RESUMO
The exact cellular and molecular mechanisms of multiple sclerosis and other autoimmune diseases have not been established. Autoimmune pathologies are known to be associated with faults in the immune system and changes in the differentiation profiles of bone marrow stem cells. This study analyzed various characteristics of experimental autoimmune encephalomyelitis (EAE) in 2D2 mice. Differentiation profiles of six hematopoietic stem cells of bone marrow were found to significantly differ in 2D2 male and female mice during the spontaneous development of EAE. In addition, we found various properties of B and T cells, CD4+ and CD8+ lymphocytes in blood and several organs (bone marrow, spleen, thymus, and lymph nodes) of 2D2 male and female mice to be considerably different. These changes in hematopoietic stem cells differentiation profiles and level of lymphocyte proliferation in various organs of 2D2 mice were found to induce the production of IgGs against DNA, myelin basic protein, and myelin oligodendrocyte glycoprotein, increasing the number of autoantibodies hydrolyzing these substrates. We compared the changes of these immunological and biochemical parameters in 2D2 mice with those of mice of two other lines (Th and C57BL/6), also prone to spontaneous development of EAE. Some noticeable and even extreme variations were found in the time-related development of parameters between male and female mice of 2D2, Th, and C57BL/6 lines. Despite some differences, mice of all three lines demonstrated the changes in hematopoietic stem cells profiles, lymphocyte content, and production of catalytic autoantibodies. Given that these changes are harmful to mice, we believe them to cause the development of experimental autoimmune encephalomyelitis.
Assuntos
Anticorpos Catalíticos , Encefalomielite Autoimune Experimental , Animais , Autoanticorpos , Medula Óssea/patologia , Diferenciação Celular , Proliferação de Células , Encefalomielite Autoimune Experimental/patologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito , Fragmentos de PeptídeosRESUMO
We have previously shown that immunization of C57BL/6 mice, prone to spontaneous development of experimental autoimmune encephalomyelitis (EAE), with three antigens (MOG35-55 , DNA-histone complex or DNA-methylated BSA complex), alters the differentiation profiles of bone marrow haematopoietic stem cells (HSCs). These are associated with the production of autoantibodies (auto-Abs) against these antigens and the formation of abzymes hydrolysing DNA, MOG, myelin basic protein (MBP) and histones. Immunization of mice with antigens accelerates the development of EAE. This work is the first to analyse the ratio of auto-Abs without and with catalytic activities at different stages of EAE development (onset, acute and remission phases) after immunization of mice with the three specific antigens. Prior to immunization and during spontaneous in-time development of EAE, the concentration of auto-Abs against MBP, MOG, histones and DNA and activities of IgG antibodies in the hydrolysis of substrates increased in parallel; correlation coefficients = +0.69-0.94. After immunization with MOG, DNA-histone complex or DNA-met-BSA complex, both positive (from +0.13 to +0.98) and negative correlations (from -0.09 to -0.69) were found between these values. Our study is the first showing that depending on the antigen, the relative amount of harmful auto-Abs without and abzymes with low or high catalytic activities may be produced only at onset and in acute or remission phases of EAE. The antigen governs the EAE development rate, whereby the ratio of auto-Abs without catalytic activity and with enzymatic activities of harmful abzymes hydrolysing MBP, MOG, histones and DNA varies strongly between different disease phases.
Assuntos
Anticorpos Catalíticos/imunologia , Antígenos/imunologia , Autoanticorpos/imunologia , Suscetibilidade a Doenças/imunologia , Encefalomielite Autoimune Experimental/etiologia , Animais , Autoantígenos/imunologia , Diferenciação Celular , Proliferação de Células , DNA/imunologia , Células-Tronco Hematopoéticas/metabolismo , Histonas/imunologia , Histonas/metabolismo , Hidrólise , Imunização , Imunoglobulina G/imunologia , CamundongosRESUMO
Exact mechanisms of autoimmune disease development are still yet unknown. However, it is known that the development of autoimmune diseases is associated with defects in the immune system, namely, the violation of the bone marrow hematopoietic stem cells (HSCs) differentiation profiles. Different characteristics of autoimmune reaction development in experimental autoimmune encephalomyelitis (EAE) prone Th mice characterizing T-lymphocytes response were analyzed using standard approaches. Profiles of several HSCs differentiation of bone marrow (BFU-E, CFU-E, CFU-GM, CFU-GEMM, T- and B-lymphocytes) of Th male and female mice during spontaneous development of EAE were noticeably different. Patterns of total lymphocytes, B- and T-cells proliferation in several different organs (bone marrow, blood, spleen, thymus, and lymph nodes) were also remarkably different. In addition, there were in time noticeable differences in their changes for some organs of male and female mice. Characters of changes in the profiles of CD4 and CD8 cells proliferation in some organs not always coincide with those for total T lymphocytes. The changes in the differentiation profiles of HSCs and the level of lymphocytes proliferation in the bone marrow and other organs were associated with the increase in the concentration of antibodies against DNA, myelin basic protein, and myelin oligodendrocyte glycoprotein, and catalytic antibodies hydrolyzing these substrates. Despite some differences in changes in the analyzed parameters, in general, the spontaneous development of EAE in male and female mice occurs to some extent in a comparable way.
