Phenotypic differences between mdx black mice and mdx albino mice. Comparison of cytokine levels in the blood.

In mdx mice, mutation in the muscle protein dystrophin gene results in the development of chronic degeneration of the muscle tissue. We performed a comparative analysis of blood cytokine levels in mdx mice, classical black mice and mice with additional genetic defect responsible for the manifestations of oculocutaneous albinism. In mdx albino mice, the total pool of cytokines (IL-10, IL-6, IL-5, IL-2, IL-1α, IL-4, IL-17, granulocyte-macrophage growth factor, TNF-α, and IFN-γ) was increased. This increase was not associated with selective release of one of the above cytokines into the blood. The fraction of pro-inflammatory cytokines (IL-6, IL-1α, TNF-α) was increased in the total pool and the percentage of antiinflammatory cytokines (IL-4) was reduced. Changes in cytokine pool probably reflect the differences in the severity of the pathological process in the muscle tissue of both genetic variations of mdx mice.

Cytokine profile of the blood in mice with normal and abnormal heart rhythm.

Differences in the pools of 10 cytokine were found in blood samples from the caudal vein of mice with normal and abnormal heart rhythm. Both groups were albino mice bred by us and differing from mdx albino mice by the absence of mutation in muscular dystrophin gene. Mice with normal heart rhythm had low IL-17 content and elevated concentrations of proinflammatory cytokines IL-6 and IL-1α in comparison with the normal (according to published data). In mice with bradyarrhythmias, increased blood levels of IL-10, IL-6, IL-5, IL-2, IL-1α, IL-17, IL-4, TNF-α, and granulocyte-macrophage colony-stimulating factor were detected. The relative content of IL-4 and IL-17 in the total cytokine pool increased. The lifespan of mice with bradyarrhythmias and cytokine hyperexpression was shorter by 2-3 months in comparison with mice without heart rhythm disturbances and moderate changes in the cytokine pool.

[Pathological changes in heart activity of mdx mice and future prospects of mdx model in cardioimmunulogy].

Dystrophin is a protein linking the cytoskeleton to a complex of transmembrane proteins that interact with the extracellular matrix. The fragility of the cardiomyocyte cell membrane resulting from the lack of dystrophin is thought to cause an excessive susceptibility to mechanical stress. Based on surface ECC we demonstrate the differences of cardiac phenotype in young (2- to 3-mo-old) and aged (over 1,5 years) dystrophin-deficient mdx mouse and normal mouse with the same genetic background. It was shown that main alterations in the mdx electrocardiogram concern primarily ventricular conduction velocity (QRS complex duration) and time of ventricular repolarization (QT interval) duration).The issue under discussion is whether dystrophin deficient mdx model can be used in research studies in cardioimmunology.

A new genetic variant of mdx mice: study of the phenotype.

Genetic selection in a colony of mdx mice (suffering from X-chromosome-linked muscular dystrophy) resulted in generation of their new genetic variant. In this new variant, the genetic, biochemical, and histological markers of muscular dystrophy are combined with signs of oculocutaneous albinism (skin and fur depigmentation), transillumination of the iris, sharply reduced pigmentation of the retinal epithelium, and increase of the eyeball refraction). Two sensorimotor tests (negative geotaxis and wire back down hanging) detected other phenotypical characteristics of albino mdx mice carrying, in addition to the mutation in the dystrophin gene exon 23 (intrinsic of the "classical" black mdx mice), an extra mutation responsible for pigmentation disorders. Slow geotaxis, despite longer wire back down hanging capacity, was regarded as aggravation of the neurological dysfunction in albino mdx mice in comparison with black mdx mice.

[Regenerative activity of muscle tissue and thymus status in young and old mdx mice with muscle injury and wound xenoplasty].

Responses of the skeletal muscle tissue and thymus to muscle injury (complete transection) and wound xenoplasty with the minced muscle tissue of newborn rats (tissue therapy) were studied in mdx mice aged 12-16 and 40-48 weeks. The muscle tissue of mdx mice has genetic defects causing chronic dystrophic processes in it. The muscle tissue of young mdx mice proved to retain a relatively high capacity for regeneration. Under conditions of tissue therapy of the wound, the formation of muscle fibers from muscle cells of the graft and active regeneration of muscle fibers in the recipient mice were observed, and no structural defects were detected in the thymus. The capacity of posttraumatic regeneration in old mdx mice was lower. The xenogenic graft was undergoing resorption, thereby suppressing regeneration of muscle fibers and causing further tissue destruction in the injured muscle. The thymus parenchyma was subject to degenerative changes such as the formation of gaps, hemorrhagic foci, and increased numbers of macrophages and mast cells.

Restoration of gastrocnemius muscle in MDX mice of different age after injury and implantation of xenogenic muscle tissue.

