[Leber's hereditary optic neuropathy]. / Leberova hereditární neuropatie optiku (LHON).

BACKGROUND: Leber's hereditary neuropathy of the optic nerve (LHON) is manifested by bilateral affection of the eyes with acute or subacute loss of vision. The disease is caused by point mutations in the mitochondrial DNA (mtDNA) and is one of the most frequent mitochondrial diseases in the population. In patients with LHON 18 different point mutations in the mtDNA were described which correlate partly with the rate of progression of the disease and the severity and prognosis of the final affection of vision. METHODS AND RESULTS: The submitted paper deals with the results of molecular genetic examinations in three families with clinical manifestations of LHON. In three patients in the first family a homoplasmic mutation of mtDNA G3460A was found. In the second family in a young man with severely impaired vision a heteroplasmic mutation G3460A was found associated with a higher ratio of mutated mtDNA molecules than in his mother who is clinically healthy. In the third family the presence of homoplasmic mutation of mtDNA in position G11778A was detected. CONCLUSIONS: The diagnosis of LHON and genetic counselling in affected families should be based on close collaboration of ophthalmological and genetic departments with specialized laboratories engaged in molecular biological diagnosis of mitochondrial diseases.

[Various manifestations of the A8344G mtDNA heteroplasmic mutation in 4 families with the MERRF syndrome]. / Rozmanitost projevu heteroplazmické mutace A8344G mtDNA ve ctyrech rodinách se syndromem MERRF.

BACKGROUND: The most frequent manifestation of mitochondrial DNA (mtDNA) mutation 8344 A-->G is MERRF syndrome (Myoclonic Epilepsy and Myopathy with Ragged Red Fibres). Less frequent symptoms include ataxia, perceptive type of deafness, cardiomyopathy or external ophthalmoplegia and mental and motor retardation in children. We describe heterogeneity of clinical symptoms and results of biochemical and molecular investigations in four families with the heteroplasmic mutation 8344 A-->G in mtDNA. METHODS AND RESULTS: In co-operation with paediatric, neurological and genetic specialists from the Czech and Slovak Republic we found in 1993-1998 at the enzymatic or molecular level more than 90 children and adults with impaired mitochondrial energy metabolism. Heteroplasmic mutation 8344 A-->G in mtDNA was found in four families. Ataxia and progressive muscle weakness appeared in the first proband with 50% of mutated copies of mtDNA in muscle at the age of 30 years. The second proband with 95% of mutated mtDNA had his first clinical symptoms--muscle hypotonia, cardiomyopathy and mental and motor retardation--in infancy while his four relatives with 25-50% mutated mtDNA lack so far clinical symptoms. In a female from the third family with 50% mutated mtDNA in muscle the disease manifested at the age of 42 years with progressive external ophthalmoplegia (PEO) and muscle weakness. In the fourth proband with 50% of mutated mtDNA in blood the disease started in infancy with spastic quadruparesis and arrested mental and motor development. Enzymatic and histochemical investigation in muscle biopsy in two probands revealed lower cytochrom c oxidase activity. Ragged-red fibres were found only in one adult patient. CONCLUSIONS: MtDNA mutation 8344 A-->G can manifest by heterogeneous symptoms. A higher percentage of mutated mtDNA is usually associated with more serious forms of the disease, but there is not always a correlation between the degree of heteroplasmy and severity of the disease or the age of the first clinical symptoms.

[Leber's hereditary optic nerve neuropathy]. / Leberova hereditárna neuropatia zrakového nervu.

The authors present a case report of 26 years old man with bilateral optic nerve neuropathy. Detection of heteroplasmic mutation of mitochondrial DNA at G3460A site confirmed the suspicion on Lebers hereditary optic nerve neuropathy (LHON). Genetic and environmental factors of the disease and various accompanying neurologic and other symptoms, which can together with the optic nerve defect participate in the development of of the LOHN clinical pattern are discussed. (Ref. 12.)

[Lactate acidosis in childhood]. / Laktátová acidóza v detském veku.

