Gut--liver axis: the impact of gut microbiota on non alcoholic fatty liver disease.

AIM: To examine the impact of gut microbiota on non alcoholic fatty liver disease (NAFLD) pathogenesis. DATA SYNTHESIS: Emerging evidence suggests a strong interaction between gut microbiota and liver. Receiving approximately 70% of its blood supply from the intestine, the liver represents the first line of defence against gut-derived antigens. Intestinal bacteria play a key role in the maintenance of gut-liver axis health. Disturbances in the homeostasis between bacteria- and host-derived signals at the epithelial level lead to a break in intestinal barrier function and may foster "bacterial translocation", defined as the migration of bacteria or bacterial products from the intestinal lumen to mesenteric lymph nodes or other extraintestinal organs and sites. While the full repertoire of gut-derived microbial products that reach the liver in health and disease has yet to be explored, the levels of bacterial lipopolysaccharide, a component of the outer membrane of Gram-negative bacteria, are increased in the portal and/or systemic circulation in several types of chronic liver diseases. Derangement of the gut flora, particularly small intestinal bacterial overgrowth, occurs in a large percentage (20-75%) of patients with chronic liver disease. In addition, evidence implicating the gut-liver axis in the pathogenesis of metabolic liver disorders has accumulated over the past ten years. CONCLUSIONS: Complex metabolic diseases are the product of multiple perturbations under the influence of triggering factors such as gut microbiota and diet, thus, modulation of the gut microbiota may represent a new way to treat or prevent NAFLD.

Bombesin: a possible role in wound repair.

During tissue regeneration and wound healing of the skin, migration, proliferation and differentiation of keratinocytes are important processes. Here we assessed the effect of a neuropeptide, bombesin, on keratinocytes during regeneration from scratch wounding. Bombesin purified from amphibian skin, is homologous of mammalian gastrin-releasing peptide and is active in mammals. Its pharmacological effects mediate various physiological activities: hypertensive action, stimulating action on gastric secretion, hyperglycemic effect or increased insulin secretion. In vitro it shows a hyperproliferative effect on different experimental models and is involved in skin repair. The aim of this study was to elucidate the effect of Bombesin in an in vitro experimental model on a mechanically injured human keratinocyte monolayer. We evaluated different mediators involved in wound repair such as IL-8, TGFbeta, IL-1, COX-2, VEGF and Toll-like receptors 2 and 4 (TLR2 and TLR4). We also studied the effects of bombesin on cell proliferation and motility and its direct effect on wound repair by observing the wound closure after mechanical injury. The involvement of the bombesin receptors neuromedin receptor (NMBR) and gastrin-releasing peptide receptor (GRP-R) was also evaluated. Our data suggest that bombesin may have an important role in skin repair by regulating the expression of healing markers. It enhanced the expression of IL-8, TGFbeta, COX-2 and VEGF. It also enhanced the expression of TLR2, while TLR4 was not expressed. Bombesin also increased cell growth and migration. In addition, we showed that NMBR was more involved in our experimental model compared to GRP-R.

Anti-apoptotic seminal vesicle protein IV inhibits cell-mediated immunity.

The in vitro effect of seminal vesicle protein IV (SV-IV) on the cytotoxic activity of human natural or acquired cellular immunity has been investigated by standard immunological procedures, a (51)Cr-release cytotoxicity assay, and labeled-ligand binding experiments. The data obtained demonstrate that: (1) fluoresceinated or [(125)I]-labeled SV-IV binds specifically to the surface of human purified non-adherent mononuclear cells (NA-MNC); (2) SV-IV suppresses the cytotoxicity of natural killer (NK) cells against K562 target cells, that of IL-2-stimulated NK (LAK) cells against DAUDI target cells, and that of VEL antigen-sensitized cytotoxic T lymphocytes (CTLs) against VEL target cells; (3) treatment of K562 target cells alone with SV-IV decreases their susceptibility to NK-induced lysis. These findings indicate that the protein SV-IV has a marked in vitro inhibitory effect on NK, LAK and CTL cytotoxicity, providing a better understanding of its immune regulatory functions.

Experimental study on vasoactive intestinal peptide (VIP) and its diaminopropane bound (VIP-DAP) analog in solution.

Bioactive peptides represent an exciting area of research in the fields of biochemistry and medicine and in particular the VIP/PACAP network appears to be of interest. Vasoactive intestinal peptide (VIP) is a pleiotropic factor that exerts a physiological regulatory influence and is involved in the pathogenesis of several human disorders. In this paper we have reported structural characterization of VIP by experimental and computational methods as well as a comparative analysis of the peptide with its transglutaminase catalyzed analog VIP-Diaminopropane (VIP-DAP).

