The predictive value of clinical, radiographic, echocardiographic variables and cardiac biomarkers for assessing risk of the onset of heart failure or cardiac death in dogs with preclinical myxomatous mitral valve disease enrolled in the DELAY study.

OBJECTIVES: To identify the predictive value on time to onset of heart failure (HF) or cardiac death of clinical, radiographic, and echocardiographic variables, as well as cardiac biomarkers N-terminal pro brain natriuretic peptide (NT-proBNP) and cardiac troponin I in dogs with preclinical myxomatous mitral valve disease (MMVD). ANIMALS: One hundred sixty-eight dogs with preclinical MMVD and left atrium to aortic root ratio ≥1.6 (LA:Ao) and normalized left ventricular end-diastolic diameter ≥1.7 were included. METHODS: Prospective, randomized, multicenter, single-blinded, placebo-controlled study. Clinical, radiographic, echocardiographic variables and plasma cardiac biomarkers concentrations were compared at different time points. Using receiving operating curves analysis, best cutoff for selected variables was identified and the risk to develop the study endpoint at six-month intervals was calculated. RESULTS: Left atrial to aortic root ratio >2.1 (hazard ratio [HR] 3.2, 95% confidence interval [95% CI] 1.9-5.6), normalized left ventricular end-diastolic diameter > 1.9 (HR: 6.3; 95% CI: 3.3-11.8), early transmitral peak velocity (E peak) > 1 m/sec (HR: 3.9; 95% CI: 2.3-6.7), and NT-proBNP > 1500 ρmol/L (HR: 5.7; 95% CI: 3.3-9.5) were associated with increased risk of HF or cardiac death. The best fit model to predict the risk to reach the endpoint was represented by the plasma NT-proBNP concentrations adjusted for LA:Ao and E peak. CONCLUSIONS: Logistic and survival models including echocardiographic variables and NT-proBNP can be used to identify dogs with preclinical MMVD at higher risk to develop HF or cardiac death.

DELay of Appearance of sYmptoms of Canine Degenerative Mitral Valve Disease Treated with Spironolactone and Benazepril: the DELAY Study.

INTRODUCTION: Efficacy of renin-angiotensin-aldosterone system (RAAS) blockade using angiotensin-converting enzyme inhibitors (ACEi) in dogs with preclinical myxomatous mitral valve disease (MMVD) is controversial. HYPOTHESIS: Administration of spironolactone (2-4 mg q 24 h) and benazepril (0.25-0.5 mg q 24 h) in dogs with preclinical MMVD, not receiving any other cardiac medications, delays the onset of heart failure (HF) and cardiac-related death. Moreover, it reduces the progression of the disease as indicated by echocardiographic parameters and level of cardiac biomarkers N-terminal pro brain natriuretic peptide (NT-proBNP) and cardiac troponin I (cTnI). ANIMALS: 184 dogs with pre-clinical MMVD and left atrium-to-aortic root ratio (LA:Ao) ≥1.6 and normalized left ventricular end-diastolic diameter (LVEDDn) ≥1.7. METHODS: This is a prospective, randomized, multicenter, single-blinded, placebo-controlled study. Primary outcome variable was time-to-onset of first occurrence of HF or cardiac death. Secondary end points included effect of treatment on progression of the disease based on echocardiographic and radiographic parameters, as well as variations of NT-proBNP and cTnI concentrations. RESULTS: The median time to primary end point was 902 days (95% confidence interval (CI) 682-not available) for the treatment group and 1139 days (95% CI 732-NA) for the control group (p = 0.45). Vertebral heart score (p = 0.05), LA:Ao (p < 0.001), LVEDDn (p < 0.001), trans-mitral E peak velocity (p = 0.011), and NT-proBNP (p = 0.037) were lower at the end of study in the treatment group. CONCLUSIONS: This study failed in demonstrating that combined administration of spironolactone and benazepril delays onset of HF in dogs with preclinical MMVD. However, such treatment induces beneficial effects on cardiac remodeling and these results could be of clinical relevance.

