Colorectal cancer and inflammatory bowel diseases share common salivary proteomic pathways.

Inflammatory bowels diseases (IBD) are high risk conditions for colorectal cancer (CRC). The discovery of IBD and CRC noninvasive protein/peptide biomarkers using saliva and feces was the aim of this study involving 20 controls, 25 IBD (12 Crohn's Disease-CD), 37 CRC. By untargeted proteomic (LTQ-Orbitrap/MS), a total of 152 proteins were identified in saliva. Absent in controls, 73 proteins were present in both IBD and CRC, being mainly related to cell-adhesion, cadherin-binding and enzyme activity regulation (g-Profiler). Among the remaining 79 proteins, 14 were highly expressed in CD and 11 in CRC. These proteins clustered in DNA replication/expression and innate/adaptive immunity. In stool, endogenous peptides from 30 different proteins were identified, two being salivary and CD-associated: Basic Proline-rich Protein 1 (PRBs) and Acidic Proline-rich Phosphoprotein. Biological effects of the PRBs-related peptides GQ-15 and GG-17 found in CD stool were evaluated using CRC cell lines. These peptides induced cell proliferation and activated Erk1/2, Akt and p38 pathways. In conclusion, the salivary proteome unveiled DNA stability and immunity clusters shared between IBD and CRC. Salivary PRB-derived peptides, enriched in CD stool, stimulate CRC cell proliferation and the pro-oncogenic RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways suggesting a potential involvement of PRBs in IBD and cancer pathogenesis.

Analytical and clinical evaluations of SNIBE Maglumi chemiluminescent immunoassay for the detection of SARS-CoV-2 antigen in salivary samples.

OBJECTIVES: In this study, we describe the analytical and clinical performances of the SNIBE Maglumi SARS-CoV-2 antigen fully-automated chemiluminescent immunoassay (MAG-CLIA) on salivary samples. METHODS: Limit of detection (LOD), linearity and precision were tested for values close to or below the declared LOD. Clinical performance of MAG-CLIA was evaluated on leftover salivary samples from the healthcare workers (HCW) surveillance program, at the University-Hospital of Padova. Salivary samples were analyzed by Lumipulse G SARS-CoV-2 Ag, and in case where the values exceeded 0.41 ng/L, further testing was conducted using TaqPathTM COVID-19 RT-PCR (Applied Biosystems, Thermo Fisher Scientific). RESULTS: The estimated MAG-CLIA LOD was 3 ng/L, with repeatability of 7.5 %. Good linearity was demonstrated by diluting two samples at 52.7 ng/L and 211.4 ng/L. Of the 228 HCW samples, 59/228 (25.9 %) were positive, 169/228 (74.1 %) were negative. MAG-CLIA SARS-CoV-2 sAg median level (and interquartile range [IQR]) was 5.03 ng/L (<0.001-35.8 ng/L) for positive and <0.001 ng/L (<0.001 ng/L) for negative samples. MAG-CLIA AUC was 0.795 (95 % CI: 0.720-0.871). Using the best cut-off, 3.5 ng/L, sensitivity and specificity were 57.1 % (95 % CI: 42.2-71.2 %) and 97.0 % (95 % CI: 93.2-99.0 %), respectively. The agreement with the molecular assay was 88.1 % (Cohen's kappa 0.606 [SE=0.066, p<0.001]). CONCLUSIONS: The analytical performances of MAG-CLIA are satisfactory, also when values below LOD were tested. In saliva samples, although specificity was elevated, clinical performance was not comparable with that on nasopharyngeal swabs (NPS).

Evaluation of a new molecular test for the detection of SARS-CoV-2 nucleic acid in salivary samples.

Background: Molecular testing is considered the gold standard for the detection of SARS-CoV-2. This study aimed to compare the performance of the P742H SARS-CoV-2 Nucleic Acid Multiplex Detection Kit in salivary samples, with respect to the 732HF Novel Coronavirus (2019-nCoV) Nucleic Acid Detection Kit and the TaqPath COVID-19 CEIVD RT-PCR Kit, used at University-Hospital of Padova, Italy. Methods: One hundred twenty-four salivary samples selfcollected by healthcare workers (HCW) during the screening program at University-Hospital of Padova, Italy, from Oct to Nov 2022, were included in the study. RNA extraction was performed by Viral DNA and RNA Extraction Kit (Technogenetics, Lodi, Italy) and amplification by P742H and 732HF (Technogenetics, Lodi, Italy). RNA was extracted using MagNa Pure 96 DNA and Viral NA Small Volume Kit (Roche, Switzerland) for TaqPath analysis (Thermo Fisher Scientific, USA).