Prognostic value of human telomerase reverse transcriptase gene expression in oral carcinogenesis.

Human telomerase reverse transcriptase (hTERT) gene expression in resected specimens of oral squamous cell carcinoma (OSCC) and their surrounding tissue, either apparently normal or clearly histologically dysplastic, was evaluated by both real-time RT-PCR and immunohisto-chemical protein analyses. The expression level of hTERT in oral dysplasia and in OSCC was markedly higher than in normal tissues. The correlation between hTERT expression in OSCC and clinico-pathological parameters or survival of OSCC patients was statistically analyzed. Our study demonstrates that there is no significant relationship between hTERT expression and classical clinico-pathological parameters. Interestingly, survival analysis showed both overexpressing cases and lower survival rate in the early stage of OSCC (p=0.03 for immunohistochemistry; p=0.04 for RT real-time PCR). The histological location of hTERT in these tumors has been discussed in the context of the cancer stem cell theory.

Exopolysaccharides production in Lactobacillus bulgaricus and Lactobacillus casei exploiting microfiltration.

The physiology of Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus casei, extensively used in the dairy industry, was studied in order to evaluate key parameters in the synthesis of exopolysaccharides and to improve their production through novel fermentation processes. Selected strains were studied in shake flasks and in fermentor experiments using glucose and lactose as main carbon sources and bacto casitone as the only complex component, in a temperature range between 35 and 42 degrees C. The production of exopolysaccharides was monitored and correlated to the growth conditions using both a colorimetric assay and chromatographic methods. Fermentor experiments in batch mode yielded 100 mg l(-1) of EPS from L. bulgaricus and 350 mg l(-1) from L. casei. Moreover, the use of a microfiltration (MF) bioreactor resulted in exopolysaccharides (EPS) concentrations threefold and sixfold those of batch experiments, respectively. The monosaccharidic composition of the two analyzed polymers differed from those previously reported. The optimization of the production of EPSs using the MF fermentation strategy could permit the use of these molecules produced by generally recognised as safe (GRAS) microorganisms in the place of other polysaccharides in the food industry.

Ultrastructural mucosal alterations and increased intestinal permeability in non-celiac, type I diabetic patients.

BACKGROUND: Increased intestinal permeability was described in several intestinal auto-immune conditions. There are very few and contradictory reports about type I diabetes mellitus, an auto-immune condition sometimes associated with celiac disease. AIMS: To investigate intestinal permeability in type I diabetes mellitus patients with no concomitant celiac disease, with a comparison to ultra-structural aspects of duodenal mucosa. PATIENTS: 46 insulin dependent diabetes mellitus, non-celiac, patients (18 females and 28 males, mean age 15.8 +/- 5.3 [S.D.] years) were enrolled. The mean duration of the disease was 5.7 years. METHODS: The morphological aspect of the small bowel mucosa, at standard light microscopy and electron transmission microscopy, along with intestinal permeability (by lactulose/mannitol test) were studied. Lactulose and mannitol urinary excretion were determined by means of high performance anion exchange chromatography-pulsed amperometric detection. RESULTS: The lactulose/mannitol ratio was 0.038 [0.005-0.176] (median and range) in 46 patients compared to 0.014 [0.004-0.027] in 23 controls: insulin dependent diabetes mellitus group values being significantly higher than those of the controls (P < 0.0001, Mann-Whitney test). Eight insulin dependent diabetes mellitus patients underwent endoscopy and biopsies were analysed by means of light microscopy and transmission electron microscopy. At the light microscopy level, none of the biopsy samples showed any sign of atrophy nor inflammation, whereas transmission electron microscopy analysis showed remarkable ultra-structural changes in six out of the eight patients. Four parameters were evaluated: height and thickness of microvilli, space between microvilli and thickness of tight junctions. CONCLUSIONS: This alteration of intestinal barrier function in non-celiac type I diabetes mellitus, frequently associated with mucosal ultra-structural alterations, could suggest that a loss of intestinal barrier function can be a pathogenetic factor in a subset of insulin dependent diabetes mellitus patients.

Intestinal permeability is decreased in anorexia nervosa.

