Walhalla

This is a poem written by Frank Graziano

Isolation of human conjunctival mast cells and epithelial cells: Tumor necrosis factor-alpha from mast cells affects intercellular adhesion molecule 1 expression on epithelial cells

PURPOSE. TO isolate and purify mast cells and epithelial cells from human cadaveric donor conjunctival tissue and to characterize interactions between these cell types in vitro. METHODS. Monodispersed cell suspensions obtained by enzymatic digestion of conjunctival tissue were applied to a single-density Percoll gradient. Epithelial cells obtained from the top layer of the gradient were cultured to confluence. Mast cells obtained from the pellet were equilibrated in culture medium and further purified using a two-step Percoll gradient. Using reverse transcriptionpolymerase chain reaction (RT-PCR), RNA from the purified mast cell preparation was probed for tumor necrosis factor-a (TNFa) message. Fluorescence activated cell sorting (FACS) analysis of intracellular immunostained mast cells was used to detect the TNFa protein. An examination for intercellular adhesion molecule 1 OCAM-1) on epithelial cells was performed after 24-hour incubations with either recombinant TNFa supernatants from calcium ionophore A23187 (Cal)-stimulated mast cells or appropriate controls using FACS analysis. RESULTS. Highly purified human conjunctival mast cells and epithelial cells (each >95%) were obtained from human cadaveric donor tissue. RT-PCR analysis of purified mast cell RNA revealed the expression of TNFa mRNA. An evaluation of mast cells for intracellular protein demonstrated positive staining for tryptase and TNFa. ICAM-1 was found on purified epithelial cells, and incubation of epithelial cell monolayers with supernatants from Cal-stimulated mast cells resulted in upregulation of this receptor. This upregulation was blocked by incubation with TNFa-neutralizing antibody. CONCLUSIONS. This work provides the methods for isolating and purifying mast cells and epithelial cells from human donor tissue and the opportunity for studying mechanisms of conjunctival inflammation by evaluating the interactions between these cells. (Invest Ophthalmol Vis Sci. 1998; 39:336-343) C onjunctival mast cells and their mediators are implicated, at least in part, in all types of allergic ocular disease. Human studies of allergic ocular disease, however, have primarily consisted of the study of pathologic biopsy specimens (demonstrating increased numbers of mast cells, eosinophils, and other inflammatory cells) and tear film evaluation for mediators and cellular infiltration

DALI:Vitamin D and lifestyle intervention for gestational diabetes mellitus (GDM) prevention: an European multicentre, randomised trial - study protocol

BACKGROUND: Gestational diabetes mellitus (GDM) is an increasing problem world-wide. Lifestyle interventions and/or vitamin D supplementation might help prevent GDM in some women. METHODS/DESIGN: Pregnant women at risk of GDM (BMI 65 29 (kg/m(2))) from 9 European countries will be invited to participate and consent obtained before 19+6 weeks of gestation. After giving informed consent, women without GDM will be included (based on IADPSG criteria: fasting glucose<5.1 mmol; 1 hour glucose <10.0 mmol; 2 hour glucose <8.5 mmol) and randomized to one of the 8 intervention arms using a 2 7 (2 7 2) factorial design: (1) healthy eating (HE), 2) physical activity (PA), 3) HE+PA, 4) control, 5) HE+PA+vitamin D, 6) HE+PA+placebo, 7) vitamin D alone, 8) placebo alone), pre-stratified for each site. In total, 880 women will be included with 110 women allocated to each arm. Between entry and 35 weeks of gestation, women allocated to a lifestyle intervention will receive 5 face-to-face, and 4 telephone coaching sessions, based on the principles of motivational interviewing. The lifestyle intervention includes a discussion about the risks of GDM, a weight gain target <5 kg and either 7 healthy eating 'messages' and/or 5 physical activity 'messages' depending on randomization. Fidelity is monitored by the use of a personal digital assistance (PDA) system. Participants randomized to the vitamin D intervention receive either 1600 IU vitamin D or placebo for daily intake until delivery. Data is collected at baseline measurement, at 24-28 weeks, 35-37 weeks of gestation and after delivery. Primary outcome measures are gestational weight gain, fasting glucose and insulin sensitivity, with a range of obstetric secondary outcome measures including birth weight. DISCUSSION: DALI is a unique Europe-wide randomised controlled trial, which will gain insight into preventive measures against the development of GDM in overweight and obese women