Emerging role of monocytes and of their intracellular calcium content in spondyloarthritis

Background. The Spondyloarthritis (SpA) are a group of a multifactorial diseases characterised by a complex interplay between an inherited background and environmental factors that lead to immune response dysregulation and inflammation of the joints, mainly the sacro-ileal. Different from rheumatoid arthritis, there are no specific biomarkers for disease activity in the SpA that could be used in clinical practice. New biomarkers discovery could be helpful for early diagnosis, monitoring of disease activity, as well as for prognosis, outcome measures, and for assessing treatment efficacy. In SpA patients, macrophages infiltrating the inflamed joints, derive from circulating monocytes, express not only inflammatory cytokines, like TNF-α, IL-1β or TGF-β, but also enzymes causing tissue destruction and remodelling, like metalloproteinases. Metalloproteinases (MMPs), MMP3 in particular, have been reported to be highly expressed in synovial tissue and in peripheral blood of SpA patients. Recent studies have showed that MMP8 and MMP9, in particular, are produced by peripheral blood mononuclear cells (PBMCs) if they are stimulated by calprotectin (S100A8/S100A9 heterodimer). The SpA synovial tissue is characterized by an increased vascularization and an infiltrate composed of nucleated polymorphs, macrophages and lymphocytes. In these cells calcium signals are essential for various cellular functions, including proliferation, differentiation, apoptosis, and gene transcription. The aims of this work are to investigate whether the TNF-α, IL-1β, TGF-β, S100A8, S100A9, MMP3, MMP8 and MMP9 mRNA expression levels and intracellular calcium ([Ca2+]i) fluxes variations in PBMCs might be associated with SpA. Methods. The study population comprised 64 patients with a diagnosis of SpA (39 males and 25 females; mean age±standard deviation: 39.5±13.2 years) and 100 healthy controls (58 males and 42 females; mean age±standard deviation: 46.68.5). Among patients, 26 (40.6%) had diagnosis of Ankylosing Spondylitis (AS), (modified New York criteria) and 38 (59.3%) had a diagnosis of Psoriatic Arthritis (PsA) (CASPAR criteria). Blood samples were collected and complete blood count, CRP, ESR, uric acid, ALT and glucose were evaluated. Relative quantification (Real Time PCR) of TNF-α, IL-1β, TGF-β, S100A8, S100A9, MMP3, MMP8 and MMP9 mRNA were performed. Intracellular calcium ([Ca2+]i) fluxes were studied in patients and controls monocyte cells by a fluorescent microscope. Results. The mRNA expression levels in PBMCs of TNF-α, IL-1β, TGF-β were similar in AS and PsA patients when compared to controls. The variations of TNF-α, TGF-β and IL-1β were correlated each other. TNF-α mRNA expression levels also show a significant correlation if patient’s relatives with SpA where found (t=-2.5386, p=0.013). MMP8 and MMP9 mRNA expression levels did not vary between controls and patients, nor they were related to disease clinical activity indices. S100A9 mRNA expression did not vary, the expression of S100A8 (F=3.29, p=0.039) was reduced in PsA patients. S100A8 and S100A9 expression levels were significantly correlated with circulating inflammatory cells and S100A8 was correlated with CRP and ESR. Monocytes from healthy controls had evident and frequent ([Ca2+]i) oscillations, while SpA patients monocytes did not. The percentage of cells exhibiting ([Ca2+]i) oscillations profile was significantly lower in AS with respect to controls (F=6.15, p=0.003). The percentage of monocytes with intracellular calcium oscillations and the studied molecules were not correlated with the type of therapy or of drug used. Conclusions. SpA associates with a reduced expression of the inflammatory S100A8 calcium binding protein and with a decreased intracellular calcium fluxes in patients' cells compared to healthy subjects, suggesting that the presence of the disease affects the "on-off" mechanisms that regulate the concentration of intracellular calcium

Spondyloarthritis: Matrix Metalloproteinasesas Biomarkers of Pathogenesis and Response to Tumor Necrosis Factor (TNF) Inhibitors

