RESUMO
We have previously reported that alloreaction can lead to activation of dendritic cells through secretion of inflammatory cytokines. Here, we addressed whether alloreaction-derived cytokines may also lead to acute myelogenous leukemia (AML) blast differentiation. With this aim, supernatant (sn) harvested from major or minor histocompatibility antigen-mismatched mixed lymphocyte reaction (MLR) were used to culture French American Bristish (FAB) type M4 or M5 AML blasts. Our results showed that the secreted factors induced upregulation of CD40, CD54, and/or HLA molecules in AML blasts. Protein fractionation, blockade experiments and exogenous cytokine reconstitution demonstrated the involvement of TNF in the upregulation of CD54, CD40 and HLA-class II molecules, and of IFNgamma in the increase of HLA-class I and class II molecule expression. But, in line of its much higher levels of secretion, TNFbeta, rather than TNFalpha, was likely to play a preponderant role in AML blast differentiation. Moreover TNFbeta and IFNgamma were also likely to be involved in the AML blast differentiation-mediated by HLA-identical donor T-cell alloresponse against recipient AML blasts. In conclusion, we show herein that upon allogeneic reaction, TNFbeta secretion contributes, in concert with IFNgamma, to increase or restore surface molecules involved in AML blast interaction with T cells.
Assuntos
Antígenos CD40/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Interferon gama/metabolismo , Leucemia Mieloide Aguda/metabolismo , Linfotoxina-alfa/metabolismo , Adulto , Idoso , Anticorpos/farmacologia , Proteínas Sanguíneas/química , Proteínas Sanguíneas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Meios de Cultura Livres de Soro , Feminino , Humanos , Imunofenotipagem , Interferon gama/imunologia , Interferon gama/farmacologia , Interleucina-1/imunologia , Interleucina-1/metabolismo , Interleucina-2/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Leucemia Mieloide Aguda/patologia , Teste de Cultura Mista de Linfócitos , Linfotoxina-alfa/farmacologia , Masculino , Pessoa de Meia-Idade , Peso Molecular , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
Here, we investigated the influence of cyclosporin A (CsA) on dendritic cell (DC) generation. With this aim, human DC were propagated from monocytes in serum-free medium with granulocyte macrophage-colony stimulating factor and interleukin-4. DC were then exposed to tumor necrosis factor alpha (TNF-alpha) for maturation. Our results show that CsA does not impair commitment of monocytes into DC, as assessed by loss of CD14 and increase of CD40 and CD1a. However, TNF-alpha-induced DC maturation was affected, as CsA-treated DC expressed lower levels of human leukocyte antigen and costimulatory molecules but sustained levels of CD1a, and less DC expressed DC-lysosomal-associated-membrane-protein (LAMP) and CD83. Accordingly, CsA inhibited the allostimulatory and accessory cell functions of DC. Surprisingly, when other maturation stimuli were used, we observed that CsA significantly inhibited maturation induced by lipopolysaccharides but not by polyribocytidylic acid or CD40 ligand, as assessed by DC phenotype and functions. Therefore, our results indicate that CsA may differentially affect DC maturation.
Assuntos
Ciclosporina/farmacologia , Células Dendríticas/imunologia , Imunossupressores/farmacologia , Apresentação de Antígeno/efeitos dos fármacos , Antígenos CD/análise , Ligante de CD40/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Relação Dose-Resposta a Droga , Antígenos HLA-DR/análise , Cinética , Lipopolissacarídeos/antagonistas & inibidores , Teste de Cultura Mista de Linfócitos , Poli I-C/antagonistas & inibidores , RNA de Cadeia Dupla/antagonistas & inibidores , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
We have investigated by flow cytometry the action of dextran sulfate (DxS) on the expression of the LFA-1 molecule in human lymphocytes. This work was undertaken because of the involvement of the LFA-1 molecule in HIV-1 induced syncytia and because of the role of DxS played in the inhibition of syncytia formation. Firstly we detected five distinct topographic regions (epitopes) on the LFA-1 molecule with a panel of 11 monoclonal antibodies (Mabs). Then we demonstrated that DxS interacts with some epitopes mainly present on the alpha chain of the LFA-1 molecule. This inhibition on the LFA-1 expressions by DxS occurs after 1-3 hr of incubation of either 4 or 37 degrees C with complete reappearance of LFA-1 within 1 hr of placing cells in fresh medium. In addition both 5 and 500 kDa have been found to have a similar influence on the inhibition of the LFA-1 expression, while non sulfated dextran have no effect. Other sulfated polyanion (SP) such as heparin and chondroitin sulfate have no effect on the LFA-1 expression. Further at 4 degrees C, DxS does not alter the expression of molecules recognized by Mab such as Leu3a (CD4), Leu2a (CD8), Leu4 (CD3) and Leu5b (CD2). However at 4 degrees C, DxS decreases the expression of CD45R molecule which is recognized by Mab Gap8.3. At 37 degrees C, we observe a decrease also in CD4 expression after DxS exposure. It has also been found that DxS decreases LFA-1 expression to the same extent regardless of the basal expression of LFA-1 in each selected cell subset (LFA-1 low, dim or bright). These results suggest that the inhibitory effect of DxS on the HIV-induced syncytium formation could be due partially to a specific steric hindrance of some LFA-1 determinants.
