Clinical trial assessing VSL#3 for the treatment of anterior resection syndrome

Background: Restoration of bowel continuity after a temporary loop ileostomy following rectal resection often produces impaired bowel function. The purpose of this clinical trial was to assess the efficacy of a probiotic, VSL#3 (VSL Pharmaceuticals Inc., Gaithersburg, MD, USA), in improving bowel function following ileostomy closure. Methods: Between March 2005 and April 2008, a prospective, double‐blind, placebo‐controlled randomized trial of a probiotic preparation was conducted across four South Australian hospitals. Sixty‐three patients who underwent a loop ileostomy reversal were randomized to receive 4‐week treatment of either probiotic therapy (n= 31) or placebo (n= 32). Bowel symptomology was collected through a patient‐completed bowel diary and the Gastrointestinal Quality of Life Index (GIQLI). Results: Completion rates of the 4‐week therapy regime were similar for both groups: 18 active versus 20 placebos. There was no statistically significant difference in the number of patients who withdrew or had adverse events in the two treatment groups. Reasons for patient withdrawal from the study were similar for both groups. Repeated measures analysis of variance showed no statistically significant difference between the GIQLI scores for the two treatment groups. Conclusions: The use of the probiotic preparation, VSL#3, did not alter the post‐operative bowel function of patients undergoing loop ileostomy reversal.     

Effect of mouth-rinse formulations on oral malodour processes in tongue-derived perfusion biofilm model

An in vitro matrix biofilm perfusion model of tongue-derived microcosms for studying volatile sulfur compound (VSC) biogenesis has been previously described. The model was modified in order to monitor H(2)S in situ by use of a specialized electrode assembly based on microbial fuel cell technology. This system was designed to give real-time measurements expressed as electrode power output, which were proportional to H(2)S levels, measured by other means. In addition to the model modifications, the aim of this study was to demonstrate the biofilm responses following single or multiple exposure to biocidal, biostatic or VSC-inhibiting active compounds used in products. Tongue-derived biofilms (n = 6 per experiment) were perfused with one-fifth strength BHI at 20 ml h(-1) pH 7.2 and pulsed with putative treatment agent, placebo and controls including Zn(2+) ions and chlorhexidine (CHX). Compared with their pre-treatment conditions, all biofilms responded to the treatments in terms of reductions in hydrogen sulfide generation (as detected by the biofilm-electrode response) and other microbial volatile organic compounds (VOCs) as detected using a selected ion flow tube mass spectrometry analyser. The microbiological analysis of the treated and control biofilms show that test products (formulations with active agents) all gave reduced cell populations compared to the control biofilm. An order of effects (magnitude and duration) suggests that both the test agent and CHX produced the strongest reductions, distinct from the responses obtained for the placebo and water controls, which were largely similar. It is concluded that the in vitro perfusion model may be used to replicate many of the activities and reactions believed to be occurring by the tongue biofilm microflora within a real mouth, including H(2)S and VOC biogenesis and their inhibition by exposure to active agents.     

Detection of minimal residual disease in acute lymphoblastic leukemia by in vitro amplification of rearranged T-cell receptor delta chain sequences

Human T-cell receptor (TCR) delta-chain diversity mainly originates from high junctional variability, since only a limited number of germline elements is available. This extraordinary diversity at the V.J junction, due to the use of two D delta elements and extensive incorporation of N nucleotides, constitutes a specific clonal marker for cell populations exhibiting rearranged TCR delta genes. To this end we amplified in vitro by polymerase chain reaction (PCR) the TCR delta junctional region of five acute lymphoblastic leukemias (ALL), isolated respective DNA fragments, and used them directly as clonospecific probes. The combination of PCR technology and hybridization to clonospecific probes permitted the detection of leukemia DNA at dilution of 1:100,000 in all five cases. Moreover, we were able to investigate one of the ALL patients 11 months after achieving continuous complete remission. Conventional Southern blot analysis failed to detect rearranged TCR genes at this stage. However, residual leukemic cells could readily be detected by PCR technique. We conclude that the strategy proposed here is a very sensitive tool to detect minimal residual disease in a significant proportion of human lymphoid neoplasias.     

