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Objectives: Aside from the outbreak of the coronavirus disease 2019 (COVID-19), serological tests are not well known for their diagnostic value. We assessed the performance of serological tests using stored sera from patients with a variety of pathologic conditions, collected before the 2020 pandemic in Italy. Methods: Rapid lateral flow tests and Enzyme-Linked Immunosorbent Assays (ELISA) that detect Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were carried out using 1150 stored human serum samples that had been collected in 2018 and 2019. The tests were also run using samples from 15 control patients who had positive or negative oral swab test results, as assessed using real-time reverse transcription-polymerase chain reaction (rRT-PCR). The urea dissociation test was employed to rule out false-positive reactivity in the two antibody detection methods. Results: The lateral flow tests revealed 21 positive samples from the stored sera: 12 for IgM, four for IgG, and five for IgM/IgG. Among the nine rRT-PCR- positive controls, six individuals presented IgG and three IgM/IgG positivity. Using the urea (6 mol/L) dissociation test, two of the twelve stored samples that had shown IgM positivity were confirmed to be positive. The ELISA test detected four IgM-positive and three IgG-positive specimens. After treatment with 4 mol/L urea, the IgM-positive samples became negative, whereas the IgG positivity persisted. All of the rRT-PCR-positive controls were found to retain IgM or IgG positivity following the urea treatment. Conclusions: Our findings highlight the limited utility of serological testing for the SARS-CoV-2 virus based on the results of specimens collected before the outbreak of the infection.

The pathogenesis of ulcerative colitis (UC) is unknown, although genetic loci and altered gut microbiota have been implicated. Up to a third of patients with moderate to severe UC require proctocolectomy with ileal pouch ano-anastomosis (IPAA). We aimed to explore the mucosal microbiota of UC patients who underwent IPAA. Methods: For microbiome analysis, mucosal specimens were collected from 34 IPAA individuals. Endoscopic and histological examinations of IPAA were normal in 21 cases, while pouchitis was in 13 patients. 19 specimens from the healthy control (10 from colonic and 9 from ileum) were also analyzed. Data were analyzed using an ensemble of software packages: QIIME2, coda-lasso, clr-lasso, PICRUSt2, and ALDEx2. Results: IPAA specimens had significantly lower bacterial diversity as compared to normal. The microbial composition of the normal pouch was also decreased also when compared to pouchitis. Faecalibacterium prausnitzii, Gemmiger formicilis, Blautia obeum, Ruminococcus torques, Dorea formicigenerans, and an unknown species from Roseburia were the most uncommon in pouch/pouchitis, while an unknown species from Enterobacteriaceae was over-represented. Propionibacterium acnes and Enterobacteriaceae were the species most abundant in the pouchitis and in the normal pouch, respectively. Predicted metabolic pathways among the IPAA bacterial communities revealed an important role of immunometabolites such as SCFA, butyrate, and amino acids. Conclusions: Our findings showed specific bacterial signature hallmarks of dysbiosis and could represent bacterial biomarkers in IPAA patients useful to develop novel treatments in the future by modulating the gut microbiota through the administration of probiotic immunometabolites-producing bacterial strains and the addition of specific prebiotics and the faecal microbiota transplantation.

The inflammatory bowel disease (IBD) is a systemic disease that is characterized by the inflammation of the gastrointestinal tract. It includes ulcerative colitis and the Crohn's disease. Presently, IBD is one of the most investigated common complex human disorders, although its causes remain unclear. Multi-omics mechanisms involving genomic, transcriptomic, proteomic, and epigenomic variations, not to forget the miRNome, together with environmental contributions, result in an impairment of the immune system in persons with IBD. Such interactions at multiple levels of biology and in concert with the environment constitute the actual engine of this complex disease, demanding a multifactorial and multi-omics perspective to better understand the root causes of IBD. This expert analysis reviews and examines the latest literature and underscores, from the perspective of systems biology, the value of multi-omics technologies as opportunities to unravel the "IBD integrome." We anticipate that multi-omics research will accelerate the new discoveries and insights on IBD in the near future. It shall also pave the way for early diagnosis and help clinicians and families with IBD to forecast and make informed decisions about the prognosis and, possibly, personalized therapeutics in the future.