The intensity of regeneration of crossed gastrocnemius muscle was evaluated in two groups of mdx mice of different age 2 weeks after implantation of crushed muscle tissue from newborn rats into the wound defect area. The effect of xenoplasty manifested in increased weight of the damaged muscle. The effect was observed in mice aging 12-16 weeks but not in those aged 40-48-weeks. Structural changes in the skeletal muscle tissue intrinsic of mdx mice and augmenting with age were detected in intact mice before the experiment. Activity of muscle fiber regeneration in intact and injured muscle of 40-48-week-old mice was significantly lower than in 12-16-week-old ones. Myoblasts of the xenogenic transplant retained viability in recipient muscles for at least 2 weeks. posttraumatic regeneration was stimulated in only 12-16-week animals. Xenoplasty was ineffective in older animals and even somewhat enhanced the destructive processes in the muscle. It seems that age-specific regeneration activity of the recipient skeletal muscle tissue should be taken into consideration in the development of effective strategy of cell therapy for progressive muscular dystrophy.

Cardiotropic effect of extracardiac transplantation of embryonic human myoblasts to mice with bradycardia: various effects of cell material.

Animals with bradycardia were detected in reproductive colony of mdx mice. Low pulse rate was associated with poor survival and predisposition to sudden death, but did not directly depend on the presence of dystrophin mutant gene or animal age. Heart rate increased in old mice with bradycardia after extracardial, intramuscular, and intravenous injection of human embryonic myoblasts. Stable normalization of the pulse was observed 2 weeks after transplantation, but early peak of heart rate was observed as early as 24 h after cell transplantation. Cell suspensions, which could contain stem cells (blood mononuclears and CD34+ lymphocytes), also corrected heart rhythm. Unlike the effect of myoblasts, cardiotropic effect of mononuclears was preceded by a period of tachycardia, while the effect of CD34+ lymphocytes was very unstable. The cardiotropic effect of myoblasts was combined with life span prolongation and certain rejuvenation in some animals. Erythrocytes and supernatant obtained during blood cell fractionation did not modify the heart rhythm in mice with bradycardia. After injection of myoblasts to mice with rare and normal pulses serum creatine kinase activity decreased with different rates. These data attest to a variety of biological effects of stem cells and/or their derivatives and to ambiguous mechanisms of these effects.

[Dysbacteriosis and immunopathological process].

The poststress translocation of intestinal microflora to the internal environment of the body is proposed to be regarded as the mechanism of neuroimmune cooperation in the realization of adaptative raction. It is pointed out that the effectiveness of the adaptive process depends on the state of microbiocenosis as a mediating factor. The inclusion of remedies for the correction of intestinal microbiocenosis into the complex therapy of patients with chronic pathology (as a specific manifestation of dysadaptive state) makes it possible to achieve results which cannot be achieved by the standard approach. We should return to the initial interpretation of I.I.Mechnikov's thesis on the basic role of dysbacteriosis in the development of the pathological process.

Hereditary muscular dystrophy in MDX mice as a homologous model for introduction of cell technologies in the treatment of progressive muscular dystrophies in humans.

Life-time monitoring of the main clinical and laboratory manifestations of hereditary muscular dystrophy in mdx mice confirmed the presence of mutation in exon 23 of dystrophin gene and the absence of this protein in skeletal muscles of mutant animals. Muscular dystrophy in mice was similar to human progressive muscle disorder, which allows the use of this model for the development of cell technologies for the treatment of hereditary muscular diseases in humans.

Xenotransplantation of embryonic precursors of human myogenesis for the correction of dystrophinopathy in mice with hereditary muscular dystrophy.

Human embryonic myogenic precursors were transplanted into muscles of mdx mice with hereditary dystrophin-deficient muscular dystrophy. Transplantation induced the synthesis of human dystrophin. The number of dystrophin-positive fibers progressively decreased, however, some of them were preserved even 5 months after transplantation. Our results indicate that xenogeneic transplantation of embryonic myogenic precursors compensates the genetic defect in dystrophin-deficient mice.

Tissue hypoxia and intestinal dysbiosis in children with tuberculosis.

We studied the role of autochthonous microflora from body cavities in the development of tissue hypoxia and instability of cell membranes. In children with tuberculosis dysbiosis manifested in nonspecific quantitative changes in the intestinal microflora and the presence of coxsackievirus antigens in the urine. DNA-containing viruses with pronounced immunosuppressive activity (e.g., herpesvirus, measles virus, and rubella virus) were found in most children. Microbiological and virological changes were accompanied by the appearance of laboratory signs for tissue hypoxia, which included inhibition of Krebs cycle dehydrogenases and alpha-glycerophosphate pathway in blood lymphocytes. Regression analysis revealed a relationship between the content of extraintestinal coxsackieviruses and inactivation of alpha-glycerophosphate dehydrogenase, succinate dehydrogenase and ratio of facultatively anaerobic bacteria in microbiocenosis, and expression of acid phosphatase and total population of malonate-positive enterobacteria, staphylococci, yeasts, and enterococci.

Pathogenetic role of dysbacteriosis in the development of complications of type 1 diabetes mellitus in children.

