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Background The novel coronavirus (COVID-19) has presented a significant and urgent threat to global health and there has been a need to identify prognostic factors in COVID-19 patients. The aim of this study was to determine whether chest CT characteristics had any prognostic value in patients with COVID-19. Methods A retrospective analysis of COVID-19 patients who underwent a chest CT-scan was performed in four medical centers. The prognostic value of chest CT results was assessed using a multivariable survival analysis with the Cox model. The characteristics included in the model were the degree of lung involvement, ground glass opacities, nodular consolidations, linear consolidations, a peripheral topography, a predominantly inferior lung involvement, pleural effusion, and crazy paving. The model was also adjusted on age, sex, and the center in which the patient was hospitalized. The primary endpoint was 30-day in-hospital mortality. A second model used a composite endpoint of admission to an intensive care unit or 30-day in-hospital mortality. Results A total of 515 patients with available follow-up information were included. Advanced age, a degree of pulmonary involvement ≥ 50% (Hazard Ratio 2.25 [95% Cl: 1.378 to 3.671], p= 0.001), nodular consolidations and pleural effusions were associated with lower 30-day in-hospital survival rates. An exploratory subgroup analysis showed a 60.6% mortality rate in patients over 75 with ≥ 50% lung involvement on a CT-scan. Conclusions Chest CT findings such as the percentage of pulmonary involvement ≥ 50%, pleural effusion and nodular consolidation were strongly associated with 30-day mortality in COVID-19 patients. CT examinations are essential for the assessment of severe COVID-19 patients and their results must be considered when making care management decisions.

Background and aims: CD4+ T cells constitute central players in inflammatory bowel diseases (IBDs), driving inflammation in the gut mucosa. Current evidence indicates that CCR9 and the integrin α4β7 are necessary and sufficient to imprint colonic homing on CD4+ T cells upon inflammation. Interestingly, dopaminergic signaling has been previously involved in leukocyte homing. Despite dopamine levels are strongly reduced in the inflamed gut mucosa, the role of dopamine in the gut homing of T cells remains unknown. Here, we study how dopaminergic signaling affects T cells upon gut inflammation. Methods: Gut inflammation was induced by transfer of naïve T cells into Rag1-/- mice or by administration of dextran sodium sulfate. T cell migration and differentiation were evaluated by adoptive transfer of congenic lymphocytes followed by flow cytometry analysis. Protein interaction was studied by bioluminescence resonance energy transfer analysis, bimolecular fluorescence complementation, and in situ proximity ligation assays. Results: We show the surface receptor providing colonic tropism to effector CD4+ T cells upon inflammation is not CCR9 but the complex formed by CCR9 and the dopamine receptor D5 (DRD5). Assembly of the heteromeric complex was demonstrated in vitro and in vivo using samples from mouse and human origin. The CCR9:DRD5 heteroreceptor was upregulated in the intestinal mucosa of IBD patients. Signaling assays confirmed that complexes behave differently than individual receptors. Remarkably, the disruption of CCR9:DRD5 assembly attenuated the recruitment of CD4+ T cells into the colonic mucosa. Conclusions: Our findings describe a key homing receptor involved in gut inflammation and introduce a new cell surface module in immune cells: macromolecular complexes formed by G protein-coupled receptors integrating the sensing of multiple molecular cues. Keywords: Chemokine Receptors; Dopaminergic Regulation; G Protein-Coupled Receptors Heteromers; Gut Tropism; Inflammatory Bowel Diseases; Inflammatory Colitis; T Cell Migration.

Highlights • CT severity score (CSS) could predict ICU admission, intubation, and mortality. • Reticular pattern in lung CT scans, could be protective against adverse outcomes. • CSS was weakly correlated with initial qSOFA score. • CSS could not predict the length of stay in hospital., Background Chest computed tomography (CT) scan is frequently used in the diagnosis of COVID-19 pneumonia. Objectives This study investigates the predictive value of CT severity score (CSS) for length-of-stay (LOS) in hospital, initial disease severity, ICU admission, intubation, and mortality. Methods In this retrospective study, initial CT scans of consecutively admitted patients with COVID-19 pneumonia were reviewed in a tertiary hospital. The association of CSS with the severity of disease upon admission and the final adverse outcomes was assessed using Pearson's correlation test and logistic regression, respectively. Results Total of 121 patients (60±16 years), including 54 women and 67 men, with positive RT-PCR tests were enrolled. We found a significant but weak correlation between CSS and qSOFA, as a measure of disease severity (r: 0.261, p = 0.003). No significant association was demonstrated between CSS and LOS. Patients with CSS>8 had at least three-fold higher risk of ICU admission, intubation, and mortality. Conclusions CSS in baseline CT scan of patients with COVID-19 pneumonia can predict adverse outcomes and is weakly correlated with initial disease severity.

Background Although an RT-PCR test is the “gold standard” tool for diagnosing an infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), chest imaging can be used to support a diagnosis of coronavirus disease 2019 (COVID-19) – albeit with fairly low specificity. However, if the chest imaging findings do not faithfully reflect the patient's clinical course, one can question the rationale for relying on these imaging data in the diagnosis of COVID-19. Aims To compare clinical courses with changes over time in chest imaging findings among patients admitted to an ICU for severe COVID-19 pneumonia. Methods We retrospectively reviewed the medical charts of all adult patients admitted to our intensive care unit (ICU) between March 1, 2020, and April 15, 2020, for a severe COVID-19 lung infection and who had a positive RT-PCR test. Changes in clinical, laboratory and radiological variables were compared, and patients with discordant changes over time (e.g. a clinical improvement with stable or worse radiological findings) were analyzed further. Results Of the 46 included patients, 5 showed an improvement in their clinical status but not in their chest imaging findings. On admission to the ICU, three of the five were mechanically ventilated and the two others received high-flow oxygen therapy or a non-rebreather mask. Even though the five patients’ radiological findings worsened or remained stable, the mean ± standard deviation partial pressure of arterial oxygen to the fraction of inspired oxygen (PaO2:FiO2) ratio increased significantly in all cases (from 113.2 ± 59.7 mmHg at admission to 259.8 ± 59.7 mmHg at a follow-up evaluation; p=0.043). Interpretation Our results suggest that in cases of clinical improvement with worsened or stable chest imaging variables, the PaO2:FiO2 ratio might be a good marker of the resolution of COVID-19-specific pulmonary vascular insult.