Background: Achalasia is a rare idiopathic disease with a complex etio-pathogenesis still unknown. This study aimed to identify microRNA (miRNA)-mRNA regulatory networks underlying achalasia. Methods: The investigation was performed in tissue specimens from 11 patients and five controls using the microarray technology followed by an integrated bioinformatics analysis. Key Results: One hundred and six miRNAs were significantly up-regulated and 64 were down-regulated in achalasia patients. The expression of the most 10 differential expressed miRNAs (miR-122-5p, miR-133a-3p, miR-504-5p, miR-187-3p, miR-133b, miR-200c-3p, miR-375, miR-200b-5p, miR-200b-3p, and miR203a) was confirmed by droplet digital PCR in an independent cohort. The interactions between the significant miRNAs and their targets uncovered 14 miRNA-mRNA interacting pairs with experimentally predicted genes (ie, FN1, ROCK2, DPYSL2), and 35 pairs with not experimentally target genes (ie, SULF1, MRVI1, PRKG1); all genes were involved in immune cell trafficking, skeletal and muscular system development, nervous system development macro-processes. Conclusion & Inferences: The mRNA–miRNA regulatory networks described in this study provide new insights in the genetic background of the disease, suggesting further investigations in novel pathogenic mechanisms.

The authors would like to indicate that Dr. Orazio Palmieri and Dr. Teresa Maria Creanza contributed equally to the work published in the International Journal of Molecular Sciences [1] [...]

Background & Aims In patients with chronic HCV-1 infection, recent evidences indicate that determination of a dinucleotide polymorphism (ss469415590, ΔG/TT) of a new gene, designated IFN λ-4, might be more accurate than the 12979860CC type of the IL28B locus in predicting sustained virological response (SVR) following peg-interferon and ribavirin. In addition, combined genotyping of different SNPs of the IL28B locus was shown to help dissect patients most prone to SVR among those with rs12979860CT. We examined whether single or combined genotyping of two IL28B SNPs, rs12979860 and rs8099917, and ss469415590 variation might improve the prediction of SVR. Results In the study cohort of 539 patients, 38% had SVR. The SNPs 12979860CC, rs8099917TT, and rs469415590TT/TT correlated significantly with SVR (68%, 50%, and 67%). Carriers of either the triplotype rs12979860CC_ss469415590TT/TT_rs8099917TT or the diplotype rs12979860CC_ss469415590TT/TT had the highest SVR rate (72%). In carriers of the rs12979860 T allele, neither the rs8099917 nor the ss469415590 improved the response prediction. After pooling this finding with data from previous studies, in rs12979860 T heterozygous individuals the co-presence of the rs8099917TT SNP was associated with improved response prediction. Conclusion In HCV-1 patients, the rs12979860 polymorphism appeared as the hit SNP better predicting response following peg-interferon and ribavirin treatment. Additional ss469415590 or rs8099917 genotyping had no added benefit for response prediction. In the subset of carriers of the rs12979860 T allele, genotyping of the rs8099917 SNP was unhelpful in the present investigation, but may inform clinical prediction of treatment response when our data were pooled with previous investigations.

Background Crohn’s disease (CD) is a pathologic condition with different clinical expressions that may reflect an interplay between genetics and environmental factors. Recently, it has been highlighted that three genetic markers, NOD2, MHC and MST1, were associated to distinct CD sites, supporting the concept that genetic variations may contribute to localize CD. Genetic markers, previously shown to be associated with inflammatory bowel disease (IBD), were tested in CD patients with the aim to better dissect the genetic relationship between ileal, ileocolonic and colonic CD and ascertain whether a different genetic background would support the three disease sites as independent entities. Methods A panel of 29 SNPs of 19 IBD loci were analyzed by TaqMan SNP allelic discrimination method both evaluating their distinct contribute and analyzing all markers jointly. Results Seven hundred and eight CD patients and 537 healthy controls were included in the study. Of the overall population of patients, 237 patients had an ileal involvement (L1), 171 a colonic localization (L2), and the 300 remaining an ileocolon location (L3). We confirmed the association for 23 of 29 variations (P < 0.05). Compared to healthy controls, 16 variations emerged as associated to an ileum disease, 7 with a colonic disease and 14 with an ileocolonic site (P < 0.05). Comparing ileum to colonic CD, 5 SNPs (17%) were differentially associated (P < 0.05). A genetic model score that aggregated the risks of 23 SNPs and their odds ratios (ORs), yielded an Area Under the Curve (AUC) of 0.70 for the overall CD patients. By analyzing each CD location, the AUC remained at the same level for the ileal and ileocolonic sites (0.73 and 0.72, respectively), but dropped to a 0,66 value in patients with colon localization. Conclusions Our findings reaffirm the existence of at least three different subgroups of CD patients, with a genetic signature distinctive for the three main CD sites.