Assuntos
Anticorpos Catalíticos/imunologia , Diferenciação Celular/genética , Encefalomielite Autoimune Experimental/imunologia , Ativação Linfocitária/imunologia , Linfócitos/imunologia , Animais , Anticorpos Catalíticos/genética , Células da Medula Óssea/imunologia , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células/genética , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Ativação Linfocitária/genética , Contagem de Linfócitos , Camundongos , Glicoproteína Mielina-Oligodendrócito/genética , Glicoproteína Mielina-Oligodendrócito/imunologia , Baço/imunologiaRESUMO
The exact mechanisms of multiple sclerosis (MS) development are still unknown, but the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice is associated with the violation of bone marrow hematopoietic stem cells (HSCs) differentiation profiles associated with the production of harmful for human's autoantibodies hydrolyzing myelin basic protein, myelin oligodendrocyte glycoprotein (MOG35-55), and DNA. It was shown that IgGs from the sera of healthy humans and autoimmune patients oxidize many different compounds due to their H2O2-dependent peroxidase and oxidoreductase activity in the absence of H2O2. Here we first analyzed the change in the relative redox activities of IgGs antibodies from the blood of C57BL/6 mice over time at different stages of the EAE development. It was shown that the peroxidase activity of mice IgGs in the oxidation of ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) is on average 6.9-fold higher than the oxidoreductase activity. The peroxidase activity of IgGs increased during the spontaneous development of EAE during 40 days, 1.4-fold. After EAE development acceleration due to mice immunization with MOG35-55 (5.3-fold), complexes of bovine DNA with methylated bovine serum albumin (DNA-metBSA; 3.5-fold), or with histones (2.6-fold), the activity was increased much faster. The increase in peroxidase activity after mice immunization with MOG35-55 and DNA-metBSA up to 40 days of experiments was relatively gradual, while for DNA-histones complex was observed its sharp increase at the acute phase of EAE (14-20 days). All data show that IgGs' redox activities can play an important role in the protection of mice from toxic compounds and oxidative stress.
Assuntos
Anticorpos Catalíticos/metabolismo , Autoanticorpos/metabolismo , Encefalomielite Autoimune Experimental/enzimologia , Células-Tronco Hematopoéticas/imunologia , Oxirredutases/metabolismo , Peroxidases/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Humanos , Peróxido de Hidrogênio/farmacologia , Imunoglobulina G/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína Básica da Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito/administração & dosagem , Oxirredução , Oxirredutases/imunologia , Fragmentos de Peptídeos/administração & dosagem , Peroxidases/imunologiaRESUMO
Experimental autoimmune encephalomyelitis (EAE)-prone C57BL/6 mice are used as a model of human multiple sclerosis. We immunize mice with myelin oligodendrocyte glycoprotein (MOG), DNA-histone and DNA-methylated bovine serum albumin (met-BSA) complexes to reveal different characteristics of EAE development including bone marrow lymphocyte proliferation and differentiation profiles of hematopoietic stem cells. Immunization of C57BL/6 mice with MOG35-55 results in the acceleration of EAE development. Anti-DNA antibodies are usually directed against DNA-histone complexes resulting from cell apoptosis. During the acute EAE phase (7-20 days after immunization), catalytic antibodies efficiently hydrolysing myelin basic protein (MBP), MOG and DNA are produced with parallel suppression of antibodies hydrolysing histones. We could show that in contrast to MOG, immunization with histone-DNA results in a reduction of proteinuria, a significant increase in anti-DNA, anti-MBP and anti-MOG antibody titres, as well as an increase in their catalytic activities for antigen hydrolysis, but slightly changes the concentration of cytokines. Contrary to MOG, DNA-histone and DNA-met-BSA only stimulated the formation of anti-DNA antibodies hydrolysing DNA with a long delay (15-20 days after immunization). Our data indicate that for C57BL/6 mice immunization with DNA-met-BSA and DNA-histone complexes may have opposing effects compared to MOG. DNA-histone stimulates the appearance of histone-hydrolysing abzymes in the acute EAE phase, while abzymes with DNase activity appear at significantly later time-points. We conclude that MOG, DNA-histone and DNA-met-BSA have different effects on numerous bone marrow, cellular, immunological and biochemical parameters of immunized mice, but all antigens finally significantly stimulate the development of the EAE.
