Clinical correlations and long-term follow-up in 100 patients with sarcoglycanopathies.

BACKGROUND AND PURPOSE: To describe a large series of patients with α, ß, and γ sarcoglycanopathies (LGMD-R3, R4, and R5) and study phenotypic correlations and disease progression. METHODS: A multicentric retrospective study in four centers in the Paris area collecting neuromuscular, respiratory, cardiac, histologic, and genetic data. The primary outcome of progression was age of loss of ambulation (LoA); disease severity was established according to LoA before or after 18 years of age. Time-to-event analysis was performed. RESULTS: One hundred patients (54 γ-SG; 41 α-SG; 5 ß-SG) from 80 families were included. The γ-SG patients had earlier disease onset than α-SG patients (5.5 vs. 8 years; p = 0.022) and ß-SG patients (24.4 years). Axial muscle weakness and joint contractures were frequent and exercise intolerance was observed. At mean follow-up of 22.9 years, 65.3% of patients were wheelchair-bound (66.7% α-SG, 67.3% γ-SG, 40% ß-SG). Dilated cardiomyopathy occurred in all sarcoglycanopathy subtypes, especially in γ-SG patients (p = 0.01). Thirty patients were ventilated and six died. Absent sarcoglycan protein expression on muscle biopsy and younger age at onset were associated with earlier time to LoA (p = 0.021 and p = 0.002). Age at onset was an independent predictor of both severity and time to LoA (p = 0.0004 and p = 0.009). The α-SG patients showed genetic heterogeneity, whereas >90% of γ-SG patients carried the homozygous c.525delT frameshift variant. Five new mutations were identified. CONCLUSIONS: This large multicentric series delineates the clinical spectrum of patients with sarcoglycanopathies. Age at disease onset is an independent predictor of severity of disease and LoA, and should be taken into account in future clinical trials.

Nemaline myopathies: State of the art.

Nemaline myopathy (NM) is one of the most common forms of congenital myopathy. The condition is defined by the histopathological finding of nemaline bodies (rods) on muscle biopsy and is associated with hypotonia and muscle weakness. The clinical spectrum encompasses lethal forms presenting in the neonatal period with profound weakness and less severe congenital diseases of later onset. NM is significantly heterogeneous from a genetic point of view, and its inheritance can be autosomal-dominant (AD), sporadic or autosomal-recessive (AR). To date, 11 genes encoding proteins of skeletal muscle thin filaments, Kelch domain-associated proteins and an unconventional myosin have been implicated in NM. The mechanisms leading to nemaline body formation and muscle weakness are still largely unclear. This report reviews the clinical, histopathological and genetic features of NM, with a focus on some of the recently discovered forms.

Chronic progressive external ophthalmoplegia: a new heteroplasmic tRNA(Leu(CUN)) mutation of mitochondrial DNA.

We sequenced all genes of mitochondrial tRNAs of a patient with chronic progressive external ophthalmoplegia with 5% ragged red fibres and 15% COX-negative fibres but without macrorearrangements of mitochondrial DNA (mtDNA). Direct sequencing showed a novel heteroplasmic G>A substitution in position 12316 of tRNA(Leu(CUN)) gene. This change destroys a highly conserved G-C base coupling in tRNA TpsiC branch. By RFLP analysis we could demonstrate different degrees of heteroplasmy in different patient's tissues. This alteration, absent in a population of 110 patients with different encephalomyopathies, can be considered pathogenic: it is the tenth tRNA(Leu(CUN)) pathogenic mutation described up to date.

Time related neutralization of two doses acetyl salicylic acid.

