Search

Therapy with mesenchymal stromal cells (MSCs) has shown promise in inflammatory bowel disease—leveraging their immunosuppressive and regenerative properties. However, the potential immunogenic complications of allogenic MSCs sourced from different tissues raise concern. Thus, we assessed the fitness and functionality of autologous intestinal MSCs as a potential platform for cellular therapy. Mucosal biopsy-derived MSCs from Crohn’s disease (n = 11), ulcerative colitis (n = 12), and controls (n = 14) were analyzed by microscopy and flow cytometry for doubling-time, morphology, differentiation potential, and immunophenotype. Gene expression, cell-subtype composition, along with surface marker and secretome changes after IFN-γ priming were measured by bulk and single-cell RNA sequencing coupled with a 30-plex Luminex panel. MSCs expanded ex vivo demonstrate canonical MSC markers, similar growth kinetics, and tripotency regardless of the patient phenotype. Global transcription patterns were similar at baseline though inflammatory bowel disease (IBD) rectal MSCs showed changes in select immunomodulatory genes. IFN-γ priming resulted in upregulation of shared immunoregulatory genes (particularly in PD-1 signaling) and overrode the transcriptional differences observed at baseline. Furthermore, MSCs secrete key immunomodulatory molecules at baseline and in response to IFN-γ including CXCL10, CXCL9, and MCP-1. Overall, MSCs from IBD patients have normal transcriptional and immunomodulatory properties with therapeutic potential and can be sufficiently expanded.

Background Crohn’s disease (CD) is a heritable chronic inflammatory disorder. Non-coding RNAs (ncRNAs) play an important role in epigenetic regulation by affecting gene expression, but can also directly affect protein function, thus having a substantial impact on biological processes. We investigated whether non-coding RNAs (ncRNA) at diagnosis are dysregulated during CD at different CD locations and future disease behaviors to determine if ncRNA signatures can serve as an index to outcomes. Methods Using subjects belonging to the RISK cohort, we analyzed ncRNA from the ileal biopsies of 345 CD and 71 non-IBD controls, and ncRNA from rectal biopsies of 329 CD and 61 non-IBD controls. Sequence alignment was done (STAR package) using Human Genome version 38 (hg38) as reference panel. The differential expression (DE) analysis was performed with EdgeR package and DE ncRNAs were identified with a threshold of fold change (FC) > 2 and FDR Results In total, we identified 130 CD specific DE ncRNAs (89 in ileum and 41 in rectum) when compared to non-IBD controls. Similarly, 35 DE ncRNAs were identified between B1 and B2 in ileum, whereas no differences among CD disease behaviors were noticed in rectum. We also found inflammation specific ncRNAs between inflamed and non-inflamed groups in ileal biopsies. Overall, we observed that expression of mir1244-2, mir1244-3, mir1244-4, and RN7SL2 were increased during CD, regardless of disease behavior, location, or inflammatory status. Lastly, we tested ncRNA expression at baseline as potential tool to predict the disease status, disease behaviors and disease inflammation at 3-year follow up. Conclusions We have identified ncRNAs that are specific to disease location, disease behavior, and disease inflammation in CD. Both ileal and rectal specific ncRNA are changing over the course of CD, specifically during the disease progression in the intestinal mucosa. Collectively, our findings show changes in ncRNA during CD and may have a clinical utility in early identification and characterization of disease progression.

Background Crohn’s disease is a lifelong disease characterized by chronic inflammation of the gastrointestinal tract. Defining the cellular and transcriptional composition of the mucosa at different stages of disease progression is needed for personalized therapy in Crohn’s. Methods Ileal biopsies were obtained from (1) control subjects (n = 6), (2) treatment-naïve patients (n = 7), and (3) established (n = 14) Crohn’s patients along with remission (n = 3) and refractory (n = 11) treatment groups. The biopsies processed using 10x Genomics single cell 5' yielded 139 906 cells. Gene expression count matrices of all samples were analyzed by reciprocal principal component integration, followed by clustering analysis. Manual annotations of the clusters were performed using canonical gene markers. Cell type proportions, differential expression analysis, and gene ontology enrichment were carried out for each cell type. Results We identified 3 cellular compartments with 9 epithelial, 1 stromal, and 5 immune cell subtypes. We observed differences in the cellular composition between control, treatment-naïve, and established groups, with the significant changes in the epithelial subtypes of the treatment-naïve patients, including microfold, tuft, goblet, enterocyte,s and BEST4+ cells. Surprisingly, fewer changes in the composition of the immune compartment were observed; however, gene expression in the epithelial and immune compartment was different between Crohn’s phenotypes, indicating changes in cellular activity. Conclusions Our study identified cellular and transcriptional signatures associated with treatment-naïve Crohn’s disease that collectively point to dysfunction of the intestinal barrier with an increase in inflammatory cellular activity. Our analysis also highlights the heterogeneity among patients within the same disease phenotype, shining a new light on personalized treatment responses and strategies.

