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BACKGROUND AIMS: Whole tumor cell lysates (TCLs) obtained from cancer cells previously killed by treatments able to promote immunogenic cell death (ICD) can be efficiently used as a source of tumor-associated antigens for the development of highly efficient dendritic cell (DC)-based vaccines. Herein, the potential role of the interferon (IFN)-inducible protein phospholipid scramblase 1 (PLSCR1) in influencing immunogenic features of dying cancer cells and in enhancing DC-based vaccine efficiency was investigated. METHODS: PLSCR1 expression was evaluated in different mantle-cell lymphoma (MCL) cell lines following ICD induction by 9-cis-retinoic acid (RA)/IFN-α combination, and commercial kinase inhibitor was used to identify the signaling pathway involved in its upregulation. A Mino cell line ectopically expressing PLSCR1 was generated to investigate the potential involvement of this protein in modulating ICD features. Whole TCLs obtained from Mino overexpressing PLSCR1 were used for DC loading, and loaded DCs were employed for generation of tumor antigen-specific cytotoxic T lymphocytes. RESULTS: The ICD inducer RA/IFN-α combination promoted PLSCR1 expression through STAT1 activation. PLSCR1 upregulation favored pro-apoptotic effects of RA/IFN-α treatment and enhanced the exposure of calreticulin on cell surface. Moreover, DCs loaded with TCLs obtained from Mino ectopically expressing PLSCR1 elicited in vitro greater T-cell-mediated antitumor responses compared with DCs loaded with TCLs derived from Mino infected with empty vector or the parental cell line. Conversely, PLSCR1 knock-down inhibited the stimulating activity of DCs loaded with RA/IFN-α-treated TCLs to elicit cyclin D1 peptide-specific cytotoxic T lymphocytes. CONCLUSIONS: Our results indicate that PLSCR1 improved ICD-associated calreticulin exposure induced by RA/IFN-α and was clearly involved in DC-based vaccine efficiency as well, suggesting a potential contribution in the control of pathways associated to DC activation, possibly including those involved in antigen uptake and concomitant antitumor immune response activation.
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Antineoplásicos , Vacunas , Proteínas de Transferencia de Fosfolípidos/genética , Proteínas de Transferencia de Fosfolípidos/metabolismo , Calreticulina/metabolismo , Muerte Celular Inmunogénica , Antineoplásicos/metabolismo , Antígenos de Neoplasias , Inmunidad , Células Dendríticas , Vacunas/metabolismoRESUMEN
Eosinophilic esophagitis (EoE) is increasingly diagnosed in patients with dysphagia. Type-2 immunity can induce EoE histopathology via non-IgE-dependent mechanisms, possibly involving IgG4 and IL-10. To elucidate the contribution of this response to EoE pathogenesis, we examined its association with clinical and histologic endpoints in adult EoE patients given a two-food elimination diet. IgG4- and IL-10-expressing cells were counted in esophageal biopsies and serum food-specific IgG4 measured at baseline and follow-up. Variables were correlated with histologic measures of disease activity. Patients exhibited significant reduction in esophageal eosinophilia and overall histology. A significant decrease in IL-10+-cell frequencies correlated with histologic changes. In contrast, a decline in serum and esophageal IgG4, while substantial, did not correlate with IL-10+-cell frequencies or histologic parameters. These results suggest a critical role of IL-10 in EoE pathogenesis. Conversely, IgG4 expression, while reflecting exposure to food antigens, is not obviously related to EoE histopathology or IL-10 expression.
