RESUMO
The resistance of Plasmodium falciparum to chloroquine (CQ) is probably mediated by point mutations in two genes: pfcrt and pfmdr1. The aim of the present study was to investigate, in patients treated with CQ, the association between host factors, such as immunity and initial level of parasitaemia, and the ability to clear P. falciparum parasites carrying the key chloroquine-resistance (CQR) mutations, pfcrt 76T and pfmdr1 86Y. Identical CQ-efficacy trials were performed in 51 young children (aged <5 years) from Kibaha, in north-western Tanzania, and 44 patients (aged 3-57 years) from Darawish, in eastern Sudan. In both areas, all the CQ-treatment failures had infections with the 76T and 86Y alleles before treatment. Although the presence of these two alleles was significantly associated with treatment failure in Sudan (P=0.001), the corresponding association in Tanzania did not reach statistical significance (P=0.1). Of the 39 patients from Darawish and 44 from Kibaha who harboured parasites with the CQR mutations, 12 and 19, respectively, managed to clear their parasitaemias. The ability to clear CQR parasites was significantly associated with the initial level of parasitaemia (with P-values of 0.05 in Tanzania and 0.01 in Sudan) and with age-- the best surrogate for protective immunity in endemic areas (with P-values of 0.02 in Tanzania and 0.001 in Sudan). These results confirm previous observations that indicated that the 76T and 86Y alleles play a role in the mechanism of CQR, although other factors, such as level of parasitaemia when treated and age, are also important. The 76T and 86Y alleles could still be used as predictive markers for CQR, in non-immune individuals and low-transmission areas.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Malária Falciparum/genética , Proteínas de Membrana/genética , Parasitemia/genética , Proteínas de Protozoários/genética , Adulto , Animais , Antimaláricos/uso terapêutico , Criança , Pré-Escolar , Cloroquina/uso terapêutico , Resistência a Medicamentos/genética , Genes MDR/genética , Genes MDR/imunologia , Humanos , Malária Falciparum/tratamento farmacológico , Proteínas de Membrana Transportadoras , Mutação , Parasitemia/imunologia , Plasmodium falciparum/genética , Sudão , TanzâniaRESUMO
Acquired multidrug resistance (MDR) remains a major challenge in the treatment of cancer with chemotherapeutic drugs. It can be mediated by the up-regulated expression of different proteins within the tumor cell membrane. Here, we used murine multidrug resistance-1 (MDR-1) as a target-antigen for the immunotherapy of cancer. We successfully demonstrated that peripheral T cell tolerance can be broken by oral administration of a DNA vaccine encoding MDR-1 and carried by attenuated Salmonella typhimurium to secondary lymphoid organs. Thus, mice, immunized orally three times at 2-wk intervals and challenged 2 wk thereafter with either MDR-1 expressing CT-26 colon carcinoma cells or MDR-1 expressing Lewis lung carcinoma cells, revealed a significant increase in life span. This was evident, when compared with animals either vaccinated with the empty control vector or challenged with the parental cell lines lacking overexpression of MDR-1. The immune response induced was antigen-specific and CD8+ T cell-mediated. The presence of the target antigen led to up-regulation of activation markers on CD8+ T cells and resulted in a strong cytotoxic T cell response as well as lysis of tumor target cells in vitro. We furthermore established the vaccine to be an effective treatment for established multi-drug-resistant tumor metastases, resulting in a significantly increased life span of experimental animals. Absence of CD8+ T cells due to in vivo depletion led to abrogation of effectiveness. Taken together, our results demonstrate that T cell tolerance against the MDR-1 self-antigen can be broken. It is anticipated that the combination of such an approach with chemotherapy could lead to more effective treatments of cancer.
