Correction to: A case of intracoronary and endovenous levosimendan as a rescue therapy for refractory cardiac arrest.

After publication of this supplement [1], it was brought to our attention that the author 'L. Vegnuti' has been erroneously omitted from the author list of the following abstract.

Latin America: the next region for haematopoietic transplant progress.

Haematopoietic cell transplant activity in the 28 countries comprising Latin America is poorly defined. We conducted a voluntary survey of members of the Latin American Bone Marrow Transplantation Group regarding transplant activity 2009-2012. Collated responses were compared with data of transplant rates from the Worldwide Network for Blood and Marrow Transplantation for other geographic regions. Several socio-economic variables were analysed to determine correlations with transplant rates. In total, 94 teams from 12 countries reported 11 519 transplants including 7033 autotransplants and 4486 allotransplants. Annual activity increased from 2517 transplants in 2009 to 3263 in 2012, a 30% increase. Median transplants rate (transplant per million inhabitants) in 2012 was 64 (autotransplants, median 40; allotransplants, median 24). This rate is substantially lower than that in North America and European regions (482 and 378) but higher than that in the Eastern Mediterranean and Asia Pacific regions (30 and 45). However, the Latin America transplant rate is 5-8-fold lower than that in America and Europe, suggesting a need to increase transplant availability. Transplant team density in Latin America (teams per million population; 1.8) is 3-4-fold lower than that in North America (6.2) or Europe (7.6). Within Latin America, there is substantial diversity in transplant rates by country partially explained by diverse socio-economic variables including per capita gross national income, health expenditure and physician density. These data should help inform future health-care policy in Latin America.

Aldosterone receptor antagonism and heart failure: insights from an outpatient clinic.

OBJECTIVE: In randomized clinical trials, aldosterone antagonists have been shown to reduce mortality and morbidity in heart failure (HF). The aim of the present study was to examine the risk-benefit profile of aldosterone antagonists in routine clinical practice. METHODS: A retrospective analysis, extending over a 1-year period, of the clinical, instrumental and laboratory data of 264 HF outpatients was performed. All patients were on a beta-blocker and an ACE-inhibitor (or angiotensin-II receptor-blocker) and 151 were taking an aldosterone antagonist. RESULTS: At baseline, subjects treated with aldosterone antagonists had a higher NYHA class, a larger left-ventricular end-diastolic volume, a worse ejection fraction and a higher systolic pulmonary arterial pressure (sPAP). During follow-up, a greater reduction in sPAP and a tendency towards improved systolic and diastolic function were observed in subjects treated with aldosterone antagonists. Moreover, clinical and laboratory parameters did not deteriorate in patients taking aldosterone antagonists. Mortality rates were similar in the two groups (8.6% vs. 8.8%, P = NS). CONCLUSIONS: The use of aldosterone antagonists in HF is associated with an improvement in cardiac function and is well tolerated. In the present study, patients administered these agents had a comparable clinical outcome to that of the control group, despite important differences in baseline risk.

The term diplegia should be enhanced. Part III: inter-observer reliability of the new rehabilitation oriented classification.

AIM: The aim of this study was to validate a recent classification of gait in children with the spastic diplegic form of cerebral palsy (CP) by checking the reliability of different scorers in assigning subject walking performance to one of the four specific patterns described in the classification. METHODS: The gait patterns of 50 children and adolescents with CP (23 males, 27 females; age range 3-17 years) were selected among patients whose videos were stored in the archives of the Pisa and Reggio Emilia Hospitals. Only video recordings of gait with homogeneous features (duration of at least 90 s, simultaneous recordings on sagittal and frontal views, and other criteria) were taken for examination. The videos were blindly scored using an observational gait scale, at first by two of the authors of the classification system (defined as ''maximum experts''), then by ten expert observers, and finally by 206 professionals of rehabilitation after a one-day training on the classification. Cohen's kappa statistics (k) and intra class correlations (ICC) were calculated. RESULTS: Kappa and ICC indicate an almost perfect agreement both between the two maximum experts and among the ten expert observers. Good results were also obtained in the group of one-day trained scorers. Only a few cases were assigned to the ''unclassified'' category. The profession of the observer (doctor or therapist) and previous knowledge of the classification had no significant influence on reliability scores. CONCLUSION: The results suggest that the proposed classification can be reliably applied, even utilizing short video recordings, to arrange diplegic children into different patterns. Further studies are needed to validate the use of this classification system for clinical and research aims.

