IL-22 and Lactobacillus delbrueckii mitigate alcohol-induced exacerbation of DSS-induced colitis.

Ulcerative colitis (UC) is characterized by cycles of active disease flare and inactive disease remission. During UC remission, IL-22 is up-regulated, acting as a hallmark of entrance into UC remission. Recently, we found that in our mouse model of binge alcohol and dextran sodium sulfate (DSS)-induced colitis, alcohol increases severity of UC pathology. In this study, we assessed not only whether alcohol influenced IL-22 expression and thereby perpetuates UC, but also whether recombinant IL-22 (rIL-22) or treatment with a probiotic could alleviate exacerbated symptoms of UC. Levels of large intestine IL-22 were significantly decreased ∼6.9-fold in DSS ethanol compared with DSS vehicle. Examination of lamina propria (LP) cells in the large intestine revealed IL-22+ γδ T cells in DSS vehicle-treated mice were significantly increased, while IL-22+ γδ T cells in DSS ethanol mice were unable to mount this IL-22 response. We administered rIL-22 and found it restored weight loss of DSS ethanol-treated mice. Colonic shortening and increased Enterobacteriaceae were also attenuated. Administration of Lactobacillus delbrueckii attenuated weight loss (p < 0.01), colon length (p < 0.001), mitigated increases in Enterobacteriaceae, increased levels of IL-22, and increased levels of p-STAT3 back to that of DSS vehicle group in DSS ethanol mice. In contrast, sole administration of L. delbrueckii supernatant was not sufficient to reduce UC exacerbation following alcohol. Our findings suggest L. delbrueckii contributes to repair mechanisms by increasing levels of IL-22, resulting in phosphorylation of STAT3, thus attenuating the alcohol-induced increases in intestinal damage after colitis.

Clinical Manifestations and Stool Load of Atypical Enteropathogenic Escherichia coli Infections in United States Children and Adults.

BACKGROUND & AIMS: There is debate whether atypical enteropathogenic Escherichia coli (aEPEC) causes disease in adults. aEPEC is commonly detected in symptomatic and asymptomatic individuals. aEPEC, in contrast to typical EPEC, lacks bundle-forming pili, altering its pathogenicity. Here, we define for the first time the clinical manifestations of sporadic aEPEC infection in United States children and adults and determine whether EPEC load correlates with disease. METHODS: This is a retrospective case-control study of 380 inpatients/outpatients of all ages. EPEC load in stools was determined by quantitative polymerase chain reaction. RESULTS: Diarrhea, vomiting, abdominal pain, and fever were more prevalent in EPEC-positive cases than in EPEC-negative controls. aEPEC infection caused mostly acute, mild diarrhea lasting for 6 to 13 days. However, some had severe diarrhea with 10 to 40 bowel movements per day or had persistent/chronic diarrhea. Fever, vomiting, and abnormal serum sodium levels were more common in children. Adults more often reported abdominal pain and longer duration of diarrhea. Symptomatic aEPEC infection was associated with leukocytosis in 24% of patients. EPEC load >0.1% was associated with symptomatic infection; however, loads varied greatly. Co-infecting pathogens did not alter diarrhea severity or EPEC load. Longitudinal data reveal that some are colonized for months to years or are repeatedly infected. CONCLUSIONS: aEPEC is associated with a wide array of symptoms in adults, ranging from asymptomatic carriage to severe diarrhea. Higher EPEC loads are associated with presence of symptoms, but bacterial load does not predict disease or severity. Future studies are needed to understand bacterial and host factors that contribute to aEPEC pathogenicity to improve diagnostic tools and clinical care.

Alcohol decreases intestinal ratio of Lactobacillus to Enterobacteriaceae and induces hepatic immune tolerance in a murine model of DSS-colitis.