In peripheral blood, DNA methylation (DNAm) patterns in inflammatory bowel disease patients reflect inflammatory status rather than disease status. Here, we studied DNAm in diseased rectal mucosa from ulcerative colitis (UC) patients, conducting a cell-type-specific EWAS in epithelial, immune and fibroblast cells to understand DNAm changes across disease states, course, and clinical outcomes. At diagnosis, rectal mucosa in UC exhibited a lower proportion of epithelial cells and fibroblasts, and higher proportion of immune cells, along with 3504, 910, and 2279 altered DNAm sites as detected in our cell-specific EWAS, respectively. While treatment had significant effects on DNAm of immune cells, its effects on fibroblasts and epithelial cells were attenuated. Those requiring colectomy exhibited cell composition and DNAm patterns at follow-up more like disease onset than patients who did not require colectomy. Collectively DNAm and gene expression analysis suggest that targeting epithelial genes involved in barrier function may improve clinical outcomes.

The use of beneficial bacteria to promote gastrointestinal heath is widely practiced, however, the mechanisms whereby many of these microbes elicit their beneficial effects remain elusive. Previously, we conducted a screen for the discovery of novel beneficial microbes and identified the potent cytoprotective effects of a strain of Lactococcus lactis subsp. cremoris. Here, we show that dietary supplementation with L. lactis subsp. cremoris induced transcript enrichment of a set of genes within the colon whose functions are associated with host cell and microbe interactions. Specifically, L. lactis subsp. cremoris induced the expression of tlr2, which we show was required for L. lactis subsp. cremoris to elicit its beneficial effects on the intestine. L. lactis subsp. cremoris did not confer beneficial effects in mice deficient in TLR-2, or deficient in its adaptor protein Myd88 in chronic gut injury models. In addition to cytoprotection, culture supernatant from L. lactis subsp. cremoris accelerated epithelial migration in a cultured epithelial cell scratch wound assay; and effect that was abrogated by a TLR-2 antagonist. Furthermore, L. lactis subsp. cremoris accelerated epithelial tissue restitution following the infliction of a colonic wound biopsy in a TLR-2 and Myd88-dependent manner. Within colonic wounds, L. lactis subsp. cremoris induced the activation of signaling pathways that function in tissue restitution following injury, including the ERK signaling pathway, and of focal adhesion complex (FAC) proteins. Together, these data demonstrate that L. lactis subsp. cremoris signals via the TLR2/MyD88-axis to confer cytoprotection and accelarated tissue restituion in the gut epithelium. These data point to evolving adaptations where beneficial gut microbes moduate innate immune signaling to excert positive influnces on host physiology.

Intestinal homeostasis is regulated in-part by reactive oxygen species (ROS) that are generated in the colonic mucosa following contact with certain lactobacilli. Mechanistically, ROS can modulate protein function through the oxidation of cysteine residues within proteins. Recent advances in cysteine labeling by the Isotope Coded Affinity Tags (ICATs) technique has facilitated the identification of cysteine thiol modifications in response to stimuli. Here, we used ICATs to map the redox protein network oxidized upon initial contact of the colonic mucosa with Lactobacillus rhamnosus GG (LGG). We detected significant LGG-specific redox changes in over 450 proteins, many of which are implicated to function in cellular processes such as endosomal trafficking, epithelial cell junctions, barrier integrity, and cytoskeleton maintenance and formation. We particularly noted the LGG-specific oxidation of Rac1, which is a pleiotropic regulator of many cellular processes. Together, these data reveal new insights into lactobacilli-induced and redox-dependent networks involved in intestinal homeostasis.