Composition of biomineral organic matrices with special emphasis on turbot (Psetta maxima) otolith and endolymph.

The soluble organic matrix (OM) of various biominerals (red coral skeleton, oyster shell, sea urchin test, turbot otolith, chicken eggshell) was extracted after demineralization with acetic acid. The protein content of the OM varies strongly from 0.02 to 1.6 microg/mg biomineral whereas proteoglycans present less variations (from 0.7 to 1.4 microg/mg biomineral). Electrophoresis of biominerals OM shows differences in their protein pattern although several bands are present in all matrices. OM of all biominerals shows carbonic anhydrase activity but no activity was detectable in the endolymph. OM of all biominerals also displays an anticalcifying activity. After separation of the OM extracts by chloroform-methanol, 80% of the anticalcifying activity was found in the methanol phase except in the urchin test. After OM precipitation with trichloracetic acid, 70% of the activities was found in the supernatants. Partial biochemical characterization suggests that the anticalcifying factor is a polyanionic and water-soluble molecule, which could be proteoglycans. The endolymph surrounding the otolith also displays an anticalcifying activity although its inhibitous activity was 50 times lower than that of the otolith OM. However, the anticalcifying activity of the endolymph is assumed by a proteic structure (80% activity precipitated with TCA treatment). Our results suggest that both carbonic anhydrase and anticalcifying activities are widespread and play a significant role in the regulation of biomineral formation. Results are discussed in relation to the calcification process that takes place at the fluid-mineral interface.

Daily variations of endolymph composition: relationship with the otolith calcification process in trout.

Ionic and organic parameters of the otolith calcification process in the trout Oncorhynchus mykiss were analysed in plasma and endolymph over the day:night cycle. Plasma pH remained constant and total CO(2) concentration was significantly lower (by 21%) during the day than at night. Calcifying parameters (total CO(2), total calcium concentration) were measured in the proximal and distal endolymphs and were unchanged in the latter during the day:night cycle, but fluctuated in the former. Non-collagenous protein and collagen concentrations in endolymph were higher (1.5- and 10-fold, respectively) during the day than at night. As there was no change in total calcium concentration, we propose that Ca(2+) increases during the dark period and was maximal by the end of the night when the total CO(2) concentration has also increased (by 14%). Measurements of endolymph pH in situ revealed significant differences between samples from proximal and distal endolymph (7.38 and 7.87, respectively), but no variation between values obtained during the day and at night. Thus, the saturation state of aragonite (Sa) in the proximal endolymph should fluctuate around unity during the day:night cycle, and CaCO(3) precipitation should occur when supersaturation is reached. The electrophoretic pattern of proximal endolymph showed variations in both major and minor components. Immunoblotting of endolymph, using a rabbit antiserum raised against the otolith soluble organic matrix revealed an increase in the expression of two proteins (65 kDa and 75 kDa) during the day period. We propose that organic matrix and calcium carbonate deposition on the otolith vary antiphasically: organic matrix deposition begins by the end of the day period, when the concentration of organic precursors is maximal in the endolymph, whereas CaCO(3) precipitation starts once the solubility of CaCO(3) is exceeded.

Otolith growth in trout Oncorhynchus mykiss: supply of Ca2+ and Sr2+ to the saccular endolymph.