Assuntos
Anticorpos Catalíticos/biossíntese , Diferenciação Celular , DNA/metabolismo , Encefalomielite Autoimune Experimental/patologia , Histonas/metabolismo , Animais , Apoptose , Peso Corporal , Proliferação de Células , Ensaio de Unidades Formadoras de Colônias , Modelos Animais de Doenças , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Hidrólise , Linfócitos/citologia , Linfócitos/metabolismo , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Proteinúria/complicações , Fatores de TempoRESUMO
A model of chronic opisthorchiasis combined with social stress is examined; this situation is more likely for humans and animals than a separate impact of the infectious factor. For this purpose, we evaluated the effects of Opisthorchis felineus ("OP" group) and 30-day social stress (confrontations between males, "SS" group) alone and in combination ("OP + SS" group) in inbred C57BL/6 male mice and compared these effects according to the parameters listed below. The animals exposed to neither factor formed the control group ("CON"). All animals were assayed for blood biochemical parameters, changes in blood cell composition, and pattern of bone marrow hematopoiesis. By the end of the experiment, we have observed crucial effects of the two factors on the blood and liver of "OP" and "OP + SS". Eosinophil and basophil counts increased and relative segmented neutrophil and monocyte counts decreased in "OP + SS" mice on the background of activated myelopoiesis, mainly determined by social stress. Despite depressed erythropoiesis, "OP" mice displayed no changes in the relative peripheral erythrocyte counts. On the contrary, social stress, which stimulated erythropoiesis in "SS" and "OP + SS" mice, was accompanied by a decrease in the relative erythrocyte counts and hematocrit. Hepatosplenomegaly was observed on the background of these two impacts. Changes in transaminase (ALT and AST) and alkaline phosphatase activities as well as an increase in cholesterol and product of lipid peroxidation suggest a pronounced destruction of the liver. Altogether, social stress exacerbates many of the assayed blood parameters in the mice infected with the liver fluke.
Assuntos
Opistorquíase/sangue , Estresse Psicológico/complicações , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Ductos Biliares/parasitologia , Células Sanguíneas/química , Análise Química do Sangue , Glicemia/análise , Proteínas Sanguíneas/análise , Medula Óssea/química , Antígenos CD13/sangue , Colesterol/sangue , Modelos Animais de Doenças , Índices de Eritrócitos , Hematócrito , Hematopoese , Células-Tronco Hematopoéticas , Contagem de Leucócitos , Fígado/parasitologia , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Opistorquíase/complicações , Opistorquíase/psicologia , Contagem de Plaquetas , Baço/patologia , Estresse Psicológico/sangueRESUMO
Immunization of experimental autoimmune encephalomyelitis (EAE)-prone C57BL/6 mice with MOG35-55 (a model used to study aspects of human multiple sclerosis) is known to lead to the production of various abzymes. The production of catalytic IgGs that can efficiently hydrolyse myelin basic protein (MBP), MOG and DNA is associated with changes in the profile of differentiation and level of proliferation of mice bone marrow haematopoietic stem cells (HSCs). As MOG simulates the production of abzymes with high DNase activity, we compared the effects of DNA and MOG immunization on EAE-prone mice. In contrast to MOG, immunization with DNA leads to a suppression of proteinuria, a decrease in the concentrations of antibodies to MOG and DNA and a reduction in abzyme production. Immunization with DNA only resulted in a significant increase in DNase activity over 40 days where it became 122-fold higher than before immunization, and fivefold higher when comparing to the maximal activity obtained after MOG treatment. DNA and MOG immunization had different effects on the differentiation profiles of HSCs, lymphocyte proliferation, and the level of apoptosis in bone marrow and other organs of mice. The data indicate that for C57BL/6 mice, DNA may have antagonistic effects with respect to MOG immunization. The usually fast immune response following MOG injection in C57BL/6 mice is strongly delayed after immunization with DNA, which is probably due to a rearrangement of the immune system following the response to DNA.
