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Crohn's disease (CD) is a complex and heterogeneous condition with no perfect preclinical model or cure. To address this, we explore adult stem cell-derived organoids that retain their tissue identity and disease-driving traits. We prospectively create a biobank of CD patient-derived organoid cultures (PDOs) from colonic biopsies of 53 subjects across all clinical subtypes and healthy subjects. Gene expression analyses enabled benchmarking of PDOs as tools for modeling the colonic epithelium in active disease and identified two major molecular subtypes: immune-deficient infectious CD (IDICD) and stress and senescence-induced fibrostenotic CD (S2FCD). Each subtype shows internal consistency in the transcriptome, genome, and phenome. The spectrum of morphometric, phenotypic, and functional changes within the "living biobank" reveals distinct differences between the molecular subtypes. Drug screens reverse subtype-specific phenotypes, suggesting phenotyped-genotyped CD PDOs can bridge basic biology and patient trials by enabling preclinical phase "0" human trials for personalized therapeutics., Competing Interests: Declaration of interests S.D. and P.G. have a patent on the methodology., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: E-cigarettes (E.cigs) cause inflammation and damage to human organs, including the lungs and heart. In the gut, E.cig vaping promotes inflammation and gut leakiness. Further, E.cig vaping increases tumorigenesis in oral and lung epithelial cells by inducing mutations and suppressing host DNA repair enzymes. It is well known that cigarette (cig) smoking increases the risk of colorectal cancer (CRC). To date, it is unknown whether E.cig vaping impacts CRC development., Methods: A mouse model of human familial adenomatous polyposis (CPC-APC) was utilized wherein a mutation in the adenomatous polyposis coli (APC) gene, CDX2-Cre-APC Min/+ , leads to the development of colon adenomas within 16 weeks. Mice were exposed to air (controls), E.cig vaping, cig, or both (dual exposure). After 4 weeks of 2-hour exposures per day (1 hour of each for dual exposures), the colon was collected and assessed for polyp number and pathology scores by microscopy. Expression of inflammatory cytokines and cancer stem cell markers were quantified. DNA damage such as double-strand DNA breaks was evaluated by immunofluorescence, western blot and gene-specific long amplicon qPCR. DNA repair enzyme levels (NEIL-2, NEIL-1, NTH1, and OGG1) were quantified by western blot. Proliferation markers were assessed by RT-qPCR and ELISA., Results: CPC-APC mice exposed to E.cig, cig, and dual exposure developed a higher number of polyps compared to controls. Inflammatory proteins, DNA damage, and cancer stemness markers were higher in E-cig, cig, and dual-exposed mice as well. DNA damage was found to be associated with the suppression of DNA glycosylases, particularly with NEIL-2 and NTH1. E.cig and dual exposure both stimulated cancer cell stem markers ( CD44 , Lgr-5, DCLK1, and Ki67) . The effect of E.cigs on polyp formation and CRC development was less than that of cigs, while dual exposure was more tumorigenic than either of the inhalants alone., Conclusion: E.cig vaping promotes CRC by stimulating inflammatory pathways, mediating DNA damage, and upregulating transcription of cancer stem cell markers. Critically, combining E.cig vaping with cig smoking leads to higher levels of tumorigenesis. Thus, while the chemical composition of these two inhalants, E.cigs and cigs, is highly disparate, they both drive the development of cancer and when combined, a highly common pattern of use, they can have additive or synergistic effects., Competing Interests: Competing interest: The authors declare no competing interest.

Virulent infectious agents such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and methicillin-resistant Staphylococcus aureus (MRSA) induce tissue damage that recruits neutrophils, monocyte, and macrophages, leading to T cell exhaustion, fibrosis, vascular leak, epithelial cell depletion, and fatal organ damage. Neutrophils, monocytes, and macrophages recruited to pathogen-infected lungs, including SARS-CoV-2-infected lungs, express phosphatidylinositol 3-kinase gamma (PI3Kγ), a signaling protein that coordinates both granulocyte and monocyte trafficking to diseased tissues and immune-suppressive, profibrotic transcription in myeloid cells. PI3Kγ deletion and inhibition with the clinical PI3Kγ inhibitor eganelisib promoted survival in models of infectious diseases, including SARS-CoV-2 and MRSA, by suppressing inflammation, vascular leak, organ damage, and cytokine storm. These results demonstrate essential roles for PI3Kγ in inflammatory lung disease and support the potential use of PI3Kγ inhibitors to suppress inflammation in severe infectious diseases.

