A Diagnostic Algorithm for Mitochondrial Disorders in Estonian Children.

Mitochondrial disorders are a heterogeneous group of disorders affecting energy production of the body. Different consensus diagnostic criteria for mitochondrial disorders in childhood are available - Wolfson, Nijmegen and modified Walker criteria. Due to the extreme complexity of mitochondrial disorders in children, we decided to develop a diagnostic algorithm, applicable in clinical practice in Estonia, in order to identify patients with mitochondrial disorders among pediatric neonatology and neurology patients. Additionally, it was aimed to evaluate the live-birth prevalence of mitochondrial disorders in childhood. During the study period (2003-2009), a total of 22 children were referred to a muscle biopsy in suspicion of mitochondrial disorder based on the preliminary biochemical, metabolic and instrumental investigations. Enzymatic and/or molecular analysis confirmed mitochondrial disease in 5 of them - an SCO2 gene (synthesis of cytochrome c oxidase, subunit 2) defect, 2 cases of pyruvate dehydrogenase complex deficiency and 2 cases of combined complex I and IV deficiency. The live-birth prevalence for mitochondrial defects observed in our cohort was 1/20,764 live births. Our epidemiological data correlate well with previously published epidemiology data on mitochondrial diseases in childhood from Sweden and Australia, but are lower than in Finland.

A novel mutation in the M1S1 gene responsible for gelatinous droplike corneal dystrophy.

PURPOSE: To identify the genetic defect in the M1S1 gene causing gelatinous droplike corneal dystrophy (GDLD) in an Estonian family. METHODS: DNA was extracted from members of a GDLD-affected family and control persons. Polymerase chain reaction followed by direct sequencing was used to detect mutations in the M1S1 gene. Sequencing results were confirmed with restriction analysis. RESULTS: Sequencing of the M1S1 gene revealed a novel mutation and a common polymorphism. All patients with GDLD were found to be homozygous for the insertion of nucleotide C in position 520 in M1S1. The mutation leads to formation of truncated protein. The mutation was excluded in 103 normal, unaffected individuals. Very close to the location where the mutation was identified in the M1S1 gene, a single-nucleotide polymorphism (518A/C) was found, changing aspartic acid to alanine at codon 173. CONCLUSIONS: The data indicate that mutation ins520C in the M1S1 gene is the primary cause of GDLD in the family studied.

Intracellular energetic units in red muscle cells.

The kinetics of regulation of mitochondrial respiration by endogenous and exogenous ADP in muscle cells in situ was studied in skinned cardiac and skeletal muscle fibres. Endogenous ADP production was initiated by addition of MgATP; under these conditions the respiration rate and ADP concentration in the medium were dependent on the calcium concentration, and 70-80% of maximal rate of respiration was achieved at ADP concentration below 20 microM in the medium. In contrast, when exogenous ADP was added, maximal respiration rate was observed only at millimolar concentrations. An exogenous ADP-consuming system consisting of pyruvate kinase (PK; 20-40 units/ml) and phosphoenolpyruvate (PEP; 5 mM), totally suppressed respiration activated by exogenous ADP, but the respiration maintained by endogenous ADP was not suppressed by more than 20-40%. Creatine (20 mM) further activated respiration in the presence of ATP and PK+PEP. Short treatment with trypsin (50-500 nM for 5 min) decreased the apparent K(m) for exogenous ADP from 300-350 microM to 50-60 microM, increased inhibition of respiration by PK+PEP system up to 70-80%, with no changes in MgATPase activity and maximal respiration rates. Electron-microscopic observations showed detachment of mitochondria and disordering of the regular structure of the sarcomere after trypsin treatment. Two-dimensional electrophoresis revealed a group of at least seven low-molecular-mass proteins in cardiac skinned fibres which were very sensitive to trypsin and not present in glycolytic fibres, which have low apparent K(m) for exogenous ADP. It is concluded that, in oxidative muscle cells, mitochondria are incorporated into functional complexes ('intracellular energetic units') with adjacent ADP-producing systems in myofibrils and in sarcoplasmic reticulum, probably due to specific interaction with cytoskeletal elements responsible for mitochondrial distribution in the cell. It is suggested that these complexes represent the basic pattern of organization of muscle-cell energy metabolism.

Four siblings with Hallervorden-Spatz disease.

