Nuclear fluorescence serum reactivity on monkey oesophagus: a new antibody for the follow-up of coeliac disease?

We have identified previously a nuclear fluorescence reactivity (NFR) pattern on monkey oesophagus sections exposed to coeliac disease (CD) patients' sera positive for anti-endomysium antibodies (EMA). The aim of the present work was to characterize the NFR, study the time-course of NFR-positive results in relation to gluten withdrawal and evaluate the potential role of NFR in the follow-up of CD. Twenty untreated, 87 treated CD patients and 15 healthy controls were recruited and followed for 12 months. Their sera were incubated on monkey oesophagus sections to evaluate the presence of NFR by indirect immunofluorescence analysis. Duodenal mucosa samples from treated CD patients were challenged with gliadin peptides, and thus the occurrence of NFR in culture supernatants was assessed. The NFR immunoglobulins (Igs) reactivity with the nuclear extract of a human intestinal cell line was investigated. Serum NFR was present in all untreated CD patients, persisted up to 151 ± 37 days from gluten withdrawal and reappeared in treated CD patients under dietary transgressions. Serum NFR was also detected in two healthy controls. In culture supernatants of coeliac intestinal mucosa challenged with gliadin peptides, NFR appeared before EMA. The Igs responsible for NFR were identified as belonging to the IgA2 subclass. The NFR resulted differently from EMA and anti-nuclear antibodies, but reacted with two nuclear antigens of 65 and 49 kDa. A new autoantibody, named NFR related to CD, was described. Furthermore, NFR detection might become a valuable tool in monitoring adherence to a gluten-free diet and identifying slight dietary transgressions.

The p85 regulatory subunit of PI3K mediates TSH-cAMP-PKA growth and survival signals.

Phosphatidylinositol 3-kinase (PI3K) is necessary for thyroid stimulating hormone (TSH)-induced cell cycle progression. To determine the molecular mechanism linking PI3K to TSH, we have identified a serine residue in p85alpha(PI3K) phosphorylated by protein kinase A (PKA) in vitro and in vivo. Expression of an alanine mutant (p85A) abolished cyclic AMP/TSH-induced cell cycle progression and was lethal in thyroid cells (FRTL-5). The aspartic version of the p85alpha(PI3K) (p85D) inhibited apoptosis following TSH withdrawal. The p85alpha(PI3K) wild type not the p85A bound PKA regulatory subunit RIIbeta in cells stimulated with cAMP or TSH. The binding of the aspartic version of p85alpha(PI3K) to RIIbeta was independent of cAMP or TSH stimulation. Similarly, binding of PI3K to p21Ras and activation of AKT, a downstream PI3K target, were severely impaired in cells expressing the p85A mutant. Finally, we found that the catalytic activity of PI3K was stimulated by TSH in cells expressing the wild-type p85alpha(PI3K) but not in cells expressing p85A. This latter mutant did not affect the epidermal growth factor-stimulated PI3K activity. We suggest that (1) TSH-cAMP-induced PKA phosphorylates p85alpha(PI3K) at serine 83, (2) phosphorylated p85alpha(PI3K) binds RIIbeta-PKA and targets PKAII to the membrane, and (3) PI3K activity and p21Ras binding to PI3K increase and activate PI3K downstream targets. This pathway is essential for the transmission of TSH-cAMP growth signals.

Phospholipid release from sonicated vesicles induced by a "critical" fatty acid concentration.

Dipalmitoyl phosphatidylcholine vesicles incubated in the presence of increasing amounts of myristic acid showed a progressive translocation of phospholipid molecules across a dialysis membrane. The rate of phospholipid translocation increased abruptly at a 'critical' value of myristic acid concentrations. The translocation rate of mixed dipalmitoyl phosphatidylcholine/myristic acid vesicles obtained by cosonicating the two components was also dependent on a 'critical' fatty acid concentration. A marked release of K+ and different responses of fluorescent probes to the fatty acid addition were observed at this concentration.

Phospholipid exchange and size enlargement in sonicated vesicles induced by a 'critical' fatty acid concentration.

Size enlargement of dipalmitoyl phosphatidylcholine vesicles was greatly accelerated in the range of the phase-transition temperatures, when fatty acid concentration was above a threshold level ('critical' concentration). This 'critical' concentration varied with the length of the fatty acid chain. The size enlargement process had second-order kinetics dependent on the vesicle concentration. Alkaline pH and low ionic strength inhibited the rate of size enlargement. Phospholipid exchange between dimyristoyl and dipalmitoyl phosphatidyl-choline vesicles increased abruptly above a 'critical' fatty acid concentration. The donor vesicles were those vesicles in which fatty acids reached the 'critical' concentration. The phospholipid exchange occurred both in fluid- and in solidstate vesicles. The 'critical' fatty acid concentration accelerating the phospholipid exchange process was lower than that accelerating the size enlargement process. The phospholipid exchange process explained in terms of a diminished hydrophobic attraction among the phospholipid molecules of the bilayer occurs via a free phospholipid molecule transfer through the aqueous phase. The size enlargement process is interpreted in terms of high fatty acid concentration in the membrane fluid domains. The membrane structure is locally perturbed inducing vesicle sticking after collision.

Effect of lipid mixing on the permeability and fusion of saturated lecithin membranes.

The effect of phospholipid mixing on the permeability properties of multilamellar lipid vesicles (MLV) was studied. In the solid state, dimyristoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DMPC/DPPC) vesicles exhibit ideal lipid miscibility; dimyristoylphosphatidylcholine/distearoylphosphatidylcholine (DMPC/DSPC) vesicles exhibit nonideal lipid miscibility at low DSPC molar fractions; dimyristoylphosphatidylcholine/dibehenoylphosphatidylcholine range of DBPC molar fractions. The rates of K+, ethylene glycol, and water diffusion from these vesicles in the solid state were measured by photometric and electrometric techniques. The following results were obtained: (1) The rate of solute diffusion, which is decreased monotonically, in DMPC/DPPC MLV, by increasing the molar fractions of DPPC, exhibits maxima at 0.2 molar fraction of DSPC in DMPC/DSPC MLV and at 0.4 molar fraction of DBPC in DMPC/DBPC MLV. (2) The activation energy of the solute diffusion process abruptly decreases in approximately the same range of lipid molar fractions where nonideal lipid miscibility is present. (3) The membrane pore radius is increased by increasing the lipid nonideal miscibility. The rate of vesicle size increase, measured by absorbance changes, is decreased monotonically in DMPC/DPPC monolamellar vesicles [small unilamellar lipid vesicles (SUV)] by increasing the molar fraction of DPPC. It, however, exhibits a maximum in DMPC/DSPC SUV at 0.15 molar fraction of DSPC. A model was suggested in which the solute diffusion and the membrane fusion processes are controlled by fractures. The average width of the fractures is increased by increasing the lipid immiscibility.