A point mutation in the integrin beta 3 cytoplasmic domain (S752-->P) impairs bidirectional signaling through alpha IIb beta 3 (platelet glycoprotein IIb-IIIa)

Agonist-induced inside-out signaling results in an increased affinity of integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) for soluble ligands (fibrinogen [Fg] and PAC1). Ligand binding to integrins initiates outside-in signaling that leads to cellular responses such as cell spreading and focal adhesion formation. A point mutation in the beta 3 cytoplasmic domain (S752-->P) is associated with blocked inside- out alpha IIb beta 3 signaling in a variant Glanzmann's thrombasthenia. This mutation was introduced into beta 3 and cotransfected into Chinese hamster ovary cells with a chimeric alpha subunit consisting of the alpha IIb extracellular and transmembrane domains and the alpha 6B cytoplasmic domain. The substitution of the alpha IIb cytoplasmic domain with that of alpha 6 led to activation of alpha IIb beta 3 to bind PAC1, mimicking inside-out signaling. This effect was reversed by the S752-->P mutation, indicating a disruption of inside-out signaling by the mutation. In addition, transfectants expressing this beta 3 variant showed reduced alpha IIb beta 3-mediated cell spreading on immobilized Fg, focal adhesion, and fibrin clot retraction, suggesting an impairment in outside-in alpha IIb beta 3 signaling. Therefore, a single point mutation in the beta 3 cytoplasmic domain impaired bidirectional signaling through alpha IIb beta 3.     

Differential effect of phorbol esters and interleukin-3 on protein kinase C isoform content and kinase activity in the FDC-P1 cell line

FD/PMA is a subclone of the interleukin-3 (IL-3)-dependent, FDC-P1 cell line, which proliferates in response to either 12-O- tetradecanoylphorbol-13 acetate (PMA) or IL-3. While several endogenous substrates were phosphorylated in response to protein kinase C (PKC) activation in FDC-P1, phospholipid-dependent phosphorylation in the FD/PMA grown in PMA was not observed. Basal, phosphatidylserine- independent, and diolein-independent phosphorylation of cytosolic substrates with molecular weights of 17, 52, 57, and 105 Kd were enhanced in FD/PMA cells grown in PMA as compared with FDC-P1 cells cultured in IL-3. Phosphorylation of a 105-Kd substrate was enhanced in the particulate fraction of FD/PMA cells maintained in PMA. The 17-Kd substrate in FD/PMA cells comigrated with a substrate phosphorylated in a PKC-dependent manner in FDC-P1 cells. Phosphorylation of the 52- and 57-Kd substrates, but not of the 17-Kd substrate, was inhibited by H-7 and staurosporine. A portion of the PMA-induced cytosolic kinase activity coeluted with PKC on diethyl aminoethyl chromatography. While FD/PMA cells cultured in PMA contained negligible PKC-dependent phosphorylation of endogenous substrates or histone, alpha and epsilon PKC isoforms were detected by Western blot analysis. PKC phosphotransferase activity was observed in FD/PMA cells grown in PMA when peptides corresponding to residues 720 to 737 of PKC-epsilon or residues 4 to 14 of myelin basic protein were used as substrates. These data indicate that maintenance of FD/PMA cells in PMA stimulates proliferation and markedly alters PKC substrate specificity. Generation of at least two phospholipid-independent kinases occurs in PMA-treated cells.     

Inducible lethal ventricular arrhythmias in swine with pacing-induced heart failure

Introduction: Rapid pacing-induced heart failure provides an excellent animal model for the study of heart failure. We studied the development of ventricular tachyarrhythmias using programmed stimulation in a pacing-induced heart failure model. We also studied action potential characteristics and the relationship between action potential and heart rate. Methods and results: Ten pigs were instrumented and were studied before the onset and every week after rapid pacing was instituted. Weekly echocardiograms and programmed stimulation were done in a sedated state. In vitro electrophysiologic studies were done on left ventricular myocardium in 4 heart-failure animals and 4 controls. All animals developed progressive heart failure with left ventricular dilatation and reduced percentage fractional shortening. No arrhythmias were induced at baseline or the first and second weeks. Ventricular fibrillation was induced in one animal on the third week and 4 animals on week 4, while there was no appreciable worsening in echocardiographic indices of ventricular dysfunction between weeks 3 and 4. Ventricular effective refractory period was unchanged during the 4 weeks. In vitro studies showed action potential prolongation in heart failure myocardium. However, action potential duration at pacing rates > 100 bpm were similar to controls. No early or delayed afterdepolarizations were observed. Conclusion: This study demonstrated an increased susceptibility to ventricular fibrillation with the development of heart failure which was not related to the degree of ventricular disfunction. Also, the normalization of action potential duration at higher heart rates suggests that the increased incidence of inducible ventricular fibrillation in this model may not be solely due to prolonged action potential duration. Received: 4 January 1999, Returned for 1. revision: 29 January 1999, 1.Revision received: 26 May 1999, Accepted: 15 June 1999