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Esofagitis Eosinofílica , Adulto , Humanos , Alérgenos , Biopsia , Esofagitis Eosinofílica/inmunología , Inmunoglobulina G , Interleucina-10RESUMEN
Phospholipid scramblase 1 (PLSCR1) is the most studied protein of the scramblase family. Originally, it was identified as a membrane protein involved in maintaining plasma membrane asymmetry. However, studies conducted over the past few years have shown the involvement of PLSCR1 in several other cellular pathways. Indeed, PLSCR1 is not only embedded in the plasma membrane but is also expressed in several intracellular compartments where it interacts with a diverse repertoire of effectors, mediators, and regulators contributing to distinct cellular processes. Although most PLSCR1 interactors are thought to be cell-type specific, PLSCR1 often exerts its regulatory functions through shared mechanisms, including the trafficking of different molecules within intracellular vesicles such as endosomes, liposomes, and phagosomes. Intriguingly, besides endogenous proteins, PLSCR1 was also reported to interact with exogenous viral proteins, thereby regulating viral uptake and spread. This review aims to summarize the current knowledge about the multiple roles of PLSCR1 in distinct cellular pathways. Video Abstract.
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Proteínas de Transferencia de Fosfolípidos , Transporte Biológico , Membrana Celular/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismoRESUMEN
BACKGROUND: Several factors may contribute to the circadian variability of clinical manifestations in asthma and allergy. Basophils play a pivotal role in allergic inflammation. However, evidence for a functional clock governing the effector function of these cells is sparse and contradictory. We have systematically sampled the 24-hour response of basophils to IgE- and non-IgE-dependent ligands in asthma to understand their possible contribution to the diurnal variations of allergic symptoms. METHODS: Leukocytes were collected every 4 hours for 24 hours from 10 patients with moderate, persistent asthma and 10 matched, nonallergic controls, and then incubated with concentrations of anti-IgE, formyl-methionyl-leucylphenylalanine (fMLP), or the Ca2+ ionophore, A23187. Histamine release (HR) was tested for time-of-day- or disease-related variability by conventional statistics and for 24-hour rhythmicity by the cosinor method. RESULTS: HR induced by anti-IgE was significantly increased at 08:00 vs. 20:00 in basophils from asthmatics but not controls. No significant differences were seen at any time in the response to A23187, while the response to fMLP was significantly higher at 08:00 vs. 20:00 in controls but not asthmatics. The basophil response to anti-IgE, but not fMLP or A23187, varied significantly across the 24 hours in asthma, and its amplitude, percent rhythm, and acrophase were comparable to those of peak expiratory flow or serum cortisol. CONCLUSION: Using an integrated statistical approach, we show that basophil responsiveness undergoes significant circadian variability and that distinct patterns of rhythmicity can be recognized depending on the signal delivered, the activation parameters assessed, and the disease status.
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Asma/inmunología , Basófilos/inmunología , Degranulación de la Célula/inmunología , Relojes Circadianos/inmunología , Liberación de Histamina/inmunología , Inmunoglobulina E/inmunología , Adulto , Prueba de Desgranulación de los Basófilos , Femenino , Humanos , MasculinoRESUMEN
Recent advances in cancer immunotherapy have clearly shown that checkpoint-based immunotherapy is effective in a small subgroup of cancer patients. However, no effective predictive biomarker has been identified so far. The major histocompatibility complex, better known in humans as human leukocyte antigen (HLA), is a very polymorphic gene complex consisting of more than 200 genes. It has a crucial role in activating an appropriate host immune response against pathogens and tumor cells by discriminating self and non-self peptides. Several lines of evidence have shown that down-regulation of expression of HLA class I antigen derived peptide complexes by cancer cells is a mechanism of tumor immune escape and is often associated to poor prognosis in cancer patients. In addition, it has also been shown that HLA class I and II antigen expression, as well as defects in the antigen processing machinery complex, may predict tumor responses in cancer immunotherapy. Nevertheless, the role of HLA in predicting tumor responses to checkpoint-based immunotherapy is still debated. In this review, firstly, we will describe the structure and function of the HLA system. Secondly, we will summarize the HLA defects and their clinical significance in cancer patients. Thirdly, we will review the potential role of the HLA as a predictive biomarker for checkpoint-based immunotherapy in cancer patients. Lastly, we will discuss the potential strategies that may restore HLA function to implement novel therapeutic strategies in cancer patients.