Assuntos
Sulfato de Dextrana/metabolismo , Leucócitos Mononucleares/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Linfócitos/metabolismo , Anticorpos Monoclonais , Especificidade de Anticorpos , Modulação Antigênica , Antígenos CD/análise , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Antígenos CD18 , Complexo CD3 , Antígenos CD4/metabolismo , Sulfatos de Condroitina/metabolismo , Dextranos/metabolismo , Epitopos , Citometria de Fluxo , Heparina/metabolismo , Humanos , Antígeno-1 Associado à Função Linfocitária/análise , Antígeno-1 Associado à Função Linfocitária/imunologia , Peso Molecular , Receptores de Antígenos de Linfócitos T/metabolismo , Temperatura , Fatores de TempoRESUMO
Recent studies have highlighted the high degree of differentiation of monocytes. Indeed, dendritic cells (DC) can be generated from monocytes, in the presence of appropriate cytokines. However, human serum is usually avoid in such cultures. Here, we report that human serum does not inhibit generation of mature DC from blood monocytes, but rather that extra-cellular pH may play an important role in the regulation of monocyte differentiation. Indeed, monocytes cultured at pH 7.4 in the presence of high concentrations of human serum developed efficiently into mature DC, as opposed with monocytes cultured at pH 7. These pH 7.4 cultured DC presented features characteristic of mature DC, at the phenotypical, functional and morphological levels. In addition, these DC were stable, with respect to their sustained expression of CD83 and CD86, upon withdrawal of cytokines. Finally, when autologous plasma was used instead of homologous serum, differentiation of monocytes into mature DC was efficient, as well. Thus, altogether, our data show the importance of extra-cellular pH on differentiation of monocyte-derived DC in the presence of human serum, which should be maintained at plasma levels.
Assuntos
Células Dendríticas/citologia , Monócitos/citologia , Antígenos CD , Sangue , Diferenciação Celular , Células Cultivadas , Meios de Cultura , Técnicas de Cultura/métodos , Citocinas/farmacologia , Células Dendríticas/imunologia , Antígenos HLA-DR , Humanos , Concentração de Íons de Hidrogênio , Monócitos/efeitos dos fármacosRESUMO
A large number of multidrug resistance (MDR) modulators, termed chemosensitizers, have been identified from a variety of chemicals, but most have been proven to be clinically toxic. Low concentrations of the pleuromutilin-derived semi-synthetic antibiotic tiamulin (0.1 to 10 microM) sensitized the three highly resistant P-glycoprotein (Pgp)-overexpressing tumor cell lines P388 (murine lymphoid leukemia), AS30-D (rat hepatoma), CEM (human lymphoblastic leukemia), and the barely resistant AS30-D/S cell lines to several MDR-related anticancer drugs. Flow cytometric analysis showed that tiamulin significantly increased the intracellular accumulation of daunomycin. When compared to reference modulating agents such as verapamil and cyclosporin A, tiamulin proved to be 1.1 to 8.3 times more efficient in sensitizing the resistant cell lines. Moreover, when given i.p. (1.6 microg/mg body weight), tiamulin increased the survival rate of adriamycin-treated mice bearing the P388/ADR25 tumor line by 29%. In the presence of an anticancer drug, tiamulin inhibited both ATPase and drug transport activities of Pgp in plasma membranes from tumor cells. Tiamulin is thus a potent chemosensitizer that antagonizes the Pgp-mediated chemoresistance in many tumor cell lines expressing the MDR phenotype at different levels and displays no toxic effects on contractile tissues at active doses, therefore providing the promise for potential clinical applications.