The term spondyloarthritis (SpA) is used to describe a group of multifactorial chronic inflammatory diseases characterized by a predisposing genetic background and clinical manifestations typically involving the sacroiliac joint. The absence of pathognomonic clinical and/or laboratory findings generally results in a delay in diagnosis and, consequently, in treatment. In addition, 20-40% of SpA patients are non-responders to tumor necrosis factor (TNF) inhibitor therapies. Given these considerations, it is important to identify biomarkers that can facilitate the diagnosis and assessment of disease activity. As inflammation plays a key role in the pathogenesis of SpA, inflammatory mediators have been investigated as potential biomarkers for diagnosing the disease and predicting response to therapy. Some investigators have focused their attention on the role of matrix metalloproteinases (MMPs), which are known to be markers of synovial inflammation that is generated in the joint in reaction to inflammatory stimuli. Several studies have been carried out to verify if serum MMPs levels could be useful to diagnose SpA, to assess disease severity, and to predict response to TNF inhibitor therapy. The current review focuses on MMPs' role in SpA pathogenesis, diagnosis and therapeutic implications

SMAD4 loss enables EGF, TGF\u3b21 and S100A8/A9 induced activation of critical pathways to invasion in human pancreatic adenocarcinoma cells

Epidermal Growth Factor (EGF) receptor overexpression, KRAS, TP53, CDKN2A and SMAD4 mutations characterize pancreatic ductal adenocarcinoma. This mutational landscape might influence cancer cells response to EGF, Transforming Growth Factor \u3b21 (TGF\u3b21) and stromal inflammatory calcium binding proteins S100A8/A9. We investigated whether chronic exposure to EGF modifies in a SMAD4-dependent manner pancreatic cancer cell signalling, proliferation and invasion in response to EGF, TGF\u3b21 and S100A8/A9. BxPC3, homozigously deleted (HD) for SMAD4, and BxPC3-SMAD4+ cells were or not stimulated with EGF (100 ng/mL) for three days. EGF pre-treated and non pretreated cells were stimulated with a single dose of EGF (100 ng/mL), TGF\u3b21 (0,02 ng/mL), S100A8/A9 (10 nM). Signalling pathways (Reverse Phase Protein Array and western blot), cell migration (Matrigel) and cell proliferation (XTT) were evaluated. SMAD4 HD constitutively activated ERK and Wnt/\u3b2-catenin, while inhibiting PI3K/AKT pathways. These effects were antagonized by chronic EGF, which increased p-BAD (anti-apoptotic) in response to combined TGF\u3b21 and S100A8/A9 stimulation. SMAD4 HD underlied the inhibition of NF-\u3baB and PI3K/AKT in response to TGF\u3b21 and S100A8/A9, which also induced cell migration. Chronic EGF exposure enhanced cell migration of both BxPC3 and BxPC3-SMAD4+, rendering the cells less sensitive to the other inflammatory stimuli. In conclusion, SMAD4 HD is associated with the constitutive activation of the ERK and Wnt/\u3b2-catenin signalling pathways, and favors the EGF-induced activation of multiple signalling pathways critical to cancer proliferation and invasion. TGF\u3b21 and S100A8/A9 mainly inhibit NF-\u3baB and PI3K/AKT pathways and, when combined, sinergize with EGF in enhancing anti-apoptotic p-BAD in a SMAD4-dependent manner

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

SARS-CoV-2 RNA identification in nasopharyngeal swabs: issues in pre-analytics.

Abstract Objectives The direct identification of SARS-CoV-2 RNA in nasopharyngeal swabs is recommended for diagnosing the novel COVID-19 disease. Pre-analytical determinants, such as sampling procedures, time and temperature storage conditions, might impact on the end result. Our aim was to evaluate the effects of sampling procedures, time and temperature of the primary nasopharyngeal swabs storage on real-time reverse-transcription polymerase chain reaction (rRT-PCR) results. Methods Each nasopharyngeal swab obtained from 10 hospitalized patients for COVID-19 was subdivided in 15 aliquots: five were kept at room temperature; five were refrigerated (+4 °C); five were immediately mixed with the extraction buffer and refrigerated at +4 °C. Every day and for 5 days, one aliquot per condition was analyzed (rRT-PCR) for SARS-CoV-2 gene E and RNaseP and threshold cycles (Ct) compared. To evaluate manual sampling, 70 nasopharyngeal swabs were sampled twice by two different operators and analyzed separately one from the other. Results A total of 6/10 swabs were SARS-CoV-2 positive. No significant time or storage-dependent variations were observed in SARS-CoV-2 Ct. Re-sampling of swabs with SARS-CoV-2 Ct lower than 33 resulted in highly reproducible results (CV=2.9%), while a high variability was observed when Ct values were higher than 33 (CV=10.3%). Conclusions This study demonstrates that time and temperature of nasopharyngeal swabs storage do not significantly impact on results reproducibility. However, swabs sampling is a critical step, and especially in case of low viral load, might be a potential source of diagnostic errors

