The cyclophilin inhibitor Debio 025 normalizes mitochondrial function, muscle apoptosis and ultrastructural defects in Col6a1-/- myopathic mice.

BACKGROUND AND PURPOSE: We have investigated the therapeutic effects of the selective cyclophilin inhibitor D-MeAla(3)-EtVal(4)-cyclosporin (Debio 025) in myopathic Col6a1(-/-) mice, a model of muscular dystrophies due to defects of collagen VI. EXPERIMENTAL APPROACH: We studied calcineurin activity based on NFAT translocation; T cell activation based on expression of CD69 and CD25; propensity to open the permeability transition pore in mitochondria and skeletal muscle fibres based on the ability to retain Ca(2+) and on membrane potential, respectively; muscle ultrastructure by electronmicroscopy; and apoptotic rates by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assays in Col6a1(-/-) mice before after treatment with Debio 025. KEY RESULTS: Debio 025 did not inhibit calcineurin activity, yet it desensitizes the mitochondrial permeability transition pore in vivo. Treatment with Debio 025 prevented the mitochondrial dysfunction and normalized the apoptotic rates and ultrastructural lesions of myopathic Col6a1(-/-) mice. CONCLUSIONS AND IMPLICATIONS: Desensitization of the mitochondrial permeability transition pore can be achieved by selective inhibition of matrix cyclophilin D without inhibition of calcineurin, resulting in an effective therapy of Col6a1(-/-) myopathic mice. These findings provide an important proof of principle that collagen VI muscular dystrophies can be treated with Debio 025. They represent an essential step towards an effective therapy for Ullrich Congenital Muscular Dystrophy and Bethlem Myopathy, because Debio 025 does not expose patients to the potentially harmful effects of immunosuppression.

Evaluation of a topical cyclosporine A prodrug on corneal graft rejection in rats.

PURPOSE: To assess the efficacy of a topical cyclosporine A (CsA), water-soluble prodrug, for promoting the survival of allogenic rat corneal grafts after penetrating keratoplasty (PKP). METHODS: Corneas of Brown-Norway rats (donors) were transplanted to Lewis rats (recipients). Transplanted rats were divided in three treatment groups: group I (PBS) and group II (0.26% Debio088) received drops five times per day. Group III received a daily intramuscular CsA injection (10 mg/kg/day). Blood CsA concentrations were measured on days 2 and 14. On day 4, 10, 13 after PKP, grafts were scored for corneal transparency, edema and extent of neovascularization. An opacity score of greater than or equal to 3 was considered as a nonreversible graft rejection process. On day 14, the experimental eyes were processed for histology. RESULTS: On day 13, 12 of the 18 corneal transplants (67%) in group I showed irreversible graft rejection. Three of 18 transplants (19%) in group II and 5 of 16 transplants (28%) in group III showed irreversible graft rejection (p=0.013/p=0.019, OR=0.14/0.06 versus vehicle). Each mean clinical score for edema, opacity, and neovessels in group II were significantly lower than those of the grafts in group I (respectively p=0.010, p=0.013, p=0.024) and III except for neovessels (respectively p=0.002, p=0.001, p=0.057). Histology confirmed the clinical results. The mean CsA blood levels for groups II and III were, respectively 54+/-141 mug/l and 755+/-319 mug/l on day 2 and 14+/-34 mug/l and 1318+/-463 mug/l on day 14. CONCLUSIONS: Debio088 CsA prodrug drops given five times daily are as effective as intramuscular injection of 10 mg/kg/day for the prevention of acute corneal graft rejection in rats.

A novel water-soluble cyclosporine A prodrug: ocular tolerance and in vivo kinetics.

The purpose of this study is to demonstrate that a novel water-soluble prodrug of cyclosporine A (CsA) intended for topical ocular administration, does not induce eye irritation in a rabbit model and is able to generate therapeutic concentrations of CsA in the precorneal area immediately after administration. The eye irritancy of the prodrug and CsA control solution was assessed by the Draize test and by confocal laser ophthalmoscopy (CLSO). Residence time and tear concentrations of prodrug and CsA in the rabbit eye were assessed by HPLC. The Draize test showed an excellent tolerance for the prodrug solution while the reference CsA oil solution induced lachrymation and irritation. The CLSO-measured corneal lesions, subsequent to treatment with the prodrug and reference solutions, were 3% and 9%, respectively. The prodrug transformed rapidly, leading to relatively stable CsA concentrations in tears with a maximal concentration of 94 microg ml(-1) over the observation period. This study demonstrated that the prodrug solution was well tolerated and that clinically significant CsA tear concentrations were achieved. UNIL088 is a promising molecule in the treatment of immune-related disorders of the eye.