Assuntos
Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Canais de Cálcio/efeitos dos fármacos , Daunorrubicina/farmacocinética , Diterpenos/farmacologia , Resistência a Múltiplos Medicamentos , Masculino , Camundongos , Camundongos Endogâmicos DBA , Ratos , Células Tumorais Cultivadas , Vimblastina/farmacocinéticaRESUMO
Canine systemic lupus erythematosus (SLE) is a disease clinically very similar to its human counterpart. But so far, no study has reported an accurate evaluation of the lymphocyte subsets in the canine disease. Here, we present a study in which lymphocyte subsets have been evaluated in the peripheral blood of 20 dogs suffering from spontaneous systemic lupus erythematosus (SLE) in active and inactive phases, before and during treatment with prednisone and levamisole. 22 healthy dogs have been used as a control population. We show that canine SLE in active phases is associated with a several lymphopenia (1050 +/- 520 10(6) cells/l versus 2130 +/- 1 020 10(6) cells/l in controls). A striking finding is the imbalance of the CD4 and CD8 subsets (respectively 56.7 +/- 10.7% and 10.9 +/- 3.8% of CD4+ and CD8+ lymphocytes versus 40.5 +/- 11.5% and 18 +/- 4.4% in controls) and a strong activation of T-cells in active phases (64.1 +/- 16.9% of 2B3+ cells versus 46.5 +/- 16.7%). Moreover, we observed a persistence of the T subset imbalance during spontaneous evolution. In contrast, the treatment induced in dogs showing a good response the correction of CD4/CD8 ratio and no clinical manifestations, whereas in low responders no such improvements were observed. Thus, this work suggests that the main immunological imbalance seen in SLE could be associated with defective suppressor cells and provides further evidence of similarity of human and dog SLE.
Assuntos
Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/veterinária , Subpopulações de Linfócitos T/imunologia , Animais , Relação CD4-CD8 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Cães , Feminino , Linfopenia/imunologia , Linfopenia/veterinária , MasculinoRESUMO
AIMS: To determine haematological parameters, urine mutagenicity (on three Salmonella typhimurium strains), and DNA damage (using the comet assay) in mononuclear leucocytes of farmers before and after a one-day spraying period of pear and apple trees with the fungicide captan in usual conditions. METHODS: Fruit growers were exposed to captan during the 1998 (n = 12) and/or the 2000 spraying seasons (n = 17). Biological samples were collected on the morning of the day of spraying (S1), the evening after spraying (S2), and the morning of the day after (S3). The UK Predictive Operator Exposure Model (UK-POEM) was used to quantify pesticide exposure intensity. RESULTS: No effect was observed on haematological parameters for these two spraying seasons. Proportions of mutagenic urine samples did not significantly differ between S1 and S2/S3 sampling points. In contrast with strains TA97a and YG1041 mainly sensitive to frameshift mutations, a positive trend was observed between the difference (S3-S1) of mutagenic power on strain TA102 detecting base-pair mutations and the exposure predicted value given by UK-POEM, mainly due to parameters related to protective clothing. No significant variations in DNA damage levels were observed between S1 and S3, nor were correlations observed with parameters of pesticide exposure. CONCLUSIONS: A one-day spraying period with captan and other pesticides does not significantly induce DNA damages in mononuclear leucocytes. In contrast, an inefficient protective clothing could correlate with an increase in urine mutagenicity as assessed by the TA102 tester strain.