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Biomarcadores de Tumor/inmunología , Antígenos HLA/inmunología , Inmunoterapia , Neoplasias/inmunología , Presentación de Antígeno/inmunología , Biomarcadores de Tumor/genética , Regulación Neoplásica de la Expresión Génica/inmunología , Humanos , Neoplasias/genética , Neoplasias/terapia , Escape del Tumor/inmunologíaRESUMEN
Immunogenic apoptosis, or more appropriately called immunogenic cell death (ICD), is a recently described form of apoptosis induced by a specific set of chemotherapeutic drugs or by physical therapeutic modalities, such as ionizing irradiation and photodynamic therapy. The peculiar characteristic of ICD is the ability to favor recognition and elimination of dying tumor cells by phagocytes in association with the release of pro-inflammatory molecules (such as cytokines and high-mobility group box-1). While in vitro and animal models pointed to ICD as one of the molecular mechanisms mediating the clinical efficacy of some anticancer agents, it is hard to clearly demonstrate its contribution in cancer patients. Clinical evidence suggests that the induction of ICD alone is possibly not sufficient to fully subvert the immunosuppressive tumor microenvironment. However, interesting results from recent studies contemplate the exploitation of ICD for improving the immunogenicity of cancer cells to use them as an antigen cargo in the development of dendritic cell (DC) vaccines. Herein, we discuss the effects of danger signals expressed or released by cancer cells undergoing ICD on the maturation and activation of immature and mature DC, highlighting the potential added value of ICD in adoptive immunotherapy protocols.
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Apoptosis/inmunología , Vacunas contra el Cáncer/inmunología , Neoplasias/terapia , Animales , Células Dendríticas/inmunología , Humanos , Inmunoterapia/métodos , Neoplasias/inmunologíaRESUMEN
The posttranscriptional mechanisms whereby RNA-binding proteins (RBPs) regulate T cell differentiation remain unclear. RBPs can coordinately regulate the expression of functionally related genes via binding to shared regulatory sequences, such as the adenylate-uridylate-rich elements (AREs) present in the 3' untranslated region (UTR) of mRNA. The RBP HuR posttranscriptionally regulates IL-4, IL-13, and other Th2 cell-restricted transcripts. We hypothesized that the ARE-bearing GATA-3 gene, a critical regulator of Th2 polarization, is under HuR control as part of its coordinate posttranscriptional regulation of the Th2 program. We report that in parallel with stimulus-induced increase in GATA-3 mRNA and protein levels, GATA-3 mRNA half-life is increased after restimulation in the human T cell line Jurkat, in human memory and Th2 cells, and in murine Th2-skewed cells. We demonstrate by immunoprecipitation of ribonucleoprotein complexes that HuR associates with the GATA-3 endogenous transcript in human T cells and found, using biotin pulldown assay, that HuR specifically interacts with its 3'UTR. Using both loss-of-function and gain-of-function approaches in vitro and in animal models, we show that HuR is a critical mediator of stimulus-induced increase in GATA-3 mRNA and protein expression and that it positively influences GATA-3 mRNA turnover, in parallel with selective promotion of Th2 cytokine overexpression. These results suggest that HuR-driven posttranscriptional control plays a significant role in T cell development and effector function in both murine and human systems. A better understanding of HuR-mediated control of Th2 polarization may have utility in altering allergic airway inflammation in human asthmatic patients.