Recent evidence has demonstrated that reactive oxygen (eg, hydrogen peroxide) can activate host cell signaling pathways that function in repair. We show that mice deficient in their capacity to generate reactive oxygen by the NADPH oxidase 2 holoenzyme, an enzyme complex highly expressed in neutrophils and macrophages, have disrupted capacity to orchestrate signaling events that function in mucosal repair. Similar observations were made for mice after neutrophil depletion, pinpointing this cell type as the source of the reactive oxygen driving oxidation-reduction protein signaling in the epithelium. To simulate epithelial exposure to high levels of reactive oxygen produced by neutrophils and gain new insight into this oxidation-reduction signaling, epithelial cells were treated with hydrogen peroxide, biochemical experiments were conducted, and a proteome-wide screen was performed using isotope-coded affinity tags to detect proteins oxidized after exposure. This analysis implicated signaling pathways regulating focal adhesions, cell junctions, and maintenance of the cytoskeleton. These pathways are also known to act via coordinated phosphorylation events within proteins that constitute the focal adhesion complex, including focal adhesion kinase and Crk-associated substrate. We identified the Rho family small GTP-binding protein Ras-related C3 botulinum toxin substrate 1 and p21 activated kinases 2 as operational in these signaling and localization pathways. These data support the hypothesis that reactive oxygen species from neutrophils can orchestrate epithelial cell-signaling events functioning in intestinal repair.

Background While very low birth weight (VLBW) infants often require multiple red blood cell transfusions, efforts to minimize transfusion-associated risks have resulted in more restrictive neonatal transfusion practices. However, whether restrictive transfusion strategies limit transfusions without increasing morbidity and mortality in this population remains unclear. Recent epidemiologic studies suggest that severe anemia may be an important risk factor for the development of necrotizing enterocolitis (NEC). However, the mechanism whereby anemia may lead to NEC remains unknown. Study design and methods The potential impact of anemia on neonatal inflammation and intestinal barrier disruption, two well-characterized predisposing features of NEC, was defined by correlation of hemoglobin values to cytokine levels in premature infants and by direct evaluation of intestinal hypoxia, inflammation and gut barrier disruption using a pre-clinical neonatal murine model of phlebotomy-induced anemia (PIA). Results Increasing severity of anemia in the preterm infant correlated with the level of IFN-gamma, a key pro-inflammatory cytokine that may predispose an infant to NEC. Gradual induction of PIA in a pre-clinical model resulted in significant hypoxia throughout the intestinal mucosa, including areas where intestinal macrophages reside. PIA-induced hypoxia significantly increased macrophage pro-inflammatory cytokine levels, while reducing tight junction protein ZO-1 expression and increasing intestinal barrier permeability. Macrophage depletion reversed the impact of anemia on intestinal ZO-1 expression and barrier function. Conclusions Taken together, these results suggest that anemia can increase intestinal inflammation and barrier disruption likely through altered macrophage function, leading to the type of predisposing intestinal injury that may increase the risk for NEC.

Animal models have played a key role in providing an understanding of the mechanisms that govern the pathophysiology of intestinal diseases. To expand on the repertoire of organisms available to study enteric diseases, we report on the use of the Drosophila melanogaster model to identify a novel function of an effector protein secreted by Vibrio parahaemolyticus, which is an enteric pathogen found in contaminated seafood. During pathogenesis, V. parahaemolyticus secretes effector proteins that usurp the host’s innate immune signaling pathways, thus allowing the bacterium to evade detection by the innate immune system. One secreted effector protein, VopA, has potent inhibitory effects on mitogen-activated protein kinase (MAPK) signaling pathways via the acetylation of critical residues within the catalytic loops of mitogen-activated protein kinase kinases (MAPKKs). Using the Drosophila model and cultured mammalian cells, we show that VopA also has potent modulating activity on focal adhesion complex (FAC) proteins, where VopA markedly reduced the levels of focal adhesion kinase (FAK) phosphorylation at Ser910, whereas the phosphorylation levels of FAK at Tyr397 and Tyr861 were markedly increased. Cultured cells expressing VopA were also impaired in their ability to migrate and repopulate areas subjected to a scratch wound. Consistently, expression of VopA in Drosophila midgut enterocytes disrupted the normal enterocyte arrangement. Finally, VopA inhibited apoptosis in both Drosophila tissues and mammalian cultured cells. Together, our data show that VopA can alter normal intestinal homeostatic processes to facilitate opportunities for V. parahaemolyticus to prolong infection within the host.