The aim of this study is to investigate the role of single-nucleotide polymorphisms (SNPs) of the glucocorticoid receptor (GR) and of the related co-chaperone FKBP5 genes in the development of glucocorticoid (GC) resistance in Crohn's disease (CD) and ulcerative colitis (UC) patients. We have developed a high-resolution DNA melting method that allows simultaneous identification of GR (BclI, N363S and ER22/23EK) and FKBP5 (rs3800373, rs1360780 and rs4713916) polymorphisms. Genotype frequencies were determined in 100 consecutive CD and 100 UC patients under GCs therapy (50 responders and 50 resisters). The variation of FKBP5 polymorphism rs4713916 (G/A), in the putative promoter region of FKBP5, is significantly associated with resistance to GC treatment in CD (responder=17% versus resister=35%; P=0.0043). No significant differences were found in UC patients. If these preliminary findings will be confirmed, the combination of GR and FKBP5 mutational analyses could help to identify subgroups of CD patients with higher chances to benefit from GC treatment.

Background: We tested several polymorphisms of genes involved in the mucosal immune system in a population of inflammatory bowel disease (IBD) patients to investigate their possible implication in disease predisposition. Methods: Polymorphisms of 3 candidate genes (PTPN22, NFkB1, and FcGRIIIA) were investigated in 649 IBD patients (343 with Crohn's disease [CD] and 306 with ulcerative colitis [UC]), 176 unaffected relatives, and 256 healthy controls. Allele and genotype frequencies were correlated with clinical characteristics and major variants of the CARD15 gene. Our findings were pooled in a meta-analysis with the available studies in the literature. Results: No significant difference for the PTPN22 and NFkB1 variants was found. In contrast, allele and genotype frequencies of the G559T allele of the FcGRIIIA gene were significantly different in CD patients compared to controls (allele T 12% versus 8%, odds ratio [OR] = 1.58, 95% confidence interval [CI] 1.06–2.35; GT genotype 23% versus 16%, OR = 1.64, 95% CI = 1.08–2.5). However, no significant overtransmission of the T allele was confirmed at the family-based analysis. For all genes, neither an interaction with CARD15 gene, nor a significant difference at genotype/phenotype analysis was demonstrated, included response to medical therapy. Conclusions: Although involved in autoimmune diseases, the PTPN22 and NFkB1 genes do not seem involved in the IBD predisposition, also according to meta-analysis results. The association with the G559T polymorphism of the FcGRIIIA gene in CD patients deserves further investigation. (Inflamm Bowel Dis 2007)

Summary Background : Anti-Saccharomyces cerevisiae mannan antibodies have been proposed as a new serological marker associated with Crohn's disease. However, their clinical value is still unclear; furthermore, a standardization of anti-S. cerevisiae mannan measurements is lacking. Aim : In this study, we aimed to assess the correlation between anti-S. cerevisiae mannan detection and specific clinical features in Crohn's disease and ulcerative colitis. Moreover, we tested the concordance of four different anti-S. cerevisiae mannan assays. Materials and methods : Serum samples from 196 patients with Crohn's disease, 197 patients with ulcerative colitis and 100 unrelated healthy controls were tested for anti-S. cerevisiae mannan with a standard enzyme-linked immunosorbent assay method (Lille) by one of the authors (VP). Subsequently, 60 randomly selected serum samples (27 Crohn's disease, 28 ulcerative colitis and five healthy controls) were tested for anti-S. cerevisiae mannan with three different commercial kits. Results : With the Lille assay, anti-S. cerevisiae mannan were detected in 100 of 196 patients with Crohn's disease (51%; P