The PANcreatic Disease ReseArch (PANDoRA) consortium: Ten years' experience of association studies to understand the genetic architecture of pancreatic cancer.

Pancreatic cancer has an incidence that almost matches its mortality. Only a small number of risk factors and 33 susceptibility loci have been identified. so Moreover, the relative rarity of pancreatic cancer poses significant hurdles for research aimed at increasing our knowledge of the genetic mechanisms contributing to the disease. Additionally, the inability to adequately power research questions prevents small monocentric studies from being successful. Several consortia have been established to pursue a better understanding of the genetic architecture of pancreatic cancers. The Pancreatic disease research (PANDoRA) consortium is the largest in Europe. PANDoRA is spread across 12 European countries, Brazil and Japan, bringing together 29 basic and clinical research groups. In the last ten years, PANDoRA has contributed to the discovery of 25 susceptibility loci, a feat that will be instrumental in stratifying the population by risk and optimizing preventive strategies.

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.

The University of Padua salivary-based SARS-CoV-2 surveillance program minimized viral transmission during the second and third pandemic wave.

BACKGROUND: The active surveillance of students is proposed as an effective strategy to contain SARS-CoV-2 spread and prevent schools' closure. Saliva for molecular testing is as sensitive as naso-pharyngeal swab (NPS), self-collected and well accepted by participants. This prospective study aimed to verify whether the active surveillance of the Padua University employees by molecular testing of self-collected saliva is an effective and affordable strategy for limiting SARS-CoV-2 spread. METHODS: A surveillance program based on self-collection of saliva every 2 weeks (October 2020-June 2021) was conducted. Among 8183 employees of the Padua University, a total of 6284 subjects voluntarily took part in the program. Eight collection points guaranteed the daily distribution and collection of barcoded salivary collection devices, which were delivered to the laboratory by a transport service for molecular testing. Quarantine of positive cases and contact tracing were promptly activated. RESULTS: Among 6284 subjects, 206 individuals were SARS-CoV-2 positive (99 by salivary testing; 107 by NPS performed for contact tracing or symptoms). The cumulative SARS-CoV-2 incidence in this cohort was 3.1%, significantly lower than that of employees not in surveillance (8.0%), in Padua (7.1%) and in the Veneto region (7.2%). Employees with positive saliva results were asymptomatic or had mild symptoms. The levels of serum antibodies after 3 months from the infection were correlated with age and Ct values, being higher in older subjects with greater viral loads. CONCLUSIONS: Salivary-based surveillance with contact tracing effectively allowed to limit SARS-CoV-2 contagion, also in a population with a high incidence.

Identification of Recessively Inherited Genetic Variants Potentially Linked to Pancreatic Cancer Risk.

Although 21 pancreatic cancer susceptibility loci have been identified in individuals of European ancestry through genome-wide association studies (GWASs), much of the heritability of pancreatic cancer risk remains unidentified. A recessive genetic model could be a powerful tool for identifying additional risk variants. To discover recessively inherited pancreatic cancer risk loci, we performed a re-analysis of the largest pancreatic cancer GWAS, the Pancreatic Cancer Cohort Consortium (PanScan) and the Pancreatic Cancer Case-Control Consortium (PanC4), including 8,769 cases and 7,055 controls of European ancestry. Six single nucleotide polymorphisms (SNPs) showed associations with pancreatic cancer risk according to a recessive model of inheritance. We replicated these variants in 3,212 cases and 3,470 controls collected from the PANcreatic Disease ReseArch (PANDoRA) consortium. The results of the meta-analyses confirmed that rs4626538 (7q32.2), rs7008921 (8p23.2) and rs147904962 (17q21.31) showed specific recessive effects (p<10-5) compared with the additive effects (p>10-3), although none of the six SNPs reached the conventional threshold for genome-wide significance (p < 5×10-8). Additional bioinformatic analysis explored the functional annotations of the SNPs and indicated a possible relationship between rs36018702 and expression of the BCL2L11 and BUB1 genes, which are known to be involved in pancreatic biology. Our findings, while not conclusive, indicate the importance of considering non-additive genetic models when performing GWAS analysis. The SNPs associated with pancreatic cancer in this study could be used for further meta-analysis for recessive association of SNPs and pancreatic cancer risk and might be a useful addiction to improve the performance of polygenic risk scores.