Antimicrobial susceptibilities of Campylobacter strains isolated from food animals in Belgium.

Campylobacter spp. are a frequent cause of diarrhoea in man, originating mostly from poultry. It has been suggested that the veterinary use of antibiotics is largely responsible for resistance in human isolates, particularly to quinolones. During a 6 month period from June to December 1998, 677 Campylobacter isolates were obtained from healthy poultry and pigs. Samples were taken at Belgian slaughterhouses. Species identification was performed by biochemical tests, multiplex PCR and SDS-PAGE of whole-cell proteins. The in vitro susceptibility to six antimicrobial drugs was determined by the agar dilution method. Campylobacter jejuni was found more often in poultry than Campylobacter coli (79% C. jejuni versus 21% C. coli). In pigs the situation was reversed (6 versus 94%). Erythromycin resistance was significantly higher (P < 0.05) in C. coli, particularly in C. coli isolated from pigs (67.2%). Alarmingly high rates of resistance to ciprofloxacin were also noted, particularly for C. coli from broilers (62.1%). The latter indicates that resistance of Campylobacter in humans could derive from animals.

Analysis of foodborne disease in Belgium in 1997.

Foodborne disease represents a major problem for public health in industrialized countries, albeit with a low lethality. Foodborne diseases are defined as a group of viral, bacterial or parasitic gastrointestinal infections transmitted by means of food. Proper food-hygiene practices and surveillance of individual diseases and in particular outbreaks are the first steps in targeting their prevention. The incidence of this illness is difficult to estimate. In the Netherlands a yearly incidence of gastrointestinal infections of 500 per 1,000 inhabitants is estimated, of which most are foodborne. To set up priorities in the actions to undertake, to establish the most frequent risks, to develop preventive efforts and to answer to international requirements, accurate data on foodborne disease from Belgium are required. In order to co-ordinate the initiatives in the Belgian context, a working group was set up in 1995. In 1997 a total of 2,013 persons with foodborne disease were identified as part of 140 outbreaks, 22 of which occurred with 10 cases or more. Salmonella Enteritidis (88 outbreaks) was identified as the main pathogen in foodborne disease, followed by S. Typhimurium (11), S. Hadar (4). Eggs and meat products were identified as the main food-items involved, although it remains difficult to obtain proper intervention studies allowing to identify the specific cause(s). In 1997, a total of 12,732 human Salmonella isolates and 5,617 Campylobacter isolates were identified by the respective national reference laboratories. Salmonella isolates from Belgium accounted in 1997 for more than a fifth of all Salmonella isolates in the EU. The final objective of the working group is the implementation of a surveillance system for all risk factors concerned with the development of food-related illness, including an early warning system and an efficient analysis of microbiological criteria relating to human health, food and food production, including livestock. An essential element of this surveillance is communication of the results, risks and measures for prevention between all the departments, institutions and public health authorities concerned.

Evolution of coronary atherosclerosis in patients with mild coronary artery disease studied by serial quantitative coronary angiography at 2 and 4 years follow-up. The Multicenter Anti-Atheroma Study (MAAS) Investigators.

