Survival of cheese-ripening microorganisms in a dynamic simulator of the gastrointestinal tract.

A mixture of nine microorganisms (six bacteria and three yeasts) from the microflora of surface-ripened cheeses were subjected to in vitro digestive stress in a three-compartment "dynamic gastrointestinal digester" (DIDGI). We studied the microorganisms (i) grown separately in culture medium only (ii) grown separately in culture medium and then mixed, (iii) grown separately in culture medium and then included in a rennet gel and (iv) grown together in smear-ripened cheese. The yeasts Geotrichum candidum, Kluyveromyces lactis and Debaryomyces hansenii, were strongly resistant to the whole DIDGI process (with a drop in viable cell counts of less than <1 log CFU mL(-1)) and there were no significant differences between lab cultures and cheese-grown cultures. Ripening bacteria such as Hafnia alvei survived gastric stress less well when grown in cheese (with no viable cells after 90 min of exposure of the cheese matrix, compared with 6 CFU mL(-1) in lab cultures). The ability of Corynebacterium casei and Staphylococcus equorum to withstand digestive stress was similar for cheese and pure culture conditions. When grow in a cheese matrix, Brevibacterium aurantiacum and Arthrobacter arilaitensis were clearly more sensitive to the overall digestive process than when grown in pure cultures. Lactococcus lactis displayed poorer survival in gastric and duodenal compartments when it had been grown in cheese. In vivo experiments in BALB/c mice agreed with the DIDGI experiments and confirmed the latter's reliability.

Modifying the protease, antiprotease pattern by elafin overexpression protects mice from colitis.

BACKGROUND & AIMS: Colonic tissues of patients with inflammatory bowel disease have been reported to have increased proteolytic activity, but no studies have clearly addressed the role of the balance between proteases and antiproteases in the pathogenesis of colitis. We investigated the role of Elafin, a serine protease inhibitor expressed by skin and mucosal surfaces in human inflammatory conditions, and the proteases neutrophil elastase (NE) and proteinase-3 (PR-3) in mice with colitis. METHODS: We studied mice with heterozygous disruptions in NE and PR-3, mice that express human elafin (an inhibitor of NE and PR-3), and naïve mice that received intracolonic adenoviral vectors that express elafin. Trinitrobenzene sulfonic acid (TNBS) or dextran sodium sulphate (DSS) was used to induce colitis. Protease, cytokine levels, and NF-κB activity were measured in colons of mice. Caco-2 and HT29 cells were studied in assays for cytokine expression, permeability, and NF-κB activity. RESULTS: Elafin expression or delivery re-equilibrated the proteolytic balance in inflamed colons of mice. In mice given TNBS or DSS, transgenic expression of elafin or disruption of NE and PR-3 protected against the development of colitis. Similarly, adenoviral delivery of Elafin significantly inhibited inflammatory parameters. Elafin modulated a variety of inflammatory mediators in vitro and in vivo and strengthened intestinal epithelial barrier functions. CONCLUSIONS: The protease inhibitor Elafin prevents intestinal inflammation in mouse models of colitis and might be developed as a therapeutic agent for inflammatory bowel disease.

ATF4 and the integrated stress response are induced by ethanol and cytochrome P450 2E1 in human hepatocytes.

BACKGROUND & AIMS: Molecular mechanisms underlying alcoholic liver disease (ALD) are still not fully understood. Activating transcription factor-4 (ATF4) is the master coordinator of the integrated stress response (ISR), an adaptive pathway triggered by multiple stressors. which can promote cell death and induce metabolic dysregulation if the stress is intense or prolonged. The aim of this study was to assess the effect of alcohol on the ISR signaling pathway in human liver cells and to define the role of cytochrome P450 2E1 (CYP2E1) in this response. METHODS: Primary cultured human hepatocytes and human HepG2 cells over-expressing CYP2E1 by adenoviral infection were exposed to ethanol (25-100mM) for 8-48h. RESULTS: Ethanol treatment of both liver cells up-regulated ATF4 as well as the pro-survival and the pro-apoptotic transcriptional program of the ISR. Indeed, in CYP2E1-expressing HepG2 cells exposed to ethanol, the expression of ISR target genes (HMOX-1, GCLC, AsnS, IGFBP-1, GADD34,CHOP, ATF3, CHAC1) was induced. Up-regulation of ATF4 and the ISR transcriptional program was decreased by addition of the anti-oxidant glutathione. Several mechanisms mediated ATF4 protein induction, including, at early times, the phosphorylation of eIF2α which controls ATF4 translation, and, at later times, increased mRNA level and increased stability of the protein. A decrease in cell survival was also observed. CONCLUSIONS: This study demonstrates that both CYP2E1 and ethanol induce ATF4 and the integrated stress response, a pathway which coordinates signals from multiple stresses, as well as established risk factors for ALD, and can display detrimental cellular effects upon prolonged activation.

