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Introduction: The disease activity associated with the drug-utilization patterns of biologic Disease Modifying Anti-Rheumatic Drugs (DMARDs) is poorly investigated in real-world studies on rheumatoid arthritis (RA) patients. To investigate the relationship between biologic DMARD initiation/discontinuations in RA patients identified in the healthcare administrative databases of Tuscany and the Disease Activity Score 28 (DAS28) reported in the medical charts.Methods: This retrospective population-based study included RA’s first-ever biologic DMARD users of the Pisa University Hospital from 2014 to 2016. Patients were followed up until 31 December 2019. We evaluated the DAS28 recorded before (T0) and after (T1) the biologic DMARD initiation and before (TD0) and after (TD1) discontinuations. Patients were classified as “off-target” (DAS28 > 3.2) or “in-target” (DAS28 ≤ 3.2). We described the disease activity trends at initiation and discontinuation.Results: Ninety-five users were included (73 women, mean age 59.6). Among 70 patients (74%) with at least three DAS28 measures, 28 (40.0%) were off-target at T0 and 38 (54.3%) in-target at T1. Thirty-three (47%) patients had at least one discontinuation, among those with at least three DAS28 assessments. In the disease activity trend, disease stability or improvement was observed in 28 out of 37 (75.7%) patients at initiation and in 24 out of 37 (64.9%) at discontinuation.Discussion: Biologic DMARD discontinuations identified in the healthcare administrative databasese of Tuscany are frequently observed in situations of controlled RA disease. Further studies are warranted to confirm that these events can be used in studies using healthcare administrative databases as proxies of treatment effectiveness.

Abstract Validation of algorithms for selecting patients from healthcare administrative databases (HAD) is recommended. This PATHFINDER study section is aimed at testing algorithms to select rheumatoid arthritis (RA) patients from Tuscan HAD (THAD) and assessing RA diagnosis time interval between the medical chart date and that of THAD. A population was extracted from THAD. The information of the medical charts at the Rheumatology Unit of Pisa University Hospital represented the reference. We included first ever users of biologic disease modifying anti-rheumatic drugs (bDMARDs) between 2014 and 2016 (index date) with at least a specialist visit at the Rheumatology Unit of the Pisa University Hospital recorded from 2013 to the index date. Out of these, we tested four index tests (algorithms): (1) RA according to hospital discharge records or emergency department admissions (ICD-9 code, 714*); (2) RA according to exemption code from co-payment (006); (3) RA according to hospital discharge records or emergency department admissions AND RA according to exemption code from co-payment; (4) RA according to hospital discharge records or emergency department admissions OR RA according to exemption code from co-payment. We estimated sensitivity, specificity, positive and negative predicted values (PPV and NPV) with 95% confidence interval (95% CI) and the RA diagnosis median time interval (interquartile range, IQR). Two sensitivity analyses were performed. Among 277 reference patients, 103 had RA. The fourth algorithm identified 96 true RA patients, PPV 0.78 (95% CI 0.70–0.85), sensitivity 0.93 (95% CI 0.86–0.97), specificity 0.84 (95% CI 0.78–0.90), and NPV 0.95 (95% CI 0.91–0.98). The sensitivity analyses confirmed performance. The time measured between the actual RA diagnosis date recorded in medical charts and that assumed in THAD was 2.2 years (IQR 0.5–8.4). In conclusion, this validation showed the fourth algorithm as the best. The time interval elapsed between the actual RA diagnosis date in medical charts and that extrapolated from THAD has to be considered in the design of future studies.

Abstract Sars-CoV-2 complications include pneumonia and acute respiratory distress syndrome (ARDS), which require intensive care unit admission. These conditions have rapidly overwhelmed healthcare systems, with detrimental effects on the quality of care and increased mortality. Social isolation strategies have been implemented worldwide with the aim of reducing hospital pressure. Among therapeutic strategies, the use of immunomodulating drugs, to improve prognosis, seems promising. Particularly, since pneumonia and ARDS are associated with a cytokine storm, drugs belonging to therapeutic classes as anti-IL-6, anti-TNF, and JAK inhibitors are currently studied. In this article, we discuss the potential advantages of the most promising pharmacological approaches.

