Probes for INS

Introduction: The role of eosinophils in intestinal inflammation and fibrosis in inflammatory bowel disease (IBD) is largely unknown. Aim: Therefore, we assessed the functional role of eosinophils in a chronic murine model of colitis and associated fibrosis via anti-CCR3 mediated eosinophil depletion. Methods: 6-8-week-old C57BL/6 RAG-/- mice received three cycles of dextran sodium sulphate (DSS) (1.75% - 2.25% - 2.25%) each interspersed with 14 days of recovery. Twice weekly, anti-CCR3 antibody (n=8), isotype (n=8) or saline injections (n=8) were given intraperitoneally. At the same timepoints, the disease activity index (DAI; mouse weight, stool consistency and presence of blood) was determined. At sacrifice, colonic damage was scored macroscopically (presence of hyperaemia, adhesions and length and degree of colon affected by inflammation). Colonic single cells were isolated and stained for flow cytometry, where eosinophils were characterized as CD45+ CD11b+ Siglec-F+ CD117- cells. Intestinal fibrosis was scored via colon weight/length, collagen deposition, using a colorimetric hydroxyproline assay and Martius Scarlet Blue staining (MSB), and COL1A1 expression by PCR.Results: Anti-CCR3 mediated eosinophil depletion resulted in decreased disease activity compared to the other DSS treated groups injected with saline or isotype, determined by the area under the curve of the DAI (74.6±18.4 vs. 127.5±42.9 and 136.9±33.6, p=0.01 and p=0.0008 respectively). The macroscopic damage score also suggested eosinophil depleted mice to be partially protected from colonic inflammation compared to the saline and isotype injected mice that received DSS (1.1±1.0 vs. 2.1±1.2 and 3.0±0.7, p=0.09 and p=0.001 respectively). Colon weight/length and hydroxyproline assay showed a trend towards increased fibrosis in the anti-CCR3 injected group compared to saline (p=0.03 and 0.07, respectively) but not isotype (p=0.3 and 0.1, respectively) injected groups. However, COL1A1 expression levels were significantly increased in the eosinophil depleted mice compared to the saline and isotype injected mice receiving DSS (43.2±11.4 vs. 23.3±8.7 and 30.1±11.0, p=0.002 and 0.04 respectively), indicating increased collagen expression. Moreover, MSB staining showed increased collagen deposition in the anti-CCR3 treated group compared to the isotype (p=0.0008), but not the saline (p=0.09) injected group exposed to DSS. Conclusions: Eosinophil depletion via intraperitoneal anti-CCR3 injections resulted in partial protection against colonic inflammation, but was associated with increased collagen expression and deposition. Caution is therefore needed when designing therapeutic interventions targeting eosinophils

In hormone receptor-positive metastatic breast cancer (HR+ MBC), endocrine resistance is commonly due to genetic alterations of ESR1, the gene encoding estrogen receptor alpha (ERα). While ESR1 point mutations (ESR1-MUT) cause acquired resistance to aromatase inhibition (AI) through constitutive activation, far less is known about the molecular functions and clinical consequences of ESR1 fusions (ESR1-FUS). This case series discusses 4 patients with HR+ MBC with ESR1-FUS in the context of the existing ESR1-FUS literature. We consider therapeutic strategies and raise the hypothesis that CDK4/6 inhibition (CDK4/6i) may be effective against ESR1-FUS with functional ligand-binding domain swaps. These cases highlight the importance of screening for ESR1-FUS in patients with HR+ MBC while continuing investigation of precision treatments for these genomic rearrangements.

