NALCN-mediated sodium influx confers metastatic prostate cancer cell invasiveness.

There is growing evidence that ion channels are critically involved in cancer cell invasiveness and metastasis. However, the molecular mechanisms of ion signaling promoting cancer behavior are poorly understood and the complexity of the underlying remodeling during metastasis remains to be explored. Here, using a variety of in vitro and in vivo techniques, we show that metastatic prostate cancer cells acquire a specific Na+ /Ca2+ signature required for persistent invasion. We identify the Na+ leak channel, NALCN, which is overexpressed in metastatic prostate cancer, as a major initiator and regulator of Ca2+ oscillations required for invadopodia formation. Indeed, NALCN-mediated Na+ influx into cancer cells maintains intracellular Ca2+ oscillations via a specific chain of ion transport proteins including plasmalemmal and mitochondrial Na+ /Ca2+ exchangers, SERCA and store-operated channels. This signaling cascade promotes activity of the NACLN-colocalized proto-oncogene Src kinase, actin remodeling and secretion of proteolytic enzymes, thus increasing cancer cell invasive potential and metastatic lesions in vivo. Overall, our findings provide new insights into an ion signaling pathway specific for metastatic cells where NALCN acts as persistent invasion controller.

Systematic Review and Meta-analysis of Histological Gastric Biopsy Aspects According to the Updated Sydney System in Children.

OBJECTIVES: A descriptive and comparative study of gastric histological aspects according to the updated Sydney classification (USC), obtained from Helicobacter pylori-positive versus H pylori-negative children referred for upper gastrointestinal endoscopy. METHODS: The Prisma method was used to perform a systematic review and meta-analysis. Selection criteria were based on following key words USC, H pylori, children, endoscopy, or biopsy. Publication biases were assessed according to the Newcastle-Ottawa Scale, and a meta-regression analysis was done. The study was registered on the PROSPERO platform. RESULTS: Between 1994 and 2017, 1238 references were found; 97 studies were retained for the systematic review with a total number of 25,867 children; 75 studies were selected for the meta-analysis concerning 5990 H pylori-infected and 17,782 uninfected children.H pylori-positive versus H pylori-negative children, according to the USC, showed significantly higher relative risk for gastric antral and corpus chronic inflammation, presence of neutrophils, and of lymphoid follicles, and gastric mucosa atrophy, whereas, intestinal metaplasia showed a significantly higher RR only in antral biopsies. The meta-regression analysis showed that H pylori-positive versus H pylori-negative children had significantly higher risk only for corpus activity according to age, recurrent abdominal pain, and geographical area of low H pylori prevalence. CONCLUSIONS: H pylori infection in children was associated with higher relative risk for gastric antral and corpus chronic inflammation, presence of neutrophils, lymphoid follicles, and rare gastric mucosa atrophy, whereas, rare intestinal metaplasia was only significantly higher in the antral area.

A Novel Anti-TRPV6 Antibody and Its Application in Cancer Diagnosis In Vitro.

Though the first discovery of TRPV6 channel expression in various tissues took place in the early 2000s, reliable tools for its protein detection in various cells and tissues are still missing. Here we show the generation and validation of rabbit polyclonal anti-TRPV6 channel antibodies (rb79-82) against four epitopes of 15 amino acids. Among them, only one antibody, rb79, was capable of detecting the full-length glycosylated form of the TRPV6 channel at around 100 kDa. The generated antibody was shown to be suitable for all in vitro applications, such as immunoblotting, immunoprecipitation, immunocytochemistry, immunofluorescence, etc. One of the most important applications is immunohistochemistry using the paraffin-embedded sections from cancer resection specimens. Using prostate cancer resection specimens, we have confirmed the absence of the TRPV6 protein in both healthy and benign hyperplasia, as well as its expression and correlation to the prostate cancer grades. Thus, the generated rabbit polyclonal anti-TRPV6 channel antibody rb79 is suitable for all in vitro diagnostic applications and particularly for the diagnosis in clinics using paraffin-embedded sections from patients suffering from various diseases and disorders involving the TRPV6 channel.