Assuntos
Anticorpos Catalíticos/biossíntese , DNA/administração & dosagem , Encefalomielite Autoimune Experimental/imunologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Glicoproteína Mielina-Oligodendrócito/administração & dosagem , Animais , Diferenciação Celular , Proliferação de Células , Ensaio de Unidades Formadoras de Colônias , DNA/imunologia , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/patologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Imunidade Humoral , Imunização/métodos , Camundongos , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito/imunologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/imunologiaRESUMO
Myelin oligodendrocyte glycoprotein (MOG) is an antigen of the myelin sheath, which may trigger immune cell responses and the production of auto-antibodies in multiple sclerosis (MS). In this study, we used MOG(35-55) -induced experimental autoimmune encephalomyelitis (EAE), a model of human MS, to assess the production of catalytically active immunoglobulin G (IgG) antibodies or abzymes which have been shown to be present in sera of patients with several autoimmune diseases. Here, we show that IgGs from the sera of control C57BL/6 mice are catalytically inactive. During development of EAE, a specific reorganization of the immune system of mice occurred leading to a condition which was associated with the generation of catalytically active IgGs hydrolysing DNA, myelin basic protein (MBP) and MOG which was associated with increased proteinuria, changes in differentiation of mice bone marrow hematopoietic stem cells (HSCs) and an increase in proliferation of lymphocytes in bone marrow, spleen and thymus as well as a significant suppression of cell apoptosis in these organs. The strongest alterations were found in the early disease phase (18-24 days after immunization) and were less pronounced in later EAE stages (40 days after EAE induction). We conclude that a significant increase in DNase and proteolytic activities of antibodies may be considered the earliest statistically significant marker of MOG-induced EAE in mice. The possible differences in immune system reorganizations during preclinical phases of the disease, acute and late EAE, leading to production of different auto-antibodies and abzymes as well other changes are discussed.
Assuntos
Proliferação de Células , Encefalomielite Autoimune Experimental/imunologia , Células-Tronco Hematopoéticas/fisiologia , Glicoproteína Mielina-Oligodendrócito/imunologia , Animais , Apoptose , Células Cultivadas , Encefalomielite Autoimune Experimental/patologia , Ativação Linfocitária , Linfócitos/fisiologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBARESUMO
A biosafety study was carried out concerning the metabolites of Streptomyces tauricus strain 19/97 M. This strain is a promising producer of biological preparations and shows antagonistic properties against Fusarium fungi, which cause Fusarium wilt disease. The strain has a pronounced biological activity against conifers, cereals and legumes. The treatment of planting material reduces infections, increases germination and furthers plant productivity. Using metabolites, we understood the culture liquid separated by filtration after the cultivation of the strain. Animals of different taxonomic affiliations were used as test objects: (CBA × C57BI/6) F1 hybrid mice (Mus musculus) (warm-blooded organisms), Daphnia magna Straus (planktonic crustaceans) and the unicellular alga Chlorella vulgaris Beijer. In the study, we were guided by the test standards for acute oral toxicity and irritation to the skin, mucous membranes of the eyes and inhalation toxicity. The research results showed that the metabolites of the strain are not acutely toxic to organisms of different taxonomic levels. The metabolites of the strain do not have an irritating effect on the skin and mucous membranes of warm-blooded animals. Based on the studies carried out, metabolites can be used for creating a fungicidal biological preparation.
RESUMO
It has been shown for the first time that polyclonal IgG abzymes (Abzs) with DNase activity from the sera of autoimmune-prone MRL/MpJ-lpr mice can be separated by isoelectric focusing into many subfractions having the isoelectric points (pI) from 4.5 to 9, with the maximal activity for Abzs with pI = 6.5-9.0. Affinity chromatography on DNA-cellulose separated DNase IgGs into many subfractions demonstrating a range of affinities for DNA and different levels of the relative DNase activities (RDA) due to intrinsically bound metals and after addition of external Mg(2+) , Mn(2+) , Ca(2+), and Mg(2+) +Ca(2+). Some fractions significantly increase RDAs in the presence of external ions (Mg(2+) + Ca(2+) > Mg(2+) > Mn(2+) > Ca(2+)), while each of this cofactor can also inhibit or have no influence on the RDAs of another fractions. It is known that complexes of DNA with histones and other proteins of apoptotic cells are the primary immunogens in systemic lupus erythematosus (SLE). Bovine serum albumin (BSA) and methylated BSA (mBSA) increase the RDAs of only some fractions, while have no effect or inhibit other IgG fractions. The ratio of the RDAs in the presence of all metal ions, BSA, and mBSA was individual for every abzyme fraction. Mn(2+) and Ca(2+) stimulated accumulation of only relaxed form of supercoiled DNA (scDNA) in the case of all subfractions, while in the presence of Mg(2+) antibodies (Abs) of some subfractions (and in the presence of Mn(2+) +Ca(2+) all subfractions) produced relaxed DNA (rDNA) and linear DNA (linDNA) in a variable extent. The data obtained show that the polyclonal Abzs of mice may be a cocktail of Abs directly to DNA, RNA, and their complexes with proteins and anti-idiotypic Abs to active centers of different nucleases. The diversity of the physicochemical and kinetic characteristics of the Abzs seems to be significantly widened when pre-diseased mice spontaneously develop the disease.