This study is aimed at describing tofacitinib and baricitinib users by characterizing their prescription and healthcare histories, drug and healthcare utilization patterns, and direct costs from a healthcare system perspective. This retrospective cohort study was performed using Tuscan administrative healthcare databases, which selected two groups of Janus kinase inhibitors (JAKi) incident users (index date) from 1st January 2018 to 31 December 2019 and from 1 January 2018 to 30 June 2019. We included patients ≥18 years old, at least 10 years of data, and six months of follow-up. In the first analysis, we describe mean time, standard deviation (SD), from the first-ever disease-modifying antirheumatic drug (DMARD) to the JAKi, and costs of healthcare facilities and drugs in the 5 years preceding the index date. In the second analysis, we assessed Emergency Department (ED) accesses and hospitalizations for any causes, visits, and costs in the follow-up. In the first analysis, 363 incident JAKi users were included (mean age 61.5, SD 13.6; females 80.7%, baricitinib 78.5%, tofacitinib 21.5%). The time to the first JAKi was 7.2 years (SD 3.3). The mean costs from the fifth to the second year before JAKi increased from 4325 € (0; 24,265) to 5259 € (0; 41,630) per patient/year, driven by hospitalizations. We included 221 incident JAKi users in the second analysis. We observed 109 ED accesses, 39 hospitalizations, and 64 visits. Injury and poisoning (18.3%) and skin (13.8%) caused ED accesses, and cardiovascular (69.2%) and musculoskeletal (64.1%) caused hospitalizations. The mean costs were 4819 € (607.5; 50,493) per patient, mostly due to JAKi. In conclusion, the JAKi introduction in therapy occurred in compliance with RA guidelines and the increase in costs observed could be due to a possible selective prescription.

Abstract Cardiovascular complications in Alzheimer's disease (AD) patients can occur years to decades prior to the onset of clinical symptoms of the disease. Donepezil represents the most effective drug in the treatment of AD. However, the potential effect of donepezil on vascular function and structure remains largely unexplored. Here, we assessed the impact of donepezil on the vascular phenotype of an established model of accelerated senescence that develops spontaneously AD, the SAMP8 mouse. Three groups of animals were included: SAMR1 (control strain), SAMP8, and SAMP8 treated with donepezil. Treatment with donepezil was administered from the 4th to the 6th month of life. At 6 months, after cognitive tests by Morris Water Maze, animals were euthanized, and their mesenteric arteries were processed for functional experiments. Untreated SAMP8 developed cognitive impairment compared to SAMR1, while donepezil treatment significantly attenuated cognitive dysfunction. SAMP8 exhibited a higher media‐to‐lumen ratio than SAMR1 and donepezil‐treated animals. Endothelial function was impaired in SAMP8 animals compared to SAMR1. The addition of vitamin C improved the vasodilatory response to acetylcholine in SAMP8. Treatment with donepezil improved endothelial function in SAMP8 animals and reduced the additional vasodilation induced by vitamin C. In conclusion, in the SAMP8 AD model, cognitive impairment is associated with endothelial dysfunction and vascular remodeling which could contribute to cardiovascular events in AD since the prodromal phases of the disease. Treatment with donepezil alleviates vascular dysfunction associated with AD through an increase in NO availability likely by counteracting inflammation and oxidative stress.

Abstract Background Gastrointestinal dysfunction can affect Parkinson’s disease (PD) patients long before the onset of motor symptoms. However, little is known about the relationship between gastrointestinal abnormalities and the development of PD. Contrary to other animal models, the human A53T alpha-synuclein (αS) transgenic mice, Line G2–3, develops αS-driven neurological and motor impairments after 9 months of age, displaying a long presymptomatic phase free of central nervous system (CNS) dysfunction. Methods To determine whether this line can be suitable to study constipation as it occurs in prodromal PD, gastrointestinal functionality was assessed in young mice through a multidisciplinary approach, based on behavioral and biochemical analysis combined with electrophysiological recordings of mouse intestinal preparations. Results We found that the A53T αS mice display remarkable signs of gastrointestinal dysfunction that precede motor abnormalities and αS pathology in the CNS by at least 6 months. Young αS mice show a drastic delay in food transit along the gastrointestinal tract, of almost 2 h in 3 months old mice that increased to more than 3 h at 6 months. Such impairment was associated with abnormal formation of stools that resulted in less abundant but longer pellets excreted, suggesting a deficit in the intestinal peristalsis. In agreement with this, electrically evoked contractions of the colon, but not of the ileum, showed a reduced motor response in both longitudinal and circular muscle layers in αS mice already at 3 months of age, that was mainly due to an impaired cholinergic transmission of the underlying enteric nervous system. Interestingly, the presence of insoluble and aggregated αS was found in enteric neurons in both myenteric and submucosal plexi only in the colon of 3 months old αS mice, but not in the small intestine, and exacerbated with age, mimicking the increase in transit delay and the contraction deficit showed by behavioral and electrical recordings data. Conclusions Gastrointestinal dysfunction in A53T αS mice represents an early sign of αS-driven pathology without concomitant CNS involvement. We believe that this model can be very useful to study disease-modifying strategies that could extend the prodromal phase of PD and halt αS pathology from reaching the brain.