A relationship between enteric microbiocenosis and severity of type 1 diabetes mellitus was detected. Microbiological analysis showed II-IV degree dysbacteriosis in all diabetic children. Long-term therapy with probiotics aimed at eradication of opportunistic microflora resulted in recovery of microbiocenosis, which was paralleled by improvement of the clinical status, regression of complications in children who were ill for a long time, and prevention of complications in children with newly detected diabetes. These results indicate the leading role of chronic enteric toxic infectious process in the development of complications of type 1 diabetes. The significance of infection in the pathogenesis of other noninfectious diseases in man is discussed.

[Level of serum antibodies to opportunistic microflora as a marker of the secondary immunodeficiency processes in children]. / Uroven' syvorotochnykh antitel k uslovnopatogennoi mikroflore kak marker protsessa formirovaniia vtorichnogo immunodefitsita u detei.

Examination of children with different noninfectious diseases resulted in obtaining the data base on the state of health of 201 children belonging to the potential risk group of the development of secondary immunodeficiency. The children were subdivided into several groups which differed by the type of immune disturbances and accompanying metabolic shifts. The level of antibodies to one of the fragments of peptidoglycan-N-acetylmuramyldipeptide was compared with the character of changes in the immune system. Different titers of serum antibodies to peptidoglycan were found to correspond to different forms of immune disturbances. The study showed that from the group with the absence of definite signs of immunodeficiency to the group with the pronounced deficiency of T lymphocytes and monocytes expressing CD14 antigen changes in the immune system increased in parallel to a rise in the concentration of alpha 2-macroglobulin, the development of hypercholesterolemia and a decreased level of antibodies to peptidoglycan. Opportunistic microflora was seemingly an important factor in the formation of definite forms of disturbances of the immune system and accompanying metabolic shifts.

[Pathogenetic role of intestinal microbiocenosis in the pathogenesis of hereditary myodystrophy]. / Patogeneticheskaia rol' kishechnogo mikrobiotsenoza v klinike nasledstvennykh miodistrofii.

The effect of the inclusion of probiotic preparations for the correction of disturbances in normal intestinal microflora into the complex therapy of patients wish Duchenne's childhood muscular dystrophy and Becker's myopathy was analyzed. Probiotic therapy made it possible to improve the clinical state of patients, manifested by an increase in muscular strength and accompanied by positive shifts in electromyographic, immunological, biochemical, hormonal characteristics. Intestinal microbiocenosis plays seemingly a certain role in the formation of hereditary pathology.

[Role of dysbacteriosis in the formation of chronic noninfectious pathology in children]. / Rol' disbakterioza v formirovanii khronicheskoi neinfektsionnoi patologii u detei.

The results of clinical observations and laboratory data make it possible to regard dysbacteriosis as an important factor in the pathogenesis of chronic noninfectious pathology in children. The adequate complex correction of intestinal dysbacteriosis on the basis of probiotic therapy facilitates the prolonged remission of the disease in children with diabetes mellitus of type 1 (DM1) and myopathy, decreases severity of late complications of DM1. A suggestion is made on the role of dysbiotic microflora in the development of chronic non-infectious pathology in children.

[Use of flow cytofluorimetry in the analysis of interactions of mononuclear phagocytes with protein antigens]. / Ispol'zovanie protochnoi tsitofliuorimetrii dlia analiza vzaimodeistviia mononuklearnykh fagotsitov s belkovymi antigenami.

Flow cytofluorometry of samples stained with fluorochrome-labeled antigen may be used to study the interactions between human blood calls and antigens. Using fluorescein isothiocyanate-labeled tuberculin (after the original method), the authors found that human blood monocytes actively bind labeled tuberculin. Studies of the concentrations from 0.1 to 12,500 ng/ml showed that saturation of the cell capacity for endocytosis is attained at certain doses, after which their surface label prevails. The share of tuberculin-binding lymphocytes was appreciably lower than that of monocytes at the same doses of the antigen. Clinical application of the method for assessing the antigen-presenting function of macrophages is discussed.

[Proliferative activity of peripheral blood lymphocytes and evaluation of the effectiveness of drugs in lichen ruber planus]. / Proliferativnaia aktivnost' limfotsitov perifericheskoi krovi i otsenka éffektivnosti lekarstvennykh preparatov pri krasnom ploskom lishae.

A total of 106 patients with lichen planus were examined for proliferative activity of peripheral blood lymphocytes and exogenous interleukin-2 effect on recovery of cyclosporin A suppressed proliferative response to mitogen. Lymphocyte proliferative activity was assessed by the level of 3H thymidine incorporation in cellular DNA. This parameter was found reduced in patients with lichen planus in comparison with healthy donors and dependent on the disease stage but not degree of dissemination. No disorders were detected in lymphocyte capacity to express receptors for interleukin-2 in the course of their activation. Tigason, prednisolone, and delagil had a marked in vitro antiproliferative effect on the lymphocytes of patients with lichen planus, tigason exerting the highest effect and prednisolone the lowest.