Assuntos
Agricultura , Captana/toxicidade , Dano ao DNA , Fungicidas Industriais/toxicidade , Exposição Ocupacional/efeitos adversos , Adulto , Captana/administração & dosagem , Frutas , Fungicidas Industriais/administração & dosagem , Humanos , Estilo de Vida , Linfócitos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Testes de Mutagenicidade , Roupa de Proteção/estatística & dados numéricos , Urina/químicaRESUMO
During pregnancy, the fetus is an hemiallograft perfectly tolerated by the mother. With the aim of elucidating the mechanism of this tolerance, we have looked to see whether lymphocyte subsets are modified by pregnancy. Using monoclonal antibodies and flow cytometry, we have studied the changes in the immune profile of normal pregnant women and compared them with non pregnant women. In pregnant women, a significant decrease in the percentage of CD3+ cells is observed in the first trimester (61.1 +/- 14.7% versus 73.8 +/- 6.8%; p less than 0.001). The same data are obtained for CD4+ cells (38.2 +/- 10.7% versus 44.0 +/- 7.0%; p less than 0.05) and CD8+ cells (22.8 +/- 5.6% versus 28.0 +/- 8.9%; p less than 0.05). On the other hand, B lymphocytes (CD19+), monocytes (Leu M3+) and natural killer (NK) cells (Leu7+) remain stable during pregnancy. CD11a+ cells decreased during the 1st and 2nd trimesters. Lastly, activated T lymphocytes (CD3+DR+, CD8+DR+) are not modified. Using absolute numbers, a significant decrease is shown only for CD3+ cells in the 2nd and 3rd trimesters and for CD11a+ cells in the 2nd trimester of pregnancy. The decrease of T lymphocyte subsets, NK and CD11a+ cells during pregnancy partially explains the tolerance for the fetus.
Assuntos
Subpopulações de Linfócitos B/química , Gravidez/imunologia , Subpopulações de Linfócitos T/química , Antígenos CD/análise , Antígenos CD/sangue , Antígenos CD/imunologia , Subpopulações de Linfócitos B/imunologia , Antígenos CD11 , Estudos de Avaliação como Assunto , Feminino , Humanos , Células Matadoras Naturais/química , Células Matadoras Naturais/imunologia , Contagem de Leucócitos , Monócitos/química , Monócitos/imunologia , Gravidez/sangue , Primeiro Trimestre da Gravidez , Segundo Trimestre da Gravidez , Terceiro Trimestre da Gravidez , Subpopulações de Linfócitos T/imunologiaRESUMO
Modification of dendritic cells (DCs) is a promising avenue for gene therapy purposes, given the versatility and the multiplicity of functions of these cells. In this study, we show that preincubation of monocyte-derived DCs with low amounts of non-infectious virion-like particles derived from the simian immunodeficiency virus (SIV(MAC) VLPs) increases up to 10-fold the efficiency of transduction by HIV-1 lentiviral vectors at low multiplicity of infections yielding up to 90% of transduced cells, in the absence of alterations of DCs behavior. This effect is restricted to DCs and specified by the viral accessory protein Vpx. Thus, preincubation with empty VLPs of SIV(MAC) can be used in transduction protocols to increase the efficacy of HIV-1-mediated modification of DCs.
Assuntos
Células Dendríticas/virologia , Terapia Genética/métodos , HIV-1/genética , Terapia Viral Oncolítica/métodos , Vírus da Imunodeficiência Símia/genética , Transdução Genética/métodos , Linhagem Celular , Células Cultivadas , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Humanos , Macrófagos/virologia , VírionRESUMO
Bone marrow aspirates from 28 healthy donors (18 adults, 10 children) were analysed by flow cytometry (FACS analyser) after purification of low density bone marrow cells (Ld BMC) on a density gradient (d = 1,077) and labeling with 23 anti-hematopoietic cells monoclonal antibodies. Based on physical properties the Ld BMC could be divided into four different populations called E, My, Mo et L including 13 +/- 8%, 33 +/- 15%, 12 +/- 5% and 42 +/- 14% of these cells respectively. The phenotypic analysis of these different populations allowed us to identify in E, erythrocytes (glycophorin A+, Rhesus D+ but negative for early erythroid differentiation markers like the transferrin receptor and/or the FA6-152 antigen); in My, the myeloid lineage (VIM2+, HLADR-); in Mo, the monocytic lineage (CD14+) and some myeloblasts (CD14-, VIM2+, HLADR+) and finally in L, an heterogeneous population including: 1) leucocyte cells, in which 27.3 +/- 9.0% are T cells labelled to the same extent by CD2, CD3, CD5 and CD6, 13.2 +/- 5.9% are B cells assessed by CD19 and CD20, 8.3 +/- 5.7% are Pré-B (CD10+), less than 5% are "natural killer" cells (CD16+ or Leu7+) and finally less than 6% are myelomonocytes (CD14+ or VIM12); 2) the erythroid lineage (Rhesus D+ = 43.7 +/- 12.9%, transferrin receptor and FA6-152+ 36.7 +/- 9.6%); 3) undifferentiated cells or progenitor cells (CD34+ = 6.5 +/- 3.5%); 4) cells unlabelled with any antibodies (approximatively 6%). We have not observed difference between adults and children bone marrow in regard to physical properties properties and all but also immunological markers. Indeed, a significant (p less than 0.02) higher proportion of B cells (CD19 and CD10) was observed in children. These data get from a large number of bone marrow could be used to quantify the imbalance of some bone marrow disorders.