Alcohol can potentiate disease in a mouse model of dextran sodium sulfate (DSS) colitis; however, the underlying mechanism remains to be established. In this study, we assessed whether the potentiated disease could be related to Enterobacteriaceae and Lactobacillus, as changes in their relative abundance can impact intestinal health. We also assessed whether the intestinal barrier is compromised after alcohol and DSS as it may increase bacterial translocation and liver inflammation. Mice were administered DSS followed by binge ethanol or water vehicle, generating four experimental groups: (Control+Vehicle, Control+Ethanol, DSS+Vehicle, DSS+Ethanol). DNA was isolated from colon and cecal contents followed by qPCR for levels of Enterobacteriaceae and Lactobacillus. Colon and liver sections were taken for histology. Intestinal epithelial cells were isolated from the colon for RNA expression. DSS+Ethanol cecal contents exhibited a 1 log increase in Enterobacteriaceae (p < .05), a 0.5 log decrease in Lactobacillus, and a 1.5 log decrease (p < .05) in the Lactobacillus:Enterobacteriaceae ratio compared to DSS+Vehicle, with similar trends in colon contents. These changes correlated with shorter colons and more weight loss. Irrespective of ethanol administration, DSS compromised the mucosal barrier integrity, however only DSS+Ethanol exhibited significant increases in circulating endotoxin. Furthermore, the livers of DSS+Ethanol mice had significantly increased levels of triglycerides, mononuclear cells, yet exhibited significantly depressed expression of liver inflammatory pathways, suggestive of tolerance induction, compared to mice receiving DSS+Vehicle. Our results suggest that ethanol after DSS colitis increases the intestinal burden of Enterobacteriaceae which may contribute to intestinal and liver damage, and the induction of immune tolerance.

Inhalation Injury: Unmet Clinical Needs and Future Research.

Pulmonary and systemic insults from inhalation injury can complicate the care of burn patients and contribute to significant morbidity and mortality. However, recent progress in diagnosis and treatment of inhalation injury has not kept pace with the care of cutaneous thermal injury. There are many challenges unique to inhalation injury that have slowed advancement, including deficiencies in our understanding of its pathophysiology, the relative difficulty and subjectivity of bronchoscopic diagnosis, the lack of diagnostic biomarkers, the necessarily urgent manner in which decisions are made about intubation, and the lack of universal recommendations for the application of mucolytics, anticoagulants, bronchodilators, modified ventilator strategies, and other measures. This review represents a summary of critical shortcomings in our understanding and management of inhalation injury identified by the American Burn Association's working group on Cutaneous Thermal Injury and Inhalation Injury in 2018. It addresses our current understanding of the diagnosis, pathophysiology, and treatment of inhalation injury and highlights topics in need of additional research, including 1) airway repair mechanisms; 2) the airway microbiome in health and after injury; and 3) candidate biomarkers of inhalation injury.

Summary of the 2018 Alcohol and Immunology Research Interest Group (AIRIG) meeting.

On January 26, 2018, the 23rd annual Alcohol and Immunology Research Interest Group (AIRIG) meeting was held at the University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado. The meeting consisted of plenary sessions with oral presentations and a poster presentation session. There were four plenary sessions that covered a wide range of topics relating to alcohol use: Alcohol and Liver Disease; Alcohol, Inflammation and Immune Response; Alcohol and Organ Injury; Heath Consequences and Alcohol Drinking. The meeting provided a forum for the presentation and discussion of novel research findings regarding alcohol use and immunology.

Alcohol enhances symptoms and propensity for infection in inflammatory bowel disease patients and a murine model of DSS-induced colitis.

Over 1.4 million Americans have been diagnosed with inflammatory bowel disease (IBD), and ulcerative colitis (UC) makes up approximately half of those diagnoses. As a disease, UC cycles between periods of remission and flare, which is characterized by intense abdominal pain, increased weight loss, intestinal inflammation, rectal bleeding, and dehydration. Interestingly, a widespread recommendation to IBD patients for avoidance of a flare period is "Don't Drink Alcohol" as recent work correlated alcohol consumption with increased GI symptoms in patients with IBD. Alcohol alone not only induces a systemic pro-inflammatory response, but can also be directly harmful to gut barrier integrity. However, how alcohol could result in the exacerbation of UC in both patients and murine models of colitis has yet to be elucidated. Therefore, we conducted a retrospective analysis of patients admitted for IBD with a documented history of alcohol use in conjunction with a newly developed mouse model of binge alcohol consumption following dextran sulfate sodium (DSS)-induced colitis. We found that alcohol negatively impacts clinical outcomes of patients with IBD, specifically increased intestinal infections, antibiotic injections, abdomen CT scans, and large intestine biopsies. Furthermore, in our mouse model of binge alcohol consumption following an induced colitis flare, we found alcohol exacerbates weight loss, clinical scores, colonic shortening and inflammation, and propensity to infection. These findings highlight alcohol's ability to potentiate symptoms and susceptibility to infection in UC and suggest alcohol as an underlying factor in perpetuating symptoms of IBD.

ABCB1 transporter and Toll-like receptor 4 in trabecular meshwork cells.