Assuntos
Anticorpos Catalíticos/metabolismo , DNA/metabolismo , Desoxirribonucleases/metabolismo , Animais , Cálcio/farmacologia , Bovinos , Celulose/análogos & derivados , Celulose/química , Cromatografia de Afinidade , DNA/química , Ativação Enzimática/efeitos dos fármacos , Focalização Isoelétrica , Magnésio/farmacologia , Manganês/farmacologia , Camundongos , Camundongos Endogâmicos MRL lprRESUMO
Abzymes (Abzs) with different enzymic activities have been detected in the sera of patients with various autoimmune (AI) diseases and in AI mice. In this work, electrophoretically homogeneous IgGs were isolated from the sera of MRL-lpr/lpr mice spontaneously developing lupus-like AI pathology. It was shown for the first time that polyclonal IgGs (pIgGs) and their isolated heavy and light chains hydrolyze different nucleoside-5'-triphosphate (NTPs), nucleoside-5'-diphosphate (NDPs), adenosine monophosphate and deoxiadenosine-5'-monophosphate (dAMP), whereas antibodies from the sera of control healthy mice were catalytically inactive. Monoclonal mouse IgGs also effectively hydrolyze nucleotides. The data demonstrate that nucleotide-hydrolyzing activity is an intrinsic property of isolated mouse pIgG and monoclonal IgG. It was shown that various markers of AI pathologies (proteinuria and antibody titers to native and denatured DNA) demonstrating spontaneous development of AI reactions increased in animals with aging and correlated with an increase in Abz relative activity in hydrolysis of nucleotides. The highest increase in AI reaction markers and in Abz enzymic activity was found in mice immunized with a DNA-protein complex.
Assuntos
Anticorpos Antinucleares/metabolismo , Anticorpos Catalíticos/metabolismo , Imunoglobulina G/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Nucleotídeos/metabolismo , Animais , Anticorpos Antinucleares/sangue , Anticorpos Catalíticos/sangue , DNA/imunologia , Modelos Animais de Doenças , Hidrólise , Imunoglobulina G/sangue , Cadeias Pesadas de Imunoglobulinas/sangue , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias Leves de Imunoglobulina/sangue , Cadeias Leves de Imunoglobulina/metabolismo , Lúpus Eritematoso Sistêmico/sangue , Camundongos , Camundongos Endogâmicos MRL lpr , Desnaturação de Ácido NucleicoRESUMO
Till yet there is no data concerning mechanisms of autoimmune diseases development. Experimental autoimmune encephalomyelitis (EAE) prone C57BL/6 (T- and B-lymphocyte response), non-autoimmune CBA, and Th mice with T cell response were immunized with myelin oligodendrocyte glycoprotein (MOG35-55) to compare different characteristics of autoimmune reaction development. Bone marrow differentiation profiles of hematopoietic stem cells (HSCs), lymphocyte proliferation in various organs associated with the production of antibodies against DNA, myelin basic protein (MBP), and MOG, as well as abzymes hydrolyzing these antigens, were analyzed before and after immunization. Profiles of HSC differentiation [BFU-E (erythroid burst-forming unit (early erythroid colonies), CFU-E (erythroid burst-forming unit (late erythroid colonies), CFU-GM (granulocytic-macrophagic colony-forming unit), and CFU-GEMM granulocytic-erythroid-megakaryocytic-macrophagic colony-forming unit] and patterns of lymphocyte proliferation in different organs (brain, spleen, thymus, and lymph nodes) were very different for C57BL/6, CBA, and Th mice. We conclude that only C57BL/6 mice were predisposed to spontaneous and MOG-induced acceleration of EAE development. CBA mice are not prone to the development of autoimmune reactions. After immunization, Th mice demonstrate changes in several parameters similar to C57BL/6 and other to CBA mice; Th mice are more prone to developing autoimmune reactions than CBA mice. Our data may be important for understanding the combined presence in mice lymphocytes with T and B cell responses for spontaneous and induced autoimmune diseases.