Background: Genetic factors and microbial imbalances play crucial roles in colorectal cancers (CRCs), yet the impact of infections on cancer initiation remains poorly understood. While bioinformatic approaches offer valuable insights, the rising incidence of CRCs creates a pressing need to precisely identify early CRC events. We constructed a network model to identify continuum states during CRC initiation spanning normal colonic tissue to pre-cancer lesions (adenomatous polyps) and examined the influence of microbes and host genetics., Methods: A Boolean network was built using a publicly available transcriptomic dataset from healthy and adenoma affected patients to identify an invariant Microbe-Associated Colorectal Cancer Signature (MACS). We focused on Fusobacterium nucleatum ( Fn ), a CRC-associated microbe, as a model bacterium. MACS-associated genes and proteins were validated by RT-qPCR, RNA seq, ELISA, IF and IHCs in tissues and colon-derived organoids from genetically predisposed mice ( CPC-APC Min+/- ) and patients (FAP, Lynch Syndrome, PJS, and JPS)., Results: The MACS that is upregulated in adenomas consists of four core genes/proteins: CLDN2/Claudin-2 (leakiness), LGR5/leucine-rich repeat-containing receptor (stemness), CEMIP/cell migration-inducing and hyaluronan-binding protein (epithelial-mesenchymal transition) and IL8/Interleukin-8 (inflammation). MACS was induced upon Fn infection, but not in response to infection with other enteric bacteria or probiotics. MACS induction upon Fn infection was higher in CPC-APC Min+/- organoids compared to WT controls. The degree of MACS expression in the patient-derived organoids (PDOs) generally corresponded with the known lifetime risk of CRCs., Conclusions: Computational prediction followed by validation in the organoid-based disease model identified the early events in CRC initiation. MACS reveals that the CRC-associated microbes induce a greater risk in the genetically predisposed hosts, suggesting its potential use for risk prediction and targeted cancer prevention.

SARS-CoV-2 infection-induced aggravation of host innate immune response not only causes tissue damage and multiorgan failure in COVID-19 patients but also induces host genome damage and activates DNA damage response pathways. To test whether the compromised DNA repair capacity of individuals modulates the severity of COVID-19 infection, we analyze DNA repair gene expression in publicly available patient datasets and observe a lower level of the DNA glycosylase NEIL2 in the lungs of severely infected COVID-19 patients. This observation of lower NEIL2 levels is further validated in infected patients, hamsters and ACE2 receptor-expressing human A549 (A549-ACE2) cells. Furthermore, delivery of recombinant NEIL2 in A549-ACE2 cells shows decreased expression of proinflammatory genes and viral E-gene, as well as lowers the yield of viral progeny compared to mock-treated cells. Mechanistically, NEIL2 cooperatively binds to the 5'-UTR of SARS-CoV-2 genomic RNA to block viral protein synthesis. Collectively, these data strongly suggest that the maintenance of basal NEIL2 levels is critical for the protective response of hosts to viral infection and disease., (© 2023. The Author(s).)

Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family of receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, ErbB2, and ErbB4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, proinflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production, and disruption of blood-brain barrier integrity in microfluidics-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof of principle for a repurposed, ErbB-targeted approach to combat emerging viruses.

E-cigarette (E-cig) inhalation affects health status by modulating inflammation profiles in several organs, including the brain, lung, heart, and colon. The effect of flavored fourth-generation pod-based E-cigs (JUUL) on murine gut inflammation is modulated by both flavor and exposure period. Exposure of mice to JUUL mango and JUUL mint for one month upregulated inflammatory cytokines, particularly TNF-α, IL-6, and Cxcl-1 (IL-8). JUUL Mango effects were more prominent than those incurred by JUUL Mint after one month of exposure. However, JUUL Mango reduced the expression of colonic inflammatory cytokines after three months of exposure. In this protocol, we detail the process of RNA isolation from the mouse colon and the use of extracted RNA in profiling the inflammatory milieu. Efficient RNA extraction from the murine colon is the most important step in the evaluation of inflammatory transcripts in the colon., Competing Interests: Competing interestsThe authors declare there is no conflict of interest., (Copyright © 2023 The Authors; This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).)