Almost 80% of people confronting COVID-19 recover from COVID-19 disease without any particular treatments. They experience heterogeneous symptoms; a wide range of respiratory symptoms, cough, dyspnea, fever, and viral pneumonia. However, some others need urgent intervention and special treatment to get rid of this widespread disease. So far, there isn't any unique drug for the potential treatment of COVID 19. However, some available therapeutic drugs used for other diseases seem beneficial for the COVID-19 treatment. On the other hand, there is a robust global concern for developing an efficient COVID-19 vaccine to control the COVID-19 pandemic sustainably. According to the WHO report, since 8 October 2021, 320 vaccines have been in progress. 194 vaccines are in the pre-clinical development stage that 126 of them are in clinical progression. Here, in this paper, we have comprehensively reviewed the most recent and updated information about coronavirus and its mutations, all the potential therapeutic approaches for treating COVID-19, developed diagnostic systems for COVID- 19 and the available COVID-19 vaccines and their mechanism of action., Graphical Abstract ga1

Purpose As of August 30th, there were in total 25.1 million confirmed cases and 845 thousand deaths caused by coronavirus disease of 2019 (COVID-19) worldwide. With overwhelming demands on medical resources, patient stratification based on their risks is essential. In this multi-center study, we built prognosis models to predict severity outcomes, combining patients’ electronic health records (EHR), which included vital signs and laboratory data, with deep learning- and CT-based severity prediction. Method We first developed a CT segmentation network using datasets from multiple institutions worldwide. Two biomarkers were extracted from the CT images: total opacity ratio (TOR) and consolidation ratio (CR). After obtaining TOR and CR, further prognosis analysis was conducted on datasets from INSTITUTE-1, INSTITUTE-2 and INSTITUTE-3. For each data cohort, generalized linear model (GLM) was applied for prognosis prediction. Results For the deep learning model, the correlation coefficient of the network prediction and manual segmentation was 0.755, 0.919, and 0.824 for the three cohorts, respectively. The AUC (95 % CI) of the final prognosis models was 0.85(0.77,0.92), 0.93(0.87,0.98), and 0.86(0.75,0.94) for INSTITUTE-1, INSTITUTE-2 and INSTITUTE-3 cohorts, respectively. Either TOR or CR exist in all three final prognosis models. Age, white blood cell (WBC), and platelet (PLT) were chosen predictors in two cohorts. Oxygen saturation (SpO2) was a chosen predictor in one cohort. Conclusion The developed deep learning method can segment lung infection regions. Prognosis results indicated that age, SpO2, CT biomarkers, PLT, and WBC were the most important prognostic predictors of COVID-19 in our prognosis model.

Background & Aims Infection with hepatitis C virus (HCV) remains a major cause of morbidity and mortality worldwide despite the recent advent of highly effective direct-acting antivirals. The envelope glycoproteins of HCV are heavily glycosylated with a high proportion of high-mannose glycans (HMGs), which serve as a shield against neutralizing antibodies and assist in the interaction with cell-entry receptors. However, there is no approved therapeutic targeting this potentially druggable biomarker. Methods The anti-HCV activity of Avaren-Fc (AvFc) was evaluated through the use of in vitro neutralization assays as well as an in vivo challenge in the PXB chimeric human liver mouse model. Drug toxicity was assessed by histopathology, serum alanine aminotransferase, and mouse body weights. Results AvFc was capable of neutralizing cell culture–derived HCV in a genotype-independent manner, with 50% inhibitory concentration values in the low nanomolar range. Systemic administration of AvFc in a histidine-based buffer was well tolerated; after 11 doses every other day at 25 mg/kg there were no significant changes in body or liver weights or in blood human albumin or serum alanine aminotransferase activity. Gross necropsy and liver pathology confirmed the lack of toxicity. This regimen successfully prevented genotype 1a HCV infection in all animals, although an AvFc mutant lacking HMG binding activity failed. Conclusions These results suggest that targeting envelope HMGs is a promising therapeutic approach against HCV infection, and AvFc may provide a safe and efficacious means to prevent recurrent infection upon liver transplantation in HCV-related end-stage liver disease patients., Graphical abstract

Background & Aims Obesity promotes the development of nonalcoholic fatty liver diseases (NAFLDs), yet not all obese patients develop NAFLD. The underlying causes for this discrepancy remain elusive. LPGAT1 is an acyltransferase that catalyzes the remodeling of phosphatidylglycerol (PG), a mitochondrial phospholipid implicated in various metabolic diseases. Here, we investigated the role of LPGAT1 in regulating the onset of diet-induced obesity and its related hepatosteatosis because polymorphisms of the LPGAT1 gene promoter were strongly associated with susceptibility to obesity in Pima Indians. Methods Mice with whole-body knockout of LPGAT1 were generated to investigate the role of PG remodeling in NAFLD. Results LPGAT1 deficiency protected mice from diet-induced obesity, but led to hepatopathy, insulin resistance, and NAFLD as a consequence of oxidative stress, mitochondrial DNA depletion, and mitochondrial dysfunction. Conclusions This study identified an unexpected role of PG remodeling in obesity, linking mitochondrial dysfunction to NAFLD., Graphical abstract