Background: Mucociliary clearance (MCC) is a vital innate defense mechanism that is impaired in people with cystic fibrosis (CF) and animal CF models. Dysfunctional MCC contributes to airway inflammation and infection, which hasten lung function decline. Most people with CF benefit from highly effective CF transmembrane conductance regulator (CFTR) modulators, but some mutations are unresponsive to currently available modulators, and even people with CF who benefit from modulator therapy may be unable to clear chronic pulmonary infections. Accordingly, CFTR-independent methods to increase MCC are needed. We previously discovered that the combination of low-dose cholinergic with βadrenergic agonists synergistically increased MCC velocity (MCCV) in ex vivo tracheal preparations from ferrets and newborn piglets. MCC was also significantly greater in tracheas from CF ferrets to a value of approximately 55% of that in wild-type animals. The MCCV increases were produced without inducing airway narrowing [1]. To further our preclinical work, we tested three hypotheses. We hypothesized that synergistic increases in MCCV by the combined agonists involve epithelial sodium channel (ENaC) inhibition, greater secretion of bicarbonate, and additivity with CFTR modulators. Methods: To test these hypotheses, we measured MCCV in excised newborn piglet tracheas with 10 µM formoterol (beta-adrenergic agonist) plus 0.3 µM methacholine (cholinergic agonist) with and without 10 µM benzamil (ENaC inhibitor) using particle tracking. Bicarbonate secretion rates were measured in tracheal mucosa of Yucatan minipigs mounted in Ussing chambers using a pH-stat method with pH electrodes and automated titrators (Metrohm Titrando 902). To assess whether the synergy agonists improved CF tissues exposed to CFTR modulators, we used high-speed digital microscopy to measure the effective diffusivity (Deff in µm2 /msec) of approximately 2-µm fluorescent polystyrene spheres (0.1%, ThermoFisher) added to the apical surface fluid layer of human CF primary nasal cell cultures (F508del homozygote) grown under air-liquid interface conditions with and without elexacaftor/ tezacaftor/ivacaftor (ELX/TEZ/IVA) (3 μM ELX, 3 μM TEZ, 10 μM IVA). Results: Baseline MCCV was 6 times as high with benzamil inhibition of ENaC (0.5 ± 0.7 mm/min to 3.0 ± 0.7 mm/min; p = 0.02, 4 piglets), but when benzamil was present during synergistically stimulated MCCV, no further increase was seen, consistent with the hypothesis that ENaC was already inhibited by the synergy agonists (MCCV: synergy agonists, 13.9 ± 1.6 mm/ min vs. synergy agonists + benzamil, 14.0 ± 1.6 mm/min; p = 0.97, n = 4 piglets, each condition). The synergy agonists increased bicarbonate secretion rates by about 83% (0.6 ± 0.2 µmol/cm2 per hour at baseline vs. 1.1 ± 0.3 µmol/cm2 per hour with synergy agonists, 5 experiments with 3 pig tracheas). Particle diffusivity in CF primary nasal cell cultures showed synergy agonists plus ELX/TEZ/IVA > synergy agonists > ELX/TEZ/IVA > no treatment. Conclusions: Results were consistent with our hypotheses. The combination of beta adrenergic plus low-dose cholinergic agonists produces synergistic increases in MCCV by inhibiting ENaC and increasing bicarbonate secretion and appears to be at least additive to the effects induced by ELX/TEZ/IVA modulator therapy.

Background In the large intestine, the multipotent stem cells are located at the base of the crypt and differentiate into three main cell types: enterocytes, goblet cells, and enteroendocrine cells. Goblet cells’ main function is the synthesis and secretion of mucins. Genetic and epigenetic changes that provide survival advantages for stem or progenitor cells resulting in the deregulation of cellular differentiation are major causes of all carcinomas. Methods Our laboratory has a large collection of colorectal cancer (CRC) cell lines, well characterised in terms of gene expression and mutations. We analysed the presence of goblet cells in CRC cell lines using the genes Mucin 2 (MUC2) and Trefoil factor 3 (TFF3). The genes both at the mRNA level and at the protein level were investigated. The effects of various transcription factors were assessed by knockdown and overexpression techniques. Results We found that most of the cell lines are unable to produce goblet cells and that the number of MUC2 and TFF3-positive cells among the goblet cell positive cell lines was quite variable. While in the normal colon, MUC2 and TFF3 are always co-expressed, but that is not always the case in the CRC cell lines. MUC2-negative and TFF3-positive cell lines appear to reflect a novel interesting subset. The investigation of several transcription factors on goblet cell differentiation showed that downregulation of Atonal homologue 1 (ATOH1) had a dramatic effect on goblet cell production, while knocking down of SAM pointed domain ETS transcription factor (SPDEF), Caudal type homeobox 1 (CDX1), and 2 (CDX2) had a modest effect. Individually, none of these factors are sufficient to trigger the goblet cell differentiation. Conclusions As a conclusion, the percentage of goblet cells differs substantially between cell lines. Classification of the cell lines reveals an interesting major subset that has TFF3 expression without expressing MUC2. ATOH1, SPDEF, CDX1, and CDX2 had a significant effect on goblet cell differentiation, but on their own, they are not sufficient to induce the goblet cell differentiation. Understanding the mechanisms of goblet cell differentiation is important for advances in the prevention and treatment of CRC.