We reported four cases of Hallervorden-Spatz disease. All four siblings (three males and one female) in the family are affected. The first symptoms of the disease were spastic paraparesis and optic atrophy followed by trunkal dystonia and lower motor neurone involvement. The average age of the onset was 4.25 years. The diagnosis was made at the ages of 17, 14, 11 and 10 years. The diagnosis was confirmed clinically, electrophysiologically and by MRI. On MRI scans all patients demonstrated hypointense areas in globus pallidus. There is neither specific treatment nor prenatal diagnosis.

Bone healing models in rat tibia after different injuries.

AIMS: Gradual elaboration of an adequate and efficient multistage method for experimental remodelling of specific wound healing process--bone repair. Comparison of clinical characteristics with the results of microanatomy, histology, electronmicroscopy and computer morphometry. MATERIAL AND METHODS: An investigation of posttraumatic bone repair after internal fracture, excision and cortical perforation was carried out on 142 young adult male Wistar rats. The repair was studied in normal and affected animals (exercises, immobilization, isolation of periost) at 1-42 days after operation. RESULTS: The posttraumatic bone callus development and the related soft tissue repair, likewise the continuous remodelling, is an ordinary process of osteohisto- and organogenese. In trained rats the blood supply and bone formation is increased, whereas in immobilized animals it is inhibited and destroyed (osteoporose, pseudoarthrosis). After the injury some characteristics of bone repair histogenese will be became evident (after the perforation the primary endosteal and secondary periosteal ossification, inhibition of endosteal bone repair after the isolation of periost etc.). CONCLUSION: The posttraumatic bone healing, like embryohistogenese, has similar repair stages in all models of the experiments as well as similar tissue and cell responses (callus formation, its replacement, bone remodelling, etc.). However, the repair process in general (order of chondrous and/or bone callus stages, etc.) is variable and dependent on the mode and degree of injury. The use of bone cortex perforation in wound healing study is more recommendable as compared to internal fracture and excision (possibility of in situ study the periost and callus tissue compartments in bone repair machinery separately).

[Post-traumatic repair of the tibia in rats]. / Posttravmaticheskiaia reparatsiia bolshebertsovo kosti u krys.

Posttraumatic bone repair was studied histologically in 88 adult male Wistar rats. Thick, semithin and thin sections were stained with standard methods and investigated (microanatomy, histology, EM). The experimental animals were divided into five main groups: 1) control; 2) trained (swimming); 3) immobilized; 4) formalized (0.2 mg/kg i.m. every day); 5) alcoholised (3% alcohol instead of drinking water). The first group was divided into animals wit periost, contacted with endost and bone marrow and animals without periost contacts (with isolated periosteum). Bone perforation causes local hemorrhage and tissue destruction. Thr first reparative changes on the first-seventh days (proliferating fibroblasts and capillary sprouts grow into the blood clot and injured area, degranulation of neutrophils and tissue basophils, appearance of activated macrophages and osteoclasts) occur in soft tissue. Intensive collagen synthesis in fibroblasts began. On the fourteenth day collagen synthesis in osteoblasts was increased (packed collagen fibrils in vacuolated cytoplasm). Posttraumatic osteohistogenesis on d 4-21 was generally completed on d 28-42 bone formation (morphogenesis) and permanent remodeling were continued. Periosteal osteogenesis and bone repair require an existence of the periosteum contact with endosteum and bone marrow (growth and differentiation of endosteal bone callus was inhibited and those of periosteal callus was arrested in groups with isolated periosteum). Similar results were achieved in i.p. heterotopic autografts of repaired tissue in diffusion chamber (with isolated periosteal osteogenetic cells appearing only by 21st day after bone injury). In trained rats bone repair was stimulated, while in immobilised and in injured animals it was inhibited and resulted in chronic inflammation of bone marrow and abscesses.

Tenascin expression patterns and cells of monocyte lineage: relationship in human gliomas.

Stromal extracellular matrix (ECM) components are thought to play an important role in regulating invasion of human gliomas. Macrophages and microglial cells may heavily influence the integrity of the extracellular compartment of gliomas, and the affected ECM may play a key role in regulating migratory activity of both tumor cells and macrophages/microglia. The aim of this investigation was to study immunohistochemically the expression patterns of four ECM components: fibronectin, laminin, collagen IV, and tenascin (TN) in human gliomas, with special attention to TN. Our main goal was to study the possible correlation between TN expression and macrophagic/microglial infiltration in gliomas. Altogether, 90 gliomas were studied. Tumors included 46 glioblastomas, 19 anaplastic gliomas, 22 low grade gliomas, and 3 pilocytic astrocytomas. Vascular TN prevailed in perinecrotic areas of glioblastomas, whereas interstitial TN was more often expressed distant from necrosis and in the ECM of anaplastic and low grade gliomas. Double staining with CD68 and anti-TN antibodies showed that macrophagic/microglial density was significantly higher in TN-positive areas of most of the glioblastomas and anaplastic gliomas, whereas microglial percentage from total number of CD68-positive cells was in most of the cases significantly higher in TN-negative areas. In addition, we saw a morphologically spatial correlation between higher densities of macrophagic/microglial infiltration and TN expression in perinecrotic areas in glioblastomas. Attachment of macrophages to TN-positive basement membrane zones of newly formed stromal blood vessels was evident. On the basis of our results, we conclude that TN may play a crucial role in regulating trafficking of cells of monocyte lineage in human gliomas.