Upon sensing DNA double-strand breaks (DSBs), eukaryotic cells either die or repair DSBs via one of the two competing pathways, i.e., non-homologous end-joining (NHEJ) or homologous recombination (HR). We show that cell fate after DSBs hinges on GIV/Girdin, a guanine nucleotide-exchange modulator of heterotrimeric Giα•βγ protein. GIV suppresses HR by binding and sequestering BRCA1, a key coordinator of multiple steps within the HR pathway, away from DSBs; it does so using a C-terminal motif that binds BRCA1's BRCT-modules via both phospho-dependent and -independent mechanisms. Using another non-overlapping C-terminal motif GIV binds and activates Gi and enhances the "free" Gβγ→PI-3-kinase→Akt pathway, which promotes survival and is known to suppress HR, favor NHEJ. Absence of GIV, or loss of either of its C-terminal motifs enhanced cell death upon genotoxic stress. Because GIV selectively binds other BRCT-containing proteins suggests that G-proteins may fine-tune sensing, repair, and survival after diverse types of DNA damage., Competing Interests: Authors declare no competing interests., (© 2023 The Author(s).)

Microbes play an important role in regulating cellular responses and the induction of chronic diseases. Infection and chronic inflammation can cause DNA damage, and the accumulation of mutations leads to cancer development. The well-known examples of cancer-associated microbes are Helicobacter pylori in gastric cancer and Fusobacterium nucleatum (Fn), Bacteroides fragilis, and E.coli NC101 in colorectal cancer (CRC). These carcinopathogens modify the expressions of the base excision repair enzymes and cause DNA damage. This chapter will show how Fn can initiate CRC through the downregulation of a critical enzyme of the base excision repair (BER) pathway that subsequently causes accumulation of DNA damage. We used the stem cell-based organoid model and enteroid-derived monolayer (EDM) from the murine and human colon to assess the impact of infection on the expression of BER enzymes on the transcriptional and translational levels and to develop other functional assays. For example, we used this EDM model to assess the inflammatory response, DNA damage response, and physiological responses, where we correlated the level of these parameters to BER enzyme levels., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Although Barrett's metaplasia of the esophagus (BE) is the only known precursor lesion to esophageal adenocarcinomas (EACs), drivers of cellular transformation in BE remain incompletely understood. We use an artificial intelligence-guided network approach to study EAC initiation and progression. Key predictions are subsequently validated in a human organoid model, in patient-derived biopsy specimens of BE, a case-control study of genomics of BE progression, and in a cross-sectional study of 113 patients with BE and EACs. Our model classified healthy esophagus from BE and BE from EACs in several publicly available gene expression data sets (n = 932 samples). The model confirmed that all EACs must originate from BE and pinpointed a CXCL8/IL8↔neutrophil immune microenvironment as a driver of cellular transformation in EACs and gastroesophageal junction adenocarcinomas. This driver is prominent in White individuals but is notably absent in African Americans (AAs). Network-derived gene signatures, independent signatures of neutrophil processes, CXCL8/IL8 expression, and an absolute neutrophil count (ANC) are associated with risk of progression. SNPs associated with changes in ANC by ethnicity (e.g., benign ethnic neutropenia [BEN]) modify that risk. Findings define a racially influenced immunological basis for cell transformation and suggest that BEN in AAs may be a deterrent to BE→EAC progression.

Multisystem inflammatory syndrome in children (MIS-C) is an illness that emerged amidst the COVID-19 pandemic but shares many clinical features with the pre-pandemic syndrome of Kawasaki disease (KD). Here we compare the two syndromes using a computational toolbox of two gene signatures that were developed in the context of SARS-CoV-2 infection, i.e., the viral pandemic (ViP) and severe-ViP signatures and a 13-transcript signature previously demonstrated to be diagnostic for KD, and validated our findings in whole blood RNA sequences, serum cytokines, and formalin fixed heart tissues. Results show that KD and MIS-C are on the same continuum of the host immune response as COVID-19. Both the pediatric syndromes converge upon an IL15/IL15RA-centric cytokine storm, suggestive of shared proximal pathways of immunopathogenesis; however, they diverge in other laboratory parameters and cardiac phenotypes. The ViP signatures reveal unique targetable cytokine pathways in MIS-C, place MIS-C farther along in the spectrum in severity compared to KD and pinpoint key clinical (reduced cardiac function) and laboratory (thrombocytopenia and eosinopenia) parameters that can be useful to monitor severity., (© 2022. The Author(s).)