Malnutrition and absence of exogenous luminal nutrients in the gastrointestinal tract affect intestinal permeability (IP) leading to an increased penetration of substances that passively cross intestinal epithelium via intercellular pathways. We hypothesised that an increase in IP could occur in patients with anorexia nervosa because of their prolonged fasting and chronic malnutrition. Therefore, we assessed IP in 14 drug-free anorexic women and 19 drug-free age-matched healthy women by means of the lactulose/mannitol (LA/MA) test. To this purpose, after an overnight fast, subjects ingested an oral solution containing 5 g lactulose and 2 g mannitol in 100 ml water. Urine specimens were collected immediately before and 30, 60, 120, 180, 240 and 300 min after the ingestion of the sugar solution. Urinary lactulose and mannitol were determined by high-performance anion exchange chromatography coupled with pulsed amperometric detection. We found that IP, as expressed by the 5-h LA/MA excretion ratio, was significantly decreased in anorexic women because of a lower urinary recovery of lactulose. Moreover, in patients, the time course of lactulose excretion significantly differs from healthy controls. These results do not confirm our hypothesis of increased IP in anorexia nervosa. Since IP reflects the anatomo-functional status of intestinal mucosa, the present findings support the idea that changes in the anatomo-physiology of intestinal mucosa occur in anorexia nervosa.

S-adenosyl-L-methionine N-ole-1-oyltaurate: pharmacokinetic of the orally administered salt in rats.

A pharmacokinetic study based on the distribution of radioactivity from the double labelled S-adenosyl-L-methionine (SAM) has been carried out by oral administration of the liposoluble stable salt [methyl-(14)C, 8-(3)H]SAM N-ole-1-oyltaurate to rats. The SAM sulfate p-toluensulfonate salt, the only SAM salt at present commercialized as drug, was chosen as reference compound to have a comparative pharmacokinetic analysis. The metabolism of the SAM is characterised by a differential use of the two labelled moieties by the various organs, liver being the most active in metabolizing the sulfonium compound with a preferential uptake of the methyl-(14)C fragment. The radioactivity detected after the administration of [methyl-(14)C, 8-(3)H]SAM N-ole-1-oyltaurate is, in all the organs examined, two times higher than the [methyl-(14)C, 8-(3)H]SAM sulfate p-toluensulfonate compound, attesting that the liposoluble [methyl-(14)C, 8-(3)H]SAM N-ole-1-oyltaurate is provided with better bioavailability.

Porins from Salmonella typhimurium accelerate human blood coagulation in vitro by selective stimulation of thrombin activity: implications in septic shock DIC pathogenesis.

The effect of porins, major hydrophobic outer membrane proteins purified from Salmonella typhimurium, on human blood coagulation was investigated. It was found that micromolar concentrations of porins accelerated markedly human blood coagulation in vitro. Using appropriate experiments, data were obtained showing that the main target of the porin-induced procoagulant effect was thrombin. A possible binding of porins with thrombin has been suggested to be the basis of this effect. The implications of this finding in the pathogenesis of the disseminated intravascular coagulation syndrome (DIC) occurring during the Gram-negative septic shock is discussed.

Innovative fermentation strategies for the production of extremophilic enzymes.

A new type of microfiltration (MF) bioreactor, developed in our laboratory, was investigated for use in improving efficiency of the production of extremophilic enzymes. In spite of the difficulties in cultivating hyperthermophiles, we achieved, in 300 h fermentation, more than 38 g/l dry weight of Sulfolobus solfataricus using a MF technique, and we demonstrated that the activity of alcohol dehydrogenase (ADH), as the reporter enzyme, was not affected by cell density. However, hyperthermophile cultivation is difficult to scale up because of evaporation and the very low growth rate. Thus, to achieve high productivity we cultivated, in the MF bioreactor, recombinant mesophilic hosts engineered for the production of two thermophilic enzymes, namely, trehalosyldextrin-forming enzyme (SsTDFE) and trehalose-forming enzyme (SsTFE) from Sulfolobus solfataricus. The traditional Luria-Bertani broth used for recombinant Escherichia coli growth was replaced with a semidefined medium. The latter was used in both the batch and the MF experiments, and the ratio of complex components (e.g., yeast extract and tryptone) to a simple carbon source (glycerol) was decreased during the fed-batch phase to further decrease the medium cost in view of industrial applications. The bioprocess developed was able to improve productivity 500 fold for rSsTFE and 60 fold for rSsTDFE with respect to the wild type cultivated in MF mode. Comparisons with another recombinant enzyme, alpha-glucosidase (rSsalphagly), from Sulfolobus solfataricus produced in our MF bioreactor are reported.

