Piscirickettsiosis or salmonid rickettsial septicaemia (SRS) caused by Piscirickettsia salmonis constitutes one of the main problems in farmed salmonid and marine fishes. Since the first reports of the disease, it has been successfully isolated and maintained in eukaryotic cellculture systems, but these systems are time-consuming, the media are costly, and eliminating heavily contaminated host cell debris is difficult. In this report, we describe a marine-based broth supplemented with L-cysteine, named AUSTRAL-SRS broth, that facilitates superior growth of P. salmonis strains. Strains reached an optical density of approximately 1.8 when absorbance was measured at 600 nm after 6 d incubation at 18°C. Several passages (n = 6) did not alter the culture kinetics. We report for the first time the purification of DNA, lipopolysaccharide (LPS) and whole membrane protein obtained from P. salmonis grown in this liquid medium, and thus provide a suitable platform to simplify the preparation of P. salmonis cells for genetic and serological studies. Moreover, the results of the cytopathic effect test showed that P. salmonis grown in AUSTRAL-SRS broth maintained their virulence properties, inducing apoptosis after 3 d. This makes the medium a good candidate for the successful growth of P. salmonis and an excellent basis for the development of low cost vaccines. KEY WORDS: Piscirickettsia salmonis · Broth · CultureResale or republication not permitted without written consent of the publisher

Trefoil factors (TFF) are secretory products of mucin producing cells. Th ey play a key role in the maintenance of the surface integrity of oral mucosa and enhance healing of the gastrointestinal mucosa by a process called restitution. TFF comprises the gastric peptides (TFF), spasmolytic peptide (TFF), and the intestinal trefoil factor (TFF). Th ey have an important and necessary role in epithelial restitution within the gastrointestinal tract. Signifi cant amounts of TFF are present in human milk. Th is study aimed to determine a possible correlation between TFF isolated from human breast milk and levels of cytokines (IL and IL) and defensins (hBD and hBD) in intestinal epithelial cells HT- treated with trefoil. Samples of human milk were collected within - weeks postpartum from healthy human mothers (--years-old) by manual breast massage, and TFF was purifi ed by ammonium sulfate precipitation, isoelectric precipitation, DEAE-chromatography, and gel fi ltration. In this work we measured the concentrations and mRNA levels of cytokines and defensins by immunoassay (ELISA) and semiquantitative RT-PCR technique, respectively. Also we measured the peroxidase activity. We present the fi rst evidence of human milk TFF purifi cation. Here we show that the presence of TFF isolated from milk strongly correlates with downregulation of IL and IL in human intestinal epithelial cells. On the other hand, TFF activated the epithelial cells in culture to produce beta defensins  (hBD) and beta defensins  (hBD). Th ese fi ndings suggest that TFF can activate intestinal epithelial cells and could actively participate in the immune system of breastfed babies by inducing the production of peptides related to innate defence, such as defensins.

Introduction: Trefoil factors are effector molecules in gastrointestinal tract physiology. They are classified into three groups: the gastric peptides (TFF1), spasmolytic peptide (TFF2) and intestinal trefoil factor (TFF3). Previous studies have shown that trefoil factors are located and expressed in human endocrine pancreas suggesting that TFF3 play a role in: a) pancreatic cells migration, b) β-cell mitosis, and c) pancreatic cells regeneration. We speculated that the presence of TFF3 in pancreas, could be associated to a possible regulation mechanism by insulin and glucose. To date, there are not reports whether the unbalance in carbohydrate metabolism observed in diabetes could affect the production or expression of TFF3.Methods: We determined the TFF3 levels and expression by immunoassay (ELISA) and semi-quantitative RT-PCR technique respectively, of intestinal epithelial cells (HT-29) treated with glucose and insulin. Also,Real Time-PCR (RTq-PCR) was done.Results: Increasing concentrations of glucose improved TFF3 expression and these levels were further elevated after insulin treatment. Insulin treatment also led to the up-regulation of human sodium/glucose transporter 1 (hSGLT1), which further increases intracellular glucose levels. Finally, we investigated theTFF3 levels in serum of diabetes mellitus type 1 (T1DM) and healthy patients. Here we shown that serum TFF3 levels were down-regulated in T1DM and this levels were up-regulated after insulin treatment. Also, the TFF3 levels of healthy donors were up-regulated 2 h after breakfast.Conclusion: Our fi ndings suggest for the fi rst time that insulin signaling is important for TFF3 optimal expression in serum and intestinal epithelial cells.