A prospective pilot study of the T-lymphocyte response to fine particulate matter exposure.

Exposure to air pollution is associated with increased morbidity and mortality. Once the fine atmospheric particulate matter (FP) is inhaled, some of its compounds can pass through the lungs and reach the bloodstream where they can come into contact with immune cells. Exposure to FP particularly affects sensitive populations such as the elderly. Aging affects the immune system, making the elderly more vulnerable. The project aims to determine the effects of FP exposure on human T cells while looking for biomarkers associated with exposure. Blood samples from 95 healthy subjects in three different age groups (20-30, 45-55 and 70-85 years) were collected to determine a potential age effect. T lymphocytes were isolated to be exposed ex vivo for 72 hours to 45 µg/mL of FP collected in Dunkirk and chemically characterized. Overexpression of the CYP1A1, CYP1B1 and CYP2S1 genes was therefore measured after exposure of the T cells to FP. These genes code for enzymes known to be involved in the metabolic activation of organic compounds such as polycyclic aromatic hydrocarbons detected in the FP sample. T-cell profiling allowed us to suggest a mixed T-helper 1/2 profile caused by exposure to FP. With regard to the influence of age, we have observed differences in the expression of certain genes, as well as an increase in interleukin-4 and -13 concentrations in the elderly. These results showed that exposure of T lymphocytes to FP causes effects on both transcriptomic and cytokine secretion levels.

Chemical characterization of fine and ultrafine PM, direct and indirect genotoxicity of PM and their organic extracts on pulmonary cells.

Particulate matter in ambient air constitutes a complex mixture of fine and ultrafine particles composed of various chemical compounds including metals, ions, and organics. A multidisciplinary approach was developed by studying physico-chemical characteristics and mechanisms involved in the toxicity of particulate atmospheric pollution. PM0.3-2.5 and PM2.5 including ultrafine particles were sampled in Dunkerque, a French industrialized seaside city. PM samples were characterized from a chemical and toxicological point of view. Physico-chemical characterization evidenced that PM2.5 comes from several sources: natural ones, such as soil resuspension and marine sea-salt emissions, as well as anthropogenic ones, such as shipping traffic, road traffic, and industrial activities. Human BEAS-2B lung cells were exposed to PM0.3-2.5, or to the Extractable Organic Matter (EOM) of PM0.3-2.5 and PM2.5. These exposures induced several mechanisms of action implied in the genotoxicity, such as oxidative DNA adducts and DNA Damage Response. The toxicity of PM-EOM was higher for the sample including the ultrafine fraction (PM2.5) containing also higher concentrations of polycyclic aromatic hydrocarbons. These results evidenced the major role of organic compounds in the toxicity of PM.

Flagellin-Mediated Protection against Intestinal Yersinia pseudotuberculosis Infection Does Not Require Interleukin-22.

Signaling through Toll-like receptors (TLRs), the main receptors in innate immunity, is essential for the defense of mucosal surfaces. It was previously shown that systemic TLR5 stimulation by bacterial flagellin induces an immediate, transient interleukin-22 (IL-22)-dependent antimicrobial response to bacterial or viral infections of the mucosa. This process was dependent on the activation of type 3 innate lymphoid cells (ILCs). The objective of the present study was to analyze the effects of flagellin treatment in a murine model of oral infection with Yersinia pseudotuberculosis (an invasive, Gram-negative, enteropathogenic bacterium that targets the small intestine). We found that systemic administration of flagellin significantly increased the survival rate after intestinal infection (but not systemic infection) by Y. pseudotuberculosis This protection was associated with a low bacterial count in the gut and the spleen. In contrast, no protection was afforded by administration of the TLR4 agonist lipopolysaccharide, suggesting the presence of a flagellin-specific effect. Lastly, we found that TLR5- and MyD88-mediated signaling was required for the protective effects of flagellin, whereas neither lymphoid cells nor IL-22 was involved.

Activation of mutated TRPA1 ion channel by resveratrol in human prostate cancer associated fibroblasts (CAF).