Kinetic and pharmacological characteristics of Ca2+ fluxes across the saccular epithelium of trout were studied using a perfused isolated inner ear. 45Ca2+influx from the Ringer solution to the endolymph was 3-4 nmoles h(-1)microl(-1) endolymph, which corresponds to a global turnover rate of the endolymph calcium of 200 % h(-1). Ca2+ entry into the proximal endolymph was faster than into the distal fluid. Net Ca2+ movement across the saccular epithelium depended on the direction and intensity of the chemical gradient of calcium between the Ringer solution and the endolymph. Increasing the calcium concentration in the Ringer solution up to 4.4 mmol l(-1) provoked an accumulation of Ca2+ in both proximal and distal endolymphs, and equilibrium was reached about 30 min after the beginning of perfusion. Perfusion with calcium-free Ringer partially emptied the proximal compartment of calcium, whereas the calcium levels in the distal endolymph did not vary during 70 min of perfusion. Verapamil (10(-5) mol l(-1)) and cyanide (CN, 10(-3) mol l(-1)) did not modify the accumulation of Ca2+ within the endolymph in the presence of a favourable calcium chemical gradient. Furthermore the relationship between Ca2+ net fluxes and the chemical calcium gradient across the saccular epithelium was linear, indicating a passive diffusional mechanism via a paracellular pathway. Similar relationships were found for Sr2+ fluxes across the saccular epithelium in the presence of positive chemical gradients (1, 2 and 4 mmol l(-1) Sr2+). In vivo experiments in which trout were intraperitoneously injected with CaCl2 solution confirmed the tight relationship between the calcium levels in plasma and endolymph (both proximal and distal). Sampling proximal and distal endolymphs in trout and turbot saccules revealed a decreasing proximo-distal calcium gradient in endolymph of both fish species. The present results strongly suggest that the endolymph is supplied with Ca2+ and Sr2+ via a paracellular pathway located in the proximal area of the saccular epithelium.

Biochemical relationships between endolymph and otolith matrix in the trout (Oncorhynchus mykiss) and turbot (Psetta maxima).

This paper compares the organic compositions of the otolith and endolymph of trout and turbot. Irrespective of the method of demineralization (0.5 M EDTA or acetic acid), trout otoliths were found to be largely composed of proteins (48%), collagens (23%), and proteoglycans (29%). Collagen was only detectable in the EDTA-insoluble (0.30 microg/mg) and in the acetic acid-soluble fractions (0.53 microg/mg). The same compounds were found in the endolymph but in different proportions (proteins 85%, collagens 12%, and proteoglycans 3%). It was shown that the distribution of these compounds was not uniform within the endolymph. Proteins, collagens, and amino acids were 4, 10, and 3 times, respectively, more concentrated in the proximal (facing the macula) than the distal side whereas proteoglycans were 10 times more concentrated at the distal side. SDS PAGE analyses of proximal and distal samples of endolymph showed similar patterns suggesting that the spatial gradient of protein is quantitative and not qualitative. SDS PAGE comparison of endolymph and otolith samples showed only two proteins with similar molecular weights. We propose that collagen and protein gradients are involved in the organic matrix formation and otolith calcification process. Endolymphs from both trout and turbot display inhibitions of in vitro calcification although these inhibitions were 50 and 80 times, respectively, less than that of the otoliths. The inhibitory factor probably plays a significant role in the regulation of otolith calcification.

Chemical composition of saccular endolymph and otolith in fish inner ear: lack of spatial uniformity.

Fish otoliths provide a record of age, growth, and environmental influences. In both trout and turbot, spatial chemical investigation of the endolymph surrounding the otolith (sagitta) showed a lack of uniformity. Proteins, PO(3-)(4), and Mg(2+) were significantly more concentrated in the proximal (facing the macula) than distal zone, whereas the opposite was observed for K(+) and total CO(2) (totCO(2)). Na(+) concentration ([Na(+)]) was 20% higher in the proximal zone in trout but not in turbot. Total Ca and Cl(-) contents were uniformly distributed in both species. We propose that the endolymphatic gradients of protein and totCO(2) concentration within the endolymph are involved in the otolithic biocalcification process. Microchemical analyses of otolith sections by wavelength dispersive spectrometry showed a lack of spatial uniformity in the K/Ca and Na/Ca ratios, whereas the Sr/Ca ratio was uniform. There is a clear relationship between endolymph and otolith [K(+)], but the interpretation of the results for [Na(+)] needs further investigation. Thus the lack of uniformity in the otolith composition must be taken into account when investigating otolith microchemistry.