Aspirin has a well established role in the prevention of arterial thrombosis. Discussion on the efficacy and safety of aspirin in the treatment and prophylaxis of thrombosis has become an important issue. In fact, hemorrhage complications are often associated with its use. On the other hand, previous studies showed unexpected thrombotic potencies associated with the presence of this drug at ultra low doses (ULD) in the circulation. In this study, we aimed to evaluate the effect of aspirin at ULD, injected 1, 2, or 3 hours after the administration of aspirin at 100 mg/kg, on hemostasis and bleeding in rats. We used an experimental model of thrombosis induced by laser beams to evaluate these effects. Platelet aggregation was determined by Cardinal and Flower method. Results from this investigation demonstrate that the neutralizing effect of aspirin at ULD did not operate significantly 1 hour after the injection of aspirin at 100 mg/kg. This effect was observed 2 and 3 hours after. The use of aspirin at ULD to neutralize the side effects of aspirin at high doses will reduce the hemorrhagic risk during extra corporeal circulation. The therapeutic benefit and safety of aspirin therapy in the treatment of cardiovascular diseases can be obtained.

Thrombogenic potential of contrast media in an experimental model of laser-induced thrombosis.

Controversy still exists about the pro- or antithrombotic side effects of contrast media used in daily medical practice. Recent reports have shown that various contrast media, including ionic compounds, have deleterious prothrombotic actions. A new evaluation of these adverse side effects is reported here, with the study of the dose-effect relationship. Two ionic (ioxaglate and diatrizoate) and two non-ionic contrast media (iopamidol and iohexol) were studied. Experiments were done on 22 groups of 5 Wistar male rats each, using a Laser Argon-induced thrombosis model in mesenteric microvessels. Three parameters were studied: the number of laser beams needed to induce platelet thrombus formation, the number of emboli, and the duration of embolization. Platelet count and platelet aggregation also were determined. Iopamidol and iohexol induced a significant rise in both the number of emboli and the duration of embolization in mesenteric microvessels at doses up to 1 mL/kg. Ioxaglate and diatrizoate also significantly increased these parameters at doses up to 2 mL/kg. All the products tested decreased platelet count, inducing a -17 to -30% variation from control values. Diatrizoate and ioxaglate inhibited platelet aggregation, while iopamidol and Iohexol behaved as activators. All non-ionic, and to a lesser extent, all ionic contrast media demonstrated prothrombotic properties.

Effects of acetyl salicylic acid therapy on an experimental thrombosis induced by laser beam.

Aspirin inhibits the synthesis of both platelet and vascular arachidonic acid metabolism which have opposite effects on platelet functions. The rationale for its clinical use as an antithrombotic drug has therefore been questioned. Therefore, we investigated the effects of acetylsalicylic acid (ASA) at 100 mg/kg on an experimental thrombosis induced by laser beams using different groups of rats that were previously treated with the same dose (100 mg/kg), according to the delay between the first and second injections. A partial occlusion was induced by laser beams in the rat mesenteric microvessels (15-25 m). The thrombus formed within seconds after the laser lesion; both it and the embolization which began within minutes after, were continuously accounted. Experiments were done on 11 groups of 5 animals each: 45 rats received a first injection of ASA at j(0) and a second injection 30 minutes before thrombosis induction at j(0)+x (x=2, 4, 6, 8, 9, 10, 12, 14 and 16 days). Different groups are defined according to the x value. The rats receiving NaCl 0.9% or a single injection of ASA at 100 mg/kg 30 minutes before thrombosis induction were used as control (Group I) and reference group (Group II) respectively. In this study, ASA treatment showed two types of results. The administration of ASA (100 mg/kg) 30 minutes before laser-induced thrombosis prevented thrombus formation. In the same way, ASA injected to rats already treated with the same dose 2 or 4 day later also demonstrated a potent antithrombotic effect. The same trends were observed with animals receiving the second injection (100 mg ASA) at j(0+8), j(0+12), j(0+14), and j(0+16). However, when injected to rats at j(0+6) and at j(0+10), ASA did not shown any effects on thrombus formation compared to the control (p>/=0.05). The same phases of ASA action were observed on the induced hemorrhagic time. The antithrombotic effects of the later second injection of ASA (100 mg/kg) were neutralized in rats previously receiving the same dose of this drug. This phenomenon seems to be periodic and is of great importance for the observance of ASA treatment.