Background and aim: Patients with Parkinson's disease (PD) are often characterized by functional gastrointestinal disorders. Such disturbances can occur at all stages of PD and precede the typical motor symptoms of the disease by many years. However, the morphological alterations associated with intestinal disturbances in PD are undetermined. This study examined the remodelling of colonic wall in 6-hydroxydopamine (6-OHDA)-induced PD rats. Methods: 8 weeks after 6-OHDA injection animals were sacrificed. Inflammatory infiltrates, collagen deposition and remodelling of intestinal epithelial barrier and tunica muscularis in the colonic wall were assessed by histochemistry, immunohistochemistry, immunofluorescence and western blot analysis. Results: 6-OHDA rats displayed significant alterations of colonic tissues as compared with controls. Signs of mild inflammation (eosinophil infiltration) and a transmural deposition of collagen fibres were observed. Superficial colonic layers were characterized by severe morphological alterations. In particular, lining epithelial cells displayed a reduced claudin-1 and transmembrane 16A/Anoctamin 1 (TMEM16A/ANO1) expression; goblet cells increased their mucin expression; colonic crypts were characterized by an increase in proliferating epithelial cells; the density of S100-positive glial cells and vimentin-positive fibroblast-like cells was increased as well. Several changes were found in the tunica muscularis: downregulation of α-smooth muscle actin/desmin expression and increased proliferation of smooth muscle cells; increased vimentin expression and proliferative phenotype in myenteric ganglia; reduction of interstitial cells of Cajal (ICCs) density. Conclusions: A pathological remodelling occurs in the colon of 6-OHDA rats. The main changes include: enhanced fibrotic deposition; alterations of the epithelial barrier; activation of mucosal defense; reduction of ICCs. These results indicate that central nigrostriatal denervation is associated with histological changes in the large bowel at mucosal, submucosal and muscular level. These alterations might represent morphological correlates of digestive symptoms in PD.

BackgroundAdverse drug event (ADEs) are a significant cause of emergency department (ED) visits and consequent hospitalization. Preventing ADEs and their related ED visits in outpatients remains a public health safety challenge. In this context, the aims of the present study were to describe the frequency, seriousness and preventability of outpatients’ ADE-related ED visits and hospitalizations in the Italian general population, and to identify the presence of potential predictors of ADE-related hospitalization.MethodsWe performed a nationwide, multicentre, observational, retrospective study based on reports of suspected ADEs collected between January 1, 2007 and December 31, 2018 in 94 EDs involved in the MEREAFaPS project. Patients’ demographic characteristics, their clinical status, suspected and concomitant drugs, ADE description, and its degree of seriousness, were collected. Causality and preventability were assessed using validated algorithms, and logistic regression analyses were used to estimate the reporting odds ratios (RORs) with 95% confidence intervals (CIs) of ADE-related hospitalization, considering the following covariates: age, sex, ethnicity, number of implicated medications, parenteral administration, presence of interaction, therapeutic error, and/or complementary and alternative medicines (CAM).ResultsWithin 12 years, 61,855 reports of suspected ADE were collected, of which 18,918 (30.6%) resulted in hospitalization (ADE defined as serious). Patients were mostly female (56.6%) and Caucasians (87.7%), with a mean age of 57.5 ± 25.0 years. 58% of patients were treated with more than two drugs, and 47% of ADEs leading to hospitalization were preventable. Anticoagulants, antibiotics, and nonsteroidal anti-inflammatory drugs (NSAIDs) were the most frequently implicated agents for ED visits and/or hospitalization, which included clinically significant ADEs, such as haemorrhage for anticoagulants, moderate to severe allergic reactions for antibiotics, and dermatologic reactions and gastrointestinal disturbances for NSAIDs. Older age (1.54 [1.48–1.60]), higher number of concomitantly taken drugs (2.22 [2.14–2.31]), the presence of drug-drug interactions (1.52 [1.28–1.81]), and therapeutic error (1.54 [1.34–1.78]), were significantly associated with an increased risk of hospitalization.ConclusionOur long-term active pharmacovigilance study in ED provided a valid estimation of ADE-related hospitalization in a representative sample of the Italian general population and can suggest further focus on medication safety in outpatients, in order to early recognise and prevent ADEs.