Assuntos
Genes MDR/genética , Imunoterapia/métodos , Linfócitos T/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Animais , Linfócitos T CD8-Positivos/fisiologia , Vacinas Anticâncer , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Neoplasias do Colo/terapia , Genes MDR/imunologia , Vetores Genéticos/biossíntese , Vetores Genéticos/genética , Imunidade/fisiologia , Imunização/métodos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Tela Subcutânea/metabolismo , Tela Subcutânea/patologia , Linfócitos T/patologia , Transdução Genética/métodos , Vacinação/métodosRESUMO
Multidrug resistance (MDR) confers resistance to anticancer drugs and reduces therapeutic efficiency. It is often characterized by the expression of the MDR1 gene product P-glycoprotein (or gp170) at the membrane of tumor cells. To further propose a potential complementary tool in cancer treatment, the sensitivity of gp170 tumor cells to NK-dependent lysis was investigated. Two kinds of cells were generated from wild-type K562 erythroleukemic cells: the first were derived from Taxol-selected cells and cloned, whereas the second were retrovirally transduced by the cDNA of the MDR1 gene. The last process was also applied to the human embryonal carcinoma cells called Tera-2 cells. First, both cloned and MDR-1 K562 cells appeared highly susceptible to naive NK cell killing. Interestingly, in addition, Tera-2 cells that were not sensitive to NK lysis could be killed when they expressed gp170 at their membranes. In previous data, we demonstrated that NK cell release of bimolecular complexes composed of perforin and platelet-activating factor (PAF) interacting with the PAF-R, which has to be expressed on the target cell membranes, were components of NK tumor cell killing. In the present study, we show that gp170 has the capacity to drive constitutive PAF-R expression on tumor cells, which could be responsible for hypersensitivity to NK lysis and accelerated cell death.
Assuntos
Citotoxicidade Imunológica , Resistencia a Medicamentos Antineoplásicos/imunologia , Glicoproteínas/fisiologia , Células Matadoras Naturais/imunologia , Fator de Ativação de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/biossíntese , Receptores Acoplados a Proteínas G/biossíntese , Subfamília B de Transportador de Cassetes de Ligação de ATP , Carcinoma Embrionário/imunologia , Carcinoma Embrionário/metabolismo , Carcinoma Embrionário/patologia , Linhagem Celular Tumoral , Membrana Celular/imunologia , Membrana Celular/metabolismo , Membrana Celular/patologia , Células Clonais , Citotoxicidade Imunológica/efeitos dos fármacos , Genes MDR/imunologia , Glicoproteínas/biossíntese , Humanos , Concentração de Íons de Hidrogênio , Células K562 , Células Matadoras Naturais/efeitos dos fármacos , Leucemia Eritroblástica Aguda/imunologia , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/patologia , Paclitaxel/farmacologia , Retroviridae/genética , Teratoma/imunologia , Teratoma/metabolismo , Teratoma/patologia , Transdução Genética , TransfecçãoRESUMO
OBJECTIVE: To observe if antibody-targeted immunonanoparticles could internalize into sensitive and multidrug resistance (MDR) cells of human hepatoma; and to study if the immunonanoparticles could reverse the MDR. METHODS: Human hepatoma-specific adriamycin-loaded human serum albumin immunonanoparticles (HAb18-ADR-HSA-NP) were incubated with human hepatoma sensitive cell line (SMMC-7721) or MDR cell line (SMMC-7721/MDR+) and the internalization of immunonanoparticles were observed by laser confocus microscopy, scanning electron microscopy and transmission electron microscopy; MTT colorimetric assay was used for assaying in vitro cytotoxicities of HAb18-ADR-HSA-NP to the resistant variant cells. Then based on these data, IC50 value of the immunonanoparticles and RF (Resistant Factor) of MDR cells were calculated. RESULTS: Laser confocus microscopy showed that many fluorescent particles (labeled immunonanoparticles) tightly adsorbed to SMMC-7721 cells and were also seen in cytoplasm of SMMC-7721 cells. When incubated with immunonanoparticles at 37 degrees C, many of immunonanoparticles were visualized in cytoplasm of SMCC-7721 or SMCC-7721/MDR+. These immunonanoparticles-contained cells exhibited damaged ultrastructures and the damage degree depended on incubation time. When the human hepatoma cells were pretreated with HAb18 antibody and incubated with immunonanoparticles, few immunonanoparticles were seen in cytoplasm of the cells, suggesting antibody-specific internalization of the immunonanoparticles. Scanning electron microscopy demonstrated specific binding of the immunonanoparticles to the resistant variant cells. That immunonanoparticles exerted enhanced cytotoxicity to the resistant variant cells was demonstrated by a decrease of RF value of MDR cells, compared with free ADR (4.4 vs. 2.1). CONCLUSION: Human hepatoma-specific adriamycin-loaded human serum albumin immunonanoparticles could specifically internalize into sensitive or multidrug resistance cells of human hepatoma via antibody direction. The immunonanoparticles could enhance the sensitivity of MDR cells to ADR cytotoxicity, suggesting its reverse effect on MDR.