The Italian multi-centre project on evaluation of MRI and other imaging modalities in early detection of breast cancer in subjects at high genetic risk.

This report presents the preliminary results of the first phase (21 months) of a multi-centre, non-randomised, prospective study, aimed at evaluating the effectiveness of contrast-enhanced magnetic resonance imaging (MRI), X-ray mammography (XM) and ultrasound (US) in early diagnosis of breast cancer (BC) in subjects at high genetic risk. This Italian national trial (coordinated by the Istituto Superiore di Sanità, Rome) so far recruited 105 women (mean age 46.0 years; median age 51.0; age range 25-77 years), who were either proven BRCA1 or BRCA2 mutation carriers or had a 1 in 2 probability of being carriers (40/105 with a previous personal history of BC). Eight cases of breast carcinomas were detected in the trial (mean age 55.3 years, median age 52.5; age range 35-70 years; five with previous personal history of BC). All trial-detected BC cases (8/8) were identified by MRI, while XM and US correctly classified only one. MRI had one false positive case, XM and US none. Seven "MRI-only" detected cancers (4 invasive, 3 in situ) occurred in both pre- (n = 2) and post-menopausal (n = 5) women. With respect to the current XM screening programmes addressed to women in the age range 50-69 years, the global incidence of BC in the trial (7.6%) was over ten-fold higher. The cost per "MRI-only" detected cancer in this particular category of subjects at high genetic risk was substantially lower than that of an XM-detected cancer in the general women population. These preliminary results confirmed that MRI is a very useful tool to screen subjects at high genetic risk for breast carcinoma, not only in pre-, but also in post-menopausal age, with a low probability of false positive cases.

[The percutaneous drainage of prostatic abscesses in the dog]. / Drenaggio percutaneo degli ascessi prostatici nel cane.

INTRODUCTION: Prostatic abscesses are an uncommon finding in the dog; they are most frequently seen in dogs over six years old, often in association with benign hyperplasia. Ultrasonography (US) is an essential technique to study prostatic conditions in the dog, because the particular anatomical site of this gland in the dog makes rectal palpation insufficient to assess even macroscopic changes. Presently, US-guided drainage makes a particularly efficient tool for treatment of this condition in these patients. We report on our personal technique of percutaneous drainage of prostatic abscesses in the dog. MATERIAL AND METHODS: Forty-five dogs of different breeds and age were examined. Some of them were given short anesthesia or mild sedation for restraining purposes, although this procedure is painless and could be performed under local anesthesia like in human patients. In man, the approach is perineal, but in the dog it is best to use an abdominal approach with right or left inguinal positions. US is necessary for correct drainage of the abscess and for monitoring throughout the procedure. DISCUSSION AND CONCLUSION: US-guided percutaneous drainage of prostatic abscesses in the dog proved to be a safe and quick tool providing excellent results. No patients exhibited any postoperative complication and we had as little as 10% relapses at 30 days. The following drainage with alcoholization of the abscessual cavity resolved the conditions definitively. This technique was particularly interesting for both its success rate and the lack of postoperative complications, which are usually quite common after conventional surgery.

[Genetic diseases:recent scientific findings and health and ethical problems]. / Malattie genetiche: recenti acquisizioni scientifiche e problemi sanitari ed etici.

Genetic diseases are very numerous, even though rare as single conditions: therefore, overall they represent a significant portion of morbidity at population level. The improvement of molecular genetic techniques has brought a great increase in the diagnostic potential toward genetic diseases, concerning either symptomatic or pre-symptomatic individuals and healthy carriers. However, this has frequently unforeseen consequences, such as a discrepancy between diagnostic and therapeutic potentials. Moreover, the development of genetic tests has raised a number of questions regarding ethical, legal e social problems. The Italian guidelines for genetic tests (available on the Internet site of Istituto Superiore di Sanità: http:@www.iss.it) have been elaborated in 1998 to define general principles for performing and managing genetic tests as well as for programming and promoting genetic testing within the public health system. In accordance with recommendations by international bodies (WHO, EU), the Guidelines give emphasis to the appropriate use of both safe and efficacious tests, the performance in laboratories with high quality standards. A further crucial point is the relationship between the health system and individuals: authonomy of decision, psychological and social assistance, as well as adequate attention to ethical and privacy problems should be guaranteed.

