Fluorescence in situ hybridization analysis of HER-2/neu in brushings of normal oral mucosa

Oncogene alterations have been clearly demonstrated to be related to the carcinogenesis and progression of oral squamous cell carcinoma (OSCC). However, the analysis of these alterations for screening and early diagnostic purposes generally requires invasive techniques for surgical removal of pathological epithelium. The aim of the present study was to assess the feasibility of fluorescence in situ hybridization (FISH) analysis of HER-2/neu amplification in oral mucosa brushings and to compare the HER-2/neu status with the history and smoking and drinking habits of healthy subjects. Cells obtained by centrifugation of oral brushings from 21 subjects (overall no. of cells: 5125) were suspended in physiological saline and fixed onto two slides for cytological evaluation and FISH analysis (dual-target, dual-color fluorescence assay) of the HER-2/neu gene and CEP17 centromere. A mean of 89.8% of the cells showed two HER-2/neu signals and a mean of 94% had two CEP17 signals at fluorescent microscopy. Finally, a mean of 96% of cells with HER-2/neu / CEP17 had a ratio equal to 1. No association between smoking and drinking habits, age and the HER-2/neu and CEP17 characteristics evaluated by FISH was found.     

L-arginine, the precursor of nitric oxide, abolishes the effect of estrogens on bleeding time in experimental uremia.

We have reported previously that conjugated estrogens that are effective in shortening the prolonged bleeding time in uremic patients are also effective on bleeding time in a rat model of uremia. Using such a rat model we have recently demonstrated that nitric oxide (NO), an endothelium-derived vasodilator, is involved in mediating the bleeding tendency of uremia. With the present study we wanted to investigate whether conjugated estrogen mixture or its active component, 17 beta-estradiol, reduce uremic bleeding by interfering with the NO pathway. Our results showed that the shortening effect of conjugated estrogen and 17 beta-estradiol on bleeding time of uremic rats was completely reversed by giving the animals the NO precursor L-arginine, but not D-arginine, which is not a precursor of NO. Dexamethasone which at variance to progesterone inhibits the process of induction of NO-forming enzyme, shortened the prolonged bleeding time of uremic rats within 4 hours from injection. This effect was eliminated by L-arginine but not D-arginine administration. The glucocorticoid receptor antagonist cortexolone prevented the shortening of bleeding time induced by dexamethasone, suggesting that a receptor-mediated mechanism is involved in the hemostatic effect of dexamethasone as previously reported for estrogens. Unlike conjugated estrogens and dexamethasone, progesterone had no effect on bleeding time. All these findings would indicate that the effect of estrogens and dexamethasone on primary hemostasis in uremia might be mediated by changes in NO synthetic pathway.     

Endoplasmic reticulum stress and unfolded protein response in inclusion body myositis muscle

Proteins in the endoplasmic reticulum (ER) require an efficient system of molecular chaperones whose role is to assure their proper folding and to prevent accumulation of unfolded proteins. The response of cells to accumulation of unfolded proteins in the ER is termed "unfolded protein response" (UPR). UPR is a functional mechanism by which cells attempt to protect themselves against ER stress, resulting from the accumulation of the unfolded/misfolded proteins. Because intracellular inclusions, containing either amyloid-beta (Abeta) or phosphorylated tau, are the characteristic feature of sporadic inclusion body myositis (s-IBM) muscle biopsies, we studied expression and immunolocalization of five ER chaperones, calnexin, calreticulin, GRP94, BiP/GRP78, and ERp72, in s-IBM and control muscle biopsies. Physical interaction of the ER chaperones with amyloid-beta precursor protein (AbetaPP) was studied by a combined immunoprecipitation/immunoblotting technique in s-IBM and control muscle biopsies, and in AbetaPP-overexpressing cultured human muscle fibers. In all s-IBM muscle biopsies, all five of the ER chaperones were immunodetected in the form of inclusions that co-localized with amyloid-beta. By immunoblotting, expression of ER chaperones was greatly increased as compared to the controls. By immunoprecipitation/immunoblotting experiments, ER chaperones co-immunoprecipitated with AbetaPP. Our studies provide evidence of the UPR in s-IBM muscle and demonstrate for the first time that the ER chaperones calnexin, calreticulin, GRP94, BiP/GRP78, and ERp72 physically associate with AbetaPP in s-IBM muscle, suggesting their playing a role in AbetaPP folding and processing.     