Background Diabetes clinical trials have shown SGLT inhibition improves cardiovascular outcomes, yet the mechanism is not fully understood. Hyperglycemia is a common finding in diabetic and non-diabetic patients presenting with ACS and is a powerful predictor of prognosis and mortality. The role of hyperglycemia in ischemia-reperfusion injury (IRI) is not fully understood, and whether the Sodium Glucose Co-Transporter 1 (SGLT1) plays a role in infarct augmentation, before and/or after reperfusion, remains to be elucidated. Purpose Investigate if SGLT1 is involved in a glucotoxicity injury during IRI and whether inhibiting SGLT1 with an SGLT1 inhibitor may reduce infarct size. Method RT-PCR and in-situ hybridization (RNAScope) combined with Immunofluorescence integrated co detection with different cell marker techniques were used to detect SGLT1 mRNA expression in Sprague-Dawley whole myocardium and isolated primary cardiomyocytes. An Ex-vivo Langendorff ischemia-reperfusion perfusion model was used to study the effect of high glucose (22mmol) on myocardium at reperfusion. Canagliflozin (CANA) a non-selective SGLT inhibitor (1μmoL/L to block the SGLT1 receptor and SGLT2 and 5nmol/L to block only the SGLT2 receptor) and Mizagliflozin a selective SGLT1 inhibitor (100nmol/L) was introduced following ischemia at two different glucose concentration concentrations at reperfusion and its effect on infarct size measured using triphenyltetrazolium chloride (TTC) staining. Results We showed that SGLT1 is homogenously expressed throughout the myocardium and is particularly evident within the vasculature. we demonstrate that hyperglycemia at reperfusion is injurious to myocardium with an increase of myocardial infarction. Our data reveal that glucose exacerbation of injury appears to be mediated via SGLT1. We have also demonstrated that high-glucose mediated injury in the isolated, perfused heart model is abrogated through the administration of a clinically available mixed SGLT2/SGLT1 inhibitor, canagliflozin, at a dose that inhibits both SGLT2 and SGLT1, but by the SGLT2-selective concentration. Conclusion We have shown that SGLT1 is present in the myocardium. Hyperglycemia appears to augment myocardial infarction and inhibition of SGLT1 attenuates this incre Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): The government of saudi Arabia

Primary bladder tumors have a high degree of malignancy. To investigate the expression of human papillomavirus type 16 (HPV-16) in primary bladder tumors and the loss of cell differentiation and to explore the significance of HPV-16 detection, it is expected to be a disease. Treatment provides a theoretical basis.Fifty-seven patients with primary bladder tumors admitted to our hospital from January 2019 to January 2022 were selected as the research subjects, and they were divided into HPV-related groups according to the human papillomavirus (HPV) infection status (n = 28) and HPV unrelated group (n = 29). The general data of patients were collected, the expression of HPV-16 in bladder tissue samples was detected, and the correlation between pathological parameters and HPV-16 expression was analyzed.Among HPV subtypes, HPV 16 subtype accounted for the highest proportion, followed by HPV-18 and HPV-6 subtypes; there was no significant difference in tumor stage (stage 1, stage a, stage 2a) between the HPV-related group and the HPV-unrelated group (stage 1, stage a, and stage 2a). P > 0.05); there was no significant difference in postoperative pathological expression (high expression and low expression) of patients (P > 0.05); there was no statistical difference in age and gender between HPV-related and HPV-unrelated groups (P > 0.05), HPV-related group and HPV-unrelated group compared daily regular drinking and smoking status, the difference was statistically significant (P < 0.05); HPV-16 expression was not correlated with tumor differentiation degree and age of patients (P > 0.05); the area under the curve (AUC) of HPV-16 for judging primary bladder tumor expression and cellular molecular deletion was 0.891, with a sensitivity of 83.94% and a specificity of 88.57%.HPV-16 is an upper, expressed in primary bladder tumors and will participate in the differentiation and loss of cells, which can provide effective guidance and basis for the diagnosis of primary bladder tumors, which is an important factor for judging the pathological stage and prognosis of patients and can provide a theoretical reference for the formulation of therapeutic measures.