Assuntos
Anticorpos Antineoplásicos/imunologia , Carcinoma Hepatocelular/patologia , Resistencia a Medicamentos Antineoplásicos/imunologia , Endocitose , Neoplasias Hepáticas/patologia , Antibióticos Antineoplásicos/farmacologia , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/biossíntese , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Genes MDR/imunologia , Humanos , Neoplasias Hepáticas/genética , Microscopia Confocal , Nanotecnologia , FenótipoRESUMO
CD20 determinant expressed on B precursors is associated with regulation of proliferation, apoptosis and maturation of these cells. The acute lymphoblastic leukemia "common" type (cALL) based on expression of CD20 is subdivided in type I and II. However, the clinical significance of CD20 expression on cALL and significance of cALL type I and II discernment are not fully elucidated. The association of CD20 expression with the expression of multidrug resistance molecule (MDR), CD34, atypical immunophenotypes of leukemia cells and response to induction therapy were determined in the group of 147 patients with acute lymphoblastic leukemia (ALL) B progenitor type (ALL-proB -14 patients) and common type (cALL-133 patients). The expression of CD20 on leukemia cells was studied routinely at diagnosis before the therapy. This expression was noted on leukemia cells of 6 ALL-proB patients (42.8%) and 66 cALL patients (49.6%). The expression of CD20 showed no association with the expression of CD34, CD22 and MDR. The reverse association was observed between CD20 expression and the presence of co-expression of myeloid (CD13, CD33, CD65, CD15) and T lymphoid determinants (CD2, CD5, CD7) on leukemia cells. The effect of induction therapy analyzed as time of blast cells cytoreduction in peripheral blood and time of reaching the complete remission showed the slower clearance of peripheral blood from blast cells associated with expression of CD20. There was no association of CD20 expression with the time of reaching the hematological remission. The above results suggested a "protective" role of CD20 against co-expression of other determinants (myeloid and lymphoid) and no association with the results of induction therapy.
Assuntos
Antígenos CD20/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Adolescente , Antígenos CD/imunologia , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Genes MDR/imunologia , Humanos , Imunofenotipagem , MasculinoRESUMO
BACKGROUND: Living-donor liver transplantation (LDLT) and subsequent immunosuppressive therapy with tacrolimus have been cornerstones in the recovery of patients from end-stage liver failure, but there has been no critical dosage regimen for tacrolimus therapy, especially the initial dosage. In this study, we examined whether the absorptive barriers, multidrug resistance protein (MDR1), or cytochrome P450 IIIA4 (CYP3A4) are important pharmacokinetic factors for tacrolimus and are prognostic indicators for LDLT outcome. METHODS: We used competitive polymerase chain reaction to evaluate the messenger ribonucleic acid (mRNA) expression levels of MDRL And Cyp3A4 in mucosal cells of the upper jejunum from a part of the Rroux-en- Y limb for biliary reconstruction during LDLT of recipients (n = 48). The tacrolimus dosage was started at an oral dose of 0.075 mg/kg every 12 hours and adjusted on the basis of its whole-blood trough level by use of a semiautomated microparticle enzyme immunoassay. RESULTS: The mRNA expression level of MDR1 (r = -0.776), but not CYP3A4 (r = -0.094), was inversely related to the concentration/dose ratio of tacrolimus. High levels of MDR1, but not CYP3A4, were strongly associated with reductions in survival rates after LDLT with the Kaplan-Meier method and log-rank statistics (P =.020 and P =.135, respectively). With use of a Cox regression procedure, high levels of MDR1 (relative risk, 12.99; 95% confidence interval, 1.64-103.23), but not CYP3A4 (relative risk, 0.93; 95% confidence interval, 0.87-1.00) appeared to be a significant prognostic indicator for poor survival. CONCLUSIONS: Intestinal MDR1 is not only a good probe with which to predict the interindividual variation in tacrolimus pharmacokinetics after LDLT but also a powerful prognostic indicator for the outcome of LDLT.