Synthesis of novel anti-inflammatory peptides derived from the amino-acid sequence of the bioactive protein SV-IV.

SV-IV is a basic, thermostable, secretory protein of low Mr (9758) that is synthesized by rat seminal vesicle (SV) epithelium under strict androgen transcriptional control. This protein is of obvious pharmacological interest because it has potent nonspecies-specific immunomodulatory, anti-inflammatory, and pro-coagulant activities. In evaluating the clinical relevance and the possible use in medicine of SV-IV, we became interested in the study of its structure-function relationships and aimed to identify in its polypeptide chain specific peptide fragments possessing the marked anti-inflammatory properties of the protein not associated with other biological activities (pro-coagulation and immunomodulation) typical of this molecule. By using two different experimental approaches (the fragmentation of the protein into peptide derivatives by chemical methods and the organic synthesis on solid phase of selected peptide fragments), data were obtained showing that in this protein: (a) the immunomodulatory activity is related to the structural integrity of the whole molecule; (b) the anti-inflammatory activity is located in the N-terminal region of the molecule, the 8-16 peptide fragment being the most active; (c) the identified anti-inflammatory peptide derivatives do not seem to possess pro-coagulant activity, even though this particular function has been located in the 1-70 segment of the molecule.

Effective production of a thermostable alpha-glucosidase from Sulfolobus solfataricus in Escherichia coli exploiting a microfiltration bioreactor.

A microfiltration (MF) membrane bioreactor was developed for an efficient production of a recombinant thermostable alpha-glucosidase (rSsGA) from Sulfolobus solfataricus MT-4. The aim of the membrane bioreactor was to improve the control of the concentration of key components in the growth of genetic engineered microorganisms, such as Escherichia coli. The influence of medium composition was studied in relation to cell growth and alpha-glucosidase production. The addition of components such as yeast extract and tryptone resulted in a higher enzyme production. High cell density cultivation of E. coli BL21(DE3) on semidefined medium, exploiting a microfiltration bioreactor, was studied in order to optimize rSsGA production. In addition to medium composition, the inducer employed (either isopropyl beta-D-thiogalactopyranoside or lactose), the induction duration, and the cultivation mode influenced both the final biomass and the enzyme yield. The MF bioreactor allowed a cell concentration of 50 g/L dry weight and a corresponding alpha-glucosidase production of 11,500 U/L. The improvement obtained in the enzyme production combining genetic engineering and the microfiltration strategy was estimated to be 2,000-fold the wild-type strain.

Synthesis and characterisation of a new class of stable S-adenosyl-L-methionine salts.

S-adenosyl-L-methionine (SAM) is an important metabolic intermediate that serves as a donor of methyl and aminopropyl groups to a variety of acceptor molecules. The molecule in vitro is unstable both in solution and in crystalline form undergoing irreversible conversion to 5'-methyltioadenosine (MTA) and homoserine lactone. Since this form of instability seems to be prevented in the cell of the living organism by bonds with macromolecules, we designed and developed a novel class of salts of SAM with large size anions to improve the stability of the sulfonium compound outside the cell. For this purpose we synthesised and characterised by NMR and IR spectroscopy anions consisting of amidic derivatives of taurine with fatty acids. Stability studies performed with the new SAM salts indicate that SAM becomes much more stable when it interacts with large size anions and in fact, more than 84% of the SAM is recovered after 36 months in lyophilized samples. The high stability of the new products widens the possibility of new therapeutic applications of SAM in human therapy.

Altered intestinal permeability to mannitol in diabetes mellitus type I.

BACKGROUND: Intestinal permeability has seldom been investigated in diabetes mellitus, even though patients frequently report gastrointestinal symptoms, and it has recently been shown that the prevalence of celiac disease associated with diabetes mellitus is higher than expected. METHODS: Intestinal permeability to cellobiose and mannitol was investigated in 31 patients affected by type I uncomplicated diabetes mellitus. Values were compared with those obtained in 32 normal subjects. RESULTS: The percentage of mannitol recovery was far higher than normal in two thirds of the investigated patients and correlated with the length of disease, even though the probes' ratio (cellobiose/mannitol) was in the normal range. CONCLUSIONS: A not previously reported increase of intestinal permeability to mannitol, clear-cut and not associated with that of the larger probe, is found in type I uncomplicated diabetes mellitus. These results may describe a primary feature of type I diabetes mellitus and the initial steps of evolution to celiac disease.