Monoclonal antibody (mAb) therapy has been previously exploited for viral infections, such as respiratory syncytial virus pneumonia and Ebolavirus disease. In the ongoing COVID-19 pandemic, early signals of efficacy from convalescent plasma therapy have encouraged research and development of anti-SARS-CoV-2 mAbs. While many candidates are in preclinical development, we focus here on anti-SARS-CoV-2 neutralizing mAbs (or mAb cocktails) that represent the late-stage clinical pipeline, i.e., those currently in Phase 2 or Phase 3 clinical trials. We describe the structure, mechanism of action, and ongoing trials for VIR-7831, LY-CoV555, LY-CoV016, BGB-DXP593, REGN-COV2, and CT-P59. We speculate also on the next generation of these mAbs.

Background: Inflammatory bowel diseases (IBDs) are generally not considered a risk factor for the development of lymphoma. When considering IBD treatments, there is good evidence supporting thiopurines (azathioprine, 6-mercaptopurine) as a risk factor for lymphoma. Conversely, the association between the use of anti-TNF agents and the development of lymphoma remains undetermined. In this systematic review, we analyzed the evidence coming from observational studies supporting an association between the use of anti-TNF drugs and lymphoma in patients with IBDs.Methods: This systematic review was performed according with MOOSE and PRISMA statements. We searched observational studies conducted on IBD patients, using MEDLINE, EMBASE, and Google Scholar, published in English language, within the period ranging from January 1st, 1999 to June 30th, 2018. An assessment of the methodologic shortcomings of selected studies was performed as well.Results: Fourteen studies met the eligibility criteria and were included in the review. Only four studies found a significant association of anti-TNF drug with lymphoma or groups of cancers including lymphoma. However, the methodologic shortcomings of all the included studies made their results unreliable, irrespectively of whether their findings supported an association or not.Conclusions: Current evidence from observational studies does not allow excluding or confirming an association of the exposure to anti-TNF treatments with lymphoma in IBD patients.

The management of visceral pain is a major clinical problem in patients affected by gastrointestinal disorders. The poor knowledge about pain chronicization mechanisms prompted us to study the functional and morphological alterations of the gut and nervous system in the animal model of persistent visceral pain caused by 2,4-dinitrobenzenesulfonic acid (DNBS). This agent, injected intrarectally, induced a colonic inflammation peaking on day 3 and remitting progressively from day 7. In concomitance with bowel inflammation, the animals developed visceral hypersensitivity, which persisted after colitis remission for up to three months. On day 14, the administration of pain-relieving drugs (injected intraperitoneally and intrathecally) revealed a mixed nociceptive, inflammatory and neuropathic pain originating from both the peripheral and central nervous system. At this time point, the colonic histological analysis highlighted a partial restitution of the tunica mucosa, transmural collagen deposition, infiltration of mast cells and eosinophils, and upregulation of substance P (SP)-positive nerve fibers, which were surrounded by eosinophils and MHC-II-positive macrophages. A significant activation of microglia and astrocytes was observed in the dorsal and ventral horns of spinal cord. These results suggest that the persistence of visceral pain induced by colitis results from maladaptive plasticity of the enteric, peripheral and central nervous systems.