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Antígenos de Superficie/fisiología , Citocinas/biosíntesis , Citocinas/genética , Factor de Transcripción GATA3/biosíntesis , Factor de Transcripción GATA3/genética , Regulación de la Expresión Génica/inmunología , Proteínas de Unión al ARN/fisiología , Células Th2/inmunología , Células Th2/metabolismo , Animales , Secuencia de Bases , Línea Celular Tumoral , Proteínas ELAV , Proteína 1 Similar a ELAV , Femenino , Humanos , Células Jurkat , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Células 3T3 NIH , Estabilidad del ARN/inmunología , Transcripción Genética/inmunologíaRESUMEN
The central role of the microbiota as a pivotal factor regulating anti-tumor immune responses has recently been appreciated. Increasing evidence has put a spotlight on the connection of microbiota to T cells, by showing impaired effector and/or memory responses in germ-free (GF) mice or in the presence of dysbiotic communities, and association with tumor growth and overall survival (OS). These observations also have significant implications for anti-tumor therapy and vaccination, suggesting that the communication between T cells and the microbiota involves soluble mediators (microbiota-derived metabolites) that influence various functions of T cells. In addition, there is growing appreciation of the role of bacterial translocation into the peritumoral milieu from the intestinal tract, as well as of locally developed tumor microbial communities, spatially separated from the gut microbiota, in shaping the tumor microbiome. Collectively, these findings have added new support to the idea that tonic inputs mirroring the existence of tumor microbiome could regulate the function of tumor-infiltrating T cells and tissue-resident memory T (TRM) cells. In this review, we focus on recent advances and aspects of these active areas of investigation and provide a comprehensive overview of the unique mechanisms that play a pivotal role in the regulation of anti-tumor immunity by the microbiota, some of which could be of particular relevance for addressing problems caused by tumor heterogeneity. It is our hope that this review will provide a theoretical foundation for future investigations in this area.
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Microbioma Gastrointestinal , Microbiota , Ratones , Animales , Linfocitos T , Diferenciación CelularRESUMEN
Pseudomonas aeruginosa (PA) is a major Gram-negative opportunistic pathogen causing several serious acute and chronic infections in the nosocomial and community settings. PA eradication has become increasingly difficult due to its remarkable ability to evade antibiotics. Therefore, epidemiological studies are needed to limit the infection and aim for the correct treatment. The present retrospective study focused on PA presence among samples collected at the San Giovanni di Dio and Ruggi D'Aragona University Hospital in Salerno, Italy; its resistance profile and relative variations over the eight years were analyzed. Bacterial identification and antibiotic susceptibility tests were performed by VITEK® 2. In the 2015-2019 and 2020-2022 timeframes, respectively, 1739 and 1307 isolates of PA were obtained from respiratory samples, wound swabs, urine cultures, cultural swabs, blood, liquor, catheter cultures, vaginal swabs, and others. During 2015-2019, PA strains exhibited low resistance against amikacin (17.2%), gentamicin (25.2%), and cefepime (28.3%); moderate resistance against ceftazidime (34.4%), imipenem (34.6%), and piperacillin/tazobactam (37.7%); and high resistance against ciprofloxacin (42.4%) and levofloxacin (50.6%). Conversely, during the 2020-2022 era, PA showed 11.7, 21.1, 26.9, 32.6, 33.1, 38.7, and 39.8% resistance to amikacin, tobramycin, cefepime, imipenem, ceftazidime, ciprofloxacin, and piperacillin/tazobactam, respectively. An overall resistance-decreasing trend was observed for imipenem and gentamicin during 2015-2019. Instead, a significant increase in resistance was recorded for cefepime, ceftazidime, and imipenem in the second set of years investigated. Monitoring sentinel germs represents a key factor in optimizing empirical therapy to minimize the spread of antimicrobial resistance.