Simultaneous analysis of the transcripts of thousands of genes by cDNA microarrays allows the identification of genetic regulatory mechanisms involved in disease pathophysiology. The circadian clock circuitry controls essential cell processes and the functioning of organ systems, which are characterized by rhythmic variations with 24-hour periodicity. The derangement of these processes is involved in the basic mechanisms of inflammatory, metabolic, degenerative and neoplastic diseases. We evaluated by genome-wide cDNA microarray analysis the transcriptome of endoscopic mucosal biopsies of patients with inflammatory bowel diseases (IBD) focusing on the expression of circadian genes in Crohn’s disease (CD) and ulcerative colitis (UC). Twenty-nine IBD patients (15 with CD and 14 with UC) were enrolled and mucosal biopsies were sampled at either inflamed or adjacent non-inflamed areas of the colon. A total of 150 circadian genes involved in pathways controlling crucial cell processes and tissue functions were investigated. In CD specimens 50 genes were differentially expressed, and 21 genes resulted up-regulated when compared to healthy colonic mucosa. In UC specimens 50 genes were differentially expressed, and 27 genes resulted up-regulated when compared to healthy colonic mucosa. Among the core clock genes ARNTL2 and RORA were up-regulated, while CSNK2B, NPAS2, PER1 and PER3 were down-regulated in CD specimens. Conversely, ARNTL2, CRY1, CSNK1E, RORA and TIPIN were up-regulated, while NR1D2 and PER3 were down-regulated in UC. In conclusion, in CD and UC patients there are differences in the expression of circadian genes between normal and diseased intestinal mucosa. The deregulated genes evidenced by transcriptome analysis in the major IBDs may play a crucial role in the pathophysiological mechanisms and may suggest novel therapeutic approaches.

Article first published online 21 April 2015. Supplemental Digital Content is Available in the Text., Background: Ulcerative colitis (UC) and Crohn's disease (CD) share some pathogenetic features. To provide new steps on the role of altered gene expression, and the involvement of gene networks, in the pathogenesis of these diseases, we performed a genome-wide analysis in 15 patients with CD and 14 patients with UC by comparing the RNA from inflamed and noninflamed colonic mucosa. Methods: Two hundred ninety-eight differentially expressed genes in CD and 520 genes in UC were identified. By bioinformatic analyses, 34 pathways for CD, 6 of them enriched in noninflamed and 28 in inflamed tissues, and 19 pathways for UC, 17 in noninflamed and 2 in inflamed tissues, were also highlighted. Results: In CD, the pathways included genes associated with cytokines and cytokine receptors connection, response to external stimuli, activation of cell proliferation or differentiation, cell migration, apoptosis, and immune regulation. In UC, the pathways were associated with genes related to metabolic and catabolic processes, biosynthesis and interconversion processes, leukocyte migration, regulation of cell proliferation, and epithelial-to-mesenchymal transition. Conclusions: In UC, the pattern of inflammation of colonic mucosa is due to a complex interaction network between host, gut microbiome, and diet, suggesting that bacterial products or endogenous synthetic/catabolic molecules contribute to impairment of the immune response, to breakdown of epithelial barrier, and to enhance the inflammatory process. In patients with CD, genes encoding a large variety of proteins, growth factors, cytokines, chemokines, and adhesion molecules may lead to uncontrolled inflammation with ensuing destruction of epithelial cells, inappropriate stimulation of antimicrobial and T cells differentiation, and inflammasome events.

Crohn’s disease (CD) is a debilitating inflammatory bowel disease (IBD) that emerges due to the influence of genetic and environmental factors. microRNAs (miRNAs) have been identified in the tissue and sera of IBD patients and may play an important role in the induction of IBD. Our study aimed to identify differentially expressed miRNAs and miRNAs with the ability to alter transcriptome activity by comparing inflamed tissue samples with their non-inflamed counterparts. We studied changes in miRNA–mRNA interactions associated with CD by examining their differential co-expression relative to normal mucosa from the same patients. Correlation changes between the two conditions were incorporated into scores of predefined gene sets to identify biological processes with altered miRNA-mediated control. Our study identified 28 miRNAs differentially expressed (p-values < 0.01), of which 14 are up-regulated. Notably, our differential co-expression analysis highlights microRNAs (i.e., miR-4284, miR-3194 and miR-21) that have known functional interactions with key mechanisms implicated in IBD. Most of these miRNAs cannot be detected by differential expression analysis that do not take into account miRNA–mRNA interactions. The identification of differential miRNA–mRNA co-expression patterns will facilitate the investigation of the miRNA-mediated molecular mechanisms underlying CD pathogenesis and could suggest novel drug targets for validation.