Ipriflavone does not alter bone apatite crystal structure in adult male rats.

We have previously found that a short-term treatment with high doses of ipriflavone increased bone density and improved the biomechanical properties of adult male rat bones, without altering their mineral composition. To determine whether this effect can be associated with alterations of bone crystal structure, we have performed X-ray diffraction analysis of bones obtained from rats treated with ipriflavone at doses that were effective in inducing favorable changes on bone density and biomechanics. Eighteen-week-old male Sprague Dawley rats were treated by oral route with either ipriflavone (200 or 400 mg/kg/day), or its vehicle for 12 weeks. The treatment was well tolerated and body weight increased to the same extent in all animals. As a measure of bone crystallinity, we examined the (310) and (002) reflections of the X-ray diffraction patterns, corresponding to the directions perpendicular and parallel to the c-axis of the crystals, respectively. No major differences were observed between ipriflavone-treated and control animals for the broadening parameter beta(1/2) for (310) and (002) peaks, as well as for lattice parameters. Therefore, a 12-week treatment with ipriflavone at high doses does not induce significant modifications of bone "crystallinity." Thus, the positive effect of ipriflavone on bone mineral density appears to be associated with an increased apatite crystal formation rather than an increase of crystal size. These results provide further evidence for the safety and usefulness of ipriflavone in the treatment of osteoporotic syndromes.

Effect of a new de-N-acetyl-lysoglycosphingolipid on some tumour models.

A new de-N-acetylated glycosphingolipid termed WILD20, a breakdown product of GM1 obtained through alkaline hydrolysis, and characterized by nuclear magnetic resonance, mass spectrometry and elementary analysis, was found to inhibit phospholipase A2 via phosphokinase C translocation blockade. The substance inhibited various tumour cell lines in vitro, in synergy with doxorubicin and cisplatin. In vivo, it showed an antitumoral effect when both the tumour cells and WILD20 were injected at the same site (peritoneal cavity). Tumour cells, incubated with WILD20, showed a dose-dependent decrease of oncogenicity without impairment of viability. WILD20 also down-regulated tumour cell adherence to laminin and fibronectin. When peritumorally administered, WILD20 impaired tumour growth and potentiated the peritumoral effects of recombinant interleukin 2. The results obtained merit exploration of the therapeutical possibilities of this agent in human cancer patients.

Investigation of potential oncogenetic effects of beta-cyclodextrin in the rat and mouse.

The results of oncogenicity studies of beta-cyclodextrin in inbred Fischer 344 rats and CD-1 outbred mice are presented. Chronic feeding of beta-cyclodextrin to Fischer 344 rats and CD-1 mice did not cause any treatment related carcinogenic effects. The only toxic effect was seen in mice as macroscopic distension of the large intestine with soft or fluid contents, histologically associated with the mucosa covered by mucous secretion containing exfoliated cells, and mucosal flattening and intestinal gland atrophy. Despite these observations, no differences between control and treated groups were observed concerning mortality, clinical observations or body weight and food consumption.

Antiplatelet effects of a new de-N-acetyl-lyso-glycosphingolipid.

Gal beta 1-->3GalN beta 1-->4Gal(3<--2 alpha Neu)beta 1-->4Glc beta-->1Sph (WILD20), a new glycosphingolipid, a breakdown product of the monosialoganglioside GM1 obtained through alkaline hydrolysis, shows dose-dependent platelet anti-aggregating properties in vitro and in vivo. This effect is agonist- and species-independent. The family of lysosphingolipids, to which the compound belongs, is present in platelets particularly after thrombin treatment. WILD20 antiplatelet effect is due to the interference with ADP or thrombin-induced aggregation, probably via phospholipase A2 (PLA2) blockade; the substance is also effective when arachidonic acid is used as an agonist. Serotonin blood levels are also reduced. The substance, orally active at dosages of 0.1-0.01 mg/kg as antiplatelets agent, prolonged bleeding time without interfering with the coagulative or fibrinolytic processes.