Assuntos
Diferenciação Celular , Encefalomielite Autoimune Experimental/metabolismo , Glicoproteína Mielina-Oligodendrócito/metabolismo , Animais , Proliferação de Células , Células-Tronco Hematopoéticas/metabolismo , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Glicoproteína Mielina-Oligodendrócito/administração & dosagemRESUMO
We have shown previously that electrophoretically and immunologically homogeneous polyclonal IgGs from the sera of autoimmune-prone MRL mice possess DNase activity. Here we have analyzed for the first time activation of DNase antibodies (Abs) by different metal ions. Polyclonal DNase IgGs were not active in the presence of EDTA or after Abs dialysis against EDTA, but could be activated by several externally added metal (Me(2+)) ions, with the level of activity decreasing in the order Mn(2+)> or =Mg(2+)>Ca(2+)> or =Cu(2+)>Co(2+)> or =Ni(2+)> or =Zn(2+), whereas Fe(2+) did not stimulate hydrolysis of supercoiled plasmid DNA (scDNA) by the Abs. The dependencies of the initial rate on the concentration of different Me(2+) ions were generally bell-shaped, demonstrating one to four maxima at different concentrations of Me(2+) ions in the 0.1-12 mM range, depending on the particular metal ion. In the presence of all Me(2+) ions, IgGs pre-dialyzed against EDTA produced only the relaxed form of scDNA and then sequence-independent hydrolysis of relaxed DNA followed. Addition of Cu(2+), Zn(2+), or Ca(2+) inhibited the Mg(2+)-dependent hydrolysis of scDNA, while Ni(2+), Co(2+), and Mn(2+) activated this reaction. The Mn(2+)-dependent hydrolysis of scDNA was activated by Ca(2+), Ni(2+), Co(2+), and Mg(2+) ions but was inhibited by Cu(2+) and Zn(2+). After addition of the second metal ion, only in the case of Mg(2+) and Ca(2+) or Mn(2+) ions an accumulation of linear DNA (single strand breaks closely spaced in the opposite strands of DNA) was observed. Affinity chromatography on DNA-cellulose separated DNase IgGs into many subfractions with various affinities to DNA and very different levels of the relative activity (0-100%) in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. In contrast to all human DNases having a single pH optimum, mouse DNase IgGs demonstrated several pronounced pH optima between 4.5 and 9.5 and these dependencies were different in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. These findings demonstrate a diversity of the ability of IgG to function at different pH and to be activated by different optimal metal cofactors. Possible reasons for the diversity of polyclonal mouse abzymes are discussed.
Assuntos
DNA/química , Animais , Doenças Autoimunes/metabolismo , Cálcio/química , Cobalto/química , Cobre/química , Ácido Edético/química , Humanos , Hidrólise , Imunoglobulina G/química , Magnésio/química , Camundongos , Camundongos Transgênicos , Níquel/química , Zinco/químicaRESUMO
BACKGROUND: Large DNA poxviruses encode a diverse family of secreted proteins that modulate host inflammatory and antiviral responses, in particular by inhibiting one of the key players of the mammalian immune system, the tumor necrosis factor (TNF). METHODS: We investigated the effects of a recombinant variola (smallpox) virus TNF-decoy receptor (VARV-CrmB) in a murine model of contact dermatitis. Our results demonstrate that the VARV-CrmB protein significantly reduces the 2,4-dinitrochlorbenzene (DNCB)-induced migration of skin leukocytes during the sensitization phase and suppresses ear oedema during the elicitation phase of the contact reaction. RESULTS: Studies focusing on the bone marrow hematopoiesis in the contact dermatitis model revealed that the epicutaneous co-application of DNCB and VARV-CrmB protein normalized the DNCBinduced effects to control levels. CONCLUSION: As an effective TNF antagonist, the VARV-CrmB protein might be conceived as a beneficial candidate for further research and development of therapeutic approaches in the field of the inflammatory skin diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Dermatite Alérgica de Contato/tratamento farmacológico , Receptores Chamariz do Fator de Necrose Tumoral/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Proteínas Virais/farmacologia , Animais , Anti-Inflamatórios/isolamento & purificação , Dermatite Alérgica de Contato/imunologia , Dinitroclorobenzeno/imunologia , Modelos Animais de Doenças , Haptenos/imunologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Receptores do Fator de Necrose Tumoral/administração & dosagem , Receptores Chamariz do Fator de Necrose Tumoral/isolamento & purificação , Vírus da Varíola , Proteínas Virais/administração & dosagemRESUMO
Animals spontaneously developing lupus-like autoimmune pathology (SLE) are very promising models to study the mechanisms of natural abzymes (Abzs) generation and their role in etiology and pathogenesis of autoimmune diseases, but Abzs from the sera of animals remain virtually unstudied. In this work, electrophoretically homogeneous IgGs were isolated from the sera of MRL/MpJ-lpr mice. It was shown for the first time that amylase activity is an intrinsic property of antibodies (Abs) and their isolated heavy and light chains. Various markers of SLE pathology (proteinuria, enhanced concentration of anti-DNA Abs) increased with spontaneous development of SLE and especially after animal immunization, correlating with the increase in Abz relative amylase activity. The highest amylase activity was found in the sera Abs of healthy mice after delivery and at the beginning of lactation; this was not correlated with markers of mouse SLE but supports the idea that pregnancy could "activate" or "trigger" autoimmune-like manifestations and Abzs production in healthy mammals. The possible differences in mechanisms of Abzs production in lactating mice and animals developing SLE are discussed.