Aspirin and fraxiparine in the prevention of laser induced thrombosis in the presence of iodinated contrast media.

The purpose of this study was to investigate the thromboembolic properties of ionic and nonionic contrast media in rats pretreated with aspirin and/or fraxiparine using an experimental model of laser induced thrombosis in the mesenteric microvessels of 17 groups of five male Wistar rats each. Two ionic (ioxaglate and diatrizoate) and two nonionic contrast media (iopamidol and iohexol), alone or associated with antithrombotic drugs (aspirin and/or fraxiparine) were studied. To evaluate the effects of these substances in this model, the number of laser beams needed to induce platelet thrombus formation, the number of emboli detached from the thrombus and the duration of embolization were quantified. Platelet aggregation induced by ADP, induced hemorrhagic time (IHT) and haemoglobin loss level were also determined. Both contrast media injected at 3 ml/kg caused a significant increase in the number of emboli and the duration of embolization (p<0.05). Pretreatment with aspirin and/or fraxiparine in the presence of ionic contrast media showed antithrombotic activities equal to those obtained when they were tested alone (p<0.05), while in the presence of nonionic contrast media, these drugs only neutralised the prothrombotic effects. There were no differences with the NaCl treated group (p>0.05). The ionic contrast media, and to a lesser extent the nonionic contrast medium: iohexol, inhibited platelet aggregation, while iopamidol behaved as an activator. The antithrombotic drugs tested in this study prevent the prothrombotic activities of contrast media therefore suggesting their use before radiographic procedures.

Fibrinogen as a factor of thrombosis: experimental study.

Epidemiological, clinical, and experimental studies have clearly demonstrated the strong association between baseline fibrinogen level and risk of thromboembolic complications. The pathogenesis of postoperative or post-traumatic thrombosis in man is associated with fibrinogen level in plasma. The purpose of this study was to evaluate the effects of fibrinogen administration on thrombus formation at different dosages. To investigate these effects, we used an experimental model of induced thrombosis in rat microcirculation. This model allows single endothelial cell destruction by laser injuries, thus leading to thrombus formation. Fibrinogen was injected intravenously via penis vein and tested at various dosages (50, 100, and 200 mg/kg), 60 minutes after injection on arterial thrombosis induction and 120 minutes after injection on venous thrombosis induction. Results showed that the administration of fibrinogen increases the number of emboli, the duration of embolization, the amplitude, and the velocity of the ex-vivo platelet aggregation induced by ADP (p < 0.05). A positive correlation between the percent of fibrinogen increase in plasma and the enhancement of thromboembolic risk in the experimented animals was observed.

Combination of two doses of acetyl salicylic acid: experimental study of arterial thrombosis.

The antithrombotic effect of high dose acetylsalicylic acid is well known, and recently, in vitro studies hinted the potent thrombotic effect of ultra-low dose of acetylsalicylic acid (<1mg/day) showing a significant decrease in bleeding time. In this study, we investigated the effect of a combination between a high and an ultra-low dosage (100 mg/kg+ 10(-30) mg/kg) on an arterial thrombosis induced by a laser beam. We used an intravital microscopic technique, allowing to evaluate (anti)-thromboembolic events at previously determined locations of microvasculature. Thrombus formation was induced by argon-laser shot. The instrumental test setup was completed with a video system, to select mesenteric arterioles with the same diameter (between 15 and 25 microm). The changes in platelet aggregability were determined by Cardinal and Flower method, and the concentration of acetylsalicylic acid in the plasma was measured by high pressure liquid chromatography. Antithrombotic effect of high dose (100 mg/kg) acetylsalicylic acid was confirmed in all results obtained. Asa injected at ultra-low dose (10(-30) mg/kg) had a potent thrombotic properties and decreased significantly the bleeding time. The subcutaneous administration of the combination of the two doses permitted to come back to the control values, and the bleeding time was shortened compared to control group.