Previous studies showed the effects of resveratrol (RES) on several cancer cells, including prostate cancer (PCa) cell apoptosis without taking into consideration the impact of the tumor microenvironment (TME). The TME is composed of cancer cells, endothelial cells, blood cells, and cancer-associated fibroblasts (CAF), the main source of growth factors. The latter cells might modify in the TME the impact of RES on tumor cells via secreted factors. Recent data clearly show the impact of CAF on cancer cells apoptosis resistance via secreted factors. However, the effects of RES on PCa CAF have not been studied so far. We have investigated here for the first time the effects of RES on the physiology of PCa CAF in the context of TME. Using a prostate cancer CAF cell line and primary cultures of CAF from prostate cancers, we show that RES activates the N-terminal mutated Transient Receptor Potential Ankyrin 1 (TRPA1) channel leading to an increase in intracellular calcium concentration and the expression and secretion of growth factors (HGF and VEGF) without inducing apoptosis in these cells. Interestingly, in the present work, we also show that when the prostate cancer cells were co-cultured with CAF, the RES-induced cancer cell apoptosis was reduced by 40%, an apoptosis reduction canceled in the presence of the TRPA1 channel inhibitors. The present work highlights CAF TRPA1 ion channels as a target for RES and the importance of the channel in the epithelial-stromal crosstalk in the TME leading to resistance to the RES-induced apoptosis.

How do they stick together? Bacterial adhesins implicated in the binding of bacteria to the human gastrointestinal mucins.

The gastrointestinal mucosal surface is the primary interface between internal host tissues and the vast microbiota. Mucins, key components of mucus, are high-molecular-weight glycoproteins characterized by the presence of many O-linked oligosaccharides to the core polypeptide. They play many biological functions, helping to maintain cellular homeostasis and to establish symbiotic relationships with complex microbiota. Mucin O-glycans exhibit a huge variety of peripheral sequences implicated in the binding of bacteria to the mucosal tissues, thereby playing a key role in the selection of specific species and in the tissue tropism displayed by commensal and pathogenic bacteria. Bacteria have evolved numerous strategies to colonize host mucosae, and among these are modulation of expression of cell surface adhesins which allow bacteria to bind to mucins. However, despite well structurally characterized adhesins and lectins, information on the nature and structure of oligosaccharides recognized by bacteria is still disparate. This review summarizes the current knowledge on the structure of epithelial mucin O-glycans and the interaction between host and commensal or pathogenic bacteria mediated by mucins.

A one-step immune-chromatographic Helicobacter pylori stool antigen test for children was quick, consistent, reliable and specific.

AIM: This French study assessed a quick, noninvasive, immuno-chromatographic, Helicobacter pylori (H. pylori) stool antigen test for detecting infections in children. METHODS: We enrolled 158 children, with a median age of 8.5 years (range eight months to 17 years), with digestive symptoms suggesting upper gastrointestinal tract disease. Upper digestive endoscopy was performed with gastric biopsy specimens for histology, a rapid urease test, culture test and quantitative real-time polymerase chain reaction. The H. pylori stool antigen test was performed twice for each child and the results were compared to the reference method. RESULTS: The reference methods showed that 23 (14.6%) of the 158 children tested were H. pylori positive. The H. pylori stool antigen test showed 91.3% sensitivity, with a 95% confidence interval (95% CI) of 86.9-95.6 and 97% specificity (95% CI 94.3-99.6), 30.84 positive likelihood ratio and 0.09 negative likelihood ratio. The test accuracy was 96.2% (95% CI 93.2-99.1). The two blinded independent observers produced identical H. pylori stool antigen test results and the Kappa coefficient for the H. pylori stool antigen test was one. CONCLUSION: The H. pylori stool antigen test was found to be a consistent, reliable, quick and specific test for detecting the H. pylori infection in children.

TRPV6 calcium channel translocates to the plasma membrane via Orai1-mediated mechanism and controls cancer cell survival.

