. Conclusion: The study revealed that patients presented in the late stage of the disease and a bulk of them did not complete the treatment. There is a need to lobby Governments to subsidize cancer treatment, especially in the area of chemotherapy treatment.

Information is limited on the factors that may affect the preputial differential epithelial cell count in healthy intact dogs. Our objectives were to establish RIs of the preputial differential epithelial cell count in dogs and investigate the potential impact of signalment, presence of gross preputial discharge or neutrophils, preputial cleaning, and normal estrogen variation on the differential cell count. We enrolled prospectively 50 client-owned, healthy dogs in our study. Preputial cytology smears were obtained, and we performed a 4-part (parabasal, intermediate, superficial, cornified) differential cell count, before and after gentle preputial cleaning. We found higher counts of fully cornified cells (median: 11–12%) compared to previous studies. The cleaning procedure significantly decreased the intermediate (p = 0.002) and increased the superficial (p = 0.006) cells. Age was positively associated with intermediate (p = 0.007) and negatively with superficial (p = 0.01) and cornified (p = 0.02) cells. The concentration of estrogens was negatively associated with the percentage of parabasal cells (p = 0.04). In healthy dogs, a variable impact may be expected in the preputial differential epithelial cell count, by the cleaning of the prepuce before sampling, the age of the dogs, and the normal variation of estrogen concentration. [ABSTRACT FROM AUTHOR]

Inflammatory bowel disease (IBD) pathogenesis involves significant contributions from genetic and environmental factors. Loss-of-function single-nucleotide polymorphisms (SNPs) in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene increase IBD risk and are associated with altered microbiome population dynamics in IBD. Expansion of intestinal pathobionts, such as adherent-invasive E. coli (AIEC), is strongly implicated in IBD pathogenesis as AIEC increases pro-inflammatory cytokine production and alters tight junction protein regulation - suggesting a potential mechanism of pathogen-induced barrier dysfunction and inflammation. We aimed to determine if PTPN2 deficiency alters intestinal microbiome composition to promote expansion of specific bacteria with pathogenic properties. In mice constitutively lacking Ptpn2, we identified increased abundance of a novel mouse AIEC (mAIEC) that showed similar adherence and invasion of intestinal epithelial cells, but greater survival in macrophages, to the IBD-associated AIEC, LF82. Furthermore, mAIEC caused disease when administered to mice lacking segmented-filamentous bacteria (SFB), and in germ-free mice but only when reconstituted with a microbiome, thus supporting its classification as a pathobiont, not a pathogen. Moreover, mAIEC infection increased the severity of, and prevented recovery from, induced colitis. Although mAIEC genome sequence analysis showed >90% similarity to LF82, mAIEC contained putative virulence genes with >50% difference in gene/protein identities from LF82 indicating potentially distinct genetic features of mAIEC. We show for the first time that an IBD susceptibility gene, PTPN2, modulates the gut microbiome to protect against a novel pathobiont. This study generates new insights into gene-environment-microbiome interactions in IBD and identifies a new model to study AIEC-host interactions.

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

Allergic airway disease (AAD) is a collective term for respiratory disorders that can be exacerbated upon exposure to airborne allergens. The contribution of fungal allergens to AAD has become well established over recent years. We conducted a comprehensive review of the literature using Preferred Reporting Items for Systematic Reviews and Meta‐Analyses guidelines to better understand the mechanisms involved in the allergic response to fungi in airway epithelia, identify knowledge gaps and make recommendations for future research. The search resulted in 61 studies for final analysis. Despite heterogeneity in the models and methods used, we identified major pathways involved in fungal allergy. These included the activation of protease‐activated receptor 2, the EGFR pathway, adenosine triphosphate and purinergic receptor‐dependent release of IL33, and oxidative stress, which drove mucin expression and goblet cell metaplasia, Th2 cytokine production, reduced barrier integrity, eosinophil recruitment, and airway hyperresponsiveness. However, there were several knowledge gaps and therefore we recommend future research should focus on the use of more physiologically relevant methods to directly compare key allergenic fungal species, clarify specific mechanisms of fungal allergy, and assess the fungal allergy in disease models. This will inform disease management and future interventions, ultimately reducing the burden of disease. [ABSTRACT FROM AUTHOR]