BACKGROUND: Secondary lactate acidosis is found in children with hypoxaemia, with impaired tissue perfusion, in hepatic and renal failure or in intoxications. Primary lactate acidosis is usually caused by hereditary metabolic disorders. The objective of the trial was to analyze the causes which lead in childhood to the development of primary hyperlactacidaemia. METHODS AND RESULTS: The authors examined during 1995-1996 the lactate and pyruvate concentration in 479 children referred by paediatric and neurological departments with a suspect hereditary metabolic disturbances. A raised lactate in blood or cerebrospinal fluid > 2.3 mmol/l was found in 230 children incl. 49 where a metabolic disorder was detected. Ten children had impaired cytochrome c oxidase, two children had a combined deficience of NADH dehydrogenase and cytochrome c oxidase, three children had a deficience of the pyruvate dehydrogenase (PDH) complex, one child had a deficience of ATP synthase and seven children suffered from impaired beta-oxidation. Glycogenosis type I, III or IX was found in 13 children. In three children organic aciduria was found, two children had an impaired urea cycle and three children impaired fructose metabolism. In five children a low level of free and total carnitene was found as a result of valproate treatment. A significant increase of the lactate level by more than 1 mmol/l during an oral glucose load was found in 11 of 16 children with impairment of the respiratory chain or PDH complex. In 58 children concurrently lactate in blood and cerebrospinal fluid assessed but no correlation of lactate levels was found. CONCLUSIONS: In patients with suspect hereditary metabolic disorders examination of lactate, pyruvate and alanine levels can be considered a screening test for detection of mitochondrial disorders. It remains difficult to reveal the cause of hyperlactacidaemia in a sick child even if a wide range of laboratory methods are used which contribute to the diagnosis of hereditary metabolic disorders.

[Mitochondrial myopathy, deafness and type 2 diabetes mellitus with tRNALeu(UUR) point mutation in mitochondrial DNA]. / Mitochondriální myopatie, hluchota a diabetes mellitus 2. typu na podklade bodové mutace tRNALeu(UUR) v mitochondriální DNA.

BACKGROUND: A heteroplasmic A3243G point mutation in tRNALeu(UUR) gene of mitochondrial DNA (mtDNA) is found in patients with MELAS syndrome (Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes), less frequently in patients with other dominating clinical features, such as deafness, diabetes mellitus type 2, hypertrophic cardiomyopathy, renal problems or inborn development defects. Present report describes histochemical, enzymatic and molecular biology studies of the family with clinical variant of meals syndrome. METHODS AND RESULTS: A 45-year-old woman with progressive muscle weakness, external ophtalmoplegia, perceptive deafness, ischemic heart disease, diabetes mellitus type 2 and hyperlactacidemia was metabolically investigated because the multiorgan problems indicated mitochondrial origin of the disease. Muscle biopsy revealed pronounced myopathic changes, ragged red fibers and decreased activity of respiratory chain enzymes - succinate cytochrome c reductase (< 5% control) and cytochrome c oxidase (< 10% control). Restriction fragment analysis of mtDNA from muscle, blood and hair follicles detected heteroplasmic A -> G mutation in the position 3243 of the tRNALeu(UUR) gene, which was more pronounced in muscle (28% of total mtDNA) than in blood (12%) or in hair follicles (10%). No mutation was found in blood and hair follicles of patient's mother and daughter. CONCLUSIONS: Diagnostics of mitochondrial diseases require close collaboration of clinicians with specialised laboratories. Treatment of mitochondrial disorders is only symptomatic, however, early diagnosis of the molecular defect is important for genetic counselling.

[DNA diagnosis of the fragile X chromosome syndrome--FRAXA using PCR]. / DNA diagnostika syndromu fragilního X chromozomu--FRAXA pomocí PCR.

BACKGROUND: Fragile X syndrome is gonosomal recessive mental retardation with the frequency 1:1000 in male population. Fragile X syndrome is caused by amplification of CGG repeat in 1. exon of FMT-1 gene. The aim of this study was to set up and validate a rapid and efficient PCR diagnosis to select FRAXA negative patients in population of mental retarded patients. METHODS AND RESULTS: In the set up phase of the method, 196 patients were diagnosed. We were using modified radioactive PCR of CGG. Obtained PCR fragments were separated on 6% denaturing PAGE. Results were correlated with Southern blot analysis using pE5.1 probe. STR-PCR was verified on a large set of patients and shows validity and efficiency of results in the case of pre- and full mutations in male hemizygous patients too. For estimation of carriers with pre- and full mutation by females modified diagnostic approach was developed. There was no difference found between results from PCR and Southern blot analysis. CONCLUSIONS: The PCR method is convenient not only for selection of FRAXA negative patients, but for diagnosis of full mutation and premutation of affected probands.

Effect of an adrenergic agonist and a cholinergic antagonist on the airway epithelium.

The ultrastructure of the rabbit tracheal epithelium was studied 30 minutes after intratracheal administration of two puffs of salbutamol and ipratropium bromide, respectively. The injury to the tracheal epithelium due to the treatment with both bronchospasmolytic drugs was considered moderate to severe. In both experimental groups, the degree of goblet cells' stimulation did not differ significantly, the ciliated cells were less damaged compared with the goblet ones and the morphological signs of the impaired self-cleaning ability were revealed.