Transient receptor potential vanilloid subfamily member 6 (TRPV6) is a highly selective calcium channel that has been considered as a part of store-operated calcium entry (SOCE). Despite its first discovery in the early 2000s, the role of this channel in prostate cancer (PCa) remained, until now, obscure. Here we show that TRPV6 mediates calcium entry, which is highly increased in PCa due to the remodeling mechanism involving the translocation of the TRPV6 channel to the plasma membrane via the Orai1/TRPC1-mediated Ca(2+)/Annexin I/S100A11 pathway, partially contributing to SOCE. The TRPV6 calcium channel is expressed de novo by the PCa cell to increase its survival by enhancing proliferation and conferring apoptosis resistance. Xenografts in nude mice and bone metastasis models confirmed the remarkable aggressiveness of TRPV6-overexpressing tumors. Immunohistochemical analysis of these demonstrated the increased expression of clinical markers such as Ki-67, prostate specific antigen, synaptophysin, CD31, and CD56, which are strongly associated with a poor prognosis. Thus, the TRPV6 channel acquires its oncogenic potential in PCa due to the remodeling mechanism via the Orai1-mediated Ca(2+)/Annexin I/S100A11 pathway.

In vitro short-term exposure to air pollution PM2.5-0.3 induced cell cycle alterations and genetic instability in a human lung cell coculture model.

Although its adverse health effects of air pollution particulate matter (PM2.5) are well-documented and often related to oxidative stress and pro-inflammatory response, recent evidence support the role of the remodeling of the airway epithelium involving the regulation of cell death processes. Hence, the overarching goals of the present study were to use an in vitro coculture model, based on human AM and L132 cells to study the possible alteration of TP53-RB gene signaling pathways (i.e. cell cycle phases, gene expression of TP53, BCL2, BAX, P21, CCND1, and RB, and protein concentrations of their active forms), and genetic instability (i.e. LOH and/or MSI) in the PM2.5-0.3-exposed coculture model. PM2.5-0.3 exposure of human AM from the coculture model induced marked cell cycle alterations after 24h, as shown by increased numbers of L132 cells in subG1 and S+G2 cell cycle phases, indicating apoptosis and proliferation. Accordingly, activation of the TP53-RB gene signaling pathways after the coculture model exposure to PM2.5-0.3 was reported in the L132 cells. Exposure of human AM from the coculture model to PM2.5-0.3 resulted in MS alterations in 3p chromosome multiple critical regions in L132 cell population. Hence, in vitro short-term exposure of the coculture model to PM2.5-0.3 induced cell cycle alterations relying on the sequential occurrence of molecular abnormalities from TP53-RB gene signaling pathway activation and genetic instability.

Alteration or adaptation, the two roads for human gastric mucin glycosylation infected by Helicobacter pylori.

Helicobacter pylori is a Gram-negative bacterium that colonizes the mucus niche of the gastric mucosa and infects more than half of the world's human population. Chronic infection may cause gastritis, duodenal ulcer, intestinal metaplasia or gastric cancer. In the stomach, H. pylori interacts with O-glycans of gastric mucins but the mechanism by which the bacteria succeed in altering the mucosa remains mainly unknown. To better understand the physiopathology of the infection, inhibitory adhesion assays were performed with various O-glycans expressed by human gastric mucins, and topographic expression of gastric mucins MUC5AC and MUC6 was analyzed for healthy uninfected individuals, for infected asymptomatic individuals and for patients infected by H. pylori and having the incomplete type of intestinal metaplasia. The glycosylation of the gastric mucosa of asymptomatic individuals infected by H. pylori was determined and compared with the glycosylation pattern found for patients with the incomplete type of intestinal metaplasia. Results show that H. pylori manages to modulate host's glycosylation during the course of infection in order to create a favorable niche, whereas asymptomatic infected individuals seem to counteract further steps of infection development by adapting their mucus glycosylation.

Overexpression of chemokine receptor CXCR2 and ligand CXCL7 in liver metastases from colon cancer is correlated to shorter disease-free and overall survival.