Genome-wide scan of long noncoding RNA single nucleotide polymorphisms and pancreatic cancer susceptibility.

Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second cancer-related cause of death by 2030. Identifying novel risk factors, including genetic risk loci, could be instrumental in risk stratification and implementation of prevention strategies. Long noncoding RNAs (lncRNAs) are involved in regulation of key biological processes, and the possible role of their genetic variability has been unexplored so far. Combining genome wide association studies and functional data, we investigated the genetic variability in all lncRNAs. We analyzed 9893 PDAC cases and 9969 controls and identified a genome-wide significant association between the rs7046076 SNP and risk of developing PDAC (P = 9.73 × 10-9 ). This SNP is located in the NONHSAG053086.2 (lnc-SMC2-1) gene and the risk allele is predicted to disrupt the binding of the lncRNA with the micro-RNA (miRNA) hsa-mir-1256 that regulates several genes involved in cell cycle, such as CDKN2B. The CDKN2B region is pleiotropic and its genetic variants have been associated with several human diseases, possibly though an imperfect interaction between lncRNA and miRNA. We present a novel PDAC risk locus, supported by a genome-wide statistical significance and a plausible biological mechanism.

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.

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

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.

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.

Genome-wide association study identifies an early onset pancreatic cancer risk locus.

Early onset pancreatic cancer (EOPC) is a rare disease with a very high mortality rate. Almost nothing is known on the genetic susceptibility of EOPC, therefore, we performed a genome-wide association study (GWAS) to identify novel genetic variants specific for patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) at younger ages. In the first phase, conducted on 821 cases with age of onset ≤60 years, of whom 198 with age of onset ≤50, and 3227 controls from PanScan I-II, we observed four SNPs (rs7155613, rs2328991, rs4891017 and rs12610094) showing an association with EOPC risk (P < 1 × 10-4 ). We replicated these SNPs in the PANcreatic Disease ReseArch (PANDoRA) consortium and used additional in silico data from PanScan III and PanC4. Among these four variants rs2328991 was significant in an independent set of 855 cases with age of onset ≤60 years, of whom 265 with age of onset ≤50, and 4142 controls from the PANDoRA consortium while in the in silico data, we observed no statistically significant association. However, the resulting meta-analysis supported the association (P = 1.15 × 10-4 ). In conclusion, we propose a novel variant rs2328991 to be involved in EOPC risk. Even though it was not possible to find a mechanistic link between the variant and the function, the association is supported by a solid statistical significance obtained in the largest study on EOPC genetics present so far in the literature.

Emerging role of monocytes and their intracellular calcium pattern in spondyloarthritis.

Spondyloarthritis (SpA) comprises multifactorial diseases characterized by a complex interplay between an inherited background and environmental factors that lead to immune response dysregulation and inflammation. Unlike for other rheumatic diseases, no specific biomarkers are available in clinical practice for diagnosing SpA. The aim of the present study was to search new potential biomarkers for SpA diagnosis by focusing on the innate immune response. An evaluation was made of the mRNA expression levels of inflammatory cytokines (TNF-α, IL-1ß, TGF-ß1, S100A8, S100A9) and matrix metalloproteinases (MMP3, MMP8, MMP9) in blood mononuclear cells of SpA patients (n = 64) with respect to controls (n = 100). In parallel, the pattern of intracellular calcium flows of blood monocytes was verified in order to ascertain whether any specific fingerprint characterizes innate immune cells in SpA patients. Inflammatory cytokines and MMPs expression levels were not correlated with SpA, while in this disease a reduced expression of the S100A8 and a decreased frequency of monocytes showing intracellular calcium flows were observed. In conclusion, no specific signs of systemic inflammation are detectable in SpA, but the disease affects the "on-off" mechanisms that regulate the concentration of intracellular calcium and calcium-related proteins. This potentially pave the way for the discovery of new biomarkers.

MALDI-TOF peptidomic analysis of serum and post-prostatic massage urine specimens to identify prostate cancer biomarkers.