BackgroundRecent evidence suggests that the IL-33/IL1RL1 axis plays a critical role in several autoimmune and inflammatory disorders; however, its mechanistic role in inflammatory bowel disease (IBD) has not been clearly defined. We investigated the contribution of IL-33 and IL1RL1 polymorphisms to IBD risk, and possible correlations with phenotype in an Italian cohort of adult and pediatric patients.MethodsWe evaluated the association of six SNPs in IL-33 and IL1RL1 genes, in 805 Crohn's disease (CD), 816 ulcerative colitis (UC), and 752 controls, using Taqman. IL-33 and IL1RL1 mRNA expression was also analyzed.ResultsSignificant allele and genotype associations with IL-33 rs3939286 were found in CD (P = 0.004; P = 0.035) and UC patients (P = 0.002; P = 0.038). After stratifying the cohort for age at diagnosis, the differences remained significant only in the IBD adult-onset. Significant associations were also obtained in CD patients with two IL1RL1 polymorphisms (rs13015714 and rs2058660, PConclusionsCommon IL-33 and IL1RL1 polymorphisms contribute to the risk of IBD in an Italian cohort of adult and pediatric patients, with some influence on sub-phenotypes.

Altered body rhythmicity and deregulated clock gene expression may cause circadian disruption, which can lead to immune dysregulation and chronic inflammatory diseases. PERIOD3 (PER3) polymorphisms have been associated with circadian disruption and changed secretion of cytokines involved in chronic inflammation. Crohn's disease (CD) and ulcerative colitis (UC) are multifactorial diseases resulting from complex interaction among environmental/microbial factors and the intestinal immune system, triggering an abnormal immune response in genetically susceptible individuals. We evaluated the influence of a polymorphism of the clock gene PER3 on susceptibility and behavior of these inflammatory bowel diseases. The rs2797685 variant of the PER3 gene was assessed in 1082 CD and 972 UC patients, 754 of whom had been diagnosed18 yrs of age, and 1311 unrelated healthy controls. Allele and genotype frequencies of rs2797685 were significantly increased in both CD (p = 1.6 × 10(-4), odds ratio [OR] = 1.38, 95% confidence interval [CI]: 1.17-1.63) and UC (p = .012, OR = 1.25, 95% CI: 1.05-1.48) patients. Difference between frequency distributions remained statistically significant after stratifying the cohort according to age at diagnosis for CD, but not for UC. Statistically significant association was found between PER3 polymorphism and use of immunosuppressive drugs in pediatric CD patients (p .001) and with stricturing and fistulizing disease behavior in adult CD patients (p = .031). In conclusion, results of this association study suggest a possible role of PER3 polymorphism in determining susceptibility to CD and UC and phenotypic characteristics of CD. In particular, the rs2797685 variant of the PER3 gene is associated with a more aggressive form of CD, highlighted by higher use of immunosuppressants and more frequent stricturing and fistulizing disease behaviors, as well as early onset of CD. This is a descriptive study, and functional data are needed to prove a causal relationship; nonetheless, involvement of the clock gene machinery in the susceptibility and the behavior of inflammatory bowel diseases may suggest new pathophysiological mechanisms and new therapeutic approaches.

BACKGROUND: Recent GWAs and meta-analyses have outlined about 100 susceptibility genes/loci for inflammatory bowel diseases (IBD). In this study we aimed to investigate the influence of SNPs tagging the genes/loci PTGER4, TNFSF15, NKX2-3, ZNF365, IFNG, PTPN2, PSMG1, and HLA in a large pediatric- and adult-onset IBD Italian cohort. METHODS: Eight SNPs were assessed in 1,070 Crohn's disease (CD), 1,213 ulcerative colitis (UC), 557 of whom being diagnosed at the age of ≤16 years, and 789 healthy controls. Correlations with sub-phenotypes and major variants of NOD2 gene were investigated. RESULTS: The SNPs tagging the TNFSF15, NKX2-3, ZNF365, and PTPN2 genes were associated with CD (P values ranging from 0.037 to 7×10(-6)). The SNPs tagging the PTGER4, NKX2-3, ZNF365, IFNG, PSMG1, and HLA area were associated with UC (P values 0.047 to 4×10(-5)). In the pediatric cohort the associations of TNFSF15, NKX2-3 with CD, and PTGER4, NKX2-3, ZNF365, IFNG, PSMG1 with UC, were confirmed. Association with TNFSF15 and pediatric UC was also reported. A correlation with NKX2-3 and need for surgery (P = 0.038), and with HLA and steroid-responsiveness (P = 0.024) in UC patients was observed. Moreover, significant association in our CD cohort with TNFSF15 SNP and colonic involvement (P = 0.021), and with ZNF365 and ileal location (P = 0.024) was demonstrated. CONCLUSIONS: We confirmed in a large Italian cohort the associations with CD and UC of newly identified genes, both in adult and pediatric cohort of patients, with some influence on sub-phenotypes.