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MITOCHONDRIAL MARKERS OF APOPTOSIS AS QUALITY ASSESSMENT IN TREATMENT OF LOCALIZED SCLERODERMA

Posted Mar 20 2011 8:45am
BY G.W. Nikolashin, W.A. Babanin, I.N. Kravchenko, P.N. Kolbasin,
L.P. Kolbasina, A.A. Soboleva, A.A. Pisarev
Department of skin and venereal diseases
Crimean State Medical University

Scleroderma - a chronic inflammatory disease of connective tissue characterized by its thickening (sclerosis), commonly affecting the skin but inadequate treatment and influence of different factors can also affect internal organs (systemic form of scleroderma) - gastrointestinal tract, heart, lungs , kidneys and synovial tissue. 


The etiology of the disease fairly complex and doesn’t well understood: scleroderma often referred to autoimmune diseases, scientists and doctors believe in multifactorial genesis, due to the interaction of adverse of exogenous and endogenous factors with a genetic predisposition to the disease are the cause of this pathology. 


Particular attention is paid to trigger action of chemical agents (industrial, domestic, alimentary) and some medicines. Thanks to modern researches deciphered some genetic mechanisms of predisposition to systemic sclerosis it was argued earlier that hereditary predisposition to this pathology. Revealed a combination of certain antigens and alleles of HLA allele with systemic sclerosis: A9, B8, B27, B35, DR1, DR3 (chronic), DR5 (subacute course), DR11, DR52, and S4A. 


Also scleroderma may occurs children of any age. Histological examination of affected skin areas marked thickening and thickening of collagen fibers of the dermis and perivascular infiltrates consisting of mononuclear cells. Scleroderma can have serious consequences. Symptoms are highly variable, the effects of scleroderma can range from almost imperceptible to the life-threatening manifestations. 


Determination of severity of pathology may also depend on the localization of the foci. With inadequate treatment the disease can pass into the systemic form. Early diagnosis and correct choice of treatment strategy may considerably reduce the clinical manifestations of scleroderma and morphological changes in organs and tissues. 


The trend increasing of morbidity, the social significance of the problems caused by frequent lesion of middle working age and possible disability during the torpid recurrent determine the relevance of further study of theoretical and practical aspects of this pathology. The interaction of various factors in the development of the disease complicates the choice of treatment methods and, apparently, sometimes reduces their effectiveness. 


Apoptosis - programmed cell death, accompanied by a set of characteristic changes: a decrease of its size, condensation and fragmentation of chromatin, compaction of the outer and cytoplasmic membranes without exiting the cell contents into the environment. Programmed death is the result of the genetic program. The main difference between necrosis and apoptosis are: the necrosis - a cell death due to her injury (chemical, thermal, X-rays, etc.). 


During necrotic cell vacuolised (structural changing of the outer membrane, the concentration gradient, water flows into the cell, all organelles are beginning to swell), the lysosome digest the entire contents of the cell, the cell bursts. Its content is released into the extracellular space, that, consequently, causes inflammation, and subsequently absorbed by phagocytes. 


The characteristic features of apoptosis, to distinguish it from necrosis, are: the transition of phosphatidylserine from the inner monolayer of the cytoplasmic membrane into the outer monolayer, the yield of cytochrome C from the intermembrane space of mitochondria in the cytoplasm, activation of cysteine proteases (caspases), the formation of reactive oxygen species, cytoplasmic membrane, the decrease of cell volume, chromatin condensation at the periphery of the nucleus, the subsequent disintegration of the nucleus into parts, fragmentation of cells in the vesicles with intracellular contents - apoptotic cells. 


Apoptotic bodies are captured by neighboring cell (phagocytes may) as in the case of necrosis. Emissions of cell contents does not occur, inflammation does not occur. Necrosis is characterized by a group of cells, apoptosis - for one. A manifestation of failure of apoptosis is the uncontrolled division of abnormal cells, i.e., the formation and growth of the tumor. At the same time, increased apoptosis can lead to premature aging, the development of cell aplasia and degeneration. 


The development of modern methods of therapy (e.g. anti-tumor) a lot of attention given to the processes of cell regulation and induction of apoptosis. The purpose of this study: identification of mitochondrial markers of apoptosis, depending on the timing of the scleroderma, development of effective therapy by creating the optimal scheme of etiotropic and pathogenetic therapy including new drugs and their combination with physical therapy to obtain and retain maximum therapeutic effect. To conduct the study was selected 60 patients with localized scleroderma. 