A mycobacterial coinfection in a dog suspected on blood smear.

A 4-year-old neutered female crossbred Shepherd was referred for a history of 10 days of anorexia, polyuria, polydipsia, polyadenomegaly, and diarrhea. On physical examination, the dog appeared quiet, responsive, and apyretic, with generalized and severe lymphadenomegaly. Hematologic abnormalities included neutrophilic leukocytosis with left shift, and lymphopenia. Blood smears revealed intracytoplasmic bacilli negatively stained with May-Grünwald-Giemsa in neutrophils and monocytes. Lymph node smears revealed pyogranulomatous adenitis with calcified deposits and many negative-staining rod structures, both within the cytoplasm of neutrophils and macrophages, and free in the background. An acid-fast stain (Ziehl-Neelsen) confirmed the diagnosis of mycobacterial infection. The dog was euthanized for public health and ethical reasons, and the postmortem examination revealed severe and generalized granulomatous and necrotizing lymphadenitis, panniculitis, and hepatitis, and infiltration of epithelioid macrophages in the lungs, colon, and spleen. Numerous acid-fast bacilli, consistent with mycobacterial infection, were observed both in the cytoplasm of epithelioid macrophages and giant cells, and free in the background. Mycobacterium bovis was first confirmed by conventional PCR of organ extracts. Mycobacterium avium was detected in a culture of the same organs. Further PCR amplifications and sequencing revealed a coinfection with 2 different species of mycobacterium, one belonging to the Mycobacterium avium complex and the other to the Mycobacterium tuberculosis complex.

Stabilization of IGFBP-1 mRNA by ethanol in hepatoma cells involves the JNK pathway.

BACKGROUND/AIMS: Insulin-like growth factor-binding protein-1 (IGFBP-1) modulates cell growth and metabolism in a variety of physiopathological conditions. The aim of this study was to determine the molecular mechanisms involved in IGFBP-1 upregulation by ethanol. METHODS: We studied IGFBP-1 regulation by ethanol at the protein, mRNA and gene promoter levels in the human hepatocarcinoma cell line, HepG2, which does not express significantly ethanol-metabolizing enzymes. RESULTS: Ethanol (35-150mM) induced the IGFBP-1 mRNA and protein up to 5-fold in a dose-dependent manner. A similar effect was observed using primary cultures of human hepatocytes. Various inhibitors of ethanol metabolism and the antioxidant N-acetylcysteine did not prevent ethanol effects. While ethanol did not modify the IGFBP-1 gene promoter activity, it elicited a 2- to 3-fold increase in IGFBP-1 mRNA half-life and this stabilization required the 5' and the 3' untranslated mRNA region. Ethanol triggered a rapid activation of c-Jun N-terminal Kinase (JNK) in HepG2 cells and IGFBP-1 induction was significantly decreased by a specific inhibitor of JNK. CONCLUSIONS: This study reveals a novel pathway of gene regulation by alcohol which involves the activation of JNK and the consequent mRNA stabilization.

Endoplasmic reticulum stress induction of insulin-like growth factor-binding protein-1 involves ATF4.

Endoplasmic reticulum (ER) stress is sensed by cells in different physiopathological conditions in which there is an accumulation of unfolded proteins in the ER. A coordinated adaptive program called the unfolded protein response is triggered and includes translation inhibition, transcriptional activation of a set of genes encoding mostly intracellular proteins, and ultimately apoptosis. Here we show that insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1), a secreted protein that modulates IGF bioavailability and has other IGF-independent effects, is potently induced during ER stress in human hepatocytes. Various ER stress-inducing agents were able to increase IGFBP-1 mRNA levels, as well as cellular and secreted IGFBP-1 protein up to 20-fold. A distal regulatory region of the human IGFBP-1 gene (-6682/-6384) containing an activating transcription factor 4 (ATF4) composite site was required for promoter activation upon ER stress. Mutation of the ATF4 composite site led to the loss of IGFBP-1 regulation. Electrophoretic mobility shift assay revealed an ER stress-inducible complex that was displaced by an ATF4 antibody. Knockdown of ATF4 expression using two specific small interfering RNAs impaired up-regulation of IGFBP-1 mRNA, which highlights the relevance of ATF4 in endogenous IGFBP-1 gene induction. In addition to intracellular proteins involved in secretory and metabolic pathways, we conclude that ER stress induces the synthesis of secreted proteins. Increased secretion of IGFBP-1 during hepatic ER stress may thus constitute a signal to modulate cell growth and metabolism and induce a systemic adaptive response.