Assuntos
Amilases/sangue , Amilases/imunologia , Autoimunidade/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Hidrólise , Imunoglobulina G/imunologia , Cinética , Camundongos , Camundongos Endogâmicos MRL lpr , Oligossacarídeos/metabolismoRESUMO
VARV-CrmB is a TNF binding protein of variola virus. VARV-CrmB protein was previously shown to be active as a TNF-antagonist in a number of in vivo and in vitro models. Here we investigated the epicutaneous effect of recombinant VARV-CrmB protein using an experimental model of muTNFinduced migration of skin leukocytes as well as colony forming activity of bone marrow cells (BMC). EpiÑutaneous applications of muTNF enhanced the number of cells migrating from skin flaps of BALB/c mice, whereas subsequent applications of VARV-CrmB protein in 30 min after muTNF, abolished that effect. Epicutaneously applied muTNF influenced the activity of committed hematopoietic progenitors causing a reduction of erythroid (BFUe+CFUe) colonies and increase of granulocyte-macrophage (CFU-GM) colonies in the colony-forming tests. VARV-CrmB, applied in combination with muTNF, demonstrated an ability to reverse this effect, namely, to increase BFUe+CFUe and reduce CFU-GM back to the control levels. Taking together, these data demonstrate the TNF-blocking properties of VARV-CrmB in vivo at epicutaneous applications. As effective TNF antagonist VARV-CrmB protein might be conceded as a beneficial candidate for future research and development of therapeutic approaches in the field of inflammatory skin diseases.
Assuntos
Pele/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Proteínas Virais/farmacologia , Administração Tópica , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Leucócitos/fisiologia , Masculino , Camundongos Endogâmicos BALB C , Receptores do Fator de Necrose Tumoral/administração & dosagem , Pele/imunologia , Fator de Necrose Tumoral alfa/administração & dosagem , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Vírus da Varíola , Proteínas Virais/administração & dosagemRESUMO
Embryonic stem cells (ESCs) offer a powerful in vitro model to study mechanisms implicated in cell fate decision. Developmental pathways by which pluripotent ESCs become committed to specific lineages are reflected in dynamic changes of signaling and transcriptional programs. However, the mechanisms that govern the regulatory intracellular networks underlying lineage fate decisions and differentiation programs remain poorly understood and differ significantly in different species. In this review we analyze the current understanding of the signaling mechanisms and transcriptional regulation of differentiation of murine and human ESCs into the mesoderm.