The surface measurement of aortic atherosclerosis: critical survey and comparison with histologic findings

Both the reproducibility of the surface measurements of aortic atherosclerosis and the agreement between gross inspective and histologic changes were evaluated. Aortas from male broad breasted white turkeys were chosen because of the high incidence of spontaneous and typical atherosclerotic lesions in this animal strain. Ten male turkeys were killed at 33 weeks of age. The aortas were removed including the iliac bifurcation and stained with Sudan III. Each aorta was processed blindly by four pathologists and a computerized planimeter to determine normal areas, sudanophilic areas and areas covered by plaques. The analysis of variance showed significant differences among the four pathologists' measurements of sudanophilic areas (P less than 0.01) and areas covered by plaques (P less than 0.001). The coefficients of variation among the four determinations made by one pathologist on the same aorta were 3.6% for total aortic area; 10.08% for sudanophilic area; 47.6% for the area covered by plaques. On each aorta histology was performed at the level where all the four pathologists recorded the same findings at inspection, namely a normal area, a sudanophilic area, and an area covered by plaques. Important discrepancies occurred between findings at inspection and those of histologic examination: the ten areas classified as "normal" by all the four pathologists at inspection were shown at histologic examination to be normal in only two cases. In one case a musculo-elastic layer and in seven cases a fibro-elastic layer were found. The ten areas classified as "sudanophilic" by all the observers showed a fibro-elastic layer in five cases, a musculo-elastic layer in two cases and normal findings in three cases. The ten areas classified as "covered by plaques" displayed a typical atherosclerotic plaque in all cases but one. In conclusion, our data indicate that the reproducibility of gross inspective methods is low. Important discrepancies exist between findings at inspection and histologic examinations. The relevance of these findings remains to be established as far as the assessment of human atherosclerosis is concerned.     

Aspirin and the prevention of experimental arterial thrombosis: difficulty in establishing unequivocal effectiveness

A trial of the efficacy of aspirin in the prevention of thrombotic occlusion of an "aortic loop" in rats was made simultaneously by two experimental surgeons. A relatively large dose of aspirin (80-100 mg/kg/day) was used, starting two days before operation. It appeared that aspirin was of limited benefit, reducing thrombotic occlusions by about 17% seven days after the insertion of the loop into the abdominal aorta. Although the average occlusion time was prolonged by about 17% in aspirin-treated animals, the separate trials gave no conclusive result. When the data from both operators were pooled, a statistically significant protection by aspirin was apparent (p = 0.02), by a two-tailed Student's t test. However, on using the powerful non-parametric randomization test, the occlusion times in control and aspirin-treated groups appeared not statistically different (p = 0.07). No significant difference was also found between control and treated groups when data were analyzed by X2 test. Independently of the statistical analysis, these data are quite similar to those obtained from aspirin trials in men surviving myocardial infarction. This finding points to the usefulness of the aorta loop as an animal model for arterial thrombosis.     

Aspirin kinetics and inhibition of platelet thromboxane generation-relevance for a solution of the "aspirin dilemma"

Six healthy male volunteers received aspirin (ASA) in a compressed (320 mg) and an enteric-coated (800 mg) formulation as single oral doses ten days apart. Ten plasma samples were obtained from each volunteer between 5 and 120 min after compressed ASA, and seven between 10 and 240 min after enteric-coated ASA. ASA was undetectable (less than 100 ng/ml) in plasma from three subjects receiving compressed ASA and two receiving the enteric-coated preparation. Plasma levels and kinetic parameters of salicylate were the same in subjects with undetectable and detectable ASA plasma levels. More than 98% inhibition of pre-drug serum TXB2 was noted in all samples collected one and four hours after either ASA preparation. TXB2 generation recovered on average by 3.5% at 24 hr with both preparations. Thus inhibition of platelet TXB2 generation occurred independently of the amount of ASA reaching the peripheral circulation. If this is due to inhibition of platelet function in the enterohepatic circulation followed by extensive first-pass deacetylation of ASA, vascular PGI2 synthesis could be spared. A better knowledge of the kinetic parameters of ASA for each of the formulations used in thrombosis prevention trials might help in solving the "aspirin dilemma".     

Post bending of the polypropylene flexible stent in mitral Hancock bioprostheses

The occurrence of stent post deformity was investigated in 38 consecutive, polypropylene flexible-stented Hancock porcine bioprostheses (PB) recovered from the mitral position because of early postoperative death or late tissue failure. The degree of deformity was assessed for each explant by calculating the triangular area obtained by projecting the apex of the stent posts on a plane and comparing it with the same area calculated for unimplanted PBs of comparable size. A significant stent deformity (reduction of the triangular area greater than 40%) was observed only in 6 PBs explanted because of tissue failure after a mean function time of 69.6 +/- 28 months. In these PBs, scanning electron microscopy of the bare surface of the stent at the point of maximal flexion showed cracks 0.7-1 microns large, which represent the morphological substrate of the so-called "creep" of polypropylene. Inward stent post bending may occur after implantation in mitral PBs and when severe, it corresponds to a definite ultrastructural plastic deformity of polypropylene.     