Assuntos
Sistema Enzimático do Citocromo P-450/imunologia , Genes MDR/imunologia , Imunossupressores/farmacocinética , Intestinos/imunologia , Falência Hepática/terapia , Transplante de Fígado , Tacrolimo/farmacocinética , Adolescente , Adulto , Feminino , Humanos , Imunossupressores/uso terapêutico , Lactente , Falência Hepática/mortalidade , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prognóstico , Análise de Sobrevida , Tacrolimo/uso terapêuticoRESUMO
Detection of intracellular antigens by flow cytometry requires effective fixation and permeabilization of the cell membrane. This study compares three fixation/permeabilization techniques: two commercial chemical reagents, the ORTHOPermeaFix (OPF) and the FIX&PERM Cell Permeabilization Kit (F&P), and a novel method based on microwave heating (MWH). They have been applied to the detection of two nuclear (p53 and rb/p105) and two cytoplasmic (bcl-2 and mdr-1/gp-170) antigens, using positive- and negative-control cell lines and peripheral blood mononuclear cells. Western blotting was performed as a control of protein expression. For the four antigens assessed, cellular morphology, discrimination between intact cells and debris, percentage of positive cells, and mean fluorescence intensity were examined. For this last parameter, the assessment of the MWH technique was performed using SD and a graphical approach inspired by the concepts described by Bland and Altman (Lancet 1986;346: 1085-7) as well as Petersen et al. (Clin Chem 1997;43: 2039-46). The statistical analysis shows that MWH is comparable to the commercial methods and that its reproducibility is also equivalent to OPF and F&P. As assessed for some of the most clinically relevant intracytoplasmic and intranuclear antigens, the MWH method appears to be a valuable and inexpensive alternative. It is worth noting that, unlike commercial reagents, MWH altered surface antigens. Interestingly, this feature, which would prevent cell selection on the basis of combined membrane and intracellular epitopes, is associated with a decrease of nonspecific background fluorescence.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Antígenos/análise , Proteínas Proto-Oncogênicas c-bcl-2/análise , Proteína do Retinoblastoma/análise , Proteína Supressora de Tumor p53/análise , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/imunologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/efeitos da radiação , Antígenos/metabolismo , Antígenos/efeitos da radiação , Linhagem Celular , Citoplasma/imunologia , Citoplasma/metabolismo , Citoplasma/efeitos da radiação , Epitopos/análise , Epitopos/metabolismo , Epitopos/efeitos da radiação , Citometria de Fluxo/métodos , Genes MDR/imunologia , Genes MDR/efeitos da radiação , Humanos , Linfócitos/imunologia , Linfócitos/metabolismo , Linfócitos/efeitos da radiação , Linfócitos/ultraestrutura , Micro-Ondas , Proteínas Nucleares/análise , Proteínas Nucleares/imunologia , Proteínas Nucleares/efeitos da radiação , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/efeitos da radiação , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes , Proteína do Retinoblastoma/imunologia , Proteína do Retinoblastoma/efeitos da radiação , Proteína Supressora de Tumor p53/imunologia , Proteína Supressora de Tumor p53/efeitos da radiaçãoRESUMO
The murine multiple drug resistance (mdr) gene, mdr1a, encodes a 170-kDa transmembrane protein that is expressed in many tissues including intestinal epithelial cells, a subset of lymphoid cells and hematopoietic cells. We report that mdr1a knockout (mdr1a-/-) mice are susceptible to developing a severe, spontaneous intestinal inflammation when maintained under specific pathogen-free animal facility conditions. The intestinal inflammation seen in mdr1a-/- mice has a pathology similar to that of human inflammatory bowel disease (IBD) and is defined by dysregulated epithelial cell growth and leukocytic infiltration into the lamina propria of the large intestine. Treating mdr1a-/- mice with oral antibiotics can both prevent the development of disease and resolve active inflammation. Lymphoid cells isolated from mice with active colitis are functionally reactive to intestinal bacterial Ags, providing evidence that there is enhanced immunologic responsiveness to the normal bacterial flora during IBD. This study is the first description of spontaneous colitis in a gene knockout mouse with an apparently intact immune system. This novel model of spontaneous colitis may provide new insight into the pathogenesis of IBD, the nature of dysregulated immune reactivity to intestinal bacterial Ags, and the potential functional role of mdr genes expressed in the cells and tissues of the colonic microenvironment.