BACKGROUND: Lower urinary tract symptoms (LUTS) and prostate specific antigen-based parameters seem to have only a limited utility for the differential diagnosis of prostate cancer (PCa). MALDI-TOF/MS peptidomic profiling could be a useful diagnostic tool for biomarker discovery, although reproducibility issues have limited its applicability until now. The current study aimed to evaluate a new MALDI-TOF/MS candidate biomarker. METHODS: Within- and between-subject variability of MALDI-TOF/MS-based peptidomic urine and serum analyses were evaluated in 20 and 15 healthy donors, respectively. Normalizations and approaches for accounting below limit of detection (LOD) values were utilized to enhance reproducibility, while Monte Carlo experiments were performed to verify whether measurement error can be dealt with LOD data. Post-prostatic massage urine and serum samples from 148 LUTS patients were analysed using MALDI-TOF/MS. Regression-calibration and simulation and extrapolation methods were used to derive the unbiased association between peptidomic features and PCa. RESULTS: Although the median normalized peptidomic variability was 24.9%, the within- and between-subject variability showed that median normalization, LOD adjustment, and log2 data transformation were the best combination in terms of reliability; in measurement error conditions, intraclass correlation coefficient was a reliable estimate when the LOD/2 was substituted for below LOD values. In the patients studied, 43 peptides were shared by the urine and serum, and several features were found to be associated with PCa. Only few serum features, however, show statistical significance after the multiple testing procedures were completed. Two serum fragmentation patterns corresponded to the complement C4-A. CONCLUSIONS: MALDI-TOF/MS serum peptidome profiling was more efficacious with respect to post-prostatic massage urine analysis in discriminating PCa.

Improving IBD diagnosis and monitoring by understanding preanalytical, analytical and biological fecal calprotectin variability.

BACKGROUND: The appropriate clinical use of fecal calprotectin (fCal) might be compromised by incomplete harmonization between assays and within- and between-subjects variability. Our aim was to investigate the analytical and biological variability of fCal in order to provide tools for interpreting fCal in the clinical setting. METHODS: Experiments were conducted to investigate the effects of temperature and storage time on fCal. Thirty-nine controls were enrolled to verify biological variability, and a case-control study was conducted on 134 controls and 110 IBD patients to compare the clinical effectiveness of three different fCal assays: ELISA, CLIA and turbidimetry. RESULTS: A 12% decline in fCal levels was observed within 24 h following stool collection irrespective of storage temperature. Samples were unstable following a longer storage time interval at room temperature. Within- and between-subjects fCal biological variability, at 31% and 72% respectively, resulted in a reference change value (RCV) in the region of 100%. fCal sensitivity in distinguishing between controls and IBD patients is satisfactory (68%), and the specificity high (93%) among young (<65 years), but not among older (≥65 years) subjects (ROC area: 0.584; 95% CI: 0.399-0.769). Among the young, assays have different optimal thresholds (120 µg/g for ELISA, 50 µg/g for CLIA and 100 µg/g for turbidimetry). CONCLUSIONS: We recommend a standardized preanalytical protocol for fCal, avoiding storage at room temperature for more than 24 h. Different cutoffs are recommended for different fCal assays. In monitoring, the difference between two consecutive measurements appears clinically significant when higher than 100%, the fCal biological variability-derived RCV.

Genetics in TNF-TNFR pathway: A complex network causing spondyloarthritis and conditioning response to anti-TNFα therapy.

OBJECTIVES: We investigated whether polymorphisms (SNPs) in the promoter region of TNFA, or in the autoinflammatory TNFRSF1A and MEFV genes, concur with HLA-B27 in enhancing the risk of Spondyloarthritis (SpA) and/or in predicting the response to anti-TNFα treatment. METHODS: 373 controls and 137 SpA (82 with Psoriatic Arthritis-PsA and 55 with Ankylosing Spondylitis- AS; 98/137 under TNFα inhibitor therapy) from the Veneto Region (Italy) were studied. TNFA polymorphisms (-1031T>C;-857C>T;-376G>A;-308G>A;-238G>A) and HLA-B27 were assayed by RT-PCR. Direct sequencing of MEFV (exons 2,3,5 and 10) and TNFRSF1A (exons 2,3,4 and 6) genes were performed. RESULTS: HLA-B27 was associated with AS (χ2 = 120.1; p = 0.000). Only the TNFA -1031T>C was singly associated with SpA, and the haplotype C/G, resulting from -1031T>C/-308G>A combination, was significantly associated with a reduced risk of SpA (OR: 0.67, CI: 0.46-0.97; p = 0.035). Two SNPs were identified in TNFRSF1A, the R92Q (Minor allele frequency-MAF = 0.034) and c.625+10A>G (MAF = 0.479). None of them was associated with SpA (p>0.05). The TNFRSF1A c.625+10 G allele was associated with late response to anti-TNFα therapy (p = 0.031). Twenty-one SNPs were identified in MEFV gene, 10 with a known potential functional significance. Variant alleles were extremely rare in our population (MAF<0.025) except for R202Q (MAF = 0.27). None was associated with SpA diagnosis (p>0.05). CONCLUSION: TNFRSF1A and MEFV gene SNPs are not associated with SpA in the North-East of Italy. AS risk appears to depend not only on HLA-B27, but also on the protective TNFA haplotype -1031C/-308G. The TNFRSF1A c.625+10A>G impacts on the response to anti-TNFα therapy.

