Zonulin upregulation is associated with increased gut permeability in subjects with type 1 diabetes and their relatives.

Zonulin, a protein that modulates intestinal permeability, is upregulated in several autoimmune diseases and is involved in the pathogenesis of autoimmune diabetes in the BB/Wor animal model of the disease. To verify the association between serum zonulin levels and in vivo intestinal permeability in patients with type 1 diabetes, both parameters were investigated in different stages of the autoimmune process. Forty-two percent (141 of 339) of the patients had abnormal serum zonulin levels, as compared with age-matched control subjects. The increased zonulin levels correlated with increased intestinal permeability in vivo and changes in claudin-1, claudin-2, and myosin IXB genes expression, while no changes were detected in ZO1 and occludin genes expression. When tested in serum samples collected during the pre-type 1 diabetes phase, elevated serum zonulin was detected in 70% of subjects and preceded by 3.5 +/- 0.9 years the onset of the disease in those patients who went on to develop type 1 diabetes. Combined, these results suggest that zonulin upregulation is associated with increased intestinal permeability in a subgroup of type 1 diabetic patients. Zonulin upregulation seems to precede the onset of the disease, providing a possible link between increased intestinal permeability, environmental exposure to non-self antigens, and the development of autoimmunity in genetically susceptible individuals.

Evaluation of a high temperature immobilised enzyme reactor for production of non-reducing oligosaccharides.

There is interest in the production of non-reducing carbohydrates due to their potential application in various industrial fields, particularly the food industry. In this paper, we describe the development of an immobilised cell bioprocess for the synthesis of non-reducing maltodextrins at high temperatures. The trehalosyl-dextrins-forming enzyme (TDFE) isolated from the thermoacidophilic archaeon Sulfolobus solfataricus (strain MT4), was recently expressed at high yields in Escherichia coli (strain Rb-791). Here, we evaluate different matrices, such as polyacrylamide gel, crude egg white, chitosan and calcium alginate for their effectiveness in immobilising whole recombinant E. coli cells subjected to prior thermal permeabilisation. Calcium-alginate based gels formed a solid biocatalyst with a good activity yield and the best enzymatic stability at the operating temperature (75 degrees C). Therefore, these beads were used to pack a glass column reactor to perform the bioconversion of interest. Optimal operating parameters were defined in relation to the substrate stream flow-rate and the substrate-to-biocatalyst ratio. The production of trehalosylmaltotetraose from maltohexaose reached equilibrium with a constant of about 2.6 at 75 degrees C. The bioreactor was exploited for production of trehalosylmaltodextrins from a commercial mixture of maltodextrins, achieving a productivity of 106.5 mg ml(-1) h(-1) (g biocatalyst)(-1) with ~40% conversion when using a 30% (w/v) solution.

Cellobiose and lactulose coupled with mannitol and determined using ion-exchange chromatography with pulsed amperometric detection, are reliable probes for investigation of intestinal permeability.

Lactulose/mannitol and cellobiose/mannitol tests are currently used in the investigation of intestinal permeability (IP) in many gastrointestinal diseases. The aim of this study was to produce a good technique for the determination and comparison of the above-mentioned sugar probes to overcome the problem caused by the presence of significant glycosuria in patients affected by particular metabolic disorders such as diabetes mellitus. Tests were performed in 25 healthy volunteers, using either cellobiose (Ce) (5 g) and mannitol (Ma) (2 g), or lactulose (La) (5 g) and mannitol (2 g), given as oral isosmolar loads. Sugars were recovered in urine collected for 5 h. Analysis was carried out by using anion-exchange chromatography (AEC) with pulsed amperometric detection (PAD). Baseline separation of the above carbohydrates was achieved within 13 min by using a Carbopac PA-100 column and linear gradient elution. Carbohydrate quantification was performed by an internal standard method. The calibration curve for each sugar is linear to 40 mM. The limit of sugar detection is 0.01 mM. Recovery of sugar probes is between 98.2 and 100%. The %La, %Ce, %Ma in urine were evaluated and their ratios (Ce/Ma and La/Ma) were calculated. No significant difference in IP parameters were shown (La/Ma to Ce/Ma 0.018+/-0.014 vs. 0.012+/-0.007; the attendant probability of the null hypothesis being P=0.0714). Ce/Ma and/or La/Ma tests result similarly reliable in the clinical investigation of IP and the described new method is also helpful in urine even with high glucose concentration, without any interference.

Trehalose production at high temperature exploiting an immobilized cell bioreactor.

The enzymatic production of trehalose from dextrins was studied as a series reaction in a packed bed reactor containing immobilized recombinant Escherichia coli cells, expressing either the Sulfolobus solfataricus (strain MT4) trehalosyl-dextrin forming enzyme (TDFE) or the trehalose-forming enzyme (TFE). The cells, subjected to thermal treatments to increase cell permeability and to inactivate the unwanted host proteins, were entrapped separately or together in a calcium alginate polymeric matrix. The biocatalyst beads were used to pack a tubular glass reactor that was operated in a recycle mode. The performances of a bioreactor containing alternate layers of EcTFE and EcTDFE alginate beads were evaluated and compared with the performance of the co-immobilized biocatalysts. The latter showed a superior throughput, therefore the bioreactor packed with the co-entrapped biocatalysts was tested for the production of trehalose from concentrated dextrin solutions (10%-30% w/v) and a conversion up to 90% was obtained. This conversion corresponded to a production of 127 g trehalose h(-1) kg(-1) of biocatalyst. The results obtained suggest that the bioprocess described may be of interest in the development of a large-scale industrial process for trehalose production at high temperature.