Background & Aims GPBAR1, also known as TGR5, is a G protein–coupled receptor activated by bile acids. Hepatic innate immune cells are involved in the immunopathogenesis of human liver diseases and in several murine hepatitis models. Here, by using genetic and pharmacological approaches, we provide evidence that GPBAR1 ligation attenuates the inflammation in rodent models of hepatitis. Material and methods Hepatitis was induced by concanavalin A (Con A) or α-galactosyl-ceramide (α-GalCer). 6b-Ethyl-3a,7b-dihydroxy-5b-cholan-24-ol (BAR501), a selective agonist of GPBAR1, was administrated by o.s. Results In the mouse models of hepatitis, the genetic ablation of Gpabar1 worsened the severity of liver injury and resulted in a type I NKT cells phenotype that was biased toward a NKT1, a proinflammatory, IFN-γ producing, NKT cells subtype. Further on, NKT cells from GPBAR1–/– mice were sufficient to cause a severe hepatitis when transferred to naïve mice. In contrast, GPBAR1 agonism rescued wild-type mice from acute liver damage and redirects the NKT cells polarization toward a NKT10, a regulatory, IL-10 secreting, type I NKT cell subset. In addition, GPBAR1 agonism significantly expanded the subset of IL-10 secreting type II NKT cells. RNAseq analysis of both NKT cells type confirmed that IL-10 is a major target for GPABR1. Accordingly, IL-10 gene ablation abrogated protection afforded by GPBAR1 agonism in the Con A model. Conclusion Present results illustrate a role for GPBAR1 in regulating liver NKT ecology. Because NKT cells are an essential component of liver immune system, our data provide a compelling evidence for a GPBAR1-IL-10 axis in regulating of liver immunity., Graphical abstract

BackgroundNumerous countries imposed strict travel restrictions, contributing to the large socioeconomic burden during the COVID-19 pandemic. The long quarantines that apply to contacts of cases may be excessive for travel policy.MethodsWe developed an approach to evaluate imminent countrywide COVID-19 infections after 0–14-day quarantine and testing. We identified the minimum travel quarantine duration such that the infection rate within the destination country did not increase compared to a travel ban, defining this minimum quarantine as “sufficient.”FindingsWe present a generalised analytical framework and a specific case study of the epidemic situation on November 21, 2021, for application to 26 European countries. For most origin-destination country pairs, a three-day or shorter quarantine with RT-PCR or antigen testing on exit suffices. Adaptation to the European Union traffic-light risk stratification provided a simplified policy tool. Our analytical approach provides guidance for travel policy during all phases of pandemic diseases.InterpretationFor nearly half of origin-destination country pairs analysed, travel can be permitted in the absence of quarantine and testing. For the majority of pairs requiring controls, a short quarantine with testing could be as effective as a complete travel ban. The estimated travel quarantine durations are substantially shorter than those specified for traced contacts.FundingEasyJet (JPT and APG), the Elihu endowment (JPT), the Burnett and Stender families’ endowment (APG), the Notsew Orm Sands Foundation (JPT and APG), the National Institutes of Health (MCF), Canadian Institutes of Health Research (SMM) and Natural Sciences and Engineering Research Council of Canada EIDM-MfPH (SMM).Research in contextEvidence before this studyEvidence from early in the pandemic indicates that border closures at the epicentre slowed global dissemination of COVID-19. As community transmission became established in many nations, studies have suggested that the benefit of strict border closures in mitigating the transmission of disease from travellers diminished. Research for community settings has shown that testing later during quarantine, rather than upon entry into quarantine, can substantially shorten the duration of quarantine needed to reduce post-quarantine transmission. In particular for international air travellers, a 14-day quarantine can effectively be shortened to five or seven days. The number of infectious COVID-19 cases that escape from these quarantines depends on the prevalence of disease in the country the traveller originated as well as the travel volume into the country.Added value of this studyWe developed a framework to identify quarantine and testing strategies that enable travel from specific origins without increasing their infection rates per capita within destinations. No prior study has evaluated the appropriate duration of quarantine necessary to prevent any rise in infection rates per capita in the destination countries as a result of travel. By accounting for prevalence, daily incidence, vaccine coverage, immunity, age demographics, and travel flow between countries, we quantified the contribution of travel towards within-country the imminent infections in the destination country under different quarantine and testing strategies. For travel between 26 European countries, our results for the pandemic situation observed on November 21, 2021 demonstrate that there are often less burdensome quarantine and testing strategies that can serve as effective alternatives to strict border closure. Specifically, these estimated sufficient quarantine durations are especially dependent on COVID-19 prevalence and immunity within the two countries. We also found that asymmetry in the travel flow, just not the volume of travel flow, can also influence the estimated sufficient quarantine durations. Using data on variants of concern, including Omicron, we found that the adequacy of a border control strategy to limit variant spread depends strongly on the geographical distribution of the variant. While our results pertain to European countries, we also provide an interactive spreadsheet that can be used to determine appropriate quarantine durations between any two countries. Moreover, our framework can also be applied at any spatial or population scale within which movement restrictions could feasibly be implemented.Implications of all available evidenceTravel quarantine and testing strategies can effectively mitigate importation and onward transmission within a country. Identifying sufficient strategies can allow countries to permit travel to and from other countries, without risking a short-term increase in infection rates. As long as the community transmission is occurring, the long-term epidemic trend within the destination country is more apt to be determined by other disease control measures, e.g., contact tracing, vaccination, and non-pharmaceutical interventions. Together, travel quarantine and other related control measures can mitigate the risk of transmission between countries, limiting the threat of variants of concern.

