Inflammation, Autoinflammation and Autoimmunity in Inflammatory Bowel Diseases.

In this review, the role of innate and adaptive immunity in the pathogenesis of inflammatory bowel diseases (IBD) is reported. In IBD, an altered innate immunity is often found, with increased Th17 and decreased Treg cells infiltrating the intestinal mucosa. An associated increase in inflammatory cytokines, such as IL-1 and TNF-α, and a decrease in anti-inflammatory cytokines, such as IL-10, concur in favoring the persistent inflammation of the gut mucosa. Autoinflammation is highlighted with insights in the role of inflammasomes, which activation by exogenous or endogenous triggers might be favored by mutations of NOD and NLRP proteins. Autoimmunity mechanisms also take place in IBD pathogenesis and in this context of a persistent immune stimulation by bacterial antigens and antigens derived from intestinal cells degradation, the adaptive immune response takes place and results in antibodies and autoantibodies production, a frequent finding in these diseases. Inflammation, autoinflammation and autoimmunity concur in altering the mucus layer and enhancing intestinal permeability, which sustains the vicious cycle of further mucosal inflammation.

New insights into SARS-CoV-2 Lumipulse G salivary antigen testing: accuracy, safety and short TAT enhance surveillance.

OBJECTIVES: The rapid, accurate and safe detection of SARS-CoV-2 is the key to improving surveillance and infection containment. The aim of the present study was to ascertain whether, after heat/chemical inactivation, SARS-CoV-2 N antigen chemiluminescence (CLEIA) assay in saliva remains a valid alternative to molecular testing. METHODS: In 2022, 139 COVID-19 inpatients and 467 healthcare workers were enrolled. In 606 self-collected saliva samples (Salivette), SARS-CoV-2 was detected by molecular (TaqPath rRT-PCR) and chemiluminescent Ag assays (Lumipulse G). The effect of sample pre-treatment (extraction solution-ES or heating) on antigen recovery was verified. RESULTS: Salivary SARS-CoV-2 antigen assay was highly accurate (AUC=0.959, 95% CI: 0.943-0.974), with 90% sensitivity and 92% specificity. Of the 254 antigen positive samples, 29 were false positives. We demonstrated that heterophilic antibodies could be a cause of false positive results. A significant antigen concentration decrease was observed after ES treatment (p=0.0026), with misclassification of 43 samples. Heat had a minimal impact, after treatment the correct classification of cases was maintained. CONCLUSIONS: CLEIA SARS-CoV-2 salivary antigen provides accurate, timely and high-throughput results that remain accurate also after heat inactivation, thus ensuring a safer work environment. This supports the use of salivary antigen detection by CLEIA in surveillance programs.

Salivary proteomic analysis in asymptomatic and symptomatic SARS-CoV-2 infection: Innate immunity, taste perception and FABP5 proteins make the difference.

BACKGROUND AND AIM: SARS-CoV-2 infection spawns from an asymptomatic condition to a fatal disease. Age, comorbidities, and several blood biomarkers are associated with infection outcome. We searched for biomarkers by untargeted and targeted proteomic analysis of saliva, a source of viral particles and host proteins. METHODS: Saliva samples from 19 asymptomatic and 16 symptomatic SARS-CoV-2 infected subjects, and 20 controls were analyzed by LC-MS/MS for untargeted peptidomic (flow through of 10 kDa filter) and proteomic (trypsin digestion of filter retained proteins) profiling. RESULTS: Peptides from 53 salivary proteins were identified. ADF was detected only in controls, while IL1RA only in infected subjects. PRPs, DSC2, FABP5, his-1, IL1RA, PRH1, STATH, SMR3B, ANXA1, MUC7, ACTN4, IGKV1-33 and TGM3 were significantly different between asymptomatic and symptomatic subjects. Retained proteins were 117, being 11 highly different between asymptomatic and symptomatic (fold change ≥2 or ≤-2). After validation by LC-MS/MS-SRM (selected reaction monitoring analysis), the most significant discriminant proteins at PCA were IL1RA, CYSTB, S100A8, S100A9, CA6, and FABP5. CONCLUSIONS: The differentially abundant proteins involved in innate immunity (S100 proteins), taste (CA6 and cystatins), and viral binding to the host (FABP5), appear to be of interest for use as potential biomarkers and drugs targets.

Salivary SARS-CoV-2 antigen rapid detection: A prospective cohort study.