The pharmacological activation of A3 receptors has shown potential usefulness in the management of bowel inflammation. However, the role of these receptors in the control of visceral hypersensitivity in the presence of intestinal inflammation has not been investigated. The effects of AR170, a potent and selective A3 receptor agonist, and dexamethasone (DEX) were tested in rats with 2,4-dinitrobenzene sulfonic acid (DNBS)-induced colitis to assess their tissue inflammatory parameters. The animals received AR170, DEX, or a vehicle intraperitoneally for 6 days, starting 1 day before the induction of colitis. Visceral pain was assessed by recording the abdominal responses to colorectal distension in animals with colitis. Colitis was associated with a decrease in body weight and an increase in spleen weight. The macroscopic damage score and tissue tumor necrosis factor (TNF), interleukin 1β (IL-1β), and myeloperoxidase (MPO) levels were also enhanced. AR170, but not DEX, improved body weight. Both drugs counteracted the increase in spleen weight, ameliorated macroscopic colonic damage, and decreased TNF, IL-1β, and MPO tissue levels. The enhanced visceromotor response (VMR) in rats with colitis was decreased via AR170 administration. In rats with colitis, AR170 counteracted colonic inflammatory cell infiltration and decreased pro-inflammatory cytokine levels, thereby relieving visceral hypersensitivity.

The role played by adenosine A2B receptors (A2BRs) in the regulation of enteric glial cell (EGC) functions remains unclear. This study was aimed at investigating the involvement of A2BRs in the control of EGC functions in a model of obesity. C57BL/6 mice were fed with standard diet (SD) or high fat diet (HFD) for eight weeks. Colonic tachykininergic contractions were recorded in the presence of BAY60-6583 (A2BRs agonist), MRS1754 (A2BRs antagonist), and the gliotoxin fluorocitrate. Immunofluorescence distribution of HuC/D, S100β, and A2BRs was assessed in whole mount preparations of colonic myenteric plexus. To mimic HFD, EGCs were incubated in vitro with palmitate (PA) and lipopolysaccharide (LPS), in the absence or in the presence of A2BR ligands. Toll-like receptor 4 (TLR4) expression was assessed by Western blot analysis. Interleukin-1β (IL-1β), substance P (SP), and glial cell derived neurotrophic factor (GDNF) release were determined by enzyme-linked immunosorbent assay (ELISA) assays. MRS1754 enhanced electrically evoked tachykininergic contractions of colonic preparations from HFD mice. BAY60-6583 decreased the evoked tachykininergic contractions, with higher efficacy in HFD mice. Such effects were blunted upon incubation with fluorocitrate. In in vitro experiments on EGCs, PA and LPS increased TLR4 expression as well as IL-1β, GDNF, and SP release. Incubation with BAY60-6583 reduced TLR4 expression as well as IL-1β, GDNF, and SP release. Such effects were blunted by MRS1754. The present results suggest that A2BRs, expressed on EGCs, participate in the modulation of enteric inflammation and altered tachykininergic responses associated with obesity, thus representing a potential therapeutic target.

Nucleotide-binding oligomerization domain leucine rich repeat and pyrin domain-containing protein 3 (NLRP3) inflammasome is pivotal in maintaining intestinal homeostasis and sustaining enteric immune responses in the setting of inflammatory bowel diseases. Drugs acting as NLRP3 blockers could represent innovative strategies for treatment of bowel inflammation. This study was performed in rats with dinitrobenzenesulfonic acid (DNBS)-induced colitis, to investigate how the direct blockade of NLRP3 inflammasome with an irreversible inhibitor (INF39) compares with Ac-YVAD-cmk (YVAD, caspase-1 inhibitor) and anakinra (IL-1β receptor antagonist), acting downstream on NLRP3 signaling. Animals with DNBS-colitis received YVAD (3 mg/kg) or anakinra (100 mg/Kg) intraperitoneally, and INF39 (25 mg/kg) or dexamethasone (DEX, 1 mg/kg) orally for 6 days, starting on the same day of colitis induction. Under colitis, there was a body weight decrease, which was attenuated by YVAD, anakinra or INF39, but not DEX. All test drugs counteracted the increase in spleen weight. The colonic shortening and morphological colonic alterations associated with colitis were counteracted by INF39, anakinra and DEX, while YVAD was without effects. Tissue increments of myeloperoxidase, tumor necrosis factor and interleukin-1β were more effectively counteracted by INF39 and DEX, than YVAD and anakinra. These findings indicate that: (1) direct inhibition of NLRP3 inflammasome with INF39 is more effective than caspase-1 inhibition or IL-1β receptor blockade in reducing systemic and bowel inflammatory alterations; (2) direct NLRP3 inhibition can be a suitable strategy for treatment of bowel inflammation.