Our aim was to analyze the potential role of chemokine receptors CXCR2 and CXCR4 signalling pathways in liver metastatic colorectal cancer (CRC) relapse. CXCR2, CXCR4, and their chemokine ligands were evaluated in liver metastases of colorectal cancer in order to study their correlation with overall and disease-free survival of patients having received, or not received, a neoadjuvant chemotherapy regimen. Quantitative RT-PCR and CXCR2 immunohistochemical staining were carried out using CRC liver metastasis samples. Expression levels of CXCR2, CXCR4, and their ligands were statistically analyzed according to treatment with neoadjuvant chemotherapy and patients' outcome. CXCR2 and CXCL7 overexpression are correlated to shorter overall and disease-free survival. By multivariate analysis, CXCR2 and CXCL7 expressions are independent factors of overall and disease-free survival. Neoadjuvant chemotherapy increases significantly the expression of CXCR2: treated group 1.89 (0.02-50.92) vs 0.55 (0.07-3.22), P = 0.016. CXCL7 was overexpressed close to significance, 0.40 (0.00-7.85) vs 0.15 (0.01-7.88), P = 0.12. We show the involvement of CXCL7/CXCR2 signalling pathways as a predictive factor of poor outcome in metastatic CRC. 5-Fluorouracil-based chemotherapy regimens increase the expression of these genes in liver metastasis, providing one explanation for aggressiveness of relapsed drug-resistant tumors. Selective blockage of CXCR2/CXCL7 signalling pathways could provide new potential therapeutic opportunities.

Usefulness of Gastric Biopsy-Based Real-Time Polymerase Chain Reaction for the Diagnosis of Helicobacter pylori Infection in Children.

OBJECTIVES: The aim of the study was to assess the usefulness of gastric biopsy-based quantitative real-time polymerase chain reaction (qPCR) for the detection of Helicobacter pylori infection and the identification of clarithromycin-resistant strains in children. METHODS: A gastric biopsy-based qPCR for the detection of H pylori infection and the identification of clarithromycin-resistant strains in children was evaluated in 62 children with infection and 341 children without infection. H pylori infection was considered by the "reference method" when culture was positive for both histology and rapid urease test (RUT). Results were compared with those obtained using the qPCR. RESULTS: The reference method versus H pylori qPCR positivity showed 95% confidence interval sensitivity 100% versus 100%, specificity 93.2% (86.9-99.4) versus 100%, positive predictive value 59.7% (47.4-71.9) versus 100%, negative predictive value 100% versus 100%, and, finally, test accuracy of 59.6% (47.3-71.8) versus 100%. Sixty-two children were found to be H pylori positive, based on the qPCR results. Among those, 31 children had both positive qPCR and culture with concordant antimicrobial susceptibility testing results, whereas 31 children had negative culture and positive qPCR. The qPCR showed a bacterial load ≥10 copies per milliliter when culture, histology, and RUT were all positive (29/31 children) versus <10 copies per milliliter when culture, histology, and RUT were all negative (25/31 children). Grades 2 and 3 histological gastritis were associated with a bacterial load ≥10 copies per milliliter for 28/35 of children versus 27/27 of grade 0 to 1 <10 copies per milliliter. CONCLUSIONS: H pylori qPCR positivity is a more precise test than the routine culture, histology, RUT alone and allows detecting low bacterial loads.

Claudin 3, 4, and 15 expression in solid tumors of lung adenocarcinoma versus malignant pleural mesothelioma.

Epithelioid malignant pleural mesothelioma (MPM) can easily be confused with lung adenocarcinomas (ACAs). In serous effusion, claudin (cldn) 3 is shown to be useful in the diagnosis of mesothelioma vs ACAs. Cldn15 is reported to be overexpressed in epithelioid mesothelioma and absent in human airway epithelium. The aim was to assess the value of cldn3 and cldn4 compared to that of BerEp4 and thyroid transcription factor-1 (TTF1) in differentiating lung ACA from epithelioid MPM and to examine the expression of cldn15 in these tumors. The expression of cldn3, cldn4, cldn15, BerEp4, and TTF1 was examined by immunohistochemistry in a total of 62 human specimen including 28 epithelioid MPMs and 34 ACAs of the lung. In lung ACA, cldn4 was strongly expressed in all 34 (100%) specimens followed by cldn3 in 33 (97%) of 34. BerEp4 was expressed in 32 (94.1%) of 34. TTF1 reacted for only 20 (58.82%) of 34 cases of lung ACA. In MPM specimens, the expression of cldn3 and4 as well as that of TTF1 was completely absent. In contrast, BerEp4 was focally expressed in 5 (17.85%) of 28 cases of epithelioid MPM. Cldn15 was strongly expressed in 53% pf epithelioid MPMs but also in 50% of lung ACAs. Its expression was moderate in normal pleura and limited in normal lung. Cldn3 and cldn4 appear to be the best performing carcinoma markers in discriminating lung ACA from mesothelioma compared with BerEp4 and TTF1. There is no differential expression of cldn15 between the 2 pathologies. However, the limited cldn15 expression in normal tissues and high expression in tumors make it an attractive candidate for cancer therapy.