The control group consisted of 20 healthy people. We have divided patients into 3 groups with varying disease duration. The first group comprised 22 patients with disease duration to 2 months. The second group - 17 patients with disease duration to 4 months old. The third - 21 patients, with a term  6 months of disease. In addition, we have examined 20 healthy individuals didn’t not suffering from this pathology, which formed the control group. We took advantage of modern technology, fast optical measurement cell, its organelles and processes occurring in it - flow cytometry. 


Currently, flow cytometry is used to identify certain cells in the samples (both bacterial and fungal infections, and the human body's own cells), susceptibility of microorganisms to antibiotics, as well as monitoring the status of viral HIV-infected patients. To account the results of treatment efficacy  we have used the following markers: CD95 - cell surface receptor, capable of running apoptosis after its interaction with Fas-ligand or antibodies to Fas, as well as APO2.7 - to determine the percentage of cells subjected to apoptosis . 


The method is based on the property of antigen APO2.7 (also known as 7A6) appears on the mitochondrial membrane of apoptotic cells. Expression of APO2.7 determined in the early stages of apoptosis. Just APO2.7 can be detected in cells after exposure of radiation, processing drugs or linking CD-95, Fas-ligand. Normal cells are negative or weakly positive for this antigen. 


For the most rapid and effective therapeutic effect we replaced and supplemented some of the preparations of standard treatment regimens as follows: "Vobenzim" (a combination of highly active enzymes of plant and animal origin, immunomodulator, anti-inflammatory preparation), "Kuprenil" (it has the ability to disrupt the synthesis of collagen, splitting the cross-links between the newly synthesized molecules of tropocollagen.  It has anti-inflammatory effect, in capsules), "Medobiotin". 


In addition, this scheme of treatment, we added a set of fisiotheraputic procedures: application Saky’s mud and phonophoresis (using polyenzyme ointment Vobe-mugos"), therapeutic massage. An analysis of the data shows that patients with localized scleroderma has been an intensification of apoptosis (increased levels of CD95 and expression of APO2.7). The degree of degradation of cellular immunity is reflected is reflected in an inverse correlation between the indices of cellular immunity (reduction of CD3 + and CD4 +) and indicators of apoptosis (increased CD95 and APO2.7). 


The intensification of the pathological process occurs due to loss of CD3 + and CD4 +. Favourable by the pathological process began appears to the end of the second week of treatment. The improving of general patient condition, decreased numbness in the lesions, paresthesiases, gradually eliminated inflammation in the edematous-erimatose phase. In the stage of induration skin becomes softer, more elastic, steel take shape plots atrophy. By the end of the course in all patients with a core group pronounced a fairly high degree of clinical improvement.


The best results were obtained in patients with patchy form of scleroderma, especially in localized process - a clinical remission 30% versus 17% of patients with advanced process (the differences are significant p <0,01), significant improvement was registered in 50% and 29% respectively ( p <0,05). In patients with scleroatrofic lichen clinical remission was achieved in 15% of cases, a significant improvement - in 56,4%. The linear form of scleroderma was more resistant to treatment, a significant improvement noted in 42% of cases compared with 50% (p <0,05). 


The analyzing of the effectiveness of complex treatment depending on the activity of sclerodermic process, we have found that clinical remission under the appearance of erythema was achieved only in three patients with isolated pockets. In the remaining patients of this group persisted longer inflammatory reaction in the form of redness and swelling in the center pockets and purple rim around the periphery. Significant improvements were registered in 45% of patients, improvement - in 48%, a slight improvement of 7%. 


Even better results were obtained in patients who were in the stage of induration after the decay process activity. Seals in the lesions completely disappeared (clinical remission 45% of patients), retained residual effects (significant improvement) 30%, partial induration - 15%. An analysis of the data obtained in patients with localized scleroderma has been a strengthening of apoptosis (raising SD95 positive lymphocytes and APO2.7).


Investigating the correlation between the level of cellular immunity indexes of apoptosis, we noted a close inverse correlation (r> -0,5) between the indices of cellular immunity (reduction of SDZ +, CD4 +) and indices of apoptosis (raising SD95 and APO2 .7). This negative correlation reflects the degree of degradation of cellular immunity. There has been an intensification of apoptosis by the death of cells CD3 + and CD4 +. 


Activity of sclerodermic process was in the clear depending on the therapy. The treatment, carried out during induration, was more effective than in the acute and subacute stage in the presence of inflammatory activity and the prevalence of skin erythema, but the state of atrophy varied little. The proposed complex treatment leads to a rapid and marked improvement and stable remission.


Read about Mitochondria


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