Hepatitis C virus infection in patients with essential mixed cryoglobulinemia.

OBJECTIVE: To study the association between hepatitis C virus (HCV) infection and essential mixed cryoglobulinemia. SETTING: Wards and clinics of the Ospedali Riuniti di Bergamo and Ospedale di Treviglio e Caravaggio, Italy. PATIENTS: Fifty-one patients with essential mixed cryoglobulinemia associated with glomerulonephritis and 45 controls with noncryoglobulinemic glomerulopathies. MEASUREMENTS: Antibodies to hepatitis C virus (anti-HCV) in sera from patients with essential mixed cryoglobulinemia and from controls, using two enzyme-linked immunosorbent assays (c100 ELISA and c22/c200 ELISA) and a recombinant immunoblot assay (4-RIBA); cryoprecipitate anti-HCV before and after use of dithiothreitol, a substance able to destroy IgM antibodies with rheumatoid factor activity, in patients with essential mixed cryoglobulinemia; serum HCV RNA by polymerase chain reaction in patients with essential mixed cryoglobulinemia. RESULTS: In patients with essential mixed cryoglobulinemia, the c22/c200 ELISA detected anti-HCV in 98% of serum samples (95% CI, 90% to 100%), whereas the rate of reactivity remained at 2% (CI, 0% to 12%) in the control group (P less than 0.0001). These results were confirmed by the 4-RIBA in 66% of patients with essential mixed cryoglobulinemia. The study of cryoprecipitate by c100 ELISA showed anti-HCV in 41% (Cl, 28% to 56%) of patients. After dithiothreitol, the rate of reactivity increased to 94% (CI, 84% to 99%; P less than 0.0001 by the McNemar paired chi-square test), suggesting that the elimination of rheumatoid factor leads to unmasking of anti-HCV in cryoprecipitate. Polymerase chain reaction detected HCV RNA in 13 of 16 sera from patients with essential mixed cryoglobulinemia. CONCLUSIONS: The extremely high prevalence of anti-HCV in serum and cryoprecipitate along with the frequently associated serum HCV RNA suggests a close relation between essential mixed cryoglobulinemia and chronic HCV infection.

Improvement of cognitive function by MAO-B inhibitor L-deprenyl in aged rats.

This study evaluated the ability of the selective MAO-B inhibitor, L-deprenyl, to reverse cognitive impairments appearing in aged rats, using the reference memory, Morris Water Maze paradigm. L-Deprenyl significantly improved learning and memory deficits associated with old age in doses of 1.25 and 5 mg/kg PO (escape latency measure) and doses of 1.25, 2.5 and 5 mg/kg PO (path length measure). L-Deprenyl also improved reversal learning impairments in doses of 1.25, 2.5 and 5 mg/kg PO, as expressed by the escape latency measure. The data suggest that L-deprenyl possesses potential cognitive enhancement abilities probably due to an increase in dopaminergic activity.

Morphological and biochemical effects of intrahepatic alcohol injection in the rabbit.

The local and systemic effects, as well as the repair mechanisms, of sterile absolute ethyl alcohol injection were evaluated at a range of doses (0.1-2.0 mL/kg body weight) in rabbit liver in order to confirm the feasibility and safety of local treatment of tumours in man. Saline injection was used in the control animals. The animals were killed at varying intervals (range: 1-30 days after injection), and the liver was studied by gross and microscopic examination. The ethyl alcohol injection was well tolerated and did not induce significant systemic side-effects. All doses could induce necrosis and none proved to be lethal. The alcohol injection produced an area of coagulation necrosis, the size of which appeared to be dose-related, and which was surrounded by granulation tissue, gradually repairing the necrotic lesion; the adjacent tissue was intact, or had signs of mild steatosis. However, at higher doses (1.0 and 2.0 mL/kg bodyweight), necrotic lesions were observed in the liver both near and remote from the site of injection. Fine needle percutaneous alcohol injection is effective in producing necrotic lesions which appear to be dose-related; at higher doses, however, an unpredictable intrahepatic diffusion may occur.