Assuntos
Células da Medula Óssea , Adolescente , Adulto , Anticorpos Monoclonais , Biomarcadores/análise , Medula Óssea/imunologia , Exame de Medula Óssea , Separação Celular , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , FenótipoRESUMO
Bone marrow aspirates from 48 healthy donors (34 adults, 14 children) were analyzed by flow cytometry (FACS Analyzer) after purification of low-density bone marrow cells (Ld BMC) on a density gradient (d = 1,077) and labelling with 23 anti-hematopoietic cell monoclonal antibodies. Based on physical properties, these Ld BMC could be divided into four different populations called E, My, Mo and L, which comprised 14% +/- 9%, 31% +/- 16%, 10% +/- 5% and 45% +/- 14% of these cells, respectively. The phenotypic analysis of these different populations enabled the identification in E, of erythrocytes (Glycophorin A+, Rhesus D+, but negative for early erythroid differentiation markers such as the transferrin receptor (Tf. R) and the FA6-152 antigen); in My of cells of the myeloid lineage (VIM2+, HLA DR-); in Mo of cells of the monocytic lineage (VIM2+, CD14+) plus some myeloblasts (VIM2+, CD14-, HLADR+) and finally in L of a heterogeneous population including: 1. T lymphocytes labelled to the same extent by CD2, CD3, CD5 and CD6 (28% +/- 10%), B lymphocytes assessed by CD19 and CD20 (12% +/- 8%), Pre-B cells (CD10+ = 8% +/- 7%), less than 5% of "natural killer" cells (CD16+ or Leu7+) and finally, less than 6% of myelomonocytes (CD14+ and/or VIM2+). 2. The erythroid lineage (rhesus D+ = 42% +/- 20%, Tf.R+ and FA6-152+ = 32% +/- 12%). 3. Undifferentiated cells or progenitor cells (CD34+ = 7% +/- 5%). 4. Cells unlabelled by any antibodies (approximately 6%). We observed no difference between bone marrow samples from adults or children, with respect to physical properties, and with all but four immunological markers. A significantly higher proportion of B cells (CD19+ and CD10+) (P less than 0.001) and undifferentiated cells (CD34+ and HLADR+) (P less than 0.02) was observed in children. These data, obtained from a large number of bone marrow samples, could be used to quantify the imbalance of some bone marrow disorders.
Assuntos
Medula Óssea/fisiologia , Citometria de Fluxo , Adolescente , Adulto , Anticorpos Monoclonais , Antígenos de Diferenciação/análise , Biomarcadores , Medula Óssea/imunologia , Células da Medula Óssea , Diferenciação Celular , Separação Celular/métodos , Eritrócitos/citologia , Feminino , Humanos , Linfócitos/imunologia , Masculino , Pessoa de Meia-Idade , FenótipoRESUMO
Immune deficiency has been reported in haemophiliac patients receiving antihaemophilic factor VIII preparations, but the mechanisms involved in the immunosuppression are not fully understood. By using the proliferative response of peripheral blood mononuclear cells to phytohaemagglutinin (PHA) as a test system, we investigated the inhibitory influence of a very high purity antihaemophilic factor (AHF) preparation on T cell proliferation and on T lymphocyte activation molecules. We observed that this preparation reduced significantly the PHA-induced mononuclear cell proliferation, independently of the monocyte concentration. The AHF preparation did not act through a cytotoxic mechanism or a steric hindrance of PHA. The AHF preparation had no effect on the immediate expression of T lymphocyte activation molecules such as CD54 (ICAM-1). In contrast, the very high purity AHF reduced the induced expression of two early T cell activation molecules: CD25 (interleukin-2 receptor) and CD71 (transferrin receptor). The very high purity AHF also had the capacity to inhibit the up-regulation of two late activation antigens, CD38 and CD11a/CD18, and to inhibit the induced expression of HLA-DR molecule, defined also as a late T cell activation molecule. The CD45R expression level, used as a control marker, was not changed after AHF exposure. The very high purity AHF therefore influenced an early step of cell proliferation. We have also shown that the immunoregulatory properties of the preparation were not restricted to the factor VIII itself, but resulted from the presence of dialysable and low molecular weight components in the preparation.