Ultrastructure of the tracheal epithelium in rabbits after acetylcholine administration.

The ultrastructure of the tracheal epithelium was studied 5 and 20 min after intravenous (i.v.) administration of 0.5 mg of acetylcholine. As a result of this treatment, the goblet cells were overstimulated and damaged, and the mechanism of their secretion was accelerated. Only a mild pathological alteration was encountered in the ciliated cells. According to our classification, the degree of secretory element damage was severe, the injury to the ciliary border was moderate. The morphological signs of the severe impairment of the self-cleaning ability of the epithelium were inspissated mucus and very numerous bacteria in the area among free cilia.

Comparison of the effect of a high and low dose of atropin on the ultrastructure of the tracheal epithelium.

The ultrastructure of rabbit tracheal epithelium was studied 20 min after injection of 0.04 and 0.5 mg of atropin. The tracheae were lined with altered ciliated pseudostratified columnar epithelium. The degree of damage to the epithelial cells was dose dependent. The injury to the tracheal epithelium due to treatment with 0.04 mg of atropin was considered mild. The administration of the higher dose of atropin caused moderately severe epithelial damage. Both experimental groups exhibited morphological signs of impaired self-cleaning ability.

An abnormally high occurrence of malformed kinocilia in the tracheal epithelium of a clinically healthy rabbit.

An abnormally high occurrence of malformed kinocilia containing axonemes with different number or arrangement of microtubules compared with the typical 9+2 pattern of motile cilia was encountered in the tracheal epithelium of one clinically healthy rabbit. The malformed cilia amounted to 6.87% of all kinocilia. Individual types of ciliary malformations were further classified. The frequency of ciliary malformations was compared with that in all other rabbits the ciliary border of which has been quantitatively evaluated in our laboratory. Using Grubbs test the extremely low probability of occurrence of such a high number of malformed kinocilia in the rabbits' population was verified. The studied rabbit suffered from a mild form of the immotile cilia syndrome, but the loss of less than 10% of moving cilia did not lead to the expression of the clinical signs of the impaired function of cilia in the organism.

[Airway epithelium and its reaction to harmful substances]. / Epitel dýchacích cest a jeho reakce na ruzné skodliviny.

The trachea and large bronchi are lined with a pseudostratified columnar ciliated epithelium. The secretory elements are represented by goblet cells. In healthy subjects the majority of goblet cells are filled with mucus, only 3% of them discharge secretion. Above the epithelium a regular ciliary border is developed. Among kinocilia with the 9 + 2 inner pattern about 1% of altered cilia are discovered. Goblet cells are the first to react to the majority of noxious substances with the exception of some inert dusts. Their number increases and the mechanism of secretion is accelerated. Damage to the ciliated cells is reflected in an impairment of the ciliary border where the average number of cilia per micron 2 declines and the ratio of altered cilia increases. To compare the effect of various noxious substances a classification of the degree of airway epithelium alteration was proposed. According to the degree of the ciliary border impairment and the functional state of the secretory elements the injury of the epithelium was classified as serious, moderately serious and mild.

Ultrastructure of rabbit tracheal epithelium after the administration of Hexabrix--a hexaiodated contrast agent for tracheobronchography.

The ultrastructure of the tracheal epithelium after the application of 1 ml of Hexabrix (a hexaiodated contrast agent for tracheobronchography) into the airways was studied. Severely altered pseudostratified columnar epithelium was found in the trachea. Eighty-one +/- 5% of the goblet cells had been stimulated to discharged mucus. Forty-three +/- 3% of them were completely exhausted and had degenerated. The ciliated cells displayed marked signs of pathological alteration up to vacuolar degeneration. The ciliary border above the epithelium was severely damaged. The mean number of cilia per micron 2 fell to 3.3 +/- 0.5, but the proportion of intact cilia did not drop below 95%. As morphological signs of impaired self-cleaning ability of the airway epithelium, a large amount of inspissated mucus and numerous bacteria were found in the area of the impaired ciliary border. Coarse grained contrast substance of supreme electron density was intermingled with the condensed mucus. The most pronounced damage to the cells was observed at the sites of direct contact of the contrast agent with the cell membranes.

Quantitative evaluation of the degree of damage to tracheal epithelium.