The lacdiNAc-specific adhesin LabA mediates adhesion of Helicobacter pylori to human gastric mucosa.

Adhesion of Helicobacter pylori to the gastric mucosa is a necessary prerequisite for the pathogenesis of H. pylori-related diseases. In this study, we investigated the GalNAcß1-4GlcNAc motif (also known as N,N'-diacetyllactosediamine [lacdiNAc]) carried by MUC5AC gastric mucins as the target for bacterial binding to the human gastric mucosa. The expression of LacdiNAc carried by gastric mucins was correlated with H. pylori localization, and all strains tested adhered significantly to this motif. Proteomic analysis and mutant construction allowed the identification of a yet uncharacterized bacterial adhesin, LabA, which specifically recognizes lacdiNAc. These findings unravel a target of adhesion for H. pylori in addition to moieties recognized by the well-characterized adhesins BabA and SabA. Localization of the LabA target, restricted to the gastric mucosa, suggests a plausible explanation for the tissue tropism of these bacteria. These results pave the way for the development of alternative strategies against H. pylori infection, using adherence inhibitors.

Interleukin-22 reduces lung inflammation during influenza A virus infection and protects against secondary bacterial infection.

Interleukin-22 (IL-22) has redundant, protective, or pathogenic functions during autoimmune, inflammatory, and infectious diseases. Here, we addressed the potential role of IL-22 in host defense and pathogenesis during lethal and sublethal respiratory H3N2 influenza A virus (IAV) infection. We show that IL-22, as well as factors associated with its production, are expressed in the lung tissue during the early phases of IAV infection. Our data indicate that retinoic acid receptor-related orphan receptor-γt (RORγt)-positive αß and γδ T cells, as well as innate lymphoid cells, expressed enhanced Il22 transcripts as early as 2 days postinfection. During lethal or sublethal IAV infections, endogenous IL-22 played no role in the control of IAV replication and in the development of the IAV-specific CD8(+) T cell response. During lethal infection, where wild-type (WT) mice succumbed to severe pneumonia, the lack of IL-22 did not accelerate or delay IAV-associated pathogenesis and animal death. In stark contrast, during sublethal IAV infection, IL-22-deficient animals had enhanced lung injuries and showed a lower airway epithelial integrity relative to WT littermates. Of importance, the protective effect of endogenous IL-22 in pulmonary damages was associated with a more controlled secondary bacterial infection. Indeed, after challenge with Streptococcus pneumoniae, IAV-experienced Il22(-/-) animals were more susceptible than WT controls in terms of survival rate and bacterial burden in the lungs. Together, IL-22 plays no major role during lethal influenza but is beneficial during sublethal H3N2 IAV infection, where it limits lung inflammation and subsequent bacterial superinfections.

The carbon footprint of a surgical pathology laboratory in France.