Duchenne and Becker muscular dystrophies: an Estonian experience.

The clinical and molecular features of 25 Duchenne (DMD), two intermediate (D/BMD) and three Becker (BMD) muscular dystrophy patients from 26 unrelated families were evaluated. Early psychomotor development was normal in patients with D/BMD and BMD. Learning to walk independently after 15 months of age was a risk sign of DMD in nine (36%) patients. Abnormality in crawling was seen in 13 (54%) patients with DMD. These boys demonstrated initial symptoms earlier than those who learned to crawl normally. Mental retardation was established in five (20%) patients with DMD. Deletions in the dystrophin gene were found in 11 families (48%). They were accumulated (9/11, 82%) in the distal region of the gene.

Infantile intranuclear rod myopathy.

This report concerns three unrelated floppy infants, two girls and one boy, each biopsied at the age of 1 month. They were hypotonic since birth and required artificial ventilation. The two girls died at the ages of 4 and 3 1/2 months, respectively, the boy is still alive at the age of 2 years, but requires assisted ventilation. Each of the three infants showed, by muscle biopsy, abundant intranuclear rods, the boy and one girl also had sarcoplasmic rods, which were not present in the other girl's muscle. Absence of sarcoplasmic rods, but the presence of intranuclear rods could also be documented in her autopsied muscle. Using an antibody against alpha-actinin, immunoelectron microscopy showed reaction of the sarcoplasmic and intranuclear rods demonstrating their Z-band origin. To our knowledge, this is the first report on rod myopathy with intranuclear rods only and of an immunoelectron microscopic demonstration of alpha-actinin in intranuclear rods. The presence of intranuclear rods in infants with nemaline myopathy also appears to indicate a grave prognosis of their rod myopathy.

Clinical significance of different antinuclear antibodies patterns in the course of primary biliary cirrhosis.

BACKGROUND/AIMS: The significance of antinuclear antibodies (ANA) in primary biliary cirrhosis (PBC) patient is still controversial in the literature. The purpose of this paper is to investigate the clinical significance of ANA in PBC patients. MATERIALS AND METHODS: Sixty-nine patients with PBC were investigated. Control groups included 21 patients with autoimmune hepatitis, 26 patients with alcoholic liver disease, 13 patients with systemic connective tissue disease and 27 healthy persons. ANA was detected by an immunofluorescence method on rat liver tissue sections and HEp-2 cells at serum dilution 1/40. RESULTS: In 48 out of 69 PBC patients (70%), ANA was positive in HEp-2 cell line, but in rat liver tissue sections only 29% of patients had positive ANA reactions. Most frequent patterns were multiple nuclear dots (MND) in 42% and perinuclear in 16%. MND-ANA was also found in two autoimmune hepatitis patients and in one systemic lupus erythematosus patient. Survival from the moment of developing first symptom(s) attributable to liver disease was longer in the ANA positive patients than ANA negative ones (p < 0.02). Despite immunosuppressive treatment, in most of ANA positive patients (73%) ANA did not disappear. Most frequent ANA patterns in autoimmune hepatitis and systemic connective tissue diseases patients were homogeneous and anticentromere, respectively. CONCLUSIONS: Immunofluorescence method on HEp-2 cell line for ANA detection is more sensitive than on rat liver tissue sections. In PBC patient's incidence of ANA, especially MND-ANA is a frequent immunological abnormality. ANA positive patients have better prognosis for survival.

[Prevalence of selective lactose malabsorption in Khants]. / Rasprostranennoct' selektivnoi mal'absorbtsii laktozy sredi khantov.

The aim of the study was to determine the prevalence of selective lactose malabsorption (SLM) in Khants, a small finno-ugric nation living in West Siberia. A total of 80 Khants from the Surgut region (Tyumen territory) were studied. The diagnosis of SLM was based on the evidence obtained at a 50 g lactose and, if possible, a 25 g + 25 g galactose-glucose loads. In 6 cases electron-microscopic examination of the duodenal mucosa was performed. The prevalence of SLM in the Khants reached 93-94% being the highest in CIS.