Intestinal wounds often occur during inflammatory and ischemic disorders of the gut. To repair damage, intestinal epithelial cells must rapidly spread and migrate to cover exposed lamina propria, events that involve redox signaling. Wounds are subject to extensive redox alterations, particularly resulting from H2O2 produced in the adjacent tissue by both the epithelium and emigrating leukocytes. The mechanisms governing these processes are not fully understood, particularly at the level of protein signaling. Crk-associated substrate, or Cas, is an important signaling protein known to modulate focal adhesion and actin cytoskeletal dynamics, whose association with Crk is regulated by Abl kinase, a ubiquitously expressed tyrosine kinase. We sought to evaluate the role of Abl regulation of Cas at the level of cell spreading and migration during wound closure. As a model, we used intestinal epithelial cells exposed to H2O2 or scratch wounded to assess the Abl-Cas signaling pathway. We characterized the localization of phosphorylated Cas in mouse colonic epithelium under baseline conditions and after biopsy wounding the mucosa. Analysis of actin and focal adhesion dynamics by microscopy or biochemical analysis after manipulating Abl kinase revealed that Abl controls redox-dependent Cas phosphorylation and localization to influence cell spreading and migration. Collectively, our data shed new light on redox-sensitive protein signaling modules controlling intestinal wound healing.

Background & Aims A Western-style diet, which is high in fat and sugar, can cause significant dyslipidemia and nonalcoholic fatty liver disease; the diet has an especially strong effect in women, regardless of total calorie intake. Dietary supplementation with beneficial microbes might reduce the detrimental effects of a Western-style diet. We assessed the effects of Lactococcus lactis subspecies (subsp) cremoris on weight gain, liver fat, serum cholesterol, and insulin resistance in female mice on a high-fat, high-carbohydrate diet. Methods Female C57BL/6 mice were fed either a high-fat, high-carbohydrate (Western-style) diet that contained 40% fat (mostly milk fat) and 43% carbohydrate (mostly sucrose) or a calorie-matched-per-gram control diet. The diets of mice were supplemented with 1 × 109 colony-forming units of L lactis subsp cremoris ATCC 19257 or Lactobacillus rhamnosus GG ATCC 53103 (control bacteria) 3 times per week for 16 weeks. Body weights were measured, and fecal, blood, and liver tissues were collected and analyzed. Livers were analyzed for fat accumulation and inflammation, and blood samples were analyzed for cholesterol and glucose levels. Mice were housed within Comprehensive Lab Animal Monitoring System cages, and respiratory exchange ratio and activity were measured. Hepatic lipid profiles of L lactis subsp cremoris–supplemented mice were characterized by lipidomics mass spectrometry analysis. Results Mice fed L lactis subsp cremoris while on the Western-style diet gained less weight, developed less hepatic steatosis and inflammation, and had a lower mean serum level of cholesterol and body mass index than mice fed the control bacteria. Mice fed the L lactis subsp cremoris had increased glucose tolerance while on the Western-style diet compared to mice fed control bacteria and had alterations in hepatic lipids, including oxylipins. Conclusions Dietary supplementation with L lactis subsp cremoris in female mice on a high-fat, high-carbohydrate (Western-style) diet caused them to gain less weight, develop less liver fat and inflammation, reduce serum cholesterol levels, and increase glucose tolerance compared with mice on the same diet fed control bacteria. L lactis subsp cremoris is safe for oral ingestion and might be developed for persons with metabolic and liver disorders caused by a Western-style diet.