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IgE-mediated release of proinflammatory mediators and cytokines from basophils and mast cells is a central event in allergic disorders. Several groups of investigators have demonstrated the presence of autoantibodies against IgE and/or FcεRI in patients with chronic spontaneous urticaria. By contrast, the prevalence and functional activity of anti-IgE autoantibodies in atopic dermatitis (AD) are largely unknown. We evaluated the ability of IgG anti-IgE from patients with AD to induce the in vitro IgE-dependent activation of human basophils and skin and lung mast cells. Different preparations of IgG anti-IgE purified from patients with AD and rabbit IgG anti-IgE were compared for their triggering effects on the in vitro release of histamine and type 2 cytokines (IL-4, IL-13) from basophils and of histamine and lipid mediators (prostaglandin D2 and cysteinyl leukotriene C4) from human skin and lung mast cells. One preparation of human IgG anti-IgE out of six patients with AD induced histamine release from basophils, skin and lung mast cells. This preparation of human IgG anti-IgE induced the secretion of cytokines and eicosanoids from basophils and mast cells, respectively. Human monoclonal IgE was a competitive antagonist of both human and rabbit IgG anti-IgE. Human anti-IgE was more potent than rabbit anti-IgE for IL-4 and IL-13 production by basophils and histamine, prostaglandin D2 and leukotriene C4 release from mast cells. Functional anti-IgE autoantibodies rarely occur in patients with AD. When present, they induce the release of proinflammatory mediators and cytokines from basophils and mast cells, thereby possibly contributing to sustained IgE-dependent inflammation in at least a subset of patients with this disorder.
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Basófilos , Dermatitis Atópica , Animales , Autoanticuerpos/farmacología , Citocinas/farmacología , Eicosanoides , Histamina , Humanos , Inmunoglobulina E , Inmunoglobulina G/farmacología , Interleucina-13/farmacología , Interleucina-4/farmacología , Leucotrieno C4 , Mastocitos , Prostaglandinas , ConejosRESUMEN
Bacterial ocular infections are a worldwide health problem and, if untreated, can damage the structure of the eye and contribute to permanent disability. Knowledge of the prevalence and antimicrobial susceptibility patterns of the main causative agents involved in ocular infections is necessary for defining an optimal antibiotic therapy. The aim of this study was to analyse bacterial species involved in ocular infections and the antimicrobial susceptibility patterns. Conjunctival swab samples were collected from patients with bacterial conjunctivitis at the University Hospital San Giovanni di Dio e Ruggi d'Aragona between January 2015 and December 2019. The identification and antibiotic sensitivity tests were performed using the VITEK 2 system. A total of 281 causative agents of ocular infections were isolated, 81.8% of which were Gram-positive bacteria. Coagulase-negative staphylococci (CoNS) were the most commonly isolated species among Gram-positive bacteria, followed by Staphylococcus aureus. In contrast, Pseudomonas spp. and Escherichia coli were the main species isolated among Gram-negative bacteria (18.2%). Overall, linezolid, teicoplanin, tigecycline and vancomycin were the most effective antimicrobials. Analysis of resistance rates over time highlighted increasing resistance for azithromycin, clarithromycin and erythromycin among CoNS, and clindamycin and erythromycin among Staphylococcus aureus. This study has identified the profiles of the major pathogens involved in ocular infection and their susceptibility patterns, which will help improve the treatments and the choice of antibiotics in ocular infections.
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Chronic inhalation of cigarette smoke is a prominent cause of chronic obstructive pulmonary disease (COPD) and provides an important source of exogenous oxidants. In addition, several inflammatory and structural cells are a source of endogenous oxidants in the lower airways of COPD patients, even in former smokers. This suggests that oxidants play a key role in the pathogenesis of COPD. This oxidative stress is counterbalanced by the protective effects of the various endogenous antioxidant defenses of the lower airways. A large amount of data from animal models and patients with COPD have shown that both the stable phase of the disease, and during exacerbations, have increased oxidative stress in the lower airways compared with age-matched smokers with normal lung function. Thus, counteracting the increased oxidative stress may produce clinical benefits in COPD patients. Smoking cessation is currently the most effective treatment of COPD patients and reduces oxidative stress in the lower airways. In addition, many drugs used to treat COPD have some antioxidant effects, however, it is still unclear if their clinical efficacy is related to pharmacological modulation of the oxidant/antioxidant balance. Several new antioxidant compounds are in development for the treatment of COPD.