AIMS: Angiographic studies on the natural course of both focal and diffuse coronary atherosclerosis have not been performed before, but can both be assessed by quantitative coronary angiography. The objective of this study was to describe the natural course of focal and diffuse coronary atherosclerosis over time. METHODS AND RESULTS: In 129 patients with mild coronary artery disease, but not on lipid-lowering medication, three coronary angiograms were made each 2 years apart. Nine hundred and sixty five angiographically diseased and non-diseased segments were analysed by quantitative coronary angiography. Mean lumen diameter and minimal lumen diameter were used as measures of diffuse and focal coronary atherosclerosis. Mean lumen diameter and minimum lumen diameter decreased by 0.02 and 0.03 mm per year. The rate of progression was similar in the angiographically non-diseased, as in the mildly and moderately diseased segments. Progression of diffuse coronary atherosclerosis was largest in severely stenosed lesions (percentage diameter stenosis > or = 50%) and in the right coronary artery with a loss of 0.19 mm and 0.16 mm in mean lumen diameter. Progression of focal disease was most prominent in new and mild lesions and the right coronary artery, with a decrease in minimum lumen diameter of 0.34 mm and 0.22 mm. In most subgroups, progression occurred gradually over time. On a per segment level, progression and the occurrence of new lesions occurred in 4.4% and 4.2%. Regression and disappearance of a lesions was found in 2.3% and 1.9%. On a per patient level, 36% were progressors, 12% had a mixed response, 36% were stable, and 16% were regressors. CONCLUSION: Diffuse and focal coronary atherosclerosis progressed at the same rate in the first and second 2 years in stenosed and non-stenosed segments. The rate of coronary atherosclerosis progression was small, but was higher for focal than for diffuse disease. A minority of lesions progressed and spontaneous regression was rare.

Effect of pimobendan on exercise capacity in patients with heart failure: main results from the Pimobendan in Congestive Heart Failure (PICO) trial.

PRIMARY OBJECTIVE: To determine the effects of pimobendan 2.5 and 5 mg daily on exercise capacity in patients with chronic heart failure. DESIGN: A randomised, double blind, placebo controlled trial of the addition of pimobendan to conventional treatment with a minimum follow up of 24 weeks. SETTING: Outpatient cardiology clinics in six European countries. PATIENTS: 317 patients with stable symptomatic heart failure, objectively impaired exercise capacity, and an ejection fraction of 45% or lower who were treated with at least an angiotensin converting enzyme inhibitor and a diuretic and who tolerated a test dose of pimobendan. RESULTS: Compared with placebo, both pimobendan 2.5 and 5 mg daily improved exercise duration (bicycle ergometry) by 6% (P = 0.03 and 0.05) after 24 weeks of treatment. At that time 63% of patients allocated to pimobendan and 59% of those allocated to placebo were alive and able to exercise to at least the same level as at entry (P = 0.5). No significant effects on oxygen consumption (assessed in a subgroup of patients) and on quality of life (assessed by questionnaire) were observed. Pimobendan was well tolerated. Proarrhythmic effects (24-hour electrocardiography) were not observed. In both pimobendan groups combined the hazard of death was 1.8 (95% confidence interval 0.9 to 3.5) times higher than in the placebo group. CONCLUSIONS: Pimobendan improves exercise capacity in patients with chronic heart failure who are also on conventional treatment. The balance between benefit and risk of treatment with this compound remains to be established however.

[Osteoma of the mastoid: apropos of 2 cases]. / Ostéome de la mastoïde: à propos de deux cas.

The authors report two cases of mastoid osteoma: it is a rare benign tumor attached to the cortex of the mastoid bone, approximatively fifty cases have been described in the english language literature and eight in the french literature. The clinical and radiological characteristics combined with histopathologic evidence demonstrate the diagnosis of mastoid osteoma, the most common type being the compact osteoma. Differential diagnosis should be considered with others benign bone-forming lesions (osteochondroma, chondroma, osteoblastoma, exostosis, fibrous dysplasia...) and with malignant lesions (osteosarcoma...). Surgical removal proposed for cosmetic deformity is a simple procedure for the vast majority of small osteomas. Early surgical intervention is recommended to avoid the evolution toward giant osteoma with potential risks of surgical complications.

Effect of cholesterol reduction by simvastatin on progression of coronary atherosclerosis: Design, baseline characteristics, and progress of the Multicenter Anti-Atheroma Study (MAAS).