Background & Aims The transient receptor potential ankyrin 1 (TRPA1) channel is highly expressed in the intestinal lamina propria, but its contribution to gut physiology/pathophysiology is unclear. Here, we evaluated the function of myofibroblast TRPA1 channels in intestinal remodeling. Methods An intestinal myofibroblast cell line (InMyoFibs) was stimulated by transforming growth factor-β1 to induce in vitro fibrosis. Trpa1 knockout mice were generated using the Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system. A murine chronic colitis model was established by weekly intrarectal trinitrobenzene sulfonic acid (TNBS) administration. Samples from the intestines of Crohn’s disease (CD) patients were used for pathologic staining and quantitative analyses. Results In InMyoFibs, TRPA1 showed the highest expression among TRP family members. In TNBS chronic colitis model mice, the extents of inflammation and fibrotic changes were more prominent in TRPA1-/- knockout than in wild-type mice. One-week enema administration of prednisolone suppressed fibrotic lesions in wild-type mice, but not in TRPA1 knockout mice. Steroids and pirfenidone induced Ca2+ influx in InMyoFibs, which was antagonized by the selective TRPA1 channel blocker HC-030031. Steroids and pirfenidone counteracted transforming growth factor-β1–induced expression of heat shock protein 47, type 1 collagen, and α-smooth muscle actin, and reduced Smad-2 phosphorylation and myocardin expression in InMyoFibs. In stenotic intestinal regions of CD patients, TRPA1 expression was increased significantly. TRPA1/heat shock protein 47 double-positive cells accumulated in the stenotic intestinal regions of both CD patients and TNBS-treated mice. Conclusions TRPA1, in addition to its anti-inflammatory actions, may protect against intestinal fibrosis, thus being a novel therapeutic target for highly incurable inflammatory/fibrotic disorders., Graphical abstract

Background & Aims Epithelial cells are joined by tight junctions (TJs) to form a cell sheet. In the stomach, epithelial cell sheet forms an essential barrier against gastric material, including gastric acid. Although the decreased expression of stomach-type claudin-18 (stCldn18), a TJ protein, is generally observed in human gastritis and gastric cancer, its pathological roles are not fully understood. We previously reported that mice lacking stCldn18 (stCldn18-/-) exhibit gastric acid leakage through TJs, which induces active gastritis at a young age. Here, we examined the gastric pathologies in mice after long-term stCldn18 deficiency. Methods The gastric pathologies in stCldn18-/- mice were sequentially analyzed from youth to old age, and compared to those in humans. To examine the relationship between stCldn18 deficiency-induced gastric pathologies and Wnt-dependent tumorigenesis, we generated Wnt1-overexpressing stCldn18-/- mice. Results StCldn18-/- mice developed chronic active gastritis at middle age, with expression of the chemoattractant CCL28. At old age, 20-30% of these mice developed gastric tumors with CXCL5 expression, indicative of EMT. In this process, spasmolytic polypeptide-expressing metaplasia (SPEM) cells appeared. Increased expressions of CD44-variants, TLR2, and CXCL5 indicated age-dependent changes in cell characteristics. Some features of the stCldn18-/- mouse gastric tumorigenesis resembled H pylori-infection-related human carcinogenesis. The gastric tumorigenesis was accelerated in Wnt1-overexpressing stCldn18-/- mice, indicating that Wnt is involved in the stCldn18-/- mouse gastric tumorigenesis. Conclusions StCldn18 deficiency induced gastric tumorigenesis in mice without H pylori infection. Our findings revealed that several signaling networks, including the cytokine-, stemness-, and Wnt-signaling pathways, may be activated under the stCldn18-deficiency-induced chronic active gastritis to accelerate the gastric tumorigenesis., Graphical abstract

Background & Aims: Innate immune dysfunction can promote chronic inflammatory diseases of the liver. For example, mice lacking the flagellin receptor Toll-like receptor 5 (TLR5) show microbial dysbiosis and predisposition to high-fat diet (HFD)-induced hepatic steatosis. The extent to which hepatocytes play a direct role in detecting bacterial products in general, or flagellin in particular, is poorly understood. In the present study, we investigated the role of hepatocyte TLR5 in recognizing flagellin, policing bacteria, and protecting against liver disease. Methods: Mice were engineered to lack TLR5 specifically in hepatocytes (TLR5ÎHep) and analyzed relative to sibling controls (TLR5fl/fl). TLR5 messenger RNA levels, responses to exogenous flagellin, elimination of circulating motile bacteria, and susceptibility of liver injury (concanavalin A, carbon tetrachloride, methionine- and choline-deficient diet, and HFD) were measured. Results: TLR5ÎHep expressed similar levels of TLR5 as TLR5fl/fl in all organs examined, except in the liver, which showed a 90% reduction in TLR5 levels, indicating that hepatocytes accounted for the major portion of TLR5 expression in this organ. TLR5ÎHep showed impairment in responding to purified flagellin and clearing flagellated bacteria from the liver. Although TLR5ÎHep mice did not differ markedly from sibling controls in concanavalin A or carbon tetrachlorideâinduced liver injury models, they showed exacerbated disease in response to a methionine- and choline-deficient diet and HFD. Such predisposition of TLR5ÎHep to diet-induced liver pathology was associated with increased expression of proinflammatory cytokines, which was dependent on the Nod-like-receptor C4 inflammasome and rescued by microbiota ablation. Conclusions: Hepatocyte TLR5 plays a critical role in protecting liver against circulating gut bacteria and against diet-induced liver disease. Keywords: Innate Immunity, TLR5, Hepatocytes, Inflammation, Steatosis