Assuntos
Fator VIII/farmacologia , Imunossupressores/farmacologia , Linfócitos T/efeitos dos fármacos , Antígenos CD/imunologia , Divisão Celular/efeitos dos fármacos , Humanos , Imunofenotipagem , Leucócitos Mononucleares/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Peso Molecular , Linfócitos T/imunologiaRESUMO
Clinical applications of flow cytometry to certain diseases of the dog and cat are now possible. The utility of such applications for diagnosis, prognosis and follow-up are illustrated here by a number of examples: feline AIDS resulting from FIV infection, Leukocyte Adhesion Deficiency in Irish setters, deep pyoderma in German shepherds, Immune-mediated Thrombocytopenia, canine Systemic Lupus Erythematosus and Leishmaniasis, Leukemia and Lymphoma.
Assuntos
Síndrome da Imunodeficiência Adquirida/diagnóstico , Doenças Autoimunes/diagnóstico , Citometria de Fluxo/métodos , Imunofenotipagem/métodos , Leishmaniose/diagnóstico , Leucemia/diagnóstico , Linfoma/diagnóstico , Pioderma/diagnóstico , Síndrome da Imunodeficiência Adquirida/virologia , Animais , Doenças Autoimunes/imunologia , Gatos , Cães , Citometria de Fluxo/veterinária , Imunofenotipagem/veterinária , Técnicas In Vitro , Leishmaniose/parasitologia , Leucemia/patologia , Linfoma/patologia , Prognóstico , Pioderma/microbiologiaRESUMO
Feline immunodeficiency virus (FIV), the causative agent of feline AIDS, induces a disease syndrome in cats characterized by a decreased lymphocyte-proliferative response to mitogens at all stages of infection and selective depletion of CD4 lymphocyte subsets. In this work, we report that peripheral blood lymphocytes isolated from FIV-infected cats undergo a spontaneous death, in vitro, according to a programmed cell death (PCD) or apoptosis. This phenomenon has also been seen in peripheral blood lymphocytes from HIV-infected humans and SIV-infected macaques. Four different techniques were used to document PCD in FIV-infected cats. DNA gel electrophoresis has shown a DNA fragmentation pattern with DNA fragments displaying sizes corresponding to multiples of oligonucleosomes DNA length unit (180 bp). Transmission electron microscopy revealed condensation of both nuclear chromatin and cytoplasm. An increase in the percentage of fragmented DNA was demonstrated by Burton's technique. In addition, flow cytometric analysis detected a cell population with condensed chromatin. The spontaneous PCD in FIV-infected cats could not be inhibited by RNA synthesis inhibitors or protein synthesis inhibitors. Our results could have implications for understanding the pathogenesis of FIV-infection and establishing specific strategies against apoptosis in cats and humans.
Assuntos
Apoptose , Vírus da Imunodeficiência Felina , Linfócitos/citologia , Linfócitos/microbiologia , Análise de Variância , Animais , Gatos , Células Cultivadas , Eletroforese em Gel de Ágar , Síndrome de Imunodeficiência Adquirida Felina/patologia , Técnicas In Vitro , Microscopia EletrônicaRESUMO
RU 41 740 (Biostim) is a glycoprotein extract obtained from Klebsiella pneumoniae. Its immunostimulating properties on monocytes have been established in vivo and in vitro. To confirm its spectrum of action at molecular level we studied its role on the modulation of four molecules involved in antigen presentation (HLA-DR, HLA-DQ), uptake of endotoxin (CD14) and activation (CD23). These four molecules are known to be modulated by interleukins IL-4 and IL-13. We found that HLA-DR, HLA-DQ, CD14 and CD23 were differentially regulated by biostim and IL-4 or IL-13. Surprisingly, Biostim inhibited the IL-4 or IL-13-induced expression of CD23, HLA-DQ and HLA-DR, while it did not have any action on these molecules by itself. We therefore hypothesize that Biostim, through the action on its receptor, could interact with the IL-4 receptor and IL-13 receptor and/or inhibit the IL-4 and IL-13 receptor transducing signal.