Transient depletion of the transcription elongation factor SPT6 in the keratinocyte has been recently shown to inhibit epidermal differentiation and stratification; instead, they transdifferentiate into a gut-like lineage. We show here that this phenomenon of transdifferentiation recapitulates Barrett's metaplasia, the only human pathophysiologic condition in which a stratified squamous epithelium that is injured due to chronic acid reflux is trans-committed into an intestinal fate. The evidence we present here not only lend support to the notion that the keratinocytes are potentially the cell of origin of Barrett's metaplasia but also provide mechanistic insights linking transient acid exposure, downregulation of SPT6, stalled transcription of the master regulator of epidermal fate TP63, loss of epidermal fate, and metaplastic progression. Because Barrett's metaplasia in the esophagus is a pre-neoplastic condition with no preclinical human models, these findings have a profound impact on the modeling Barrett's metaplasia-in-a-dish., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

The ongoing pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), necessitates strategies to identify prophylactic and therapeutic drug candidates for rapid clinical deployment. Here, we describe a screening pipeline for the discovery of efficacious SARS-CoV-2 inhibitors. We screen a best-in-class drug repurposing library, ReFRAME, against two high-throughput, high-content imaging infection assays: one using HeLa cells expressing SARS-CoV-2 receptor ACE2 and the other using lung epithelial Calu-3 cells. From nearly 12,000 compounds, we identify 49 (in HeLa-ACE2) and 41 (in Calu-3) compounds capable of selectively inhibiting SARS-CoV-2 replication. Notably, most screen hits are cell-line specific, likely due to different virus entry mechanisms or host cell-specific sensitivities to modulators. Among these promising hits, the antivirals nelfinavir and the parent of prodrug MK-4482 possess desirable in vitro activity, pharmacokinetic and human safety profiles, and both reduce SARS-CoV-2 replication in an orthogonal human differentiated primary cell model. Furthermore, MK-4482 effectively blocks SARS-CoV-2 infection in a hamster model. Overall, we identify direct-acting antivirals as the most promising compounds for drug repurposing, additional compounds that may have value in combination therapies, and tool compounds for identification of viral host cell targets.

E-cigarette usage continues to rise, yet the safety of e-cigarette aerosols is questioned. Using murine models of acute and chronic e-cigarette aerosol inhalation, murine colon transcriptomics, and murine and human gut-derived organoids in co-culture models, we assessed the effects of e-cigarette use on the gut barrier. Histologic and transcriptome analyses revealed that chronic, but not acute, nicotine-free e-cigarette use increased inflammation and reduced expression of tight junction (TJ) markers. Exposure of murine and human enteroid-derived monolayers (EDMs) to nicotine-free e-cigarette aerosols alone or in co-culture with bacteria also causes barrier disruption, downregulation of TJ protein, and enhanced inflammation in response to infection. These data highlight the harmful effects of "non-nicotine" component of e-cigarettes on the gut barrier. Considering the importance of an intact gut barrier for host fitness and the impact of gut mucosal inflammation on a multitude of chronic diseases, these findings are broadly relevant to both medicine and public health., Competing Interests: S.D. and P.G. have patents on methodology to prepare enteroid monolayers and functional assays related to the gut barrier. The authors have declared that no other conflict of interest exists., (© 2021 The Author(s).)

Organoids have revolutionized cancer research as highly adaptable models that enable an array of experimental techniques to interrogate tissue morphology and function. Because they preserve the genetic, phenotypic, and behavioral traits of their source tissue, organoids have gained traction as the most relevant models for drug discovery, tracking therapeutic response and for personalized medicine. As organoids are indisputably becoming a mainstay of cancer research, this review specifically addresses how colon-derived organoids can be perfected as multidimensional, scalable, reproducible models of healthy, pre-neoplastic and neoplastic conditions of the colon and for use in high-throughput "Phase-0" human clinical trials-in-a-dish., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Tumorigenesis in different segments of the intestinal tract involves tissue-specific oncogenic drivers. In the colon, complement component 3 (C3) activation is a major contributor to inflammation and malignancies. By contrast, tumorigenesis in the small intestine involves fatty acid-binding protein 1 (FABP1). However, little is known of the upstream mechanisms driving their expressions in different segments of the intestinal tract. Here, we report that the RNA-binding protein DDX5 binds to the mRNA transcripts of C3 and Fabp1 to augment their expressions posttranscriptionally. Knocking out DDX5 in epithelial cells protected mice from intestinal tumorigenesis and dextran sodium sulfate (DSS)-induced colitis. Identification of DDX5 as a common upstream regulator of tissue-specific oncogenic molecules provides an excellent therapeutic target for intestinal diseases., (© 2020 Abbasi et al.)