Creatine kinase isoenzyme BB concentrations in cerebrospinal fluid in asphyxiated preterm neonates.

Creatine kinase isoenzyme BB was determined in cerebrospinal fluid (CSF) in 79 preterm neonates using an original enzyme-linked immunosorbent assay. The criterion for inclusion was an Apgar score of 7 or less at 5 min of life. Neurological examination was performed on day 2 and day 5 of life. CSF was obtained on the same days. Lumbar puncture was performed on 41 of these babies on day 2 and in 39 on day 5 of life (one baby underwent lumbar puncture twice). All babies had clinical features of hypoxic-ischemic encephalopathy (HIF) which was classified according to Sarnat and Sarnat. The control group consisted of 90 asphyxiated term babies and 30 adults without CNS pathology. The concentration of CK-BB in cerebrospinal fluid (mean +/- SD) was significantly higher (p < 0.0005) in preterm (168.0 +/- 2) than in term babies (29.0 +/- 3.1) and healthy adults (5.3 +/- 1.2). Our results demonstrate the possibility of using the classification system of Sarnat and Sarnat for assessment of the severity of brain damage not only in term, but also in preterm babies. Neonates with HIE stages II and III showed markedly higher CK-BB values than those with HIE I on day 2 (p < 0.025) and day 5 (p < 0.05) of life. CK-BB values were markedly higher in preterm babies with none of some primitive responses (head turning, Babkin's reflex, palmar grasp). The mean concentration of CK-BB was higher in neonates with retarded psychomotor development compared with those with normal development (p < 0.05) on day 3, and after 6 and 9 months. At 12 months of age no significant difference in median CK-BB concentration was detected between neonates with normal and developmental disturbances.

Lactose malabsorption in Khants in western Siberia.

BACKGROUND: We wanted to estimate the prevalence of selective lactose malabsorption (SLM) in Khants, a small Finno-Ugric population living in Western Siberia who have traditionally consumed no milk in adulthood. METHODS: A total of 80 Khants, aged 8-57 years and living on the middle reaches of the River Ob, were studied. The diagnosis was based on a lactose tolerance test; general malabsorption was excluded by a glucose-galactose tolerance test whenever possible. In six subjects electronmicroscopic examination of the duodenal mucosa was performed. RESULTS: The prevalence of SLM in the Khants was 94%. CONCLUSION: This is the highest prevalence found in the previous Soviet Union.

[The isolation and characteristics of monoclonal antibodies to the structural proteins of Coxsackie B5 virus]. / Poluchenie i kharakteristika monoklonal'nykh antitel k strukturnym belkam virusa Koksaki B5.

Five hybridomas secreting monoclonal antibodies (MCA) to Coxsackie B5 virus were generated by fusion of Sp2/0 myeloma cells with BALB/c mouse immune splenocytes. Most MCA belonged to the IgG and one to the IgM class. Out of 4 MCA binding the structural protein VP2, two MCA showed type specificity in neutralization test and cross-reactivity with other enteroviruses, one MCA reacted with all enteroviruses tested, and the one showing type specificity among Coxsackie B group "recognized" an epitope on poliovirus type 1 as well. The MCA belonging to VP4 proved to be Coxsackie B group specific. It was assumed that MCA could be used for studies of the antigenic structure and serodifferentiation of Coxsackie B5 virus.

Ovarian carcinomas express a sperm acrosomal antigen, defined by monoclonal antibody 9H8.

A new monoclonal antibody (MAb 9H8, IgM class) reactive with human ovarian carcinoma has been raised after immunizing C57BL/6 mice with bovine sperm. Immunohistological studies indicated that 20/21 serous ovarian adenocarcinomas expressed 9H8-defined antigen but it was absent in benign ovarian tumors (0/11). 1/11 of breast carcinomas and 5/5 of rectal carcinomas expressed this antigen, although to a considerably lesser degree. Tumors of lung, skin, brain and mesothelium were negative. The antigen was also expressed in embryonic skin, in renal collecting tubule cells and in saliva. In bovine, human and mouse sperm the antigen is confined to the acrosomal region. The molecular weight of this antigen was determined by Western blot analysis and gel filtration. In SDS-PAGE the antigen ran as a broad band barely entering the 7% gel, indicating an apparent molecular weight > 300 kDa. In the absence of detergents and reducing agents this glycoprotein forms larger complexes (> 1,500 kDa) as determined by gel filtration on Sephacryl S300. The epitope contains carbohydrate structures recognized by lectin PNA (peanut agglutinin).