Background Although therapeutics in Crohn’s disease (CD) have dramatically improved, current approaches targeting the immune system have limited success at a population level. Thus, there is a need for future therapeutics to target different pathways/cell types. Recently, patient derived stem cells have shown promise in helping to better understand the molecular basis of disease. We used patient derived organoids (PDOs), produced from both CD and healthy patients’ pinch biopsies, to determine the ability of PDOs to functionally represent the human intestinal epithelium, and to better understand the role of the epithelium in CD. Methods Mucosal biopsies from the terminal ileum were obtained from pediatric subjects (CD = 16, healthy controls = 13) by colonoscopy. We performed bulk RNA-seq on the freshly isolated ileal crypts (IC), and on the corresponding PDOs grown from the IC stem cells. The sequencing was performed on an Illumina HiSeq system in a single batch. The raw FastQ files were aligned with reference genome Hg38 using STAR package. Differential expression analysis was performed on the read counts using DEseq package. We compared the organoid gene expression to that of the IC for each patient, along with the differential analysis between groups. Results On average, 7.5M read counts were obtained for each IC and organoid sample. Of the 19,900 genes tested against Hg38, we found 15,554 genes expressed in either IC or organoids. Of those, 14,149 (91%) were expressed in both IC and organoids, 1,091 (7%) were expressed only in IC, and 314 (2%) expressed only in organoids. We compared PDOs from healthy controls to CD cases and the differentially expressed genes were identified using cutoff of log2FC > ±1.2 and statistically significant threshold p < 0.05. Compared to healthy controls, CCL5 and CXCL5 genes were up-regulated in CD, whereas PDGFB, IL-33 and ILDR2 genes were down-regulated. Conclusion We demonstrate that PDOs are 91% similar in gene expression to in vivo epithelium and thus are an excellent patient-specific in vitro epithelial model system. We have further used this model to show the CD specific transcriptional defects in the epithelium. Our data suggests that the epithelium is responsible, in part, in promoting neutrophil recruitment and inflammation through increases in CCL5 and CXCL5 expression, and changes in the Th1/Th2 profile through downregulation of IL-33 expression. Dysregulation of the cytoskeleton and barrier formation are both likely affected by down regulation of PDGFB and ILDR2, respectively, resulting in a negative effect on intestinal barrier function and restitution.

Several proteins endogenously produced during the process of intestinal wound healing have demonstrated prorestitutive properties. The presence of serum amyloid A1 (SAA1), an acute-phase reactant, within inflamed tissues, where it exerts chemotaxis of phagocytes, is well recognized; however, a putative role in intestinal wound repair has not been described. Herein, we show that SAA1 induces intestinal epithelial cell migration, spreading, and attachment through a formyl peptide receptor 2–dependent mechanism. Induction of the prorestitutive phenotype is concentration and time dependent and is associated with epithelial reactive oxygen species production and alterations in p130 Crk-associated substrate staining. In addition, using a murine model of wound recovery, we provide evidence that SAA1 is dynamically and temporally regulated, and that the elaboration of SAA1 within the wound microenvironment correlates with the influx of SAA1/CD11b coexpressing immune cells and increases in cytokines known to induce SAA expression. Overall, the present work demonstrates an important role for SAA in epithelial wound recovery and provides evidence for a physiological role in the wound environment.

Many common inflammatory disorders are characterized by the infiltration of neutrophils across epithelial lined (mucosal) surfaces resulting in disruption of critical barrier function that protects from microbes and noxious agents. In such conditions, disease symptoms are complex but directly related to leukocyte effects on the barrier and epithelial cell function. It is now highly regarded that cellular factors such as cytokines and receptor–ligand interactions mediating adhesion of leukocytes to epithelial cells have potent effects on epithelial homeostasis, defined by coordinated proliferation, migration, differentiation, and regulated cell shedding. Certain cytokines, for example, not only alter leukocyte interactions with epithelia through changes in expression of adhesion molecules but also affect barrier function through alterations in the composition and dynamics of intercellular junctions. In particular, inflammation-induced loss of many tight junction molecules, in part, can account for dysregulated cellular proliferation, migration, survival, and barrier function. This review will highlight how neutrophils interact with epithelial cells with particular focus on adhesion molecules involved and signaling events that play roles in regulating mucosal homeostasis and pathobiology. A better understanding of these molecular events may provide new ideas for therapeutics directed at attenuating consequences of pathologic inflammation of mucosal surfaces.