Interaction of cholera toxin with gangliosides: differential effects of the oligosaccharide of ganglioside GM1 and of micellar gangliosides.

Ultraviolet difference absorption spectra of cholera toxin and its B protomer produced by the oligosaccharide moiety of the monosialoganglioside GM1 were measured as a function of the oligosaccharide concentration. In the presence of oligosaccharide, the spectrum is characterized by three peaks at 282, 288, and 292 nm. A linear increase in difference absorption was observed at these wavelengths vs. oligosaccharide concentration; a saturation effect occurred when the molar ratio of oligosaccharide to cholera toxin was higher than 5. The features of the spectra indicated that the binding with the oligosaccharide affected the environment of tryptophan and tyrosine residues of protomer B. In good agreement with the above results, circular dichroic spectra indicated also a local effect of the binding, mostly restricted to protomer B, while the residues of protomer A remained largely unperturbed. Difference absorption spectra were also measured for cholera toxin in the presence of ganglioside and detergent micelles. The employed gangliosides GD1a and GT1b, unable to bind cholera toxin, interact with the protein by way of contaminating traces of GM1. The preparations of GD1a and GT1b contained 0.8-1.0% (w/w) and 0.4-0.5% (w/w) of GM1, respectively. The results obtained with ganglioside GD1a and GT1b in contrast with the observations made with the oligosaccharide of GM1 indicated a major conformational change of the toxin structure. Upon comparison of the conformational change induced by ganglioside micelles with that induced by sodium dodecyl sulfate it may be suggested that the ganglioside micelle, behaving as a detergent, alters the structure of the toxin such as to induce the penetration of protomer A into the lipid milieu.(ABSTRACT TRUNCATED AT 250 WORDS)

A 31P NMR study on uptake and metabolism of hexose monophosphates in rat diaphragm muscle.

Phosphorus nuclear magnetic resonance spectroscopy was used to study uptake and metabolic conversion of glycolytic intermediates in rat diaphragm muscles. The resonances of several phosphorus-containing metabolites were identified in the intact tissues and in their ethanolic extracts. Experiments on muscles preincubated with glucose 6-phosphate or glucose 1-phosphate indicated that: 1) both substrates penetrate into the tissue and actively participate in glycolytic reactions; 2) glucose 1-phosphate is completely converted into other metabolites, including glucose 6-phosphate and its products; 3) preincubation with either hexose monophosphate in the presence or in the absence of insulin produced the same set of phosphorylated metabolites. Addition of insulin to preincubation media induced a conspicuous spread of chemical shifts in the band arising from phosphorylated sugars in tissues incubated with glucose 1-phosphate but not in those treated with glucose 6-phosphate. The different responses to insulin exhibited by the 31P n.m.r. spectral profiles of tissues incubated with the two substrates substantiate the hypothesis that glucose 6-phosphate and its products may undergo their metabolic conversions in the tissue along distinct intracellular enzymatic pathways, on which insulin would exert different regulatory effects. This study indicates that 31P n.m.r. may provide a useful approach to the elucidation of metabolic processes involving sugar phosphates in intact tissues.

Micellar gangliosides mediate the lipid insertion of cholera toxin protomer A.

The topology of the interaction of cholera toxin with ganglioside and detergent micelles was studied with the technique of hydrophobic photolabelling. Cholera toxin alpha and gamma polypeptide chains appear to penetrate into the hydrophobic core of ganglioside micelles. Micelles of SDS cause the labelling also of the beta polypeptide chains, while Triton X-100 micelles have little ability to mediate the labelling of the toxin. The specific reduction of the alpha-gamma disulfide bond allows the penetration of the alpha polypeptide chain into Triton X-100 micelles, but does not affect the interaction of cholera toxin with either ganglioside or SDS micelles. Thus, ganglioside micelles appear to cause a conformational change of the native toxin, such as to induce the penetration of the alpha chain into the micelle hydrophobic core.