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Antioxidantes , Enfermedad Pulmonar Obstructiva Crónica , Animales , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Humanos , Oxidantes/uso terapéutico , Estrés Oxidativo , Enfermedad Pulmonar Obstructiva Crónica/etiología , Enfermedad Pulmonar Obstructiva Crónica/patología , FumadoresRESUMEN
Streptococcus mutans (S. mutans) is considered the main causative agent of dental caries. The study aims to evaluate the antimicrobial activity of a natural plant product, pure 4,5''-dihydroxy-anthraquinone-2-carboxylic acid (Rhein) against S. mutans. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to determine the viability of immortalized human keratinocytes (HaCaT) following treatment with Rhein. Assay for antimicrobial activity and the time-killing test were performed to evaluate Rhein effects against planktonic S. mutans. The effect of different concentrations of Rhein on biofilm biomass and the metabolism of biofilm cells were evaluated through crystal violet and MTT assays. Further, Rhein-treated biofilms were viewed by confocal laser scanning microscopy. Rhein effects on acid production and acid environment tolerance were also assessed. The minimum inhibitory concentration (MIC) of Rhein, exerting bacteriostatic action on 90% of planktonic S. mutans (MIC90), was 5.69 µg/mL. MIC and sub-MIC concentrations of Rhein affected the metabolism of biofilm cells and disrupted biofilm biomass with minimal biofilm eradication concentrations (MBEC) inducing 50% (MBEC50) and 90% eradication (MBEC90) of 6.31 and > 50 µg/mL, respectively. Confocal images displayed a significant reduction in biofilm biomass following treatment with increasing concentrations of the compound. Rhein also reduced the virulence of the biofilm by affecting acid production and acid tolerance. Conversely, active concentrations of Rhein did not affect HaCaT cell viability. Together, these findings indicate that Rhein, a natural product that counteracts the virulence of S. mutans, may represent a novel therapeutic option for dental caries.
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Caries Dental , Streptococcus mutans , Antraquinonas , Antibacterianos/farmacología , Biopelículas , Caries Dental/tratamiento farmacológico , Humanos , Pruebas de Sensibilidad MicrobianaRESUMEN
Immunogenic cell death (ICD) in cancer represents a functionally unique therapeutic response that can induce tumor-targeting immune responses. ICD is characterized by the exposure and release of numerous damage-associated molecular patterns (DAMPs), which confer adjuvanticity to dying cancer cells. The spatiotemporally defined emission of DAMPs during ICD has been well described, whereas the epigenetic mechanisms that regulate ICD hallmarks have not yet been deeply elucidated. Here, we aimed to examine the involvement of miRNAs and their putative targets using well-established in vitro models of ICD. To this end, B cell lymphoma (Mino) and breast cancer (MDA-MB-231) cell lines were exposed to two different ICD inducers, the combination of retinoic acid (RA) and interferon-alpha (IFN-α) and doxorubicin, and to non ICD inducers such as gamma irradiation. Then, miRNA and mRNA profiles were studied by next generation sequencing. Co-expression analysis identified 16 miRNAs differentially modulated in cells undergoing ICD. Integrated miRNA-mRNA functional analysis revealed candidate miRNAs, mRNAs, and modulated pathways associated with Immune System Process (GO Term). Specifically, ICD induced a distinctive transcriptional signature hallmarked by regulation of antigen presentation, a crucial step for proper activation of immune system antitumor response. Interestingly, the major histocompatibility complex class I (MHC-I) pathway was upregulated whereas class II (MHC-II) was downregulated. Analysis of MHC-II associated transcripts and HLA-DR surface expression confirmed inhibition of this pathway by ICD on lymphoma cells. miR-4284 and miR-212-3p were the strongest miRNAs upregulated by ICD associated with this event and miR-212-3p overexpression was able to downregulate surface expression of HLA-DR. It is well known that MHC-II expression on tumor cells facilitates the recruitment of CD4+ T cells. However, the interaction between tumor MHC-II and inhibitory coreceptors on tumor-associated lymphocytes could provide an immunosuppressive signal that directly represses effector cytotoxic activity. In this context, MHC-II downregulation by ICD could enhance antitumor immunity. Overall, we found that the miRNA profile was significantly altered during ICD. Several miRNAs are predicted to be involved in the regulation of MHC-I and II pathways, whose implication in ICD is demonstrated herein for the first time, which could eventually modulate tumor recognition and attack by the immune system.