Pharmacokinetic and pharmacodynamic re-evaluation of a genetic-guided warfarin trial.

PURPOSE: A previous trial failed to demonstrate the superiority of a demographic-genetic algorithm in predicting warfarin (W) dose over a standard clinical approach. The purpose of the present study is to re-analyse the results in subgroups of patients with differing baseline sensitivity to W, integrated with additional pharmacokinetic data. METHODS: The original trial allocated 180 treatment-naïve patients with non-valvular atrial fibrillation to a control arm (CTL, n = 92) or a genetic-guided arm (GEN, n = 88). Before starting anticoagulation treatment, all patients were genotyped for CYP2C9, VKORC1 and CYP4F2 variants and classified into four quartiles (Q1, Q2, Q3, Q4) according to the algorithm-predicted W maintenance dose. International normalised ratios (INR) and plasma concentrations of S-warfarin [S-W]s and R-warfarin [R-W]s were measured at baseline and on days 5, 7, 9, 12, 15 and 19 of therapy. RESULTS: In the lowest dose quartile (Q1), the number of INRs > 3 and mean INR values on days 5 and 7 were significantly higher in CTL than in GEN. In Q3 and Q4, the mean INR values reached therapeutic level (> 2) 2 days later in CTL than in GEN. During follow-up, the mean time courses of INRs and [S-W]s in GEN were remarkably stable in all dose quartiles. Thus, mean changes from starting to final doses were significantly smaller in GEN than in CTL. Plasma concentrations of R-W (a partially active enantiomer) steadily increased from day 5 to day 19 in all Qs in both CTL and GEN, except in the Q1 CTL group, due to the marked dose reduction required. CONCLUSIONS: This analysis showed that the demographic-genetic algorithm used to predict the W dose can identify patients with differing degrees of sensitivity to W and to 'normalise' their average anticoagulant responses. The progressive rise in [R-W]s throughout the 19-day follow-up indicates that the (partial) contribution of R-W to the W anticoagulant effect changes continually during the early phase of treatment.

PDAC-derived exosomes enrich the microenvironment in MDSCs in a SMAD4-dependent manner through a new calcium related axis.

Tumor genetics and escape from immune surveillance concur in the poor prognosis of PDAC. In this study an experimental model was set up to verify whether SMAD4, deleted in about 55% PDAC and associated with poor prognosis, is involved in determining immunosuppression through Exosomes (Exo). Potential mechanisms and mediators underlying SMAD4-dependent immunosuppression were evaluated by studying intracellular calcium (Fluo-4), Exo-miRNAs (microarray) and Exo-proteins (SILAC). Two PDAC cell lines expressing (BxPC3-SMAD4+) or not-expressing (BxPC3) SMAD4 were used to prepare Exo-enriched conditioned media, employed in experiments with blood donors PBMCs. Exo expanded myeloid derived suppressor cells (gMDSC and mMDSC, flow cytometry) and altered intracellular calcium fluxes in an SMAD4 dependent manner. BxPC3-SMAD4+, but mainly BxPC3 Exo, increased calcium fluxes of PBMCs (p = 0.007) and this increased intracellular calcium trafficking characterized mMDSCs. The analysis of de-regulated Exo-miRNAs and transfection experiments revealed hsa-miR-494-3p and has-miR-1260a as potential mediators of SMAD4-associated de-regulated calcium fluxes. Eleven main biological processes were identified by the analysis of SMAD4-associated de-regulated Exo-proteins, including translation, cell adhesion, cell signaling and glycolysis. A reverse Warburg effect was observed by treating PBMCs with PDAC-derived Exo: BxPC3 Exo induced a higher glucose consumption and lactate production than BxPC3-SMAD4+ Exo. CONCLUSION: PDAC-derived Exo from cells with, but mainly from those without SMAD4 expression, create an immunosuppressive myeloid cell background by increasing calcium fluxes and glycolysis through the transfer of SMAD4-related differentially expressed miRNAs and proteins.

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.