BACKGROUND AND AIM: SARS-CoV-2 quick testing is relevant for the containment of new pandemic waves. Antigen testing in self-collected saliva might be useful. We compared salivary and naso-pharyngeal swab (NPS) SARS-CoV-2 antigen detection by a rapid chemiluminescent assay (CLEIA) and two different point-of-care (POC) immunochromatographic assays, with results of molecular testing. METHODS: 234 patients were prospectively enrolled. Paired self-collected saliva (Salivette) and NPS were obtained to perform rRT-PCR, chemiluminescent (Lumipulse G) and POC (NPS: Fujirebio and Abbott; saliva: Fujirebio) for SARS-CoV-2 antigen detection. RESULTS: The overall agreement between NPS and saliva rRT-PCR was 78.7%, reaching 91.7% at the first week from symptoms. SARS-CoV-2 CLEIA antigen was highly accurate in distinguishing positive and negative NPS (ROC-AUC = 0.939, 95%CI:0.903-0.977), with 81.6% sensitivity and 93.8% specificity. This assay on saliva reached the optimal value within 7 days from symptoms onset (Sensitivity: 72%; Specificity: 97%). Saliva POC antigen was limited in sensitivity (13%), performing better in NPS (Sensitivity: 48% and 66%; Specificity: 100% and 99% for Espline and Abbott respectively), depending on viral loads. CONCLUSIONS: Self-collected saliva is a valid alternative to NPS for SARS-CoV-2 detection by molecular, but also by CLEIA antigen testing, which is therefore potentially useful for large scale screening.

Vitamin D Prevents Pancreatic Cancer-Induced Apoptosis Signaling of Inflammatory Cells.

Combined approaches based on immunotherapy and drugs supporting immune effector cell function might increase treatment options for pancreatic ductal adenocarcinoma (PDAC), vitamin D being a suitable drug candidate. In this study, we evaluated whether treatment with the vitamin D analogue, calcipotriol, counterbalances PDAC induced and SMAD4-associated intracellular calcium [Ca2+]i alterations, cytokines release, immune effector function, and the intracellular signaling of peripheral blood mononuclear cells (PBMCs). Calcipotriol counteracted the [Ca2+]i depletion of PBMCs induced by SMAD4-expressing PDAC cells, which conditioned media augmented the number of calcium flows while reducing whole [Ca2+]i. While calcipotriol inhibited spontaneous and PDAC-induced tumor necrosis factor alpha (TNF-α) release by PBMC and reduced intracellular transforming growth factor beta (TGF-ß), it did not counteract the lymphocytes proliferation induced in allogenic co-culture by PDAC-conditioned PBMCs. Calcipotriol mainly antagonized PDAC-induced apoptosis and partially restored PDAC-inhibited NF-κB signaling pathway. In conclusion, alterations induced by PDAC cells in the [Ca2+]i of immune cells can be partially reverted by calcipotriol treatment, which promotes inflammation and antagonizes PBMCs apoptosis. These effects, together with the dampening of intracellular TGF-ß, might result in an overall anti-tumor effect, thus supporting the administration of vitamin D in PDAC patients.

Peptidomic and proteomic analysis of stool for diagnosing IBD and deciphering disease pathogenesis.

Background The sensitivities and specificities of C-reactive protein (CRP) and faecal calprotectin (fCal), as recommended for inflammatory bowel diseases (IBD) diagnosis and monitoring, are low. Our aim was to discover new stool protein/peptide biomarkers for diagnosing IBD. Methods For peptides, MALDI-TOF/MS (m/z 1000-4000) was performed using stools from an exploratory (34 controls; 72 Crohn's disease [CD], 56 ulcerative colitis [UC]) and a validation (28 controls, 27 CD, 15 UC) cohort. For proteins, LTQ-Orbitrap XL MS analysis (6 controls, 5 CD, 5 UC) was performed. Results MALDI-TOF/MS spectra of IBD patients had numerous features, unlike controls. Overall, 426 features (67 control-associated, 359 IBD-associated) were identified. Spectra were classified as control or IBD (absence or presence of IBD-associated features). In the exploratory cohort, the sensitivity and specificity of this classification algorithm were 81% and 97%, respectively. Blind analysis of the validation cohort confirmed 97% specificity, with a lower sensitivity (55%) paralleling active disease frequency. Following binary logistic regression analysis, IBD was independently correlated with MALDI-TOF/MS spectra (p < 0.0001), outperforming fCal measurements (p = 0.029). The IBD-correlated m/z 1810.8 feature was a fragment of APC2, homologous with APC, over-expressed by infiltrating cells lining the surface in UC or the muscularis-mucosae in CD (assessed by immunohistochemistry). IBD-associated over-expressed proteins included immunoglobulins and neutrophil proteins, while those under-expressed comprised proteins of the nucleic acid assembly or those (OLFM4, ENPP7) related to cancer risk. Conclusions Our study provides evidence for the clinical utility of a novel proteomic method for diagnosing IBD and insight on the pathogenic role of APC. Moreover, the newly described IBD-associated proteins might become tools for cancer risk assessment in IBD patients.