The Multicenter Anti-Atheroma Study (MAAS) is a 2 + 2-year, placebo-controlled trial to evaluate the effect of simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme a (HMG-CoA) reductase inhibitor, on progression and regression of coronary atherosclerosis in patients with established coronary artery disease. This paper describes the aims, methodology, and baseline data. Patients with at least two coronary segments visibly involved with atherosclerosis, in whom an angiogram was carried out according to the standards required for quantitative analysis, were selected provided that the serum total cholesterol was between 5.5 and 8.0 mmol/L and fasting triglycerides were lower than 4 mmol/L. Between march 1988 and October 1989, 383 eligible patients of both sexes aged 30-67 years were randomized in 11 European clinics. Patients received either 20 mg oral simvastatin or placebo daily for 2 years in addition to dietary counseling. The primary outcome measures are the change in the mean absolute width and in the mean of the minimal width of segments analyzed quantitatively by coronary angiography performed before and after 2 and 4 years of trial medication. To this end, at least 5 coronary artery segments are analyzed in each angiogram using matched view. The 2-year analysis was completed on 89% of eligible patients in February 1992. The trial was initially designed with a 2-year treatment period. To allow for the possibility to extend this, the decision was taken to keep all patients on the original medication allocation until all 2-year angiograms had been analyzed. Based on a predefined decision rule, an independent committee then recommended extension of treatment with another 2 years, to be concluded by a third angiogram. Of the patients enrolled initially, 81% continued. Four-year follow-up will be completed late 1993 and final results are expected mid 1994.

Malotilate reduces collagen synthesis and cell migration activity of fibroblasts in vitro.

Diisopropyl-1,3-dithiol-2-ylidenemalonate (malotilate) was studied for and compared with cyanidanol, hydrocortisone and colchicin on its impact on fibroblast cultures. Under in vitro conditions, malotilate specifically reduces collagen synthesis of fibroblasts. In addition, malotilate is an efficient inhibitor of fibroblast migration as tested by a chemotaxis assay in a modified Boyden chamber. Our data may support the notion that malotilate is of potential interest for interfering with fibrotic processes.

Protective effect of malotilate against galactosamine intoxication in the rat.

The influence of malotilate was tested in a model of galactosamine (GalN) intoxication in the rat; two different experiments were carried out: (1) an acute intoxication (GalN at a single dose of 500 mg/kg s.c.): after pretreatment with malotilate (100 and 200 mg/kg p.o.) a marked reduction of hepatocellular injury, as shown by decrease of ASAT, ALAT and GlDH, was observed; (2) a subacute intoxication, GalN at 3 x 300 mg/kg s.c. for 3 consecutive days, which induced a more moderate hepatic necrosis: malotilate, 200 mg/kg/day p.o. for 4 days beginning the day before the first GalN injection, decreased significantly the disturbances of the biochemical measured parameters (plasma total proteins, prothrombin time, fibrinogen level, BSP clearance and hepatic total lipids) and accelerates the return to normal. Pathology studies revealed that pretreatment with malotilate increased markedly the number of mitoses observed in the hepatic cells after intoxication. In addition, the time of appearance of these mitoses was earlier in malotilate treated animals. From these studies, it can be concluded that malotilate protects against hepatocellular injury induced by GalN in the rat and that it stimulates the regenerative capacity of the liver after intoxication.

Effect of malotilate on chronic liver injury induced by carbon tetrachloride in the rat.

The effect of malotilate, a new drug proposed for the treatment of chronic liver diseases, was studied in carbon tetrachloride (CCl4)-induced chronic liver injury in the rat. Treatment with CCl4 (0.5 ml/kg twice per week, intraperitoneally for 6 or 9 weeks) led to marked necrosis, steatosis and fibrosis, as shown by both biochemical and histological examinations, and a significant decrease of the bromosulfophtaleine (BSP) clearance test. Malotilate (50 mg/kg p.o., 5 days per week given simultaneously with CCl4 for 6 weeks), suppressed the increase of plasma aminotransferase activity and decreased significantly the accumulation of lipid and collagen in the liver; histology confirmed this protective effect of malotilate. The BSP clearance test returned to normal values and the rise in hepatic collagen synthesis activity in the malotilate-treated and intoxicated rats was reduced as compared with intoxicated control rats. The same effect was found when malotilate (100 mg/kg, p.o., 5 days per week), was given for 3 weeks to rats already intoxicated during the 6 previous weeks. Malotilate was able to prevent the increase of hepatic alterations that appeared during the last 3 weeks of CCl4 intoxication. These results show clearly that malotilate can markedly reduce the hepatic disorders induced by a chronic CCl4 intoxication in the rat.