Inflammation and pancreatic cancer: molecular and functional interactions between S100A8, S100A9, NT-S100A8 and TGFβ1

BACKGROUND: In order to gain further insight on the crosstalk between pancreatic cancer (PDAC) and stromal cells, we investigated interactions occurring between TGF\u3b21 and the inflammatory proteins S100A8, S100A9 and NT-S100A8, a PDAC-associated S100A8 derived peptide, in cell signaling, intracellular calcium (Cai2+) and epithelial to mesenchymal transition (EMT). NF-\u3baB, Akt and mTOR pathways, Cai2+ and EMT were studied in well (Capan1 and BxPC3) and poorly differentiated (Panc1 and MiaPaCa2) cell lines. RESULTS: NT-S100A8, one of the low molecular weight N-terminal peptides from S100A8 to be released by PDAC-derived proteases, shared many effects on NF-\u3baB, Akt and mTOR signaling with S100A8, but mainly with TGF\u3b21. The chief effects of S100A8, S100A9 and NT-S100A8 were to inhibit NF-\u3baB and stimulate mTOR; the molecules inhibited Akt in Smad4-expressing, while stimulated Akt in Smad4 negative cells. By restoring Smad4 expression in BxPC3 and silencing it in MiaPaCa2, S100A8 and NT-S100A8 were shown to inhibit NF-\u3baB and Akt in the presence of an intact TGF\u3b21 canonical signaling pathway. TGF\u3b21 counteracted S100A8, S100A9 and NT-S100A8 effects in Smad4 expressing, not in Smad4 negative cells, while it synergized with NT-S100A8 in altering Cai2+ and stimulating PDAC cell growth. The effects of TGF\u3b21 on both EMT (increased Twist and decreased N-Cadherin expression) and Cai2+ were antagonized by S100A9, which formed heterodimers with TGF\u3b21 (MALDI-TOF/MS and co-immuno-precipitation). CONCLUSIONS: The effects of S100A8 and S100A9 on PDAC cell signaling appear to be cell-type and context dependent. NT-S100A8 mimics the effects of TGF\u3b21 on cell signaling, and the formation of complexes between TGF\u3b21 with S100A9 appears to be the molecular mechanism underlying the reciprocal antagonism of these molecules on cell signaling, Cai2+ and EMT

A Randomized Trial of Pharmacogenetic Warfarin Dosing in Naive Patients with Non-Valvular Atrial Fibrillation

Genotype-guided warfarin dosing have been proposed to improve patient's management. This study is aimed to determine whether a CYP2C9- VKORC1- CYP4F2-based pharmacogenetic algorithm is superior to a standard, clinically adopted, pharmacodynamic method. Two-hundred naive patients with non-valvular atrial fibrillation were randomized to trial arms and 180 completed the study. No significant differences were found in the number of out-of-range INRs (INR3.0) (p = 0.79) and in the mean percentage of time spent in the therapeutic range (TTR) after 19 days in the pharmacogenetic (51.9%) and in the control arm (53.2%, p = 0.71). The percentage of time spent at INR>4.0 was significantly lower in the pharmacogenetic (0.7%) than in the control arm (1.8%) (p = 0.02). Genotype-guided warfarin dosing is not superior in overall anticoagulation control when compared to accurate clinical standard of care

L’E-learning et les nouvelles technologies de l'image dans les travaux pratiques d‘Histologie en Médecine vétérinaire : Impacts sur la motivation et la maîtrise de l'apprentissage.

Additional file 8: Table S2. Free prostate specific antigen (fPSA), total PSA (tPSA), free to total PSA (f/tPSA) and prostate cancer antigen3 (PCA3) median and IQR values for the four classifications utilized in the PCa study

Emerging role of monocytes and of their intracellular calcium content in spondyloarthritis