The gut barrier separates trillions of microbes from the largest immune system in the body; when compromised, a "leaky" gut barrier fuels systemic inflammation, which hastens the progression of chronic diseases. Strategies to detect and repair the leaky gut barrier remain urgent and unmet needs. Recently, a stress-polarity signaling (SPS) pathway has been described in which the metabolic sensor, AMP-kinase acts via its effector, GIV (also known as Girdin) to augment epithelial polarity exclusively under energetic stress and suppresses tumor formation. Using murine and human colon-derived organoids, and enteroid-derived monolayers (EDMs) that are exposed to stressors, we reveal that the SPS-pathway is active in the intestinal epithelium and requires a catalytically active AMP-kinase. Its pharmacologic augmentation resists stress-induced collapse of the epithelium when challenged with microbes or microbial products. In addition, the SPS-pathway is suppressed in the aging gut, and its reactivation in enteroid-derived monolayers reverses aging-associated inflammation and loss of barrier function. It is also silenced during progression of colorectal cancers. These findings reveal the importance of the SPS-pathway in the gut and highlights its therapeutic potential for treating gut barrier dysfunction in aging, cancer, and dysbiosis., (© 2020 Ghosh et al.)

Background and Aims: Tumor necrosis factor (TNF) is a major pathogenic effector and a therapeutic target in inflammatory bowel disease (IBD), yet the basis for TNF-induced intestinal epithelial cell (IEC) death is unknown, because TNF does not kill normal IECs. Here, we investigated how chronic nuclear factor (NF)- κB activation, which occurs in human IBD, promotes TNF-dependent IEC death in mice., Methods: Human IBD specimens were stained for p65 and cleaved caspase-3. C57BL/6 mice with constitutively active IKKβ in IEC (Ikkβ(EE) IEC ), Ripk1 D138N/D138N knockin mice, and Ripk3 -/- mice were injected with TNF or lipopolysaccharide. Enteroids were also isolated from these mice and challenged with TNF with or without RIPK1 and RIPK3 inhibitors or butylated hydroxyanisole. Ripoptosome-mediated caspase-8 activation was assessed by immunoprecipitation., Results: NF-κB activation in human IBD correlated with appearance of cleaved caspase-3. Congruently, unlike normal mouse IECs that are TNF-resistant, IECs in Ikkβ(EE) IEC mice and enteroids were susceptible to TNF-dependent apoptosis, which depended on the protein kinase function of RIPK1. Constitutively active IKKβ facilitated ripoptosome formation, a RIPK1 signaling complex that mediates caspase-8 activation by TNF. Butylated hydroxyanisole treatment and RIPK1 inhibitors attenuated TNF-induced and ripoptosome-mediated caspase-8 activation and IEC death in vitro and in vivo., Conclusions: Contrary to common expectations, chronic NF-κB activation induced intestinal crypt apoptosis after TNF stimulation, resulting in severe mucosal erosion. RIPK1 kinase inhibitors selectively inhibited TNF destructive properties while preserving its survival and proliferative properties, which do not require RIPK1 kinase activity. RIPK1 kinase inhibition could be a potential treatment for IBD., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Congenital tufting enteropathy (CTE) is an autosomal recessive disease characterized by severe intestinal failure in infancy and mutations in the epithelial cell adhesion molecule ( EPCAM ) gene. Previous studies of CTE in mice expressing mutant EpCAM show neonatal lethality. Hence, to study the cellular, molecular, and physiological alterations that result from EpCAM mutation, a tamoxifen-inducible mutant EpCAM enteroid model has been generated. The presence of mutant EpCAM in the model was confirmed at both mRNA and protein levels. Immunofluorescence microscopy demonstrated the reduced expression of mutant EpCAM. Mutant enteroids had reduced budding potential as well as significantly decreased mRNA expression for epithelial lineage markers ( Mucin 2, lysozyme, sucrase-isomaltase ), proliferation marker Ki67, and secretory pathway transcription factors ( Atoh1 , Hnf1b) . Significantly decreased numbers of Paneth and goblet cells were confirmed by staining. These findings were correlated with intestinal tissue from CTE patients and the mutant mice model that had significantly fewer Paneth and goblet cells than in healthy counterparts. FITC-dextran studies demonstrated significantly impaired barrier function in monolayers derived from mutant enteroids compared with control monolayers. In conclusion, we have established an ex vivo CTE model. The role of EpCAM in the budding potential, differentiation, and barrier function of enteroids is noted. Our study establishes new facets of EpCAM biology that will aid in understanding the pathophysiology of CTE and role of EpCAM in health and disease. NEW & NOTEWORTHY Here, we develop a novel ex vivo enteroid model for congenital tufting enteropathy (CTE) based on epithelial cell adhesion molecule ( EPCAM) gene mutations found in patients. With this model we demonstrate the role of EpCAM in maintaining the functional homeostasis of the intestinal epithelium, including differentiation, proliferation, and barrier integrity. This study further establishes a new direction in EpCAM biology that will help in understanding the detailed pathophysiology of CTE and role of EpCAM.