Assuntos
Diferenciação Celular/genética , Linhagem da Célula/genética , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Humanos , Transdução de Sinais/genética , Fatores de Transcrição/metabolismoRESUMO
It was shown that IgGs from the sera of 2-7-month-old control non-autoimmune (CBA x C57BL)F1 and BALB/c mice and 2-3-month-old autoimmune prone MRL-lpr/lpr mice (conditionally healthy mice) are catalytically inactive. During spontaneous development of deep systemic lupus erythematosus (SLE)-like pathology a specific reorganization of immune system of these mice leads to conditions associated with a production of IgGs hydrolyzing DNA, ATP and polysaccharides with low catalytic activities (conditionally pre-diseased mice).A significant increase in DNase, ATPase and amylase IgG relative activities associated with a transition from pre-diseased to deep diseased mice is correlated with additional changes in differentiation and proliferation of mice bone marrow haematopoietic stem cells (HSCs) and lymphocyte proliferation in different organs. The highest increase in all abzyme activities was found in mice immunized with DNA, which in comparison with pre-diseased and diseased mice are characterized by a different profile of HSC differentiation and by a suppression of cell apoptosis. Abzyme activities in the serum of pregnant females were comparable with those for pre-diseased mice, but the profile of HSC differentiation and cell apoptosis levels in pregnant and pre-diseased mice were quite different. Right after the beginning of lactation (4 days after delivery) and in a late time of lactation (14 days after delivery) there was an observed increase in cell apoptosis and two different stages of significant change in the HSC differentiation profiles; the first stage was accompanied with a significant increase and the second with a remarkable decrease in abzyme activities. Overall, all mouse groups investigated are characterized by a specific relationship between abzyme activities, HSC differentiation profiles, levels of lymphocyte proliferation, and cell apoptosis in different organs. From our point of view, the appearance of ATPase, DNase activities may be considered the earliest statistically significant marker of mouse spontaneous SLE and a further significant increase in their activities correlates with the appearance of SLE visible markers and with an increase in concentrations of anti-DNA Abs and urine protein. However, development of autoimmune (AI)-reactions and the increase in the sera anti-DNA antibodies (Abs) and in the abzyme activities in pregnant and lactating mice do not associate with SLE visible markers and proteinuria. The possible differences in immune system reorganizations during pre-disease, disease, pregnancy and lactation leading to production of different auto-antibodies and abzymes are discussed.
Assuntos
Anticorpos Catalíticos/imunologia , Doenças Autoimunes/imunologia , Ensaio de Unidades Formadoras de Colônias , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Adenosina Trifosfatases/metabolismo , Amilases/metabolismo , Animais , Apoptose , Bovinos , Proliferação de Células , Desoxirribonucleases/metabolismo , Feminino , Células-Tronco Hematopoéticas/enzimologia , Imunoglobulina G/isolamento & purificação , Lactação , Linfócitos/citologia , Camundongos , Camundongos Endogâmicos MRL lpr , Especificidade de Órgãos , GravidezRESUMO
A great number of authors consider as "stem cell disorders" the following immunopathologies: immunodeficiency, lymphoproliferative diseases, systemic and organ-specific autoimmune diseases. A participation of early hemopoietic precursors in immunopathology development was analyzed on the next models: age-related immunodeficiency in CBF1 mice, autoimmune hemolytic anemia in NZB mice and leukemia in AKR mice. NZB mice have an augmented number of CFUs in S-phase, as a prerequisit for elevated sensitivity to Rauscher leukemia virus, as well to autoimmune disorder development. The increased stem cell proliferation (45% of CFUs in S-phase) in AKR mice is accompanied by changes in CFU redistribution: a decrease of CFU number in bone marrow and their increase in spleen. The hallmark of "disregulation" in bone marrow of old mice is the augmentation of erythroid precursors and the decrease in the number of myeloid precursors (GM-CFU and M-CFU), in other words the shift of differentiation to erythropoiesis. The augmentation of stem cell proliferation is accompanied by the increase in the number of erythroid precursors to the detriment of myeloid precursors. Therefore, the disturbances in the regulation of proliferation and differentiation of CFUs may contribute to the formation of the immunopathologies. The correction of HSC proliferation and differentiation may be one of the important approaches to the treatment of immune disorders.
RESUMO
Last years there are some reports about influence of erythron cells and immune system. It especially concerns with immunomodulation effect of cells of erythroid series and main humoral erythropoiesis regulator - erythropoietin. Much is known about the influence of cytokines of lymphoid and macrophagal origin on hemopoiesis, and in particular on erythroid compartment, and on functional activity of polypotent hematopoietic stem cell (PHSC). Published data allow to suggest the competitive relationships between erythroid and granulocyte-macrophagal series, and between erythroid and lymphoid series. Probably, interrelations between immune and erythroid systems are needed to consider from two points of view. First of all, cytokines should be considered as regulatory factors of proliferative and differentiated activity of both PHSC, and committed precursors of erythropoiesis. On the other hand, taking into account immunomodulating effects of erythroid cell series, the changes in erythron system during immunogenesis may be feedback mechanisms with immunoregulatory effect. All above gives principally new insights in the problem of regulation of immune system functions in norm and pathological states, as well as in the problem of immunocorrection of the latter, where factors, influencing on the intensity of erythropoiesis may be used as treatment agents.