The surgical anatomy of hearts with no direct communication between the right atrium and the ventricular mass--so-called tricuspid atresia

Seventy-six hearts were studied having no direct communication between the right atrium and the ventricular mass. The different cardiac chamber combinations producing so-called "tricuspid atresia" were considered. The nature of the atrioventricular valve atresia, the morphology of the ventricular mass, the size and position of the interventricular communication, the ventriculoarterial connection, and the presence of subarterial outflow tract obstruction were all analyzed. The majority of cases were of the "classical" type, i.e., absent right atrioventricular connection with the left atrium connected to the left ventricle. In another group there was absence of the right atrioventricular connection but the left atrium drained into the morphologically right ventricle, which was left-sided. In a third group both atrial chambers connected with the ventricular mass but some structure, an imperforate valve or muscular partition, completely blocked the flow pathway through the right side of the heart. All the hearts described are candidates for the Fontan procedure with either an atrioventricular or an atriopulmonary conduit. The surgical options would not be affected by the observed variability at the atrioventricular junction but would be dictated by the feasibility of incorporating the subpulmonary ventricle within the pulmonary circulation.     

The secret life of the mitral valve

In secondary mitral regurgitation, the concept that the mitral valve (MV) is an innocent bystander, has been challenged by many studies in the last decades. The MV is a living structure with intrinsic plasticity that reacts to changes in stretch or in mechanical stress activating biohumoral mechanisms that have, as purpose, the adaptation of the valve to the new environment. If the adaptation is balanced, the leaflets increase both surface and length and the chordae tendineae lengthen: the result is a valve with different characteristics, but able to avoid or to limit the regurgitation. However, if the adaptation is unbalanced, the leaflets and the chords do not change their size, but become stiffer and rigid, with moderate or severe regurgitation. These changes are mediated mainly by a cytokine, the transforming growth factor‐β (TGF‐β), which is able to promote the changes that the MV needs to adapt to a new hemodynamic environment. In general, mild TGF‐β activation facilitates leaflet growth, excessive TGF‐β activation, as after myocardial infarction, results in profibrotic changes in the leaflets, with increased thickness and stiffness. The MV is then a plastic organism, that reacts to the external stimuli, trying to maintain its physiologic integrity. This review has the goal to unveil the secret life of the MV, to understand which stimuli can trigger its plasticity, and to explain why the equation “large heart = moderate/severe mitral regurgitation” and “small heart = no/mild mitral regurgitation” does not work into the clinical practice.     

Lumacaftor/ivacaftor improves liver cholesterol metabolism but does not influence hypocholesterolemia in patients with cystic fibrosis

BackgroundCystic fibrosis (CF) patients have reduced intestinal absorption of sterols and, despite enhanced endogenous synthesis, low plasma cholesterol. Lumacaftor/ivacaftor CFTR protein modulator therapy is used to improve the clinical outcome of CF patients homozygous for F508del mutation (homo-deltaF508). Aim of the study is to evaluate the cholesterol metabolism and hepatobiliary injury/function in adult homo-deltaF508 patients, before and after lumacaftor/ivacaftor treatment. Baseline parameters in homo-deltaF508 patients were compared to those in CF patients compound heterozygous for F508del mutation and another severe mutation (hetero-deltaF508).MethodsCholesterol metabolism was evaluated measuring plasma phytosterols and cholestanol, as intestinal absorption markers, and lathosterol, as liver biosynthesis marker. We quantified serum vitamin E, as nutritional marker. We evaluated liver injury by aspartate aminotransferase (AST) and alanine transaminase (ALT), biliary injury by γ-glutamyltransferase (γGT) and AP, and the liver function by bilirubin and albumin.ResultsBefore the treatment, homo-deltaF508 patients (n = 20) had significantly lower cholesterol and vitamin E compared to hetero-deltaF508 (n = 20). Lumacaftor/ivacaftor treatment caused: 1) further reduction of cholesterol; 2) lathosterol reduction, suggesting a normalization of endogenous synthesis; 3) cholestanol and vitamin E increment, indicating an improvement of lipid digestion/absorption. Vitamin E difference (after-before treatment) was positively associated to treatment months. Alkaline phosphatase was also reduced.ConclusionsThese data suggest an effect of lumacaftor/ivacaftor on cholesterol metabolism and enterohepatic flux in CF patients. However, lumacaftor/ivacaftor does not promote the increase of cholesterol serum concentration that on the contrary declines. Further studies are needed to research the real mechanism causing this reduction.