Rubella virus (RUBV), a positive-strand RNA virus, replicates its RNA within membrane-associated replication complexes (RCs) in the cytoplasm of infected cells. RNA synthesis is mediated by the nonstructural proteins (NSPs) P200 and its cleavage products, P150 and P90 (N and C terminal within P200, respectively), which are processed by a protease residing at the C terminus of P150. In this study of NSP maturation, we found that early NSP localization into foci appeared to target the membranes of the endoplasmic reticulum. During maturation, P150 and P90 likely interact within the context of P200 and remain in a complex after cleavage. We found that P150-P90 interactions were blocked by mutational disruption of an alpha helix at the N terminus (amino acids [aa] 36 to 49) of P200 and that these mutations also had an effect on NSP targeting, processing, and membrane association. While the P150-P90 interaction also required residues 1700 to 1900 within P90, focus formation required the entire RNA-dependent RNA polymerase (aa 1700 to 2116). Surprisingly, the RUBV capsid protein (CP) rescued RNA synthesis by several alanine-scanning mutations in the N-terminal alpha helix, and packaged replicon assays showed that rescue could be mediated by CP in the virus particle. We hypothesize that CP rescues these mutations as well as internal deletions of the Q domain within P150 and mutations in the 5′ and 3′ cis -acting elements in the genomic RNA by chaperoning the maturation of P200. CP's ability to properly target the otherwise aggregated plasmid-expressed P200 provides support for this hypothesis.

A proline-rich region (PRR) within the rubella virus (RUBV) P150 replicase protein that contains three SH3 domain-binding motifs (PxxPxR) was investigated for its ability to bind cell proteins. Pull-down experiments using a glutathioneS-transferase–PRR fusion revealed PxxPxR motif-specific binding with human p32 protein (gC1qR), which could be mediated by either of the first two motifs. This finding was of interest because p32 protein also binds to the RUBV capsid protein. Binding of p32 to P150 was confirmed and was abolished by mutation of the first two motifs. When mutations in the first two motifs were introduced into a RUBV cDNA infectious clone, virus replication was significantly impaired. However, virus RNA synthesis was found to be unaffected, and subsequent immunofluorescence analysis of RUBV-infected cells revealed co-localization of p32 and P150 but little overlap of p32 with RNA replication complexes, indicating that p32 does not participate directly in virus RNA synthesis. Thus, the role of p32 in RUBV replication remains unresolved.

The P150 and P90 replicase proteins of rubella virus (RUBV), a plus-strand RNA Togavirus, produce a unique cytoplasmic fiber network resembling microtubules. Pharmacological and mutagenic approaches were used to determine if these fibers functioned in virus replication. The pharmacological approach revealed that microtubules were required for fiber formation, but neither was necessary for virus replication. Through the mutagenic approach it was found that α-helices near both termini of P150 were necessary for fiber assembly and infectivity, but fiber formation and viability could not be correlated because most of these mutations were lethal. The N-terminal α-helix of P150 affected both proteolytic processing of P150 and P90 from the P200 precursor and targeting of P200, possibly through directing conformational folding of P200. Finally, we made the unexpected discovery that RUBV genomes can spread from cell-to-cell without virus particles, a process that we hypothesize utilizes RUBV-induced cytoplasmic projections containing fibers and replication complexes.

The rubella virus (RUBV) nonstructural replicase proteins (NSPs), P150 and P90, are proteolytically processed from a P200 precursor. To understand the NSPs' function in the establishment of virus RNA replication complexes (RCs), the NSPs were analyzed in virus-infected cells or cells transfected with NSP-expressing plasmids. In infected cells, P150 was localized in cytoplasmic foci at 24 hpi and in cytoplasmic fibers, unique to RUBV, by 48 hpi. RCs, marked by dsRNA, colocalized with P150-foci, but only occasionally with the endosome/lysosome marker LAMP-2, indicating that RNA synthesis occurs at other sites rather than exclusively in endosomes/lysosomes as was previously thought. An expressed cleavage-deficient form of P200 also localized to cytoplasmic foci, suggesting that the precursor is required for targeting to sites of RC establishment. P150 was found to be the determinant of fiber formation and the NSP membrane-binding domain was mapped to the N-terminus of P150.

The hepatitis C virus (HCV) is a leading cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma and is also a common indication for liver transplantation. Interferon alfa monotherapy leads to a sustained virologic response in only 10% to 15% of HCV-infected patients. The sustained virologic response rates can be increased to approximately 40% with interferon alfa-2b and ribavirin combination therapy for up to 48 weeks. However, recent clinical trials demonstrated that pegylated interferon in combination with ribavirin can improve the response rates even further, with more than 50% of patients having a sustained virologic response to treatment. Although new therapies are emerging, significant progress must be made to reduce the morbidity and mortality from HCV infection.