Assuntos
Adjuvantes Imunológicos/farmacologia , Proteínas de Bactérias/farmacologia , Interleucina-13/farmacologia , Interleucina-4/farmacologia , Monócitos/efeitos dos fármacos , Ligação Competitiva/efeitos dos fármacos , Células Cultivadas , Antígenos HLA-DQ/efeitos dos fármacos , Antígenos HLA-DR/efeitos dos fármacos , Humanos , Klebsiella pneumoniae/imunologia , Receptores de Lipopolissacarídeos/efeitos dos fármacos , Monócitos/imunologia , Receptores de IgE/efeitos dos fármacosRESUMO
Polyspecific IgG given intravenously at high dose (IVIG) are increasingly used as an immunomodulating therapy in autoimmune diseases. However, very few studies have dealt with the action of IVIG on the expression of the leukocyte markers. During a clinical trial in which 13 young and healthy women received IVIG to prevent unexplained recurrent abortions we have evaluated by flow cytometry the action of IVIG on 17 clusters of leukocyte differentiation (CD). We found that the IVIG perfusions (0.5 g/kg) induced an increase in the number of polymorphonuclear and monocyte cells in the peripheral blood. This effect lasted 8 days. The IVIG treatment had no effect upon T cell populations stained with antibodies specific for CD2, CD3, CD4, CD8 and on CD4+CD45RA+, CD4+CD29+, CD8+CD28+, CD8+CD28- subpopulations. A weak decrease in the B cell number was observed. The most striking phenomenon was the decrease in the number of CD56+ cells, whereas CD16+ and CD57+ cells were unaltered. By the double-staining technique we showed that CD56+CD16+ cells became CD56-CD16+ cells. Moreover, IVIG decrease the expression level of the LFA-1 molecule on monocytes and lymphocytes. The other adhesion molecules studied remained steady (CD11b, CD49d, CD49e, CD29, CD28, and CD62L). This study has shown that IVIG have no effect on 15 of 17 CD used but downmodulate two adhesion molecules playing a key role in the immune system.
Assuntos
Aborto Habitual/sangue , Imunoglobulinas Intravenosas/farmacologia , Adulto , Antígenos CD/análise , Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos T/análise , Antígenos de Diferenciação de Linfócitos T/fisiologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Antígeno CD56 , Feminino , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Contagem de Leucócitos/efeitos dos fármacos , Antígeno-1 Associado à Função Linfocitária/análise , Antígeno-1 Associado à Função Linfocitária/fisiologia , Linfócitos/química , Monócitos/química , Gravidez/sangue , Subpopulações de Linfócitos T/química , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologiaRESUMO
In order to discover some biological markers of acute graft-versus-host disease (aGVHD), we have studied the percentage of peripheral monocytes and T lymphocytes bearing HLA-DR and HLA-DQ class II molecules. This study included 25 allogeneic BMT in children, either with (n = 10) or without (n = 15) aGVHD. Within 2 months after transplantation, a higher percentage of DQ+ and DR+ monocytes and of DQ+ T lymphocytes was observed in patients without aGVHD compared with patients with aGVHD. The most discriminating marker was the strong increase in the percentage of DQ+ monocytes in patients without aGVHD (P = 0.001). In a sequential study, we observed a low percentage of DQ+ and DR+ peripheral blood mononuclear cells (PBMC) as long as the clinical manifestations of aGVHD continued. We speculate if the modulation of DQ and DR molecules on PBMC after BMT is a consequence of the action of some lymphokines, and if it plays a role in the regulation of the acute GVH reaction. We conclude that MHC class II molecules on peripheral mononuclear cells may be reliable biological markers for the diagnosis of aGVHD.