Background. The Spondyloarthritis (SpA) are a group of a multifactorial diseases characterised by a complex interplay between an inherited background and environmental factors that lead to immune response dysregulation and inflammation of the joints, mainly the sacro-ileal. Different from rheumatoid arthritis, there are no specific biomarkers for disease activity in the SpA that could be used in clinical practice. New biomarkers discovery could be helpful for early diagnosis, monitoring of disease activity, as well as for prognosis, outcome measures, and for assessing treatment efficacy. In SpA patients, macrophages infiltrating the inflamed joints, derive from circulating monocytes, express not only inflammatory cytokines, like TNF-α, IL-1β or TGF-β, but also enzymes causing tissue destruction and remodelling, like metalloproteinases. Metalloproteinases (MMPs), MMP3 in particular, have been reported to be highly expressed in synovial tissue and in peripheral blood of SpA patients. Recent studies have showed that MMP8 and MMP9, in particular, are produced by peripheral blood mononuclear cells (PBMCs) if they are stimulated by calprotectin (S100A8/S100A9 heterodimer). The SpA synovial tissue is characterized by an increased vascularization and an infiltrate composed of nucleated polymorphs, macrophages and lymphocytes. In these cells calcium signals are essential for various cellular functions, including proliferation, differentiation, apoptosis, and gene transcription. The aims of this work are to investigate whether the TNF-α, IL-1β, TGF-β, S100A8, S100A9, MMP3, MMP8 and MMP9 mRNA expression levels and intracellular calcium ([Ca2+]i) fluxes variations in PBMCs might be associated with SpA. Methods. The study population comprised 64 patients with a diagnosis of SpA (39 males and 25 females; mean age±standard deviation: 39.5±13.2 years) and 100 healthy controls (58 males and 42 females; mean age±standard deviation: 46.68.5). Among patients, 26 (40.6%) had diagnosis of Ankylosing Spondylitis (AS), (modified New York criteria) and 38 (59.3%) had a diagnosis of Psoriatic Arthritis (PsA) (CASPAR criteria). Blood samples were collected and complete blood count, CRP, ESR, uric acid, ALT and glucose were evaluated. Relative quantification (Real Time PCR) of TNF-α, IL-1β, TGF-β, S100A8, S100A9, MMP3, MMP8 and MMP9 mRNA were performed. Intracellular calcium ([Ca2+]i) fluxes were studied in patients and controls monocyte cells by a fluorescent microscope. Results. The mRNA expression levels in PBMCs of TNF-α, IL-1β, TGF-β were similar in AS and PsA patients when compared to controls. The variations of TNF-α, TGF-β and IL-1β were correlated each other. TNF-α mRNA expression levels also show a significant correlation if patient’s relatives with SpA where found (t=-2.5386, p=0.013). MMP8 and MMP9 mRNA expression levels did not vary between controls and patients, nor they were related to disease clinical activity indices. S100A9 mRNA expression did not vary, the expression of S100A8 (F=3.29, p=0.039) was reduced in PsA patients. S100A8 and S100A9 expression levels were significantly correlated with circulating inflammatory cells and S100A8 was correlated with CRP and ESR. Monocytes from healthy controls had evident and frequent ([Ca2+]i) oscillations, while SpA patients monocytes did not. The percentage of cells exhibiting ([Ca2+]i) oscillations profile was significantly lower in AS with respect to controls (F=6.15, p=0.003). The percentage of monocytes with intracellular calcium oscillations and the studied molecules were not correlated with the type of therapy or of drug used. Conclusions. SpA associates with a reduced expression of the inflammatory S100A8 calcium binding protein and with a decreased intracellular calcium fluxes in patients' cells compared to healthy subjects, suggesting that the presence of the disease affects the "on-off" mechanisms that regulate the concentration of intracellular calcium.Introduzione. Le Spondiloartriti (SpA) sono un gruppo di malattie multifattoriali caratterizzate da una complessa interazione tra fattori genetici ed ambientali che determinano una disregolazione del sistema immunitario e l’attivazione di processi infiammatori a livello articolare, in particolar modo nelle articolazioni sacro-iliache. A differenza dell’artrite reumatoide, nelle SpA non esistono dei biomarcatori specifici di attività di malattia che vengono utilizzati nella pratica clinica. Pertanto, la ricerca di nuovi biomarcatori potrebbe essere d’ausilio per una diagnosi precoce e per un adeguato monitoraggio dell’attività di malattia, oltre che essere impiegati come fattori prognostici, misure di outcome e strumenti di valutazione dell’efficacia di trattamento.Nei pazienti con SpA, i macrofagi che infiltrano