Background & Aims: Microbial molecular products incite intestinal inflammation by activating Toll-like receptors (TLRs) and inflammasomes of the innate immune system. This systemâs contribution to esophageal inflammation is not known. Gram-negative bacteria, which dominate the esophageal microbiome in reflux esophagitis, produce lipopolysaccharide (LPS), a TLR4 ligand. TLR4 signaling produces pro-interleukin (IL)1β, pro-IL18, and NOD-like receptor protein 3 (NLRP3), which prime the NLRP3 inflammasome. Subsequent NLRP3 inflammasome activation cleaves caspase-1, inducing secretion of proinflammatory cytokines and pyroptosis (inflammatory cell death). We explored LPS effects on NLRP3 inflammasome priming and activation in esophageal cells. Methods: We exposed esophageal squamous and Barrettâs epithelial cells to LPS and measured the following: (1) TLR4, pro-IL1β, pro-IL18, and NLRP3 expression; (2) caspase-1 activity; (3) tumor necrosis factor-α, IL8, IL1β, and IL18 secretion; (4) lactate dehydrogenase (LDH) release (a pyroptosis marker); and (5) mitochondrial reactive oxygen species (ROS). As inhibitors, we used acetyl-Tyr-Val-Ala-Asp-CHO for caspase-1, small interfering RNA for NLRP3, and (2-(2,2,6,6,-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride for mitochondrial ROS. Results: Squamous and Barrettâs cells expressed similar levels of TLR4, but LPS induced TLR4 signaling that increased tumor necrosis factor-α and IL8 secretion only in Barrettâs cells. Barrettâs cells treated with LPS showed increased expression of pro-IL18, pro-IL1β, and NLRP3, and increased mitochondrial ROS levels, caspase-1 activity, IL1β and IL18 secretion, and LDH release. Acetyl-Tyr-Val-Ala-Asp-CHO, NLRP3 small interfering RNA, and Mito-TEMPO all blocked LPS-induced IL1β and IL18 secretion and LDH release. Conclusions: In Barrettâs cells, LPS both primes and activates the NLRP3 inflammasome, causing secretion of proinflammatory cytokines and pyroptosis. By triggering molecular events promoting inflammation, the esophageal microbiome might contribute to inflammation-mediated carcinogenesis in Barrettâs esophagus. Keywords: IL1β, Pyroptosis, Esophageal Squamous Cell, GERD, Cytokine

Background and Aims Bowel function requires coordinated activity of diverse enteric neuron subtypes. Our aim was to define gene expression in these neuron subtypes to facilitate development of novel therapeutic approaches to treat devastating enteric neuropathies, and to learn more about enteric nervous system function. Methods To identify subtype–specific genes, we performed single-nucleus RNA-seq on adult mouse and human colon myenteric plexus, and single-cell RNA-seq on E17.5 mouse ENS cells from whole bowel. We used immunohistochemistry, select mutant mice, and calcium imaging to validate and extend results. Results RNA-seq on 635 adult mouse colon myenteric neurons and 707 E17.5 neurons from whole bowel defined seven adult neuron subtypes, eight E17.5 neuron subtypes and hundreds of differentially expressed genes. Manually dissected human colon myenteric plexus yielded RNA-seq data from 48 neurons, 3798 glia, 5568 smooth muscle, 377 interstitial cells of Cajal, and 2153 macrophages. Immunohistochemistry demonstrated differential expression for BNC2, PBX3, SATB1, RBFOX1, TBX2, and TBX3 in enteric neuron subtypes. Conditional Tbx3 loss reduced NOS1-expressing myenteric neurons. Differential Gfra1 and Gfra2 expression coupled with calcium imaging revealed that GDNF and neurturin acutely and differentially regulate activity of ∼50% of myenteric neurons with distinct effects on smooth muscle contractions. Conclusion Single cell analyses defined genes differentially expressed in myenteric neuron subtypes and new roles for TBX3, GDNF and NRTN. These data facilitate molecular diagnostic studies and novel therapeutics for bowel motility disorders.

Real-time reverse transcription polymerase chain reaction (RT-PCR) is regarded as one of the most useful and powerful tools for characterizing hematopoietic stem cells (HSCs), because samples of extremely small cell numbers can be analyzed. The expression levels determined by RT-PCR are based on relative quantification; therefore, the selection of an appropriate reference gene with a relatively stable expression level under most conditions is crucial. Here, we determined that beta2-microglobulin (B2m) is an appropriate reference gene for analyzing mouse HSCs by a novel method using single-cell RT-PCR. Clonally sorted HSCs were subjected to RT reactions with exogenous RNA fragments and then to real-time PCR. Next, the relative gene expression levels of 4 well-known housekeeping genes were quantified in each single cell sample based on the threshold cycle of exogenous RNA. The analysis revealed that B2m expression was reproducibly detected in almost all HSCs and that B2m had the most stable expression level among the compared genes, even after the cells had been cultured under various conditions. Thus, our results indicate that B2m can reliably be used as a reference gene for the relative quantification of expression levels in HSCs across various conditions. Furthermore, our work proposes a novel method for the selection of appropriate reference genes.