The KCNE2 single transmembrane-spanning voltage-gated potassium (Kv) channel β subunit is ubiquitously expressed and essential for normal function of a variety of cell types, often via regulation of the KCNQ1 Kv channel. A polymorphism upstream of KCNE2 is associated with reduced lung function in human populations, but the pulmonary consequences of KCNE2 gene disruption are unknown. Here, germline deletion of mouse Kcne2 reduced pulmonary expression of potassium channel α subunits Kcnq1 and Kcnb1 but did not alter expression of other Kcne genes. Kcne2 colocalized and coimmunoprecipitated with Kcnq1 in mouse lungs, suggesting the formation of pulmonary Kcnq1-Kcne2 potassium channel complexes. Kcne2 deletion reduced blood O2, increased CO2, increased pulmonary apoptosis, and increased inflammatory mediators TNF-α, IL-6, and leukocytes in bronchoalveolar lavage (BAL) fluids. Consistent with increased pulmonary vascular leakage, Kcne2 deletion increased plasma, BAL albumin, and the BAL:plasma albumin concentration ratio. Kcne2-/- mouse lungs exhibited baseline induction of the reperfusion injury salvage kinase pathway but were less able to respond via this pathway to imposed pulmonary ischemia/reperfusion injury (IRI). We conclude that KCNE2 regulates KCNQ1 in the lungs and is required for normal lung function and resistance to pulmonary IRI. Our data support a causal relationship between KCNE2 gene disruption and lung dysfunction.-Zhou, L., Köhncke, C., Hu, Z., Roepke, T. K., Abbott, G. W. The KCNE2 potassium channel β subunit is required for normal lung function and resilience to ischemia and reperfusion injury.

Reactive oxygen species such as H2O2 are believed to play a prominent role in the injury and loss of transport function that affect the intestinal epithelium in inflammatory conditions such as inflammatory bowel diseases. Defects in intestinal epithelial ion transport regulation contribute to dysbiosis and inflammatory phenotypes. We previously showed that H2O2 inhibits Ca2+-dependent Cl- secretion across intestinal epithelial cells (IECs) via a phosphatidylinositol 3-kinase (PI3K)- and extracellular signal-regulated kinase (ERK)-dependent mechanism that occurs, at least in part, through inhibition of the basolateral Na+-K+-2Cl- cotransporter NKCC1. NKCC1 governs Cl- entry into crypt IECs and thus plays a critical role in maintaining the driving force for Cl- secretion. Electrolyte transport consumes large amounts of cellular energy, and direct pharmacological activation of the cellular energy sensor AMP-activated protein kinase (AMPK) has been shown to inhibit a number of ion transport proteins. Here, we show that H2O2 activates AMPK in human IEC lines and ex vivo human colon. Moreover, we demonstrate that the inhibitory effect of H2O2 on Ca2+-dependent Cl- secretion and NKCC1 activity is AMPK-dependent. This inhibitory effect is associated with a physical interaction between AMPK and NKCC1, as well as increased phosphorylation (Thr212,217) of NKCC1, without causing NKCC1 internalization. These data identify a key role for AMPK-NKCC1 interaction as a point of convergence for suppression of colonic epithelial ion transport by inflammatory reactive oxygen species.NEW & NOTEWORTHY H2O2 inhibition of intestinal epithelial Ca2+-dependent Cl- secretion involves recruitment of AMP-activated protein kinase (AMPK) downstream of ERK and phosphatidylinositol 3-kinase signaling pathways, physical interaction of AMPK with the Na+-K+-2Cl- cotransporter NKCC1, and AMPK-dependent suppression of NKCC1-mediated electrolyte influx without causing NKCC1 internalization. It is intriguing that, in human intestinal epithelial cell lines and human colon, H2O2 activation of AMPK increased phosphorylation of NKCC1 residues required for promoting, not inhibiting, NKCC1 activity. These data identify an elevated complexity of AMPK regulation of NKCC1 in the setting of an inflammatory stimulus.