Quantitative evaluation of the injury to the tracheal epithelium was carried out under various experimental conditions. The damage of the epithelium was classified in three groups, according its severity. In several impaired epithelium all the goblet cells were stimulated to release their secretion. The mechanism of mucus discharge was maximally accelerated. Over 90% of the exhausted goblet cells degenerated. In average only 1 cilium/micron 2 was left in the ciliary border. Over 10% of the remaining cilia displayed signs of alteration. As morphological signs of impairment of the self-cleaning ability of the epithelium, large amounts of inspissated mucus and numerous bacteria were observed in the area of the damaged ciliary border. In moderately severe epithelial injury about 80% of the goblet cells were stimulated and half of them degenerated. The mean number of cilia in the ciliary border was 3/micron 2. The signs of impairment of the self-cleaning ability of the epithelium were less pronounced. In mild damage, about half of the goblet cells were stimulated and the degenerated ones formed less than 10% of all the mucous-secreting elements. The number of kinocilia per micron 2 fell on the average by one third. No signs of the self-cleaning ability impairment were recorded.

[The effect of a single therapeutic dose of Intussin on the ultrastructure of the epithelium of the respiratory tract]. / Vliv aplikace jedné terapeutické dávky Intussinu na ultrastrukturu epitelu dýchacích cest.

The effect of administration of a single therapeutic dose of Intussin on the ultrastructure of the rabbit tracheal epithelium was investigated. Twenty minutes after the application of five drops of Intussin only slight reaction of the pseudostratified ciliated epithelium was recorded. The ciliated cells manifested only slight signs of pathological alteration. Stimulation of 36 +/- 2% of goblet cells and degeneration of 7 +/- 2% of these cells were recorded. The average number of kinocilia per 1 microns2 of the ciliary border area was significantly (P less than 0.005) reduced to 6.7 +/- 0.5. However 97 +/- 2.7% of kinocilia remained intact. There was slight but significant (P less than 0.005) increase in number of pathological and degenerated kinocilia. As morphological signs of impaired mucus flow clumps and layers of inspissated secretion and numerous bacteria were found in the area of the ciliary border. Owing to the slight damage to the ciliary border and to the low degree of stimulation of the goblet cells the impairment of the self cleaning ability of the epitselium was obviously due to the ciliostatic action of some component of this preparation.

Effect of bronchoalveolar lavage on the ultrastructure of the tracheal epithelium in rabbits.

Serious damage to the tracheal epithelium due to bronchoalveolar lavage (BAL) was recorded. Immediately after BAL 99 +/- 2% of goblet cells were exhausted and degenerated. Their regeneration began 24 h after BAL resulting in hyperplasia of goblet cells with the formation of endoepithelial mucous glands. The most pronounced injury to the ciliated cells was apparent 2 h after BAL. BAL markedly impaired the ciliary border. The mean number of cilia/microns 2 fell to 1.5 +/- 0.3/microns 2, then gradually rose to 7.5 +/- 0.5/microns 2 (controls 9.7 +/- 0.5/microns 2). The morphological signs of impairment of the self-cleaning ability of the epithelium were the most pronounced after 24 h and were still present at the end of the experiment.

The effect of Hexabrix tracheobronchography on the ultrastructure of the airway epithelium.

The ultrastructure of the airway epithelium was studied after experimental Hexabrix tracheobronchography. Serious damage to the tracheal epithelium due to Hexabrix was recorded. 81 +/- 5% of goblet cells were stimulated to discharge mucus. 43 +/- 3% were completely exhausted and degenerated. The ciliated cells displayed vacuolar degeneration or severe pathological alteration. The average number of cilia/micron 2 fell to 3.3 +/- 0.5. As morphological signs of impaired self-cleaning ability the inspissated mucus and numerous bacteria appeared in the area of the disorganized ciliary border.

Regeneration of changes produced in rabbit tracheal epithelium by saline lavage of the airways.

The authors studied the course of the repair of changes induced in the rabbit tracheal epithelium by saline lavage of the airways. The tracheal epithelium was examined 2, 24, 48 and 72 hours after treatment. Saline lavage stimulated the goblet cells to instantaneous discharge of their secretion. 2 hours after treatment 98 +/- 3% of the goblet cells were completely exhausted and had degenerated. Repair of the changes began 24 hours after lavage and was associated with massive differentiation of new goblet cells resulting in hyperplasia of the mucus-secreting elements with formation of endoepithelial mucous glands. The most pronounced injury to the ciliated cells was apparent 2 hours after lavage, then the degree of alteration of these cells gradually decreased. Saline lavage markedly impaired the ciliary border. The mean number of kinocilia per micron2 fell to 1.5 +/- 0.3. In subsequent phases the number of kinocilia rose gradually to 7.5 +/- 0.5/micron2. This value was still significantly lower (P less than 0.005) compared with controls. The first signs of impairment of the self-cleaning ability of the epithelium were recorded 2 hours after lavage. The most pronounced disturbances of the mucus flow were observed after 24 hours. At the end of the experimental period small clumps of condensed mucus and rather numerous bacteria were still present in the area of the ciliary border.