Background & Aims: Cathelicidin (LL-37 in human and mCRAMP in mice) represents a family of endogenous antimicrobial peptides with anti-inflammatory effects. LL-37 also suppresses collagen synthesis, an important fibrotic response, in dermal fibroblasts. Here, we determined whether exogenous cathelicidin administration modulates intestinal fibrosis in two animal models of intestinal inflammation and in human colonic fibroblasts. Methods: C57BL/6J mice (n = 6 per group) were administered intracolonically with a trinitrobenzene sulphonic acid (TNBS) enema to induce chronic (6â7 weeks) colitis with fibrosis. We administered mCRAMP peptide (5 mg/kg every 3 day, week 5â7) or cathelicidin gene (Camp)-expressing lentivirus (107 infectious units week 4) intracolonically or intravenously, respectively. We then infected 129Sv/J mice with Salmonella typhimurium orally to induce cecal inflammation with fibrosis. Camp-expressing lentivirus (107 infectious units day 11) was administered intravenously. Results: TNBS-induced chronic colitis was associated with increased colonic collagen (col1a2) mRNA expression. Intracolonic cathelicidin (mCRAMP peptide) administration or intravenous delivery of lentivirus-overexpressing cathelicidin gene significantly reduced colonic col1a2 mRNA expression in TNBS-exposed mice compared with vehicle administration. Salmonella infection also caused increased cecal inflammation associated with collagen (col1a2) mRNA expression that was prevented by intravenous delivery of Camp-expressing lentivirus. Exposure of human primary intestinal fibroblasts and human colonic CCD-18Co fibroblasts to transforming growth factor-β1 (TGF-β1) and/or insulin-like growth factor 1 induced collagen protein and mRNA expression, which was reduced by LL-37 (3â5 μM) through a MAP kinase-dependent mechanism. Conclusions: Cathelicidin can reverse intestinal fibrosis by directly inhibiting collagen synthesis in colonic fibroblasts. Keywords: Antimicrobial Peptide, Collagen, Inflammatory Bowel Disease

Background & Aims Hepatic infiltration of neutrophils is a hallmark of steatohepatitis; however, the role of neutrophils in the progression of steatohepatitis remains unknown. Methods A clinically relevant mouse model of steatohepatitis induced by high-fat diet (HFD) plus binge ethanol feeding was used. Liver fibrosis was examined. In vitro cell culture was used to analyze the interaction of hepatic stellate cells (HSCs) and neutrophils. Results HFD plus one binge ethanol (HFD+1B) feeding induced significant hepatic neutrophil infiltration, liver injury, and fibrosis. HFD plus multiple binges of ethanol (HFD+mB) caused more pronounced liver fibrosis. Microarray analyses showed that the most highly activated signaling pathway in this HFD+1B model was related to liver fibrosis and HSC activation. Blockade of chemokine (C-X-C motif) ligand 1 or intercellular adhesion molecule-1 expression reduced hepatic neutrophil infiltration and ameliorated liver injury and fibrosis. Disruption of the p47phox gene (also called neutrophil cytosolic factor 1), a critical component of reactive oxygen species producing nicotinamide adenine dinucleotide phosphate-oxidase in neutrophils, diminished HFD+1B–induced liver injury and fibrosis. Co-culture of HSCs with neutrophils, but not with neutrophil apoptotic bodies, induced HSC activation and prolonged neutrophil survival. Mechanistic studies showed that activated HSCs produce granulocyte-macrophage colony-stimulating factor and interleukin-15 to prolong the survival of neutrophils, which may serve as a positive forward loop to promote liver damage and fibrosis. Conclusions The current data from a mouse model of HFD plus binge ethanol feeding suggest that obesity and binge drinking synergize to promote liver fibrosis, which is partially mediated via the interaction of neutrophils and HSCs. Microarray data in this article have been uploaded to NCBI’s Gene Expression Omnibus (GEO accession number: GSE98153)., Graphical abstract

Graphical abstract Nde1 (red) is localised to the GFAP-positive (green) stem cell of the subventricular zone of the lateral ventricle, and its overexpression in neural stem cells promotes neuronal differentiation while inhibiting astroglial differentiation., Highlights • This is the most detailed morphological documentation of Nde1 and Ndel1 both in vivo and in vitro. • This is the first histological study of Nde1 expression in the healthy human cortex. • We demonstrate the localization of Nde1 and Ndel1 in neurogenic regions of the adult mouse brain. • Overexpression of Nde1 and Ndel1 leads to the fate specification changes of neural stem cells., Mental and neurological illnesses affect one in four people. While genetic linkage analyses have shown an association of nuclear distribution factor E (NDE1, or NudE) and its ohnolog NDE-like 1 (NDEL1, or Nudel) with mental disorders, the cellular mechanisms remain unclear. In the present study, we have demonstrated that Nde1 and Ndel1 are differentially localised in the subventricular zone (SVZ) of the forebrain and the subgranular zone (SGZ) of the hippocampus, two regions where neurogenesis actively occurs in the adult brain. Nde1, but not Ndel1, is localized to putative SVZ stem cells, and to actively dividing progenitors of the SGZ. The influence of these proteins on neural stem cell differentiation was investigated by overexpression in a hippocampal neural stem cell line, HCN-A94. Increasing Nde1 expression in this neural stem cell line led to increased neuronal differentiation while decreasing levels of astroglial differentiation. In primary cultured neurons and astrocytes, Nde1 and Ndel1 were found to have different but comparable subcellular localizations. In addition, we have shown for the first time that Nde1 is heterogeneously distributed in cortical astrocytes of human brains. Our data indicate that Nde1 and Ndel1 have distinct but overlapping distribution patterns in mouse brain and cultured nerve cells. They may function differently and therefore their dosage changes may contribute to some aspects of mental disorders.