Study questionAre melatonin receptors (melatonin receptor 1A (MR1A) and melatonin receptor 1B (MR1B)) expressed in human endometrium and endometriotic tissue, and does melatonin affect endometrial cell proliferation?Summary answerMelatonin receptors are expressed in human eutopic endometrium, endometriomas and peritoneal lesions, although to different extents, and melatonin treatment attenuated estradiol-induced endometrial epithelial cell proliferation in culture.What is known alreadyMelatonin decreased endometriotic lesion volume in a rat model of endometriosis. Melatonin treatment reduced pain scores in and analgesic use by women with endometriosis.Study design, size, durationBasic science study using human endometrial tissue and an endometrial epithelial cell line.Participants/materials, setting, methodsMeasurement of melatonin receptor expression (mRNA and protein) in women with surgically confirmed endometriosis (endometrioma (n = 20) or peritoneal lesion (n = 11) alone) and women without surgical evidence of endometriosis (control, n = 15). Collection of endometrial and endometriotic tissue samples, gynecologic history and demographic information. Quantification of estradiol (1.0 nM) and melatonin (0.1 nM-1.0 μM) ± estradiol-induced endometrial epithelial cell proliferation in cultures of endometrial epithelial cells (CRL-1671) following 24 and 48 hours of culture.Main results and the role of chanceMR1A and MR1B were localized by immunohistochemistry in glandular epithelial cells of endometrial biopsies from women with and without endometriosis. Both receptors were expressed in eutopic and ectopic endometrial tissue. mRNA expression of MR1A and MR1B was significantly greater in peritoneal lesions than in either endometriomas or eutopic endometrium. However, protein expression of MR1A was decreased in peritoneal lesions compared to control eutopic endometrium, whereas MR1B expression did not differ between the groups. Melatonin (0.1 nM-1.0 μM) treatment inhibited estradiol (1.0 nM)-induced endometrial epithelial cell proliferation at 48 hours but not 24 hours of culture.Limitations, reasons for cautionBeneficial effects of melatonin seen in culture have yet to be comprehensively evaluated in women with endometriosis.Wider implications of the findingsOur data suggest that melatonin may be useful as an adjunct to current endometriosis treatments.Study funding/competing interest(s)This study was supported by the Canadian Institutes of Health Research (grant MOP142230 to W.G.F.). A.A.M. is supported by a resident research grant through the Physicians Services Incorporated Foundation. The authors have no conflicts of interest.

Butyrate is important for regulating energy status and cellular metabolism. Beta-caryophyllene (BCP) is a plant compound which may exert potentially beneficial effects on intestinal epithelial cells, such as barrier integrity and modulation of nutrient utilization. The goal of this preliminary study was to investigate the effects of BCP on butyrate utilization and metabolism in intestinal epithelial cells. Caco 2 cells were used as a model for the intestinal barrier. Cells were seeded in multi-well plates with hanging inserts (n = 4) and grown for 18 d. On d 19, 1 of 4 treatments was added to triplicate wells per plate. Treatments included vehicle control (VC), 40 μM BCP, 2 mM butyrate (BTR), and butyrate plus BCP (BB). Treatments were added to the apical side and incubated for 24 and 48 h. Transepithelial electrical resistance (TEER; ohms/cm2) readings and supernatant samples from both the apical and basolateral sides of the cell layer were taken at each time point, and butyrate and β- hydroxybutyrate (BHB) concentrations measured. Butyrate utilization was calculated as absolute nmol lost. Statistical analyses were performed using lm and emmeans packages of R, with treatment, time and side (of cell layer) as fixed effects. All wells started with the same statistical TEER values on h 0. After 24 and 48 h BCP showed no changes in TEER (P > 0.05). Compared with VC, the mean TEER for BTR and BB was greater at 24 h (579 vs 878 and 1,008 ± 35, respectively; P < 0.001). After 48 h, all groups returned to baseline, except BB which had a greater TEER (883 ± 35; P < 0.001) compared with other treatments. After 24 and 48 h BCP showed no changes for both sides of the cell layer (P > 0.05). Overall, absolute BHB concentrations increased from 24 to 48 h in all treatments (P < 0.05). Production of BHB in each of the VC and BC groups was less than 15 nmol at each time point. For BB and BTR groups, more nmol BHB was detected on the basolateral side (31.9 and 22.2 ± 1.3) vs apical (14.0 and 10.4 ± 1.3, respectively; P < 0.001) after 24 h. After 48 h, BHB was also greater on the basolateral side (58.2 and 45.2 ± 1.3 nmol for BB and BUT, respectively) vs apical side (30.2 and 24.0 ± 1.3 nmol; P < 0.001). When looking at BTR utilization, the BB treatment had greater butyrate disappearance than BTR in 24 h (254 vs 169 ± 12.6 nmol; P = 0.0005) and 48h (707 vs 573 ± 12.6 nmol; P < 0.0001). BCP may promote the metabolism of butyrate by intestinal epithelial cells. More research is needed to understand the mechanism of how BCP increases cellular butyrate utilization. [ABSTRACT FROM AUTHOR]