Non-steroidal anti-inflammatory drugs (NSAIDs) can damage the small intestine, mainly through an involvement of enteric bacteria. This study examined the pathophysiology of NSAID-associated intestinal lesions in a rat model of diclofenac-enteropathy and evaluated the effect of rifaximin on small bowel damage. Enteropathy was induced in 40-week old male rats by intragastric diclofenac (4 mg/kg BID, 14 days). Rifaximin (delayed release formulation) was administered (50 mg/kg BID) 1 h before the NSAID. At the end of treatments, parameters dealing with ileal damage, inflammation, barrier integrity, microbiota composition, and TLR-NF-κB-inflammasome pathway were evaluated. In addition, the modulating effect of rifaximin on NLRP3 inflammasome was tested in an in vitro cell system. Diclofenac induced intestinal damage and inflammation, triggering an increase in tissue concentrations of tumor necrosis factor and interleukin-1β, higher expression of TLR-2 and TLR-4, MyD88, NF-κB and activation of caspase-1. In addition, the NSAID decreased ileal occludin expression and provoked a shift of bacterial phyla toward an increase in Proteobacteria and Bacteroidetes abundance. All these changes were counterbalanced by rifaximin co-administration. This drug was also capable of increasing the proportion of Lactobacilli, a genus depleted by the NSAID. In LPS-primed THP-1 cells stimulated by nigericin (a model to study the NLRP3 inflammasome), rifaximin reduced IL-1β production in a concentration-dependent fashion, this effect being associated with inhibition of the up-stream caspase-1 activation. In conclusion, diclofenac induced ileal mucosal lesions, driving inflammatory pathways and microbiota changes. In conclusion, rifaximin prevents diclofenac-induced enteropathy through both anti-bacterial and anti-inflammatory activities.

Purpose: Luteolin exerts beneficial effects against obesity-associated comorbidities, although its influence on vascular dysfunction remains undetermined. We examined the effects of luteolin on endothelial dysfunction in a mouse model of diet-induced obesity.Methods: Standard diet (SD) or high-fat diet (HFD)-fed mice were treated daily with luteolin intragastrically. After 8 weeks, body and epididymal fat weight, as well as blood cholesterol, glucose, and triglycerides were evaluated. Endothelium-dependent relaxations of resistance mesenteric vessels was assessed by a concentration-response curve to acetylcholine, repeated upon Nw-nitro-L-arginine methylester (L-NAME) or ascorbic acid infusion to investigate the influence of nitric oxide (NO) availability and reactive oxygen species (ROS) on endothelial function, respectively. Intravascular ROS production and TNF levels were measured by dihydroethidium dye and ELISA, respectively. Endothelial NO synthase (eNOS) and superoxide dismutase 1 (SOD1), as well as microRNA-214-3p expression were examined by Western blot and RT-PCR assays, respectively.Results: HFD animals displayed elevated body weight, epididymal fat weight and metabolic indexes. Endothelium-dependent relaxation was resistant to L-NAME and enhanced by ascorbic acid, which restored also the inhibitory effect of L-NAME, suggesting a ROS-dependent reduction of NO availability in HFD vessels. Moreover, media-lumen ratio, intravascular superoxide anion and TNF levels were increased, while vascular eNOS, SOD1, and microRNA-214-3p expression were decreased. In HFD mice, luteolin counteracted the increase in body and epididymal fat weight, and metabolic alterations. Luteolin restored vascular endothelial NO availability, normalized the media-lumen ratio, decreased ROS and TNF levels, and normalized eNOS, SOD1 and microRNA-214-3p expression.Conclusion: Luteolin prevents systemic metabolic alterations and vascular dysfunction associated with obesity, likely through antioxidant and anti-inflammatory mechanisms.

Inflammation-related intestinal diseases are a set of various conditions presenting an overactive enteric immune system. A continuous overproduction of pro-inflammatory cytokines and a decreased production of anti-inflammatory modulators are generally observed, while morpho-functional alterations of the enteric nervous system lead to intestinal secretory and motor dysfunctions. The factors at the basis of these conditions are still to be totally identified and current therapeutic strategies are aimed only at achieving and maintaining remission states, by using therapeutic tools like aminosalicylates, corticosteroids, immunomodulators, biological drugs (i.e., monoclonal antibodies), and eventually surgery. Recent reports described a key role of purinergic mediators (i.e., adenosine and its nucleotides ATP and ADP) in the regulation of the activity of immune cells and enteric nervous system, showing also that alterations of the purinergic signaling are linked to pathological conditions of the intestinal tract. These data prompted to a series of investigations to test the therapeutic potential for inflammation-related intestinal conditions of compounds able to restore or modulate an altered purinergic signaling within the gut. This review provides an overview on these investigations, describing the results of preclinical and/or clinical evaluation of compounds able to stimulate or inhibit specific P2 (i.e., P2X7) or P1 (i.e., A2A or A3) receptor signaling and to modify the adenosine levels through the modulation of enzymes activity (i.e., Adenosine Deaminase) or nucleoside transporters. Recent developments in the field are also reported and the most promising purine-based therapeutic strategies for the treatment of inflammation-related gastrointestinal disorders are schematically summarized.