Electronic (e)-cigarettes theoretically may be safer than conventional tobacco. However, our prior studies demonstrated direct adverse effects of e-cigarette vapor (EV) on airway cells, including decreased viability and function. We hypothesize that repetitive, chronic inhalation of EV will diminish airway barrier function, leading to inflammatory protein release into circulation, creating a systemic inflammatory state, ultimately leading to distant organ injury and dysfunction. C57BL/6 and CD-1 mice underwent nose only EV exposure daily for 3-6 mo, followed by cardiorenal physiological testing. Primary human bronchial epithelial cells were grown at an air-liquid interface and exposed to EV for 15 min daily for 3-5 days before functional testing. Daily inhalation of EV increased circulating proinflammatory and profibrotic proteins in both C57BL/6 and CD-1 mice: the greatest increases observed were in angiopoietin-1 (31-fold) and EGF (25-fold). Proinflammatory responses were recapitulated by daily EV exposures in vitro of human airway epithelium, with EV epithelium secreting higher IL-8 in response to infection (227 vs. 37 pg/ml, respectively; P < 0.05). Chronic EV inhalation in vivo reduced renal filtration by 20% ( P = 0.017). Fibrosis, assessed by Masson's trichrome and Picrosirius red staining, was increased in EV kidneys (1.86-fold, C57BL/6; 3.2-fold, CD-1; P < 0.05), heart (2.75-fold, C57BL/6 mice; P < 0.05), and liver (1.77-fold in CD-1; P < 0.0001). Gene expression changes demonstrated profibrotic pathway activation. EV inhalation altered cardiovascular function, with decreased heart rate ( P < 0.01), and elevated blood pressure ( P = 0.016). These data demonstrate that chronic inhalation of EV may lead to increased inflammation, organ damage, and cardiorenal and hepatic disease.

During Helicobacter pylori infection, T cells are recruited to the gastric mucosa, but the host T-cell response is not sufficient to clear the infection. Some of the recruited T cells respond in a polarized manner to a Th1 response, while others become anergic. We have previously shown that T-cell anergy may be induced during infection by the interaction of T cells with B7-H1, which is up-regulated on the gastric epithelium during H. pylori infection. Recently, regulatory T (Treg) cells with a CD4(+) CD25(high) FoxP3(+) phenotype were found at an increased frequency in the gastric mucosa of biopsy specimens from H. pylori-infected patients. While Treg cells are important in maintaining tolerance, they can also suppress immune responses during infection. In this study, we examined the induction of the Treg phenotype when naïve T cells were incubated with gastric epithelial cells exposed to H. pylori. The frequency of this phenotype was markedly decreased when B7-H1 was blocked with monoclonal antibodies or its expression was blocked with small interfering RNA. The functional role of these Treg cells was assessed in proliferation assays when the cells were cocultured with activated T cells, which effectively decreased proliferation of the cells.