le articolazioni e che derivano principalmente dai monociti circolanti non esprimono solo citochine infiammatorie come TNF-α, IL-1β o TGF-β ma anche enzimi coinvolti nel rimodellamento tissutale come le metallo proteinasi di matrice (MMPs). La metalloproteinasi di matrice 3 (MMP-3), infatti, è riconosciuta come una molecola altamente espressa nel tessuto sinoviale e nel sangue periferico dei pazienti con SpA. Studi recenti hanno evidenziato che le metallo proteinasi di matrice 8 e 9 (MMP8 e MMP9) vengono prodotte dalle cellule mononucleate derivate da sangue periferico (PBMCs) quando vengono stimolate da calprotectina (eterodimero formato dalle proteine S100A8 e S100A9).Vi è poi una crescente evidenza del ruolo patogenetico nelle Spa svolto dalle cellule appartenenti all’immunità innata quali macrofagi, mastociti e neutrofili; il tessuto sinoviale dei pazienti con SpA infatti è caratterizzato da una elevata vascolarizzazione e quindi da una forte infiltrazione delle cellule immunitarie. In queste cellule i segnali di calcio intracellulare sono essenziali nella regolazione di numerose funzioni cellulari incluse proliferazione, differenziazione, apoptosi e trascrizione genica. Lo scopo di questo lavoro è stato quello di analizzare i livelli di espressione genica delle molecole TNF-α, IL-1β, TGF-β, S100A8, S100A9, MMP3, MMP8 e MMP9 e di valutare se le variazione dei flussi di calcio intracellulare ([Ca2+]i) nei PBMCs potrebbero essere associati alla presenza di SpA. Metodi.La popolazione studiata comprendeva 64 pazienti con diagnosi di SpA (età media ± deviazione standard: 39.5 ± 13.2 anni; 39 maschi, 25 femmine) e 100 controlli sani (età media ± deviazione standard: 46.6 ± 8.5 anni; 58 maschi, 42 femmine). Tra i pazienti, 26 (40.6%) presentavano spondilite anchilosante (AS) e 38 (59.3%) artrite psoriasica (PsA), con diagnosi formulata sulla base dei criteri rispettivamente di New York e CASPAR. Per ciascun soggetto arruolato, sono stati raccolti i dati demografici e fisiologici, la storia clinica e familiare. Sono stati raccolti poi, campioni di sangue, al fine di valutare l’emocromo e la VES, e di determinare i livelli di PCR, acido urico, prealbumina, alanina aminotransferasi (ALT) e glucosio. Per ciascun soggetto è stata effettuata un’analisi di espressione genica relativa (Real Time PCR) di TNF-α, IL-1β, TGF-β, S100A8, S100A9, MMP3, MMP8 e MMP9. La determinazione dei flussi di calico intracellulare ([Ca2+]i) nei pazienti e nei controlli è stata effettuate mediante microscopio ad epifluorescenza. Risultati. I livelli di espressione genica relativa nei PBMCs delle citochine infiammatorie of TNF-α, IL-1β, TGF-β erano simili nei pazienti con AS e PsA se comparati ai livelli di espressione nei controlli sani. Le variazioni dei livelli di espressione di TNF-α, TGF-β e IL-1β correlavano tra di loro. I livelli di espressione di TNF-α però risultavano correlati in maniera diretta, nei pazienti, con la presenza di una familiarità per la malattia (t=-2.5386, p=0.013). I livelli di espressione di MMP8 e MMP9 non risultavano essere associati con la diagnosi di SpA e non correlavano con gli indici clinici di attività di malattia.Anche i livelli di espressione di S100A9 non risultavano essere associati con la diagnosi di SpA mentre i livelli di espressione di S100A8 (F=3.29, p=0.039) erano ridotti nei pazienti con PsA.I livelli di espressione di S100A8 e S100A9 correlavano in maniera significativa con il numero di cellule infiammatorie circolanti e S100A8 correlava con i valori di PCR e VES. Dall’analisi dei dati ottenuti dai controlli sani e dai pazienti risultava evidente come la maggior parte dei monociti dei soggetti di controllo presentassero pulsazioni regolari di calcio intracellulare a differenza delle cellule ottenute da pazienti.I pazienti affetti da AS presentavano una ridotta percentuale di monociti con oscillazioni dei flussi di calcio intracellulare rispetto ai controlli sani (F=6.15, p=0.003). La percentuale di monociti con variazione di calcio intracellulare e l’ espressione delle molecole studiate non risultavano essere correlati ne con il tipo di terapia ne con il tipo di farmaco utilizzato. Conclusioni. In conclusione, I risultati di questo studio hanno evidenziato che nei pazienti con SpA vi è una ridotta espressione della proteina legante calcio S100A8 e vi è un decremento dei flussi di calcio intracellulare rispetto ai controlli sani, suggerendo che la presenza della malattia influenza i meccanismi "on-off" che regolano la concentrazione di calcio intracellulare

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-β), 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-β, might result in an overall anti-tumor effect, thus supporting the administration of vitamin D in PDAC patients