This study reports a new immunomagnetic bead-based microfluidic system for the rapid detection of influenza A virus infection by performing a simple two-step diagnostic process that includes a magnetic bead-based fluorescent immunoassay (FIA) and an end-point optical analysis. With the incorporation of monoclonal antibody (mAb)-conjugated immunomagnetic beads, target influenza A viral particles such as A/H1N1 and A/H3N2 can be specifically recognized and are bound onto the surface of the immunomagnetic beads from the specimen sample. This is followed by labeling the fluorescent signal onto the virus-bound magnetic complexes by specific developing mAb with R-phycoerythrin (PE). Finally, the optical intensity of the magnetic complexes can be analyzed immediately by the optical detection module. Significantly, the limit of detection (LOD) of this immunomagnetic bead-based microfluidic system for the detection of influenza A virus in a specimen sample is approximately 5 × 10−4 hemagglutin units (HAU), which is 1024 times better than compared to conventional bench-top systems using flow cytometry. More importantly, the entire diagnostic protocol, from the purification of target viral particles to optical detection of the magnetic complexes, can be automatically completed within 15 min in this immunomagnetic bead-based microfluidic system, which is only 8.5% of the time required when compared to a manual protocol. As a whole, this microfluidic system may provide a powerful platform for the rapid diagnosis of influenza A virus infection and may be extended for diagnosis of other types of infectious diseases with a high specificity and sensitivity.

Background & Aims: Gastric dendritic cells (DCs) control the adaptive response to infection with Helicobacter pylori, a major risk factor for peptic ulcer disease and gastric cancer. We hypothesize that DC interactions with the gastric epithelium position gastric DCs for uptake of luminal H pylori and promote DC responses to epithelial-derived mediators. The aim of this study was to determine whether the gastric epithelium actively recruits DCs using a novel co-culture model of human gastric epithelial spheroids and monocyte-derived DCs. Methods: Spheroid cultures of primary gastric epithelial cells were infected with H pylori by microinjection. Co-cultures were established by adding human monocyte-derived DCs to the spheroid cultures and were analyzed for DC recruitment and antigen uptake by confocal microscopy. Protein array, gene expression polymerase chain reaction array, and chemotaxis assays were used to identify epithelial-derived chemotactic factors that attract DCs. Data from the co-culture model were confirmed using human gastric tissue samples. Results: Human monocyte-derived DCs co-cultured with gastric spheroids spontaneously migrated to the gastric epithelium, established tight interactions with the epithelial cells, and phagocytosed luminally applied H pylori. DC recruitment was increased upon H pylori infection of the spheroids and involved the activity of multiple chemokines including CXCL1, CXCL16, CXCL17, and CCL20. Enhanced chemokine expression and DC recruitment to the gastric epithelium also was observed in H pylori–infected human gastric tissue samples. Conclusions: Our results indicate that the gastric epithelium actively recruits DCs for immunosurveillance and pathogen sampling through chemokine-dependent mechanisms, with increased recruitment upon active H pylori infection. Keywords: Stomach, Organoid, Mononuclear Phagocyte, In Vitro Model

Background & Aims After invading intestinal epithelial cells, enteric bacteria encounter phagocytes, but little is known about how phagocytes internalize the bacteria to generate host responses. BAI1 (brain angiogenesis inhibitor 1) binds and internalizes Gram-negative bacteria through an engulfment and cell motility protein 1 (ELMO1)/Ras-related C3 botulinum toxin substrate 1 (Rac1)-dependent mechanism. We delineate the role of ELMO1 in host inflammatory responses after enteric infection. Methods ELMO1-depleted murine macrophage cell lines, intestinal macrophages, and ELMO1-deficient mice (total or myeloid-cell specific) were infected with Salmonella enterica serovar Typhimurium. The bacterial load, inflammatory cytokines, and histopathology were evaluated in the ileum, cecum, and spleen. The ELMO1-dependent host cytokines were detected by a cytokine array. ELMO1-mediated Rac1 activity was measured by pull-down assay. Results The cytokine array showed a reduced release of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein 1 (MCP-1), by ELMO1-depleted macrophages. Inhibition of ELMO1 expression in macrophages decreased Rac1 activation (∼6-fold) and reduced internalization of Salmonella. ELMO1-dependent internalization was indispensable for TNF-α and MCP-1. Simultaneous inhibition of ELMO1 and Rac function virtually abrogated TNF-α responses to infection. Activation of nuclear factor κB, extracellular signal-regulated kinases 1/2, and p38 mitogen-activated protein kinases were impaired in ELMO1-depleted cells. Bacterial internalization by intestinal macrophages completely depended on ELMO1. Salmonella infection of ELMO1-deficient mice resulted in a 90% reduction in bacterial burden and attenuated inflammatory responses in the ileum, spleen, and cecum. Conclusions These findings suggest a novel role for ELMO1 in facilitating intracellular bacterial sensing and the induction of inflammatory responses after infection with Salmonella.