Background: Air-liquid interface (ALI) and organoid culture are key techniques for differentiating human airway epithelial cells (HAECs). The efficiency and robustness of these assays often depends on the quality of primary-isolated cells, but primary cell isolation workflows, with which the user controls the choice of isolation method, cell culture medium, and culture format, may reduce reproducibility. Therefore, an optimized, standardized workflow can enhance and support isolation of epithelial cells from diseased donors with potentially rare cystic fibrosis (CF) mutations or particularly sensitive cell populations. We have developed a standardized workflow for isolation and culture of freshly derived airway epithelial cells. Methods: Briefly, HAECs isolated from primary tissue were expanded in PneumaCult-Ex Plus Medium for 1 week and then seeded into Corning Transwell inserts and expanded until confluency. The cells were then differentiated in PneumaCult-ALI Medium for at least 4 weeks. To assess differentiation efficiency in ALI culture, the cells were immunostained to detect Muc5AC, acetylated tubulin, and ZO-1 to identify goblet cells, ciliated cells, and apical tight junctions, respectively, as well as SARS-CoV-2 cell entry targets angiotensin-converting enzyme 2 and transmembrane serine protease 2. Ion transport and barrier function of the ALI culturesand response to CF transmembrane conductance regulator (CFTR) correctors were also measured. In addition, freshly derived HAECs were seeded into Corning Matrigel domes in the presence of PneumaCult Airway Organoid Seeding Medium. One week later, the medium was changed to PneumaCult Airway Organoid Differentiation Medium and maintained for an additional 3 weeks to promote cell differentiation. These airway organoids were then treated with CFTR corrector VX-809 for 24 hours, followed by 6-hour treatment with amiloride, forskolin, and genistein to induce organoid swelling. Results: Our results demonstrate that ALI cultures derived from CF donors displayed partial rescue of CFTR across multiple passages after treatment with VX-809. Airway organoids were found to express functional CFTR, as evidenced by forskolin treatment, which induced a 64 ± 14% (n = 1 donor) greater organoid area than in vehicle control-treated airway organoids. Airway organoids derived from CF donors displayed a loss of forskolininduced swelling, which could be partially re-established with VX-809 treatment (29 ± 9%, n = 3). Conclusions: In summary, the PneumaCult workflow supports robust, efficient culture of primary-airway epithelial cells that can be used as physiologically relevant models suitable for CF research, CFTR corrector screening, and studying airway biology.

Background: Early after primary infection, HIV reservoirs are established within multiple tissues, including the brain. As these viral reservoirs are not targeted by antiretroviral therapy (cART), we require robust methods of detection, quantification, and characterization of these viral reservoirs in human tissues. Our recent work developed a multi-component imaging methodology that characterizes and quantifies viral reservoirs within the brain. Methods: The imaging methodology demonstrated utilizes the simultaneous staining of brain tissue from HIV-infected donors using DNAscope, RNAscope, and antibodies for HIV-DNA, HIV-mRNA, and either viral or host proteins, respectively. The panel of patients included in these analyses varied in cART regimen, viral load, years living with HIV, and neurocognitive status, all contrasted to age-matched tissues from uninfected patients. Results: Our group demonstrated that cART is sufficient to reduce the size of the viral reservoirs within the brains of HIV patients. We also found that about half of the cells positive for HIV-DNA expressed HIV-mRNA, and only about one-third expressed viral proteins. HIV proteins varied in expression and bystander uptake by uninfected cells but could provide insight into bystander toxicity. Conclusions: The results found were present irrespective of cART regimen and systemic viral replication but suggested that these viral reservoirs are a major barrier to curing HIV and treating associated neurocognitive disorders.

Nociceptive input to the spinal cord is transmitted by primary afferent neurons in the dorsal root ganglia (DRG). A subset of DRG neurons has the ability to attenuate nociceptive transmission through expression of the µ-opioid receptor. The relationship between algesic and analgesic properties of individual DRG neurons has not yet been evaluated in the human. By using a combination of six different 4-Plex in-situ hybridization experiments, we were able to identify three different main nociceptive populations. First, we detected a population poly-responsive, poly-modulated small-diameter nociceptors that co-express TRPV1, OPRM1, and SCN11A, and in most cases additionally SCN10A and P2RX3 and in subpopulations additionally TAC1, TRPA1, TRPM8, OPRD1, PIEZO2, and/or OPRL1. Second, we detected a medium-sized set of multimodal putative nociceptors with a very similar molecular profile, apart from a lack of expression of TRPA1. Both nociceptor types do express the µ-opioid receptor (encoded by OPRM1) and their activity and input into the spinal cord likely can be attenuated by clinically used µ-receptor agonists. The third population consists of small-diameter neurons that have a molecular expression profile characteristic for nociceptive neurons, such that they express TRPV1 together with SCN11A, SCN10A, P2RX3, PIEZO2, OPRD1, and partially TRPA1. The most distinguishable characteristic of this population is the lack of expression of the µ-opioid receptor and thus are non-susceptible to clinically used opioids. Further molecular characterization of these nociceptive populations might reveal attractive molecular candidates for targeting different types of pain indications.