Apigenin can exert beneficial actions in the prevention of obesity. However, its putative action on obesity-associated bowel motor dysfunctions is unknown. This study examined the effects of apigenin on colonic inflammatory and motor abnormalities in a mouse model of diet-induced obesity.Male C57BL/6J mice were fed with standard diet (SD) or high-fat diet (HFD). SD or HFD mice were treated with apigenin (10 mg/Kg/day). After 8 weeks, body and epididymal fat weight, as well as cholesterol, triglycerides and glucose levels were evaluated. Malondialdehyde (MDA), IL-1β and IL-6 levels, and let-7f expression were also examined. Colonic infiltration by eosinophils, as well as substance P (SP) and inducible nitric oxide synthase (iNOS) expressions were evaluated. Motor responses elicited under blockade of NOS and tachykininergic contractions were recorded in vitro from colonic longitudinal muscle preparations.When compared to SD mice, HFD animals displayed increased body weight, epididymal fat weight and metabolic indexes. HFD mice showed increments in colonic MDA, IL-1β and IL-6 levels, as well as a decrease in let-7f expression in both colonic and epididymal tissues. HFD mice displayed an increase in colonic eosinophil infiltration. Immunohistochemistry revealed an increase in SP and iNOS expression in myenteric ganglia of HFD mice. In preparations from HFD mice, electrically evoked contractions upon NOS blockade or mediated by tachykininergic stimulation were enhanced. In HFD mice, Apigenin counteracted the increase in body and epididymal fat weight, as well as the alterations of metabolic indexes. Apigenin reduced also MDA, IL-1β and IL-6 colonic levels as well as eosinophil infiltration, SP and iNOS expression, along with a normalization of electrically evoked tachykininergic and nitrergic contractions. In addition, apigenin normalized let-7f expression in epididymal fat tissues, but not in colonic specimens.Apigenin prevents systemic metabolic alterations, counteracts enteric inflammation and normalizes colonic dysmotility associated with obesity.

Collagen detection in histological sections and its quantitative estimation by computer-aided image analysis represent important procedures to assess tissue localization and distribution of connective fibers. Different histochemical approaches have been proposed to detect and quantify collagen deposition in paraffin slices with different degrees of satisfaction. The present study was performed to compare the qualitative and quantitative efficiency of three histochemical methods available for collagen staining in paraffin sections of colon. van Gieson, Sirius Red and Sirius Red/Fast Green stainings were carried out for collagen detection and quantitative estimation by morphometric image analysis in colonic specimens from normal rats or animals with 2,4-dinitrobenzenesulfonic acid (DNBS) induced colitis. Haematoxylin/eosin staining was carried out to assess tissue morphology and histopathological lesions. Among the three investigated methods, Sirius Red/Fast Green staining allowed to best highlight well-defined red-stained collagen fibers and to obtain the highest quantitative results by morphometric image analysis in both normal and inflamed colon. Collagen fibers, which stood out against the green-stained non-collagen components, could be clearly appreciated, even in their thinner networks, within all layers of normal or inflamed colonic wall. The present study provides evidence that, as compared with Sirius Red alone or van Gieson staining, the Sirius Red/Fast Green method is the most sensitive, in terms of both qualitative and quantitative evaluation of collagen fibers, in paraffin sections of both normal and inflamed colon.