Background: Surveillance of winter respiratory viral illness has been carried out for nearly 30 years using a clinical diagnosis by general practitioners as part of the Scottish Sentinel General Practice (SSGP) network. Contemparaneous laboratory diagnosis has not been available previously. Objectives: To assess the proportion of influenza-like illness (ILI) attributable to influenza, respiratory syncytial virus (RSV) and picornavirus infection during the winter season. To compare the influenza PCR data with serology of paired blood samples. Study design: Combined nose and throat swabs, from patients with ILI attending 15 general practices across Scotland, were submitted to the laboratory in virus PCR sample solution (VPSS). The extracted nucleic acid was tested using a multiplex reverse-transcription polymerase chain reaction (RT-PCR) assay. Serological analysis was performed on paired serum samples using complement fixation assays. The rate of influenza virus positivity was compared with reports of ILI obtained from the SSGP network. Results: Of 240 samples received at the laboratory, 132 (55%) were PCR positive for influenza A virus. There were nine (3.8%) picornavirus and three (1.2%) RSV PCR positives, two (0.8%) were dual influenza A/picornavirus infections. Ninety four (39.2%) were negative for all viruses tested. Results on paired sera from 89 patients showed a rising titre to influenza A in 48 of the 57 PCR positive samples (84.2%). One PCR negative patient displayed a significant rising titre to influenza A. Virological data paralleled the SSGP data but was available at least a week earlier. Conclusions: Influenza A infection was detected in the majority of patients with ILI; picornavirus infection was also shown to be an important cause of illness. PCR is a rapid and sensitive method for respiratory virus surveillance. Serology is slow, insensitive and difficult to interpret at low titres.

Background & Aims Upon intestinal epithelial damage a complex wound healing response is initiated to restore epithelial integrity and defend against pathogenic invasion. Epithelium-derived Indian Hedgehog (Ihh) functions as a critical sensor in this process. Signaling occurs in a paracrine manner because the receptor for Ihh is expressed only in the mesenchyme, but the exact Hedgehog target cell has remained elusive. The aim of this study was to elucidate further the nature of this target cell in the context of intestinal inflammation. Methods Hedgehog activity was modulated genetically in both cell type–specific and body-wide models and the resulting animals were analyzed for gene expression profiles and sensitivity for dextran sodium sulfate (DSS) colitis. To characterize the Hedgehog target cell, Gli1-CreERT2-Rosa26-ZsGreen animals were generated, which express ZsGreen in all Hedgehog-responsive cells. These cells were characterized using flow cytometry and immunofluorescence. Results Loss of Indian Hedgehog from the intestinal epithelium resulted in a rapid increase in expression of inflammation-related genes, accompanied by increased influx of immune cells. Animals with epithelium-specific deletion of Ihh or lacking the Hedgehog receptor Smoothened from Hedgehog target cells were more sensitive to DSS colitis. In contrast, specific deletion of Smoothened in the myeloid compartment did not alter the response to DSS. This suggests that Hedgehog signaling does not repress intestinal immunity through an effect on myeloid cells. Indeed, we found that Hedgehog-responsive cells expressed gp38, smooth muscle actin, and desmin, indicating a fibroblastic nature. Ihh signaling inhibited expression of C-X-C motif chemokine ligand 12 (CXCL12) in fibroblasts in vitro and in vivo, thereby impairing the recruitment of immune cells. Conclusions We show that epithelium-derived Indian Hedgehog signals exclusively to fibroblasts in the intestine. Loss of Ihh leads to a rapid immune response with up-regulation of fibroblast-derived CXCL12, and migration of immune cells into the lamina propria., Graphical abstract

Background: Influenza (Flu) and respiratory syncytial virus (RSV) are important viral pathogens that cause lower respiratory tract infections and severe exacerbations of asthma. Molecular biological techniques are permitting a rapid and accurate diagnosis of infections caused by respiratory pathogens, and have typically been applied to upper respiratory samples. Sputum induction provides an opportunity to directly sample secretions from the lower respiratory tract. Objectives/study design: To determine the role of induced sputum reverse-transcription polymerase chain reaction (RT-PCR) in the detection of respiratory pathogens and compare this with detection using serology and immunofluorescent antigen (IFA) testing, we recruited 49 adults from emergency room with exacerbations of asthma. After a medical assessment and spirometry, sputum was induced using ultrasonically nebulised normal saline. Sputum was assayed using IFA and RT-PCR for flu and RSV. Flu serology was performed acutely and at convalescence, 4 � /5 weeks later. Results: Influenza A or B was detected in 24% of the samples by PCR, significantly more than the nine cases detected using serology and the one case using IFA (P B/0.05). RSV was detected in 37% of samples using PCR and 20% by IFA (P B/0.05). Conclusion: The combination of induced sputum and RT-PCR provides a useful means of detecting respiratory infection. The technique is safe in both adults and children, and RT-PCR is more sensitive than conventional serology and IFA. The improved sensitivity of induced sputum RT-PCR also permits a more rapid diagnosis and the opportunity of early administration of effective treatments. # 2002 Elsevier Science B.V. All rights reserved.

The major concern for severe acute respiratory syndrome (SARS), caused by the SARS-associated coronavirus (SARS-CoV), is the lack of diagnostic and therapeutic agents. Using a phage display technology in a chicken system, high-affinity monoclonal antibody fragments against the SARS-CoV spike protein were characterized. Ten truncated spike protein gene fragments were expressed in Escherichia coli cells. Following the immunization of chickens with these recombinant spike proteins, two single-chain variable fragment (scFv) antibody libraries were established with short or long linkers to contain 5x10(7) and 9x10(6) transformants, respectively. After four rounds of panning selection, the scFv antibodies of randomly chosen clones were demonstrated by Coomassie blue staining, and verified by western blot analysis. In a comparison of nucleotide sequences with the chicken germline gene, we found that all clones varied in the complementarity-determining regions, that two scFv antibodies reacted significantly with SARS-CoV-infected Vero cells, and that those two specific scFv antibodies recognized the same region of the spike protein spanning amino acid residues 750-1000. In conclusion, the results suggest that the chicken scFv phage display system can be a potential model for mass production of high-affinity antibodies against the SARS-CoV spike protein.