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BACKGROUND: Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T lymphocytes expressing a semi-invariant α/ß T-cell receptor (TCR). The physiological functions of these cells, which are particularly abundant in normal liver and mucosal sites, have become clear only in recent years, but their role in most human diseases is still unknown. Since the cellular origin and etiopathogenesis of most T-lymphomas are still elusive, we decided to explore the presence of MAIT cells in biopsies from these neoplasms. METHODS: Sixteen biopsies obtained from patients with a T-cell lymphoma diagnosis were analyzed via immunofluorescence staining using an anti-Vα7.2 antibody and the MR1-antigen tetramer. Positive cases were subjected to a polymerase chain reaction for the detection of Vα7.2-Jα33, Vα7.2-Jα20, or Vα7.2-Jα12 rearrangements, followed by sequencing of the CDR3α region. RESULTS: CD3+/Vα7.2+ and CD3+/MR1-Ag-tetramer+ cells were found in 4 of 16 samples analyzed. The identification of specific TCR rearrangements confirmed the presence of these cells in all four samples. PCR and sequencing results documented the presence of multiple clones of MAIT cells in each positive sample. CONCLUSIONS: MAIT cells are frequently found in T-cell lymphomas. More in-depth studies and a larger number of samples are needed to better clarify the contribution of MAIT cells to this rare neoplasm.
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Corynebacterium striatum (C. striatum) is an emerging multidrug-resistant (MDR) pathogen associated with nosocomial infections. In this scenario, we screened the antimicrobial activity of the anthelmintic drugs doramectin, moxidectin, selamectin and niclosamide against 20 C. striatum MDR clinical isolates. Among these, niclosamide was the best performing drug against C. striatum. Niclosamide cytotoxicity was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay on immortalized human keratinocyte cells (HaCaT). After 20 h of treatment, the recorded 50% cytotoxic concentration (CC50) was 2.56 µg/mL. The antibacterial efficacy was determined via disc diffusion, broth microdilution method and time-killing. Against C. striatum, niclosamide induced a growth inhibitory area of 22 mm and the minimum inhibitory concentration that inhibits 90% of bacteria (MIC90) was 0.39 µg/mL, exhibiting bactericidal action. The biofilm biomass eradicating action was investigated through crystal violet (CV), MTT and confocal laser scanning microscopy (CLSM). Niclosamide affected the biofilm viability in a dose-dependent manner and degraded biomass by 55 and 49% at 0.39 µg/mL and 0.19 µg/mL. CLSM images confirmed the biofilm biomass degradation, showing a drastic reduction in cell viability. This study could promote the drug-repurposing of the anthelmintic FDA-approved niclosamide as a therapeutic agent to counteract the C. striatum MDR infections.
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The autoimmune enteropathy, coeliac disease (CD), is triggered by ingestion of gluten-containing grains. We recently reported that the chemokine receptor CXCR3 serves as a receptor for specific gliadin peptides that cause zonulin release and subsequent increase in intestinal permeability. To explore the role of CXCR3 in the immune response to gliadin, peripheral blood mononuclear cells from both patients with CD and healthy controls were incubated with either pepsin-trypsin-digested gliadin or 11 α-gliadin synthetic peptides in the presence or absence of a blocking anti-CXCR3 monoclonal antibody. Supernatants were analysed for interleukin-6 (IL-6), IL-8, IL-10, IL-13, IP-10 (CXCL10), tumour necrosis factor-α and interferon-γ. Gliadin broadly induced cytokine production irrespective of the clinical condition. However, IL-8 production occurred only in a subgroup of individuals and cells of the phagocytic lineage were the main source. Induction of IL-8 was reproduced by one of a comprehensive panel of synthetic α-gliadin peptides and was abrogated when CXCR3 was blocked before stimulation with either gliadin or this peptide in the CD group but not in the control group, suggesting that gliadin-induced IL-8 production was CXCR3-dependent gliadin induced IL-8 production only in CD.