Recent evidence indicates an involvement of P2X7 purinergic receptor (P2X7R) in the fine tuning of immune functions, as well as in driving enteric neuron apoptosis under intestinal inflammation. However, the participation of this receptor in the regulation of enteric neuromuscular functions remains undetermined. This study was aimed at investigating the role of P2X7Rs in the control of colonic motility in experimental colitis.Colitis was induced in rats by 2,4-dinitrobenzenesulfonic acid. P2X7R distribution was examined by immunofluorescence analysis. The effects of A804598 (selective P2X7R antagonist) and BzATP (P2X7R agonist) were tested on contractions of longitudinal smooth muscle evoked by electrical stimulation or by carbachol in the presence of tetrodotoxin.P2X7Rs were predominantly located in myenteric neurons, but, in the presence of colitis, their expression increased in the neuromuscular layer. In normal preparations, A804598 elicited a negligible increase in electrically induced contractions, while a significant enhancement was recorded in inflamed tissues. In the presence of Nω-propyl-L-arginine (NPA, neuronal nitric oxide synthase inhibitor) the A804598 effects were lost. P2X7R stimulation with BzATP did not significantly affect electrical-induced contractions in normal colon, while a marked reduction was recorded under inflammation. The inhibitory effect of BzATP was antagonized by A804598, and it was also markedly blunted by NPA. Both P2X7R ligands did not affect carbachol-induced contractions.The purinergic system contributes to functional neuromuscular changes associated with bowel inflammation via P2X7Rs, which modulate the activity of excitatory cholinergic nerves through a facilitatory control on inhibitory nitrergic pathways.

5HTTLPR polymorphism of serotonin transporter yields short (S) and long (L) alleles. SS and LS genotypes are associated with reduced expression of serotonin transporter. This cross-sectional study investigated the association of 5HTTLPR with symptom severity of irritable bowel syndrome (IBS). Patients with IBS (Rome III) and healthy controls were included. Genomic DNA was extracted from saliva, and 5HTTLPR alleles were assessed by polymerase chain reaction. IBS symptom severity was evaluated by means of IBS-SSS questionnaire. Two hundreds and four IBS patients (159 females; mean age: 39.6±12.3 years; 106 with constipation: C-IBS; 98 with diarrhea: D-IBS) and 200 healthy controls (154 females; mean age: 40.4±15.8 years) were enrolled. The overall IBS-SSS value was higher in LS/SS than LL patients (319.0±71.5 versus 283.8±62.3; P = 0.0006). LS/SS patients had also higher values of abdominal pain (59.7±21.0 versus 51.0±18.8; P = 0.020) and bowel dissatisfaction (80.1±23.9 versus 70.5±22.8; P = 0.035). The overall IBS-SSS values in C-IBS and D-IBS patients were 317.2±68.3 and 296.1±71.4, respectively (P = 0.192), with significantly higher values for abdominal distension (65.0±24.4 versus 51.4±24.8; P = 0.0006), but not for bowel dissatisfaction (80.5±21.7 versus 72.9±25.7; P = 0.138). Frequencies of 5HTTLPR genotypes did not differ significantly when comparing IBS patients (overall or upon stratification in C-IBS and D-IBS) with healthy controls. In conclusion, the LS and SS genotypes are significantly correlated with IBS symptom severity, although their possible direct causal role remains to be proven. In addition, the present findings do not support an association of 5HTTLPR with IBS or its clinical presentation in terms of bowel habit predominance.

The pathogenesis of diverticular disease (DD) is thought to result from complex interactions among dietary habits, genetic factors and coexistence of other bowel abnormalities. These conditions lead to alterations in colonic pressure and motility, facilitating the formation of diverticula. Although electrophysiological studies on smooth muscle cells (SMCs) have investigated colonic motor dysfunctions, scarce attention has been paid to their molecular abnormalities, and data on SMCs in DD are lacking. Accordingly, the main purpose of this study was to evaluate the expression patterns of molecular factors involved in the contractile functions of SMCs in the tunica muscularis of colonic specimens from patients with DD.By means of immunohistochemistry and image analysis, we examined the expression of Cx26 and Cx43, which are prominent components of gap junctions in human colonic SMCs, as well as pS368-Cx43, PKCps, RhoA and αSMA, all known to regulate the functions of gap junctions and the contractile activity of SMCs. The immunohistochemical analysis revealed significant abnormalities in DD samples, concerning both the expression and distribution patterns of most of the investigated molecular factors.This study demonstrates, for the first time, that an altered pattern of factors involved in SMC contractility is present at level of the tunica muscularis of DD patients. Moreover, considering that our analysis was conducted on colonic tissues not directly affected by diverticular lesions or inflammatory reactions, it is conceivable that these molecular alterations may precede and predispose to the formation of diverticula, rather than being mere consequences of the disease.