Serum concentration of the aminoterminal procollagen type III peptide in the rat reflects early formation of connective tissue in experimental liver cirrhosis.

A specific and sensitive radioimmunoassay for the rat aminoterminal procollagen type III peptide (PIIIP) was developed which allowed easy and sequential measurement of this peptide in the serum of individual animals. PIIIP in sera of 1-week-old rats was high (60 +/- 15.4 ng, 1 SD) falling to 15.7 +/- 4.3 ng/ml (1 SD) at 7 weeks and 6.7 +/- 2.6 ng/ml (1 SD) at 12 weeks of age. Adult animals (above 6 months of age) showed serum PIIIP levels in the narrow range of 2.5 +/- 2.33 ng/ml (2.5 SD). CCl4-induced liver damage in adult rats produced an elevated serum PIIIP (median 9.1; range 2.6-45.2 ng/ml) already after 2 weeks, rising to a mean of 33.8 ng/ml (range 22.0-47 ng/ml) after 6 weeks of continued CCl4-intoxication. In the same animals at 6 weeks, hepatic hydroxyproline was almost 5 times higher in the CCl4-group (mean 493.2; range 343.1-582.3 micrograms/100 mg dry weight) when compared with controls (109 +/- 14 micrograms/100 mg dry weight, 1 SD). These results are in complete analogy to those reported for PIIIP in sera of growing children, healthy human adults and patients with fibrogenic liver disease. Elevated serum PIIIP in rats with experimental liver fibrosis predicts the deposition of excess hepatic collagen. This novel serum test allows, for the first time, to assess altered PIIIP metabolism and hepatic fibrogenesis in individual animals as early as 2 weeks after the start of the experiment. It also reflects growth-related changes of type III collagen metabolism.

Antinecrotic effect of 3-palmitoyl-(+)-catechin against liver damage induced by galactosamine or ethanol in the rat.

The antinecrotic potential of a new drug, 3-palmitoyl-(+)-catechin (PC), which is a derivative of (+)-cyanidanol-3, was studied in two different experimental models of necrosis of the liver in the rat: acute hepatitis induced by galactosamine and liver damage induced by a combination of chronic ethanol feeding and hypoxia. The extent of liver damage was assessed by histological examination and by measuring blood hepatic enzyme and bilirubin levels. Intraperitoneal and oral treatments with PC were carried out at different dosages and times, before acute galactosamine intoxication. PC treatment decreased the extent of diffuse necrosis and inflammation in liver tissue and reduced galactosamine-induced biochemical deterioration. The lowest active doses of PC were 25 mg/kg, i.p. and 500 mg/kg by mouth. In experiments with ethanol, we confirmed that a 6-h period of hypoxia produced necrosis of the liver in rats undergoing chronic treatment with ethanol. Simultaneous treatment with PC (80 mg/kg/day) for 21 days during ethanol feeding gave significant protection against histological and biochemical deterioration induced by ethanol and hypoxia. The anti-necrotic effect of PC in two models, which are recognized as producing part of the biochemical and/or histological deterioration induced by viruses and ethanol in man, indicates that it is a potentially useful agent for the treatment of necrosis of the human liver.

[An ELISA study of the intestinal and serum anti-LPS antibody response in BALB/c mice fed with various mutants of "Salmonella typhi-murium" (author's transl)]. / Etude, par la méthode ELISA, du titre des anticorps sériques et intestinaux anti-LPS chez la souris BALB/c vaccinée à l'aide de mutants "rough" et "smooth" de Salmonella typhi-murium.

The specific anti-LPS serum antibody response in BALB/c mice, before and after an oral booster with different non-pathogenic strains of Salmonella typhi-murium, was measured by an ELISA technique. Serum response to LPS was found to depend on the length of the LPS polysaccharide moiety in "rough" mutants. The M206 "smooth" mutant induced a less marked anti-LPS response. The immunogenicity of the polysaccharidic "core" appears to be modified by the O antigen. The specific antibody response in the gut only becomes marked following after booster of Ra, Rc and M206 mutants.