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Enfermedad Celíaca/metabolismo , Gliadina/inmunología , Interleucina-8/biosíntesis , Receptores CXCR3/metabolismo , Enfermedad Celíaca/inmunología , Humanos , Interleucina-8/inmunología , Leucocitos Mononucleares , Péptidos/inmunología , Receptores CXCR3/inmunologíaRESUMEN
BACKGROUND: Celiac disease (CD) is an autoimmune enteropathy triggered by the ingestion of gluten. Gluten-sensitive individuals (GS) cannot tolerate gluten and may develop gastrointestinal symptoms similar to those in CD, but the overall clinical picture is generally less severe and is not accompanied by the concurrence of tissue transglutaminase autoantibodies or autoimmune comorbidities. By studying and comparing mucosal expression of genes associated with intestinal barrier function, as well as innate and adaptive immunity in CD compared with GS, we sought to better understand the similarities and differences between these two gluten-associated disorders. METHODS: CD, GS and healthy, gluten-tolerant individuals were enrolled in this study. Intestinal permeability was evaluated using a lactulose and mannitol probe, and mucosal biopsy specimens were collected to study the expression of genes involved in barrier function and immunity. RESULTS: Unlike CD, GS is not associated with increased intestinal permeability. In fact, this was significantly reduced in GS compared with controls (P = 0.0308), paralleled by significantly increased expression of claudin (CLDN) 4 (P = 0.0286). Relative to controls, adaptive immunity markers interleukin (IL)-6 (P = 0.0124) and IL-21 (P = 0.0572) were expressed at higher levels in CD but not in GS, while expression of the innate immunity marker Toll-like receptor (TLR) 2 was increased in GS but not in CD (P = 0.0295). Finally, expression of the T-regulatory cell marker FOXP3 was significantly reduced in GS relative to controls (P = 0.0325) and CD patients (P = 0.0293). CONCLUSIONS: This study shows that the two gluten-associated disorders, CD and GS, are different clinical entities, and it contributes to the characterization of GS as a condition associated with prevalent gluten-induced activation of innate, rather than adaptive, immune responses in the absence of detectable changes in mucosal barrier function.
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Enfermedad Celíaca/inmunología , Enfermedad Celíaca/patología , Hipersensibilidad/inmunología , Hipersensibilidad/patología , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Permeabilidad , Adulto , Alérgenos/inmunología , Femenino , Perfilación de la Expresión Génica , Glútenes/inmunología , Humanos , MasculinoRESUMEN
The mammalian target of rapamycin (mTOR) and the phosphatidylinositol-3-kinase (PI3K)/protein kinase B or Akt (PKB/Akt) signaling pathways are considered as two but somewhat interconnected significant immune pathways which play complex roles in a variety of physiological processes as well as pathological conditions. Aberrant activation of PI3K/Akt/mTOR signaling pathways has been reported to be associated in a wide variety of human diseases. Over the past few years, growing evidence in in vitro and in vivo models suggest that this sophisticated and subtle cascade mediates the orchestration of the immune response in health and disease through exposure to probiotics. An expanding body of literature has highlighted the contribution of probiotics and PI3K/Akt/mTOR signaling pathways in gastrointestinal disorders, metabolic syndrome, skin diseases, allergy, salmonella infection, and aging. However, longitudinal human studies are possibly required to verify more conclusively whether the investigational tools used to understand the regulation of these pathways might provide effective approaches in the prevention and treatment of various disorders. In this Review, we summarize the experimental evidence from recent peer-reviewed studies and provide a brief overview of the causal relationship between the effects of probiotics and their metabolites on the components of PI3K/Akt/mTOR signaling pathways and human disease.