Assuntos
Transplante de Medula Óssea/imunologia , Doença Enxerto-Hospedeiro/imunologia , Antígenos HLA-DQ/análise , Antígenos HLA-DR/análise , Leucócitos Mononucleares/imunologia , Doença Aguda , Adolescente , Criança , Pré-Escolar , Feminino , Doença Enxerto-Hospedeiro/diagnóstico , Doença Enxerto-Hospedeiro/etiologia , Humanos , Masculino , Monócitos/imunologia , Linfócitos T/imunologia , Fatores de Tempo , Transplante Homólogo/imunologiaRESUMO
Multidrug resistance of cancer (MDR) is the major cause of failure of chemotherapy. The typical MDR phenotype is due to the overexpression of membrane proteins among which the main representative is P-glycoprotein (Pgp) encoded by the MDR1 gene. Many attempts to modulate MDR by chemosensitizers have been unsuccessful in human therapy due to their intrinsic toxic effects. In an effort to modulate the MDR phenotype efficiently we designed an antisense and a transcriptional decoy strategy targeting the TATA-less human MDR1 gene promoter. The choice of the start point of transcription in a multiple start site window is related to an upstream MED-1 cis-element, the sequence and configuration of which are specific to human MDR1 gene expressed in Pgp-overproducing cancer cells. A 12mer antisense oligodeoxynucleotide (ODN) and a 12mer double-stranded ODN, both containing the MED-1 sequence, were designed and efficiently vectorized into the nucleus with the chimerical MPG peptide. A synthetic cellular model (NIH-EGFP) and highly resistant human CEM/VLB0.45 leukemia cells, significantly responded to transfection with the ODN/MPG complex. The level of EGFP fluorescence in NIH-EGFP cells decreased, and thus its production, and viability of CEM/VLB0.45 cells decreased by 63% in the presence of vinblastine, revealing that their resistance to the anticancer drug was reversed. These results open new insights into transcriptional decoy and anti-gene therapies of MDR cancers that overproduce Pgp. Gene Therapy (2000) 7, 1224-1233.
Assuntos
Resistência a Múltiplos Medicamentos/genética , Endodesoxirribonucleases/genética , Genes MDR/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Regiões Promotoras Genéticas/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Vetores Genéticos , Humanos , Fenótipo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Células Tumorais CultivadasRESUMO
To acquire some biological markers associated with the occurrence of acute graft-versus-host disease (aGVHD) after allogeneic bone marrow transplant (BMT) in children, we have studied the lymphocyte subset reconstitution and the percentage of peripheral blood mononuclear cells bearing HLA-DR and HLA-DQ class II molecules. This study included 37 allogeneic BMT: either with (n = 17) or without (n = 20) aGVHD. Within 2 months after transplantation, we observed that patients with aGVHD had a unique mononuclear cell profile characterized by (i) a significant increase in the percentages of CD8bright+CD28- T cells (P = 0.05) and CD3+ T cells (P = 0.001), (ii) an important decrease in the percentage of CD56+ cells (P = 0.0001), and (iii) a decrease in the percentages of HLA-DQ+ and HLA-DR+ monocytes (P = 0.001) and HLA-DQ+ T lymphocytes (P = 0.0001), in comparison with patients without aGVHD. Moreover, statistical studies indicate that there was a positive correlation between CD8bright+CD28- and CD3+ T cells, whereas CD3+ T cells were negatively correlated to CD56+ cells. We did not find any statistical correlation between the percentages of HLA-DQ+ or HLA-DR+ cells and the percentages of these lymphocyte subsets. Therefore, in this study done in children, we suggest that patients with (i) less than 20% of DQ+ monocytes, (ii) less than 25% of CD56+ lymphocytes, and (iii) an enhanced percentage of CD8bright+CD28- T cells are strongly associated with aGVHD. Unfortunately, these biological markers of a GVHD may not precede the clinical manifestations of the disease.
Assuntos
Transplante de Medula Óssea/imunologia , Doença Enxerto-Hospedeiro/imunologia , Subpopulações de Linfócitos/imunologia , Doença Aguda , Adolescente , Biomarcadores , Transplante de Medula Óssea/efeitos adversos , Criança , Pré-Escolar , Feminino , Humanos , Imunofenotipagem , Lactente , Masculino , Transplante HomólogoRESUMO
Decidual large granular lymphocytes (DLGL) are the most abundant lymphoid cell type found in the first trimester maternal decidua. The function of DLGL remains controversial, although freshly isolated DLGL have been shown to exert a weak NK activity. We report here the phenotypic characterization of two DLGL subpopulations by immunofluorescence, using mAb against CD56, PEN5 as well as adhesion molecules potentially involved in cell-cell contact between DLGL and trophoblasts. DLGL are CD56bright and express the CD2, CD11a, CD18, CD38 and CD50 molecules, dimly the CD54 molecule, and poorly the CD102 and CD69 molecules. A strong expression of the polysialylated form of N-CAM (or CD56) was also observed on the surface of DLGL. Finally, 40% of CD56bright DLGL cells express the PEN5 epitope, which is selectively expressed on CD56dim cells NK in the peripheral blood. No other phenotypic difference was detected between the CD56brightPEN5+ and the CD56brightPEN5- DLGL populations. Our results show that DLGL are heterogeneous and suggest that the CD56brightPEN5+ DLGL subset belongs to the classical NK cell lineage.