Isolation of Salmonella wien heat-labile enterotoxin.

An enterotoxic activity has been identified in culture filtrates of Salmonella wien. The enterotoxin causes fluid accumulation in rabbit ligated ileal loops, firm induration and erythema in rabbit skin and morphological alteration in chinese hamster ovary (CHO) cell cultures; it was revealed by treatment with calcium phosphate gel, and purified on DEAE-Sephadex A-50 and BioGel A-1.5 m. The enterotoxic activity was eluted from the BioGel column in two peaks. Approximately 50-70% of the enterotoxic activity of the first peak, corresponding to the excluded volume, was resistant to heating at 75 degrees C for 30 min, while the activity of the second peak was completely destroyed by this treatment. From the heat-labile peak a protein, in homogeneous form, was isolated exploiting its affinity towards agarose gel filtration media. This protein, with enterotoxic activity was also present as shown by SDS-PAGE, in the first peak, eluted from the Bio-Gel column, where it appears to be closely associated with cell wall or membrane components and thus protected from heat denaturation. The isolated enterotoxin is stable in alkaline conditions but it is sensitive to acidic pH values; moreover, it stimulates adenylate cyclase in cell culture systems. Thus, it appears to possess properties similar to both cholera toxin and the heat-labile enterotoxin of Escherichia coli. These results indicate that the enterotoxin is a protein in nature and it is postulated that it may participate in the pathogenesis of S. wien infection.

Interaction of GMI ganglioside with bovine serum albumin: formation and isolation of multiple complexes.

The binding of ganglioside GM1 to bovine serum albumin has been studied by using absorption and fluorescence properties of the protein chromophores. Differences in the ultraviolet absorption spectrum and in fluorescence quenching, as well as a marked shift of the wavelength at the fluorescence maximum provide information about the binding of this ganglioside to albumin. Ultracentrifugal studies showed that there are two forms of the GM1-protein complexes which differ markedly in their molecular weight. These two forms have been separated on this basis, by a chromatographic sieving procedure, and designated as complexes I and II. Both complexes are characterized by a GM1: protein ratio of one ganglioside micelle per albumin polypeptide chain. Complex II polymerizes slowly and irreversibly to a dimer, complex I. These results have been correlated with the optical studies in order to draw limited inferences as to the environment of the binding sites on the native protein. The interaction between GM1 micelles and albumin is mostly hydrophobic and the two complexes are actually mixed ganglioside-protein micelles. At submicellar concentrations of ganglioside a binding of ganglioside GM1 to albumin also occurs. This process is due, however, to an aspecific, reversible adhesion of GM1 molecules on the albumin surface with no apparent perturbation of the albumin structure.

The role of the reactive disulfide bond in the interaction of cholera-toxin functional regions.

The chemical reactivity of disulfide bonds towards reducing agents, in the absence of denaturing conditions, in cholera toxin has been studied. Treatment of the toxin with dithiothreitol or other mercaptans gave selective reduction of one of the six disulfide bonds of the protein. This reactive disulfide links two distinct functional regions of the toxin, fragment alpha, which activates adenylate cyclase, and fragment gammabeta5, which recognizes the cell surface receptors. Upon reduction, the two fragments remain bound together and the secondary structure of the protein is retained. The two functional regions have been separated and purified only by methods based on charge differences. When mixed together, purified alpha and purified gammabeta5 fragments spontaneously and rapidly re-form the disulfide bond. However, reduction of the disulfide bond is an absolute requirement for freeing the catalytic site of the alpha functional region. Thus, while other non-covalent binding regions are involved in maintaining cholera toxin molecular structure, the reactive disulfide bond may play a role in the mechanism of cell intoxication.