[Monoclonal antibodies to neuron-specific enolase in phenotyping lung cancer]. / Monoklonal'nye antitela k neironspetsificheskoi énolaze v fenotipirovanii raka legkogo.

Morphological assessment of two samples of monoclonal antibodies (mcAB) to gamma-unit of neurospecific human enolase (NSE) is performed. It is established that mcABs interact in the tissue sections with neurons of the brain and spinal cord, gangliocytes of the central and peripheral nervous system this proving their specificity. 56 cases of lung carcinoma are studied using these mcABs; endocrine carcinoma was found in 11 cases, nonendocrine--in 45 cases. Neuroendocrine differentiation is revealed in 8 of 45 cases of non-small cell carcinoma; in all observations of small cell carcinoma NSE was found.

[Cerebral creatine kinase isoenzyme in the cerebrospinal fluid: importance of its measuring for prognosis of the disease outcome in cerebral ischemic disorders]. / Mozgovoi izoénzim kreatinkinazy v spinnomozgovoi zhidkosti: vazhnost' opredeleniia soderzhaniia dlia prognozirovaniia iskhoda bolezni pri ishemicheskikh narusheniiakh golovnogo mozga.

The concentration of cerebral isoenzyme of creatine kinase was measured repeatedly in the cerebrospinal fluid (CSF) in 84 patients with ischemic disorders of brain circulation. Of these, 14 patients presented with transitory ischemic attacks, 20 patients with regressing deficit, and 50 with brain infarction. The patients' health status and functional capacities were estimated on discharge from the hospital. It was discovered that in each patient, the maximum level of cerebral isoenzyme in the CSF was in a good agreement with the clinical course of the disease and neurological status in the survived patients. It may be concluded that measurements of the concentration of the enzyme in the CSF makes it possible to draw more valid prognostic conclusions in patients with ischemic disorders of brain circulation.

[Monoclonal antibodies IGR to surface antigens of neutrophilic granulocytes in human blood]. / Monoklonal'nye antitela IGR k poverkhnostym antigenam neitrofil'nykh granulotsitov krovi cheloveka.

Four monoclonal antibodies (MAbs) from series IGR to human peripheral blood neutrophilic granulocyte cell surface antigens were obtained by the conventional hybridoma technique. Specificity of MAbs AGR was determined to various leukemic cell lines and human peripheral blood cells. Overlapping in characteristics of antigens (molecular weight, localization, expression on induced leukemic cell line HL-60) to MAbs IGR-1 4C7, IGR-1 5B6 and IGR-2 IA6 suggests their identity. These, apparently, cannot be analogous to the well known granulocyte cell surface glycoproteins LFA-1, CR-3, p150, 95 or GP 130. The characteristics of MAbs IGR-1 and IGR-2 permit concluding that the antibodies should be useful in normal and leukemic myelomonocytic cell linear differentiation studies.

[Monoclonal antibodies IGR to nuclear antigens of neutrophilic granulocytes of human blood]. / Monoklonal'nye antitela IGR k iadernym antigenam neitrofil'nykh granulotsitov krovi cheloveka.

Four monoclonal antibodies (Mabs) of series IGR-1 and IGR-2 to nuclear antigens of neutrophilic granulocytes of human peripheral blood were obtained. Mabs IGR-1 2B8 and IGR-1 6B5 are bound to their specific antigens in the nuclei of all the investigated human cell lines. These Mabs were also specific for metaphase chromosomes of cell lines HL-60 and U-937. Investigations on the ultrastructural level showed that Mabs IGR-1 6B5 reacted with the HL-60 nuclear heterochromatin region. Mabs IGR-1 3D3 and IGR-2 2F1 manifested high specificity only for the nuclei of mature neutrophils and of plasma cells.

[Electron microscopic studies of pseudointima in synthetic prostheses]. / Elktronno-mikroskopicheskoe issledovanie psevdointimy sinteticheskikh protezov.

Thirty one prostheses made of Lavsan (therylene) or teflon-lavsan that had been functioning in human patients for 16 months to 21 years were examined. No continuous endothelial lining was found present on the pseudointimal surface of any of the prostheses. The major cell types in a formed, well-vascularized pseudointima were fibroblasts and smooth-muscle cells. Cells of the macrophage series were involved in forming the granulation tissue seen around fibers of the prostheses.