Carcinogenicity study of doxefazepam administered in the diet to Sprague-Dawley rats.

Groups of 50 male and 50 female Sprague-Dawley rats were given food containing sufficient doxefazepam, a benzodiazepine derivative, to ensure intakes of 0, 3, 10, or 30 mg/kg/day. These dosages respectively correspond to 2, 20, and 60 times the mean daily hypnotic dose level of an adult man. Rats were treated for 104 weeks and then euthanized. An extensive autopsy was performed on those animals that died intercurrently and on euthanized animals. The chronic administration of doxefazepam did not influence the survival of the rats. No treatment-related changes in clinical signs and body weight gains occurred and malignant tumor rates were similar in controls and treated animals. A significant linear trend in the incidence of hepatocellular neoplasms, primarily benign, was observed in the female treated groups. This higher incidence was not associated to a higher occurrence of focal hyperplasia or other preneoplastic lesions in treated rats. The brain, a target organ for the pharmacological activity of doxefazepam, was carefully examined to search for microscopic foci of proliferative cells. A total of 12 and 6 malignant gliomas were observed in male and female rats, respectively; only two were noticed at autopsy. These tumors were mainly of the oligodendroglioma type commonly found in aged rats. Their incidence was slightly higher in treated rats, but results were not of statistical significance. The overall evaluation of the present study indicates that doxefazepam is noncarcinogenic in rats. However, the increase in liver adenomas found here as well as in previous bioassays with similar drugs and the lack of reliable historical data on the incidence of brain tumors in benzodiazepine-treated rodents suggest that additional experimental and epidemiological studies should be undertaken to exhaustively assess the toxic potential of this widely used class of drugs.

Toxicological evaluations of the benzodiazepine doxefazepam.

1-(2-Hydroxyethyl)-3-hydroxyl-7-chloro-1,3-dihydro-5-(O-fluorophenyl)-2H - 1,4-benzodiazepin-2-one (doxefazepam, SAS 643, Doxans) was investigated in a series of toxicological studies. Oral LD50 values were greater than 2000 mg/kg in mice, rats and dogs, while endoperitoneal LD50 values were 746 and 544 mg/kg in the mice and rats, respectively, and greater than 1000 mg/kg in the dogs. Subacute and chronic studies in rats and dogs evidenced a transient ataxia after administration of the test compound, which was dose-dependent in the subacute experiment, and occurred only at the highest dose in the chronic studies. No pathological findings were registered at necropsy or in microscopic observations, except an increase of liver weight at the highest dosage in the chronic study in the rat. Doxefazepam did not exert any teratogenic effects in rats and rabbits. Moreover in rats it did not alter the reproductive performance. The mutagenic studies did not reveal any mutagenic potential. In the cancerogenicity study in rats doxefazepam did not show positive carcinogenic potential.

[Angiographic and clinical evaluation of the topical administration of nitrate derivatives in distal obstructive arteriopathies of the legs. Initial experiences]. / Valutazione angiografica e clinica dell'applicazione topica di nitroderivati nelle arteriopatie ostruttive distali degli arti inferiori. Prime esperienze.

The vasodilator effect obtained by the topic use of a nitroglycerin compound (TNG) has been angiographically tested on 11 patients affected by distal arteriopathy of the legs. All patients presented arteriosclerotic vascular lesions; two of them also suffered from diabetic angiopathy. Arteriography of the legs has proved to be a very important tool in the evaluation of the patients' response to the administration of the drug. An hour after 80 mg of TNG had been applied to the skin of the examined leg, arteriography showed a marked dilation, especially of the muscular arteries and the undamaged tracts of the arteries of the legs. Angiographic evaluation of the functional blood supply thus obtained provides prognostic information and helps in the choice of the subsequent therapy. Furthermore, the clinical efficacy of prolonged treatment with this drug has been tested in 10 out of the 11 patients. They all responded with an immediate and persistent hemodynamic improvement, evaluated according to Fontaine's classification.