OBJECTIVES: The health sector contributes to climate disruption through greenhouse gas (GHG) emissions. It accounts for 8% to 10% of France's GHG emissions. Although the medical community has been alerted to the problem, more data are needed. This study aimed to determine the carbon footprint of a surgical pathology laboratory. METHODS: The study was conducted in the surgical pathology laboratory at Saint Vincent hospital (Lille) in 2021. It represented 17,242 patient cases corresponding to 54,124 paraffin blocks. The 17 staff members performed cytology, immunohistochemistry, and in situ hybridization. The study included all inputs, capital equipment, freight, travel, energy consumption, and waste. Carbon emission factors were based on the French Agence De l'Environnement et de la Maîtrise de l'Energie database. RESULTS: In 2021, the pathology laboratory's carbon footprint was 117 tons of CO2 equivalent (t CO2e), corresponding to 0.5% of Saint Vincent hospital's total emissions. The most significant emissions categories were inputs (60 t CO2e; 51%), freight associated with inputs (24 t CO2e; 20%), and travel (14 t CO2e; 12%). Waste and energy generated 10 t CO2e (9%) and 9 t CO2e (8%), respectively. CONCLUSIONS: The pathology laboratory's carbon footprint was equivalent to the yearly carbon impact of 11 French inhabitants. This footprint is dominated by inputs and associated freight. This suggests an urgent need to develop ecodesign and self-sufficiency in our routine practices.

Trpv6 channel targeting using monoclonal antibody induces prostate cancer cell apoptosis and tumor regression.

TRPV6 calcium channel is a prospective target in prostate cancer (PCa) since it is not expressed in healthy prostate while its expression increases during cancer progression. Despite the role of TRPV6 in PCa cell survival and apoptotic resistance has been already established, no reliable tool to target TRPV6 channel in vivo and thus to reduce tumor burden is known to date. Here we report the generation of mouse monoclonal antibody mAb82 raised against extracellular epitope of the pore region of the channel. mAb82 inhibited TRPV6 currents by 90% at 24 µg/ml in a dose-dependent manner while decreasing store-operated calcium entry to 56% at only 2.4 µg/ml. mAb82 decreased PCa survival rate in vitro by 71% at 12 µg/ml via inducing cell death through the apoptosis cascade via activation of the protease calpain, following bax activation, mitochondria enlargement, and loss of cristae, Cyt C release, pro-caspase 9 cleavage with the subsequent activation of caspases 3/7. In vivo, mice bearing either PC3Mtrpv6+/+ or PC3Mtrpv6-/-+pTRPV6 tumors were successfully treated with mAb82 at the dose as low as 100 µg/kg resulting in a significant reduction tumor growth by 31% and 90%, respectively. The survival rate was markedly improved by 3.5 times in mice treated with mAb82 in PC3Mtrpv6+/+ tumor group and completely restored in PC3Mtrpv6-/-+pTRPV6 tumor group. mAb82 showed a TRPV6-expression dependent organ distribution and virtually no toxicity in the same way as mAbAU1, a control antibody of the same Ig2a isotype. Overall, our data demonstrate for the first time the use of an anti-TRPV6 monoclonal antibody in vitro and in vivo in the treatment of the TRPV6-expressing PCa tumors.

Interleukin-22 is produced by invariant natural killer T lymphocytes during influenza A virus infection: potential role in protection against lung epithelial damages.

Invariant natural killer T (iNKT) cells are non-conventional lipid-reactive αß T lymphocytes that play a key role in host responses during viral infections, in particular through the swift production of cytokines. Their beneficial role during experimental influenza A virus (IAV) infection has recently been proposed, although the mechanisms involved remain elusive. Here we show that during in vivo IAV infection, mouse pulmonary iNKT cells produce IFN-γ and IL-22, a Th17-related cytokine critical in mucosal immunity. Although permissive to viral replication, IL-22 production by iNKT cells is not due to IAV infection per se of these cells but is indirectly mediated by IAV-infected dendritic cells (DCs). We show that activation of the viral RNA sensors TLR7 and RIG-I in DCs is important for triggering IL-22 secretion by iNKT cells, whereas the NOD-like receptors NOD2 and NLRP3 are dispensable. Invariant NKT cells respond to IL-1ß and IL-23 provided by infected DCs independently of the CD1d molecule to release IL-22. In vitro, IL-22 protects IAV-infected airway epithelial cells against mortality but has no role on viral replication. Finally, during early IAV infection, IL-22 plays a positive role in the control of lung epithelial damages. Overall, IAV infection of DCs activates iNKT cells, providing a rapid source of IL-22 that might be beneficial to preserve the lung epithelium integrity.