ABSTRACT Epithelial colonization is a critical first step in bacterial pathogenesis. Staphylococcus aureus can utilize several host factors to associate with cells, including α5β1 integrin and heparan sulfate proteoglycans, such as the syndecans. Here, we demonstrate that a partner protein of both integrins and syndecans, the host membrane adapter protein tetraspanin CD9, is essential for syndecan-mediated staphylococcal adhesion. Fibronectin is also essential in this process, while integrins are only critical for post-adhesion entry into human epithelial cells. Treatment of epithelial cells with CD9-derived peptide or heparin caused significant reductions in staphylococcal adherence, dependent on both CD9 and syndecan-1. Exogenous fibronectin caused a CD9-dependent increase in staphylococcal adhesion, whereas blockade of β1 integrins did not affect adhesion but did reduce the subsequent internalization of adhered bacteria. CD9 disruption or deletion increased β1 integrin-mediated internalization, suggesting that CD9 coordinates sequential staphylococcal adhesion and internalization. CD9 controls staphylococcal adhesion through syndecan-1, using a mechanism that likely requires CD9-mediated syndecan organization to correctly display fibronectin at the host cell surface. We propose that CD9-derived peptides or heparin analogs could be developed as anti-adhesion treatments to inhibit the initial stages of staphylococcal pathogenesis. IMPORTANCE Staphylococcus aureus infection is a significant cause of disease and morbidity. Staphylococci utilize multiple adhesion pathways to associate with epithelial cells, including interactions with proteoglycans or β1 integrins through a fibronectin bridge. Interference with another host protein, tetraspanin CD9, halves staphylococcal adherence to epithelial cells, although CD9 does not interact directly with bacteria. Here, we define the role of CD9 in staphylococcal adherence and uptake, observing that CD9 coordinates syndecan-1, fibronectin, and β1 integrins to allow efficient staphylococcal infection. Two treatments that disrupt this action are effective and may provide an alternative to antibiotics. We provide insights into the mechanisms that underlie staphylococcal infection of host cells, linking two known adhesion pathways together through CD9 for the first time.

Renewable alternatives to fossil diesel (FD) including fatty acid methyl ester (FAME) biodiesel have become more prevalent. However, toxicity of exhaust material from their combustion, relative to the fuels they are displacing has not been fully characterised. This study was carried out to examine particle toxicity within the lung epithelium and the role for polycyclic aromatic hydrocarbons (PAHs). Exhaust particles from a 20% (v/v) blend of FAME biodiesel had little impact on primary airway epithelial toxicity compared to FD derived particles but did result in an altered profile of PAHs, including an increase in particle bound carcinogenic B[a]P. Higher blends of biodiesel had significantly increased levels of more carcinogenic PAHs, which was associated with a higher level of stress response gene expression including CYP1A1, NQO1 and IL1B. Removal of semi-volatile material from particulates abolished effects on airway cells. Particle size difference and toxic metals were discounted as causative for biological effects. Finally, combustion of a single component fuel (Methyl decanoate) containing the methyl ester molecular structure found in FAME mixtures, also produced more carcinogenic PAHs at the higher fuel blend levels. These results indicate the use of FAME biodiesel at higher blends may be associated with an increased particle associated carcinogenic and toxicity risk.

Expression of activated Ras, RasV12, provides Drosophila cultured cells with a proliferation and survival advantage that simplifies the generation of continuous cell lines. Here, we used lineage-restricted RasV12 expression to generate continuous cell lines of muscle, glial, and epithelial cell type. Additionally, cell lines with neuronal and hemocyte characteristics were isolated by cloning from cell cultures established with broad RasV12 expression. Differentiation with the hormone ecdysone caused maturation of cells from mesoderm lines into active muscle tissue and enhanced dendritic features in neuronal-like lines. Transcriptome analysis showed expression of key cell-type-specific genes and the expected alignment with single-cell sequencing and in situ data. Overall, the technique has produced in vitro cell models with characteristics of glia, epithelium, muscle, nerve, and hemocyte. The cells and associated data are available from the Drosophila Genomic Resource Center.