PURPOSE: The aqueous humor nourishes the avascular tissues of the anterior segment, and the trabecular meshwork (TM) plays a role in the efflux of endogenous substances and xenobiotics from the aqueous humor. ATP (ATP)-binding cassette (ABC) transporter superfamily members respond to stressors such as hypoxia, cytokine signaling, and aging. The innate immune system within the TM, particularly Toll-like receptor 4 (TLR4) and its ligands, e.g., low-molecular-weight hyaluronic acid (LMW-HA) and lipopolysaccharide (LPS), plays a significant role in maintaining a normal environment in the anterior chamber. We hypothesize that the innate immune system may interact with ATP-binding cassette sub-family members ABCB1 (p-glycoprotein and multidrug resistance protein 1) to detoxify xenobiotics from the aqueous humor and in the TM. METHODS: Cell lysates of human TM cells, RAW 264.7 macrophages, and PC12 cells were subjected to western blot analysis. The TM cells were positive for TLR4, ABCB1, and CYP3A5 and were negative for the ABCC1 transporter. Human TM cells and RAW 264.7 macrophages were plated on eight-well chamber slides at 5,000 cells/well overnight in 10% fetal bovine serum (FBS) cell growth medium. The medium was changed to 0.1% FBS 2 h before treatment. Cells were challenged with 1 and 10 mM lactate, 100 ng LMW-HA (20 kDa), 100 ng high-molecular-weight HA (HMW-HA, 1,000 kDa), 100 ng LPS, and/or 100 µM naloxone for 0.5, 1, 2, and 4 h. Calcein acetyoxymethyl ester (calcein AM; 0.25 µM) was added for 30 min as the reporting molecule. After calcein AM was administered, it was cleaved by an esterase into a fluorescent product that is normally transported out of the cell by ABCB1. Positive controls were 100 µM verapamil and 50 µM digoxin. After the challenge, the TM cells were fixed at 4 °C in 3% paraformaldehyde for 15 min, mounted with Vectashield and 4',6-diamidino-2-phenylindole (DAPI) mounting medium, and analyzed by a masked observer using a Leica confocal microscope and software. RESULTS: Verapamil, an ABCB1 inhibitor, significantly (p<0.001) increased fluorescent calcein retention in the cytoplasm of the TM and RAW 264.7 cells compared to the PBS control. Digoxin, an ABCB1 activator, increased calcein efflux (p<0.001). Lactate reduced ABCB1 activity. HMW-HA significantly (p<0.001) reduced ABCB1 activity, whereas LMW-HA decreased ABCB1 activity, and the HA effects were blocked by naloxone (p<0.001), a TLR4 inhibitor. LPS alone did not change ABCB1 activity whereas dephosphorylated LPS significantly (p<0.001) enhanced ABCB1 activity in the TM cells. ß-amyloid significantly reduced ABCB1 activity, and the ß-amyloid effects were blocked by naloxone. CONCLUSIONS: TM cells are responsive to ABCB1 inhibitors and activators. ABCB1 functional activity is affected by TLR4 agonists suggesting that modulation of TLR4 is important in ABCB1 function. The innate immune inflammatory response in the TM may play a role in the ABCB1 detoxification of potentially harmful constituents in the aqueous humor.

sCD44 overexpression increases intraocular pressure and aqueous outflow resistance.

PURPOSE: CD44 plays major roles in multiple physiologic processes. The ectodomain concentration of the CD44 receptor, soluble CD44 (sCD44), is significantly increased in the aqueous humor of primary open-angle glaucoma (POAG). The purpose of this study was to determine if adenoviral constructs of CD44 and isolated 32-kDa sCD44 change intraocular pressure (IOP) in vivo and aqueous outflow resistance in vitro. METHODS: Adenoviral constructs of human standard CD44 (Ad-CD44S), soluble CD44 (Ad-sCD44), and empty viral cDNA were injected into the vitreous of BALB/cJ mice, followed by serial IOP measurements. Overexpression of CD44S and sCD44 was verified in vitro by enzyme-linked immunosorbent assay (ELISA) and western blot analysis. Anterior segments of porcine eyes were perfused with the isolated sCD44. sCD44-treated human trabecular meshwork (TM) cells and microdissected porcine TM were examined by confocal microscopy and Optiprep density gradient with western blot analysis to determine changes in lipid raft components. RESULTS: Intravitreous injection of adenoviral constructs with either Ad-CD44S or Ad-sCD44 vectors caused prolonged ocular hypertension in mice. Eight days after vector injection, Ad-CD44S significantly elevated IOP to 28.3±1.2 mmHg (mean±SEM, n=8; p<0.001); Ad-sCD44 increased IOP to 18.5±2.6 mmHg (n=8; p<0.01), whereas the IOP of uninjected eyes was 12.7±0.2 mmHg (n=16). The IOP elevation lasted more than 50 days. Topical administration of a γ-secretase inhibitor normalized Ad-sCD44-induced elevated IOP. sCD44 levels were significantly elevated in the aqueous humor of Ad-CD44S and Ad-sCD44 eyes versus contralateral uninjected eyes (p<0.01). Anterior segment perfusion of isolated 32-kDa sCD44 significantly decreased aqueous outflow rates. Co-administration of isolated sCD44 and CD44 neutralizing antibody or of γ-secretase inhibitor significantly enhanced flow rates. sCD44-treated human TM cells displayed cross-linked actin network formation. Optiprep density gradient and western blot analysis of human TM cells treated with sCD44 showed decreased annexin 2 expression and increased phosphorylated annexin 2 and caveolin 1 expression. CONCLUSIONS: Our data suggest that sCD44 increases outflow resistance in vivo and in vitro. Viral overexpression of both CD44S and sCD44 is sufficient to cause ocular hypertension. Infusion of sCD44 in porcine anterior segment eyes significantly decreased flow rates. Notably, sCD44 enhanced cross-linked actin network formation. The elevated sCD44 levels seen in POAG aqueous humor may play an important causative role in POAG pathogenesis.