Thromboembolic complications several days after a single-dose administration of aspirin.

The antithrombotic properties of acetyl salicylic acid (ASA) used at current doses are largely demonstrated. However, our previous study showed unexpected thrombotic potencies associated with the use of this drug. In this study we investigate the effect of aspirin on an experimental thrombosis induced by laser beams, according to its in vivo plasma concentration. Experiments were done on nine groups of seven Wistar male rats. The groups are defined by the delay between aspirin administration time and the laser-induced thrombosis time. Results from this study showed an enhancement of thromboembolic complications when thrombosis was induced 8 or 10 days after aspirin administration; the number of emboli and the duration of embolization are increased, compared to the control group. The prothrombotic properties of ASA demonstrated in this study, might limit its therapeutic benefit and might explain thromboembolic complications observed in some ASA-treated patients. These results also suggest a biological monitoring several days after aspirin administration to patients.

Action of neurotransmitters: acetylcholine, serotonin, and adrenaline in an experimental arterial thrombosis induced by oxygen free radicals.

It is well known that high stress and particularly an enhancement of plasma catecholamines and myocardial infarction have a close relation. In addition, adrenaline is presented as a prothrombogenic agent in vivo. The role of the other agents such as serotonin or acetylcholine, in the development of arterial thrombosis is somewhat uncertain, although, the role of each of them is often considered at the level of vascular regulation only. Therefore, the present study was designed to investigate the effects of three neurotransmitters on experimental arterial thrombosis model induced by generation of free radicals. The results demonstrate that intravenously injection of adrenaline or serotonin (1 ng/kg) stimulated arterial thrombosis formation, whereas injection of high dose of acetylcholine (5 mg/kg) slackened the thrombosis formation.

Vasoconstrictive response of rat mesenteric arterioles following infusion of cross-linked, polymerized, and conjugated hemoglobin solutions.

Infusion of hemoglobin-based oxygen-carrying solutions (HBOCs) produce an immediate rise in blood pressure with most solutions, both in animals and humans, as a result of systemic and pulmonary vasoconstriction. Autoregulation of the O2 supply by the microvasculature has been proposed as a phenomenon involved in the vasoconstriction elicited by HBOCs. Nevertheless, little is known about the ability of various HBOCs to induce constriction in the microcirculation according to their specific physicochemical properties (viscosity, molecular weight, P50, etc.). This study was therefore designed to assess the effects of three HBOCs, that is, bis(3.5-dibromosalicyl) fumarate-crosslinked hemoglobin (alphaalpha-Hb), dextran-benzene-tetracarboxylate-conjugated hemoglobin (Hb-Dex-BTC) and o-raffinose-oligomerized hemoglobin (o-raffinose-Hb), on the vascular tone of rat mesenteric arterioles (diameter, 15-25 microm) viewed microscopically in moderate hemodilution conditions. The effects of HBOCs were compared to those elicited by a reference solution of hydroxyethyl starch (HES-200) infused in the same conditions. In each experimental group, a fall in arteriolar diameter was observed 2 min and 5 min after infusion of the solution. The maximum changes were observed in Hb-Dex-BTC and o-raffinose-Hb groups, in which diameter decreased from 6.9 +/- 0.5% and 5.2 +/- 0.7%, respectively, 2 min after infusion. The changes in arteriolar diameter induced by Hb-Dex-BTC and o-raffinose-Hb were significantly higher than those elicited by HES-200 and alphaalpha-Hb. In conclusion, our data indicate that moderate hemodilution with HBOCs induces instantaneous constriction in rat mesenteric arterioles, with amplitudes depending on both pharmacological and physicochemical properties of the hemoglobin solution infused.