AIM: To describe the prevalence, diagnosis, treatment, and outcomes of end stage liver disease (ESLD) patients with severe epistaxis thought to be severe upper gastrointestinal hemorrhage (UGIH). METHODS: This observational single center study included all consecutive patients with ESLD and epistaxis identified from consecutive subjects hospitalized with suspected UGIH and prospectively enrolled in our databases of severe UGIH between 1998 and 2011. RESULTS: A total of 1249 patients were registered for severe UGIH in the data basis, 461 (36.9%) were cirrhotics. Epistaxis rather than UGIH was the bleeding source in 20 patients. All patients had severe coagulopathy. Epistaxis was initially controlled in all cases. Fifteen (75%) subjects required posterior nasal packing and 2 (10%) embolization in addition to correction of coagulopathy. Five (25%) patients died in the hospital, 12 (60%) received orthotopic liver transplantation (OLT), and 3 (15%) were discharged without OLT. The mortality rate was 63% in patients without OLT. CONCLUSION: Severe epistaxis in patients with ESLD is (1) a diagnosis of exclusion that requires upper endoscopy to exclude severe UGIH; and (2) associated with a high mortality rate in patients not receiving OLT.

Junctional adhesion molecule-A (JAM-A) is a tight junction protein that plays a key role in regulation of intestinal mucosal homeostasis. Previous studies have shown that loss of JAM-A decreases be...

Rubella virus (RUBV) replicates slowly and to low titre in vertebrate cultured cells, with minimal cytopathology. To determine whether a cellular stress response is induced during such an infection, the formation of Ras-GAP-SH3 domain-binding protein (G3BP)-containing stress granules (SGs) in RUBV-infected cells was examined. Late in infection, accumulation of G3BP granules was detected, albeit in fewer than half of infected cells. Active virus RNA replication was required for induction of these granules, but they were found to differ from SGs induced by arsenite treatment both in composition (they did not uniformly contain other SG proteins, such as PABP and TIA-1) and in resistance to cycloheximide treatment. Thus, bona fide SGs do not appear to be induced during RUBV infection. The distribution of G3BP, either on its own or in granules, did not overlap with that of dsRNA-containing replication complexes, indicating that it played no role in virus RNA synthesis. However, G3BP did co-localize with viral ssRNAs in perinuclear clusters, suggesting an interaction that could possibly be important in a post-replicative role in virus replication, such as encapsidation.

The rubella virus capsid protein (C) has been shown to complement a lethal deletion (termed ΔNotI) in P150 replicase protein. To investigate this phenomenon, we generated two lines of Vero cells that stably expressed either C (C-Vero cells) or C lacking the eight N-terminal residues (CΔ8-Vero cells), a construct previously shown to be unable to complement ΔNotI. In C-Vero cells but not Vero or CΔ8-Vero cells, replication of a wild-type (wt) replicon expressing the green fluorescent protein (GFP) reporter gene (RUBrep/GFP) was enhanced, and replication of a replicon with ΔNotI (RUBrep/GFP-ΔNotI) was rescued. Surprisingly, replicons with deleterious mutations in the 5′ and 3′ cis -acting elements were also rescued in C-Vero cells. Interestingly, the CΔ8 construct localized to the nucleus while the C construct localized in the cytoplasm, explaining the lack of enhancement and rescue in CΔ8-Vero cells since rubella virus replication occurs in the cytoplasm. Enhancement and rescue in C-Vero cells were at a basic step in the replication cycle, resulting in a substantial increase in the accumulation of replicon-specific RNAs. There was no difference in translation of the nonstructural proteins in C-Vero and Vero cells transfected with the wt and mutant replicons, demonstrating that enhancement and rescue were not due to an increase in the efficiency of translation of the transfected replicon transcripts. In replicon-transfected C-Vero cells, C and the P150 replicase protein associated by coimmunoprecipitation, suggesting that C might play a role in RNA replication, which could explain the enhancement and rescue phenomena. A unifying model that accounts for enhancement of wt replicon replication and rescue of diverse mutations by the rubella virus C protein is proposed.