Identification of telomerase RNAs from filamentous fungi reveals conservation with vertebrates and yeasts.

Telomeres are the nucleoprotein complexes at eukaryotic chromosomal ends. Telomeric DNA is synthesized by the ribonucleoprotein telomerase, which comprises a telomerase reverse transcriptase (TERT) and a telomerase RNA (TER). TER contains a template for telomeric DNA synthesis. Filamentous fungi possess extremely short and tightly regulated telomeres. Although TERT is well conserved between most organisms, TER is highly divergent and thus difficult to identify. In order to identify the TER sequence, we used the unusually long telomeric repeat sequence of Aspergillus oryzae together with reverse-transcription-PCR and identified a transcribed sequence that contains the potential template within a region predicted to be single stranded. We report the discovery of TERs from twelve other related filamentous fungi using comparative genomic analysis. These TERs exhibited strong conservation with the vertebrate template sequence, and two of these potentially use the identical template as humans. We demonstrate the existence of important processing elements required for the maturation of yeast TERs such as an Sm site, a 5' splice site and a branch point, within the newly identified TER sequences. RNA folding programs applied to the TER sequences show the presence of secondary structures necessary for telomerase activity, such as a yeast-like template boundary, pseudoknot, and a vertebrate-like three-way junction. These telomerase RNAs identified from filamentous fungi display conserved structural elements from both yeast and vertebrate TERs. These findings not only provide insights into the structure and evolution of a complex RNA but also provide molecular tools to further study telomere dynamics in filamentous fungi.

sCD44 internalization in human trabecular meshwork cells.

PURPOSE: To determine whether soluble CD44 (sCD44), a likely biomarker of primary open-angle glaucoma (POAG), is internalized in cultured human trabecular meshwork (TM) cells and trafficked to mitochondria. METHODS: In vitro, 32-kD sCD44 was isolated from human sera, biotinylated, and dephosphorylated. TM cells were incubated for 1 hour at 4°C with biotinylated albumin (b-albumin), biotin-labeled sCD44 (b-sCD44), or hypophosphorylated biotin-labeled sCD44 (-p b-sCD44) in the presence or absence of unlabeled sCD44, hyaluronic acid (HA), and a selected 10-mer HA binding peptide. The slides were warmed for 1 or 2 hours at 37°C, and 125 nM MitoTracker Red was added for the last 20 minutes of the incubation. The cells were washed, fixed, incubated with anti-biotin antibody and FITC-labeled goat anti-mouse antibody, and examined under a confocal microscope. RESULTS: TM cell membranes were positive for b-sCD44 after 4°C incubation. When the temperature was raised to 37°C, b-sCD44 or -p b-sCD44 appeared in the cytoplasm. The internalization of b-sCD44 was blocked by excess unlabeled sCD44, HA, and a 10-mer HA-binding peptide. Double label experiments with b-sCD44 or -p b-sCD44 and MitoTracker Red indicated partial overlap. The percent co-localization of MitoTracker Red at 2 hours and FITC -p b-sCD44 was 17.4% (P < 0.001) and for FITC b-sCD44 was 11.7% (P < 0.001) compared with b-albumin. The influence of putative CD44 phosphorylation sites on mitochondrial trafficking was determined by TargetP 1.1. CONCLUSIONS: sCD44 is internalized by TM cells and trafficked in part to mitochondria, which may be a factor in the toxicity of sCD44 in the POAG disease process.