RESUMO
PURPOSE: The biological association between chronic lymphocytic thyroiditis (CLT) and differentiated thyroid cancer (DTC) has not been elucidated yet. The aim of the study was to assess whether the presence of CLT exerts any influence on clinical or histological presentation of DTC. METHODS: Nine hundred and seven consecutive patients with DTC treated in the years 1998-2016 were divided into two groups according to the presence or absence of concomitant CLT. The statistical differences were analysed. RESULTS: Out of 907 patients included in the study, 331 were diagnosed with DTC and CLT (studied group), while 576 patients with DTC but without CLT constituted a control group. The distribution of papillary and follicular thyroid cancer did not differ. In CLT group, the prevalence of pT1 was greater than for pT2-pT4 DTC (P = 0.0003; OR = 1.69, 95% CI 1.27-2.24) compared to controls (68.3 vs. 56.1%, respectively). The presence of multifocal lesions was similar. The thyroid capsule infiltration without extrathyroidal invasion (P < 0.0001; OR = 0.21, 95% CI 0.14-0.31) was more frequent in the studied group, unlike extracapsular invasion, which was significantly more often present in patients with DTC but without CLT (P = 0.004; OR = 1.66; 95% CI 1.17-2.34) as well as nodal involvement (P = 0.048; OR = 0.65, 95% CI 0.42-0.99). CONCLUSIONS: The collected data indicate a protective role of CLT in preventing the spread of the DTC. The presence of CLT might limit tumour growth to the primary site.
Assuntos
Adenocarcinoma Folicular/diagnóstico , Doença de Hashimoto/diagnóstico , Câncer Papilífero da Tireoide/diagnóstico , Neoplasias da Glândula Tireoide/diagnóstico , Adenocarcinoma Folicular/epidemiologia , Adenocarcinoma Folicular/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Diferenciação Celular/fisiologia , Estudos de Coortes , Feminino , Doença de Hashimoto/epidemiologia , Doença de Hashimoto/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Estadiamento de Neoplasias/tendências , Estudos Retrospectivos , Câncer Papilífero da Tireoide/epidemiologia , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/epidemiologia , Neoplasias da Glândula Tireoide/patologia , Adulto JovemRESUMO
Regulatory T cells (Tregs) represent a small subpopulation of CD4+ cells. Tregs are characterized by the expression of transcription factor Forkhead box protein 3 (FoxP3), also known as scurfin. Tregs are modulators of adaptive immune responses and play an important role in maintaining tolerance to self-antigens, providing the suppression associated with tumour microenvironment as well. These immunomodulatory properties are the main reason for the development of numerous therapeutic strategies, designed to inhibit the activity of cancer cells. However, due to Treg subpopulation diversity and its many functional pathways, the role of these cells in the cancer development and progression is still not fully understood.
Assuntos
Imunoterapia/métodos , Neoplasias/imunologia , Linfócitos T Reguladores/imunologia , Autoantígenos/imunologia , Vacinas Anticâncer , Fatores de Transcrição Forkhead/metabolismo , Humanos , Tolerância Imunológica , Imunomodulação , Masculino , Evasão Tumoral , Microambiente TumoralRESUMO
AIM: to determine the number of sjTREC in type 2 diabetic patients and to analyze the potential factors affecting thymic output. The subjects of this study were 82 people: group I (n=68) - type 2 diabetic patients; group II (n=14) - healthy people. Measurements were taken: fasting glycemia, HbA1c, 1,5-AG (1,5-anhydro-D-glucitol), total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides, C-peptide and interleukin-7. Quantification of sjTREC concentration in peripheral blood mononuclear cells was made by real time PCR. The concentration of sjTREC/10(5) PBMC was significantly lower in the group of diabetes as compared to control, while the level of IL-7 did not change between both groups. sjTREC levels are similar in obese group, but higher in patients with a short duration of diabetes, with better glycemic control (with low levels of fasting glycemia, HbA1c and higher 1,5-AG). Analysis of HbA1c covariance, indicator of chronic hyperglycemia, is a factor which primarily determines sjTREC differences between the groups, regardless of other factors. DM2 modifies sjTREC number in the peripheral blood. The level of sjTREC concentration is affected by DM2 duration, the degree of glycemic control. Thereby, chronic hyperglycemia leads primarily to metabolism deteriorations in the thymus, which results in decrease in its functional activity.
Assuntos
Colesterol/sangue , Diabetes Mellitus Tipo 2/sangue , Interleucina-7/sangue , Obesidade/sangue , Adulto , Idoso , Glicemia , Peptídeo C/sangue , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Diabetes Mellitus Tipo 2/patologia