The expanding use of chemotherapy in curative cancer treatment has simultaneously resulted in a substantial and growing cohort of cancer survivors with prolonged disability from chemotherapy-induced peripheral neuropathy (CIPN). CIPN is associated with several commonly prescribed chemotherapeutics, including taxanes, platinum-based drugs, vinca alkaloids, bortezomib and thalidomide. These distinct classes of chemotherapeutics, with their varied neurotoxic mechanisms, often cause patients to suffer from a broad profile of neuropathic symptoms including chronic numbness, paraesthesia, loss of proprioception or vibration sensation and neuropathic pain. Decades of investigation by numerous research groups have provided substantial insights describing this disease. Despite these advances, there is currently no effective curative or preventative treatment option for CIPN and only the dual serotonin-norepinephrine reuptake inhibitor Duloxetine is recommended by clinical guidelines for the symptomatic treatment of painful CIPN.In this review, we examine current preclinical models, with our analysis focused on translational relevance and value.Animal models have been pivotal in achieving a better understanding of the pathogenesis of CIPN. However, it has been challenging for researchers to develop appropriate preclinical models that are effective vehicles for the discovery of translatable treatment options.Further development of preclinical models targeting translational relevance will promote value for preclinical outcomes in CIPN studies.

Methods: Global and neuronal specific IL-1R1 reporter mice, RNA sequencing analysis, and double-immunofluorescent labeling were used to map and validate nIL-1R1 expression. NF-κB/IL-1R1 co-reporter mice were utilized to detect IL-1R1 and NF-κB expression following intracerebroventricular (i.c.v.) IL-1 injections. Basescope in situ hybridization was utilized to detect splice variants of IL-1R Accessory Protein (IL-1AcP). Unpredictable foot shock (6x shocks over 1hr for 6d) was employed as a chronic stress paradigm. Results: IL-1R1 is expressed in subsets of glutamatergic or serotonergic neurons, with highest expression in the dentate gyrus (DG) and dorsal raphe nucleus (DRN). I.c.v. IL-1β injection reveals nIL-1R1 does not signal through the canonical NF-κB pathway, whereas endothelia and ventricular IL-1R1s do. We identified that neurons of the DG and DRN express the alternatively spliced IL-1RAcP Isoform B (IL-1RAcPb). Additional results suggest that nIL-1R1 may become reactive to IL-1 when neuronal expression of IL-1RAcPb shifts to the canonical IL-1RAcP following stress. Consequently, nIL-1R1 mediates activation of microglia near nIL-1R1 neurons. Conclusions: These data suggest that regional specific nIL-1R1 may require a culmination of stress and inflammatory signals to unlock nIL-1R1 signaling. Overall, these data provide a map of nIL-1R1 and its corresponding accessory protein in the brain along with a potential output of nIL-1R1 signaling.

Although generally regarded as degradatory enzymes, certain proteases are also signaling molecules that specifically control cellular functions by cleaving protease-activated receptors (PARs). The four known PARs are members of the large family of G protein-coupled receptors. These transmembrane receptors control most physiological and pathological processes and are the target of a large proportion of therapeutic drugs. Signaling proteases include enzymes from the circulation, from immune, inflammatory epithelial and cancer cells, as well as from commensal and pathogenic bacteria. Advances in our understanding of the structure and function of PARs provide insights into how diverse proteases activate these receptors to regulate physiological and pathological processes in most tissues and organ systems. The realization that proteases and PARs are key mediators of disease, coupled with advances in understanding the atomic level structure of PARs and their mechanisms of signaling in subcellular microdomains, has spurred the development of antagonists, some of which have advanced to the clinic. Herein we review the discovery, structure and function of this receptor system, highlight the contribution of PARs to homeostatic control, and discuss the potential of PAR antagonists for the treatment of major diseases.