RESUMO
Neuronal factor semaphorin-3A is viewed as immune suppressant of peripheral T lymphocytes, but it can also negatively affect activity of the thymus, the central organ of the immune system. The study examined the effects of this factor on proliferative activity and apoptosis of mouse thymocytes in vitro. Semaphorin-3A inhibited spontaneous and mitogen-stimulated proliferative activity of thymocytes producing no effect on the development of apoptosis in these cells. Flow cytometry revealed expression of semaphorin-3A receptors neuropilin-1 and plexin-A1 on thymocyte membranes. Approximately 13% thymocytes simultaneously expressed both receptors. The study suggests that semaphorin-3A, which is constitutively synthesized in thymic stroma in vivo, can play the role of inhibitory factor during thymocyte maturation.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Semaforina-3A/farmacologia , Timócitos/citologia , Timócitos/efeitos dos fármacos , Animais , Citometria de Fluxo , Masculino , Camundongos , Timo/citologia , Timo/efeitos dos fármacosRESUMO
We studied effects of semaphorin 3A, keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and their combinations on the proliferative activity of cortical (cTEC1-2) and medullary (mTEC3-10) thymus epithelium cell lines. Semaphorin 3A inhibited the proliferative activity of epithelial cells, while HGF and KGF, in contrast, exerted a stimulating effect. The effect of KGF and semaphorin 3A on different cell lines depended on the expression of receptors for these two factors. When the combination of two factors was used, semaphorin 3A was able to neutralize the stimulating effect of HGF and KGF. It can be assumed that semaphorin 3A synthesized in the thymus stroma, can act as a functional antagonist of HGF and KGF and have an inhibitory effect when these drugs are administered into the body for the therapeutic purpose of restoring thymus functions.
Assuntos
Proliferação de Células/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Fator 7 de Crescimento de Fibroblastos/farmacologia , Fator de Crescimento de Hepatócito/farmacologia , RNA Mensageiro/genética , Semaforina-3A/farmacologia , Animais , Linhagem Celular , Combinação de Medicamentos , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Fator 7 de Crescimento de Fibroblastos/genética , Fator 7 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuropilina-1/genética , Neuropilina-1/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Semaforina-3A/genética , Semaforina-3A/metabolismo , Transdução de Sinais , Timo/citologia , Timo/metabolismoRESUMO
Immunomodulatory properties of S. pyogenes protein M111 were studied on the model of Gurov strain and its isogenic mutant not expressing M protein. Mouse resident peritoneal macrophages were incubated with bacteria and generation of nitroxide and superoxide anions and production of IL-6, IL-10, and IL-17 were evaluated. Protein M111 modified macrophage response: it exhibited antiphagocytic activity, prevented ROS formation, and stimulated the production of anti-inflammatory cytokine IL-10. The results suggested that this protein could serve in the bacteria as a factor suppressing the host defense forces and promoting the realization of the strategy beneficial for pathogens - escape from the host immune defense.
Assuntos
Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Transporte/genética , Deleção de Genes , Evasão da Resposta Imune , Macrófagos Peritoneais/microbiologia , Streptococcus pyogenes/genética , Animais , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Transporte/imunologia , Expressão Gênica , Interleucina-10/biossíntese , Interleucina-10/imunologia , Interleucina-17/biossíntese , Interleucina-17/imunologia , Interleucina-6/biossíntese , Interleucina-6/imunologia , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Óxidos de Nitrogênio/imunologia , Óxidos de Nitrogênio/metabolismo , Fagocitose , Cultura Primária de Células , Streptococcus pyogenes/imunologia , Streptococcus pyogenes/patogenicidade , Superóxidos/imunologia , Superóxidos/metabolismoRESUMO
We studied the anti-angiogenic properties of sutent (SU11248) and celecoxib in human endothelial cell line EA. hy 926 in vitro. Sutent 0.05-0.5 microg/ml suppressed their proliferation and migration depending on dose while celecoxib did the same at 5.0 microg/ml. We were the first to demonstrate that endothelial cell incubation was followed by increase in 5'-nucleotidase activity in the presence of sutent while celecoxib did not produce such effect. It may be suggested that elevated 5'-nucleotidase concentration at the membranes of endothelial cells might in turn contribute to the pool of extracellular adenosine to stimulate antiinflammatory effect. Our data also contribute to the knowledge about the anti-angiogenic properties of sutent and celecoxib.
Assuntos
5'-Nucleotidase/metabolismo , Inibidores da Angiogênese/farmacologia , Anti-Inflamatórios/farmacologia , Células Endoteliais/efeitos dos fármacos , Indóis/farmacologia , Pirazóis/farmacologia , Pirróis/farmacologia , Sulfonamidas/farmacologia , Celecoxib , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase 2/farmacologia , Relação Dose-Resposta a Droga , Células Endoteliais/enzimologia , Humanos , SunitinibeRESUMO
Two types of monoclonal antibodies (MABs) against human thyroid peroxidase (TPO) have been obtained, which interact with spatially separated conformational epitopes of the antigen (Ka values are in the range 10(8)-10(9) M(-1)). The binding site of MAB F8 is in the immunodominant region of the TPO molecule, in the vicinity of the autoantigenic determinants, whereas the epitope specific for MAB A1 lies outside this location. Both MABs retain the ability to form immune complexes after solid-phase immobilization and chemical modification with a biotin derivative. The above properties suggest that MABs A1 and F8 may be used in immunoaffinity chromatography (isolation and purification of TPO from natural sources) and immunoassays for determinations of TPO (in biological fluids) and TPO autoantibodies (in human blood serum).
Assuntos
Anticorpos Monoclonais , Iodeto Peroxidase/imunologia , Complexo Antígeno-Anticorpo , Biotina , Cromatografia de Afinidade , Enzimas Imobilizadas , Epitopos , Humanos , ImunoensaioRESUMO
A system for quantitative determinations of human thyroid peroxidase (TPO) in biological fluids has been obtained, based on the use of enzyme-linked immunosorbent assay. Immunochemical properties of TPO were studied under variable conditions, and a new method for isolating the protein from microsomes, mitochondria, and cytosol of thyroid glands of patients with diverse thyroid diseases was developed. The procedure involves solubilization of subcellular fractions with detergents, their sonication, two sequential runs of chromatography (on sorbents with immolbilized monoclonal antibodies against TPO and goat anti-human immunoglobulin antibodies), treatment with ribonuclease, and dialysis. Highly purified preparations of intact TPO and a product of its limited trypsinolysis are expected to be used as research tools and components of high-sensitivity immunoassays.
Assuntos
Cromatografia de Afinidade/métodos , Ensaio de Imunoadsorção Enzimática , Iodeto Peroxidase/análise , Iodeto Peroxidase/isolamento & purificação , Glândula Tireoide/enzimologia , Anticorpos Monoclonais/imunologia , Humanos , Iodeto Peroxidase/imunologia , Frações Subcelulares/enzimologia , Glândula Tireoide/citologiaRESUMO
We studied long-lasting consequences of the low-doses irradiation on the immune system of 71 clean-up workers who participated in the emergency work after the Chernobyl Plant accident in 1986 and 25 healthy donors from Belarus. In sera of the workers the level of autoantibodies to thyroid gland antigens (thyroglobulin and microsomal fraction of thyroid gland) was increased in 48% of cases, the level of autoantibodies to lens oculis antigen was increased in 44% of cases; the level of circulating immune complexes was elevated in 55%, and the serum level of thyroglobulin in 60% of people. Immunological disorders were found without any definite clinical evidences of diseases and this allows us to consider the examined contingent as a group of risk for the development of autoimmune pathology in the future.
Assuntos
Doenças Autoimunes/etiologia , Centrais Elétricas , Lesões por Radiação/imunologia , Liberação Nociva de Radioativos , Adulto , Idoso , Autoanticorpos/sangue , Autoanticorpos/imunologia , Autoantígenos/imunologia , Doenças Autoimunes/sangue , Doenças Autoimunes/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Tempo , UcrâniaRESUMO
The growth of syngeneic transplantable hepatoma 22a is not accompanied by alteration of functional activity of T-lymphocytes in the spleens of tumor-bearing hosts as evidenced by means of PHA-blasttransformation test. The contents of T-lymphocytes in the spleens of tumor-bearers was non changed. The cytotoxic activity of the splenocytes towards hepatoma 22a cells was increased either in vivo (Winn-test) or in vitro (cytotoxic test) as compared with splenocytes of intact mice. By means of the test of inhibition of peritoneal macrophage adherence, the increased level of sensibilization of splenocytes towards tissue antigens was shown in mice hearing hepatoma 22a.
Assuntos
Neoplasias Hepáticas Experimentais/imunologia , Baço/imunologia , Animais , Células Cultivadas , Testes Imunológicos de Citotoxicidade , Imunidade Celular , Neoplasias Hepáticas Experimentais/patologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C3H , Transplante de Neoplasias , Baço/patologia , Linfócitos T/imunologia , Fatores de Tempo , Transplante IsogênicoRESUMO
A study was made of the ability of purified human C-reactive protein (CRP) in pentameric (pCRP) and subunit (sCRP) form to induce the production of tumour necrosis factor alpha (TNF-alpha) in macrophage-like cell line P388D1, as well as the combined effects of CRP and recombinant human TNF-alpha on the proliferation of L929 cell line. Both pCRP and sCRP induced TNF production by P388D1 cells, pCRP was inhibitory per se for L929 cells, while sCRP was not, when present in cultures for 72 h without actinomycin D (AD). In the presence of AD, sCRP was inhibitory as well, even when the period of incubation was reduced to 18 h. The presence in CRP of melittin-like sequence known to be responsible for the activation of cellular phospholipases is shown herein. Together with literature data, indication that CRP does influence the catalytic activity of purified phospholipase D, suggests that CRP exerts effect on P388D1 and L929 cells via regulation of cell phospholipases. The results also indicate that TNF effects in vivo can be modulated in different ways by conformational variants of CRP, whose occurrence and ratio depend on a phase of inflammatory reaction.
Assuntos
Proteína C-Reativa/farmacologia , Fator de Necrose Tumoral alfa/toxicidade , Animais , Divisão Celular/efeitos dos fármacos , Dactinomicina/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Humanos , Células L , Camundongos , Conformação Proteica , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Recombinantes/toxicidade , Fatores de TempoRESUMO
The inactivation kinetics of a human thyroid peroxidase protein fraction upon sonication (ultrasound frequency 27 kHz, power 60 W/cm2) of the enzyme solution in 15 mM phosphate buffer, pH 7.5, was studied. To quantitatively characterize the dependence of the slowest stage of the human thyroid peroxidase inactivation on temperature (36.0-50.4) degrees C, an effective constant of ultrasound inactivation rate Kin(US) was used. From the temperature dependence of Kin(US) at temperatures below 43 degrees C, the activation energy was estimated to be 8.11 kcal/mol. It was shown that the rate of human thyroid peroxidase inactivation strongly depends on the concentration of total protein in solution: the kin(US) value decreases more than sixfold in the protein concentration range from 0.2 to 0.8 mg/ml. It was also shown that poly(2-aminodisulfide-4-nitrophenol), its complexes with human serum albumin as well as the complexes human serum albumin--poly(gallic acid disulfide) substantially inhibit the ultrasound-induced inactivation of the enzyme and can be its effective stabilizers in the ultrasound cavitation field. This confirms the suggestion that active free radicals HO., O2.- and HO2. play a key role in the inactivation of human thyroid peroxidase. A general scheme of the inactivation of human thyroid peroxidase is proposed, which represents a chain of successive and parallel reversible and irreversible elementary steps.
Assuntos
Iodeto Peroxidase/química , Ultrassom , Antioxidantes/química , Depressão Química , Dissulfetos/química , Humanos , Cinética , Fenóis/químicaRESUMO
A biochemical system of radioimmunoassay of human thyroid peroxidase (TPO) with the use of specific autoantibodies was developed for the first time. This system includes lyophilized preparations of human autoantibodies to TPO, radioiodinated TPO, standards prepared from pure TPO, and the solid-phase protein A as a precipitating agent. An analytical system was used to study the TPO distribution in fractions of thyroid tissue homogenate. Differences in TPO concentrations in mitochondria, microsomes, and supernatant fluid depending on thyroid pathology and tissue storage conditions were found. The behavior of immobilized anti-TPO-autoantibodies in immunoaffinity chromatography of this microsomal antigen was studied.
Assuntos
Autoanticorpos/análise , Iodeto Peroxidase/análise , Radioimunoensaio/métodos , Cromatografia de Afinidade/métodos , Humanos , Iodeto Peroxidase/imunologia , Radioisótopos do Iodo , Frações Subcelulares/enzimologia , Glândula Tireoide/enzimologiaRESUMO
Levels of hepatic autoantigens, anti-hepatic antibodies and immune complexes in sera of C3HA mice bearing syngeneic weakly-immunogenic hepatoma 22a were measured. The level of autoantigens increased during tumor growth, while autoantibodies were not identified at all stages. Circulating immune complexes were shown to bind heterologous complement at early stages of hepatoma growth, but later they did not. Bound immune complexes were identified in the kidney by means of immunofluorescence. Blastogenic reaction of normal splenic cells did not change when cells were cultivated with serum of tumor-bearing mice obtained throughout tumor growth. The proliferative activity of tumor-bearers' lymphocytes decreased during cultivation with serum taken at the terminal stages of tumor growth only. However, there was no correlation with the presence of free hepatic antigen and immune complexes in sera of hepatoma-bearing mice.
Assuntos
Complexo Antígeno-Anticorpo/análise , Neoplasias Hepáticas Experimentais/imunologia , Linfócitos/imunologia , Animais , Autoanticorpos/análise , Autoantígenos/análise , Proteínas do Sistema Complemento/imunologia , Imunofluorescência , Imunoglobulinas/análise , Rim/imunologia , Fígado/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C3HRESUMO
Thymic involution accompanies the growth of many human and animals tumors but the precise mechanism of this phenomenon is unknown. We tried to elucidate the role of apoptosis as a possible mechanism of thymic involution during tumor growth. The mice were inoculated subcutaneously with syngeneic hepatoma 22a cells. Starting from the 3rd week after tumor inoculation, progressive thymic involution was observed. Histological studies revealed distinct delymphatization of the thymic cortex, reduced numbers of big lymphocytes in the subcapsular zone and of mitoses in the cortex, and an increased amount of mast cells. This was followed by decreased DNA synthesis in vitro as measured from 3H-thymidine incorporation. The number of pycnoses in the cortex was two to three times that in the thymus of control animals. DNA gel electrophoresis did not reveal any signs of apoptosis in the thymocytes without preincubation. Within 2 h of in vitro incubation of the thymocytes taken on the 7th day after the tumor inoculation, spontaneous apoptosis was more expressed than in the thymocytes from intact mice. This may reflect different induction mechanisms of apoptosis in the thymocytes from the tumor bearing and control mice. We propose that the mechanism of thymic involution during tumor growth is related to inhibition of thymocytes' proliferation, impaired differentiation and enhanced intrathymic death caused by cytokine release from the nonlymphoid thymic population.
Assuntos
Apoptose , Neoplasias Hepáticas Experimentais/patologia , Timo/patologia , Animais , Contagem de Células , Divisão Celular , DNA/biossíntese , Masculino , Camundongos , Transplante de Neoplasias , Tamanho do Órgão , Linfócitos T/metabolismo , Linfócitos T/patologia , Timo/metabolismo , Transplante IsogênicoRESUMO
A method for automatic registration of NBT test in human blood leukocytes is proposed. It can be used both in experimental and clinical studies. The principle of the method consists in formazan extraction from neutrophils free from red cells or leukocyte suspension by 2 M KOH and dimethylsulfoxide. Parallel studies carried out by the proposed method and the routine histochemical method of whole blood smears showed a good correlation of the results (r = 0.832). A technique of measuring O2- anion production, based on the proposed method, is suggested.
Assuntos
Testes Hematológicos/métodos , Nitroazul de Tetrazólio , Automação , Histocitoquímica , HumanosRESUMO
Bifidobacterium bifidum 791 (commercially available as B. bifidum BIM B-733D) cell-surface biopolymers (BPs) interact selectively with human serum thyroid peroxidase (TPO) and thyroglobulin (Tg) autoantibodies (anti TPO and anti Tg, respectively). BPanti-TPO and BPanti-Tg were isolated from the soluble fraction of B. bifidum BIM B-733D by affinity chromatography with anti-TPO or anti-Tg, respectively. Homogeneity of affinity eluates (AEanti-TPO and AEanti-Tg) was tested by size exclusion chromatography. For each AE, the elution profiles generated on the basis of absorbance at 280 nm do not conform to ELISA data for functional activity characteristic of BPs. Moreover, high functional activity was detected in chromatographic fractions that had significantly different molecular weights and no absorbance at 280 nm, which suggests a non-protein (carbohydrate) nature of BPanti-TPO and BPanti-Tg. The semi-preparative size exclusion chromatography of AEanti-TPO and AEanti-Tg with detection by refractometer gave 5,000-7,000 Da fractions containing substances that interact selectively with either anti TPO (BPanti-TPO) or anti-Tg (BPanti-Tg) according to ELISA data. Analysis by two-dimensional NMR spectroscopy including a 1H, 13C-heteronuclear single-quantum coherence experiment indicated that both substances are linear α-1,6-glucans. For the first time, an immunological similarity (molecular mimicry) of glycopolymers of B. bifidum BIM B-733D and human thyroid proteins, TPO and Tg, was shown. On the whole, our data point to a possible role of bifidobacteria in the pathogenesis of autoimmune thyroid diseases (ATD). The main requirements for triggering/acceleration or prevention/abrogation of ATD by bifidobacteria through molecular mimicry mechanism are hypothesised to be (1) genetic predisposition to ATD and (2) intestinal epithelium penetration by α-1,6-glucan.
Assuntos
Antígenos de Bactérias/imunologia , Autoanticorpos/metabolismo , Doenças Autoimunes/etiologia , Bifidobacterium/imunologia , Polissacarídeos Bacterianos/imunologia , Doenças da Glândula Tireoide/etiologia , Antígenos de Bactérias/química , Antígenos de Bactérias/isolamento & purificação , Antígenos de Bactérias/metabolismo , Doenças Autoimunes/microbiologia , Bifidobacterium/química , Cromatografia de Afinidade , Cromatografia em Gel , Humanos , Iodeto Peroxidase/imunologia , Espectroscopia de Ressonância Magnética , Peso Molecular , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/isolamento & purificação , Polissacarídeos Bacterianos/metabolismo , Ligação Proteica , Tireoglobulina/imunologia , Doenças da Glândula Tireoide/microbiologiaRESUMO
During recent years, researchers have been focusing on the concept of an infectious etiology of autoimmune diseases. The most discussed theory is molecular mimicry, i.e. the emergence of autoreactive clones of T- and B-lymphocytes as a result of cross-immune response to homologous bacterial or viral antigen. Information on the role of probiotic microorganisms (PM) in the molecular mechanisms of autoimmune thyroid diseases (ATD) is limited. Using proteins and immunogenic peptides databanks and relevant computer programs, the homology between the amino acid sequences of thyroid peroxidase (TPO) and thyroglobulin (Tg), which are potential B- and T-cell epitopes of these antigens, and proteins of bifidobacteria and lactobacilli was established. Moreover, we have found components of cells of Bifidobacterium bifidum 791, Bifidobacterium adolescentis 94 BIM, Bifidobacterium longum B379M and Lactobacillus plantarum B-01 that selectively bind human antibodies to TPO (anti-TPO) and antibodies to Tg (anti-Tg) and compete with natural antigens for the binding of anti-TPO and anti-Tg in ELISA. Additionally, a three-fold difference was observed between the probability of detecting antibodies (Abs) to the antigens of L. plantarum B-01 and B. bifidum 791 in serum samples containing and those not containing anti-TPO. On the whole, our data are arguments in favour of the assumption of the possible role of PM of the genera Bifidobacterium and Lactobacillus in triggering ATD by the mechanism of molecular mimicry. The data obtained in silico and in vitro should be proven by use of animal models and clinical studies for extrapolations to the whole body. Possible antigenic properties of components/proteins of bifidobacteria and lactobacilli, selectively binding anti-TPO and anti-Tg should be taken into consideration. Natural human Abs to these bacterial components are probably able to cross-react with the TPO and Tg in the ELISA for detection of anti-TPO and anti-Tg, which are serologic markers of ATD. It can lead to unspecific false positive results and, hence, to an incorrect diagnosis.
Assuntos
Proteínas de Bactérias/imunologia , Bifidobacterium/imunologia , Iodeto Peroxidase/imunologia , Lactobacillus plantarum/imunologia , Tireoglobulina/imunologia , Tireoidite Autoimune/etiologia , Sequência de Aminoácidos , Anticorpos Antibacterianos/sangue , Autoanticorpos/sangue , Autoantígenos/imunologia , Autoantígenos/isolamento & purificação , Linfócitos B/imunologia , Proteínas de Bactérias/química , Bifidobacterium/metabolismo , Reações Cruzadas/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito T/imunologia , Humanos , Imunoglobulinas/sangue , Imunoglobulinas/imunologia , Iodeto Peroxidase/química , Lactobacillus plantarum/metabolismo , Mimetismo Molecular , Probióticos/efeitos adversos , Homologia de Sequência de Aminoácidos , Tireoglobulina/química , Glândula Tireoide/imunologia , Tireoidite Autoimune/diagnóstico , Tireoidite Autoimune/imunologiaRESUMO
Lactate modulated functional activity of macrophages from intact mice and animals with transplantable syngeneic hepatoma 22a. Sodium lactate in a concentration of 6.5 mM in vitro increased functional activity of resident peritoneal macrophages from intact mice. The growth of transplantable syngeneic hepatoma 22a was associated with the absence of correlation between nitrite synthesis by peritoneal macrophages and serum lactate concentration. Therefore, this agent cannot be considered as a systemic activator of macrophages.
Assuntos
Carcinoma Hepatocelular/imunologia , Ácido Láctico/farmacologia , Neoplasias Hepáticas/imunologia , Macrófagos Peritoneais/efeitos dos fármacos , Nitritos/metabolismo , Animais , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C3HRESUMO
Vascular endothelial growth factor is not only angiogenic, but also immunoregulatory factor. For evaluation of the possibility of its direct interaction with mouse thymocytes we studied the effect of vascular endothelial growth factor on proliferation and apoptosis of thymocytes and expression of genes for the corresponding receptor on these cells. Vascular endothelial growth factor modulated mitogen-induced proliferation of thymocytes and stimulated spontaneous apoptosis in intact thymus cells. Thymocytes express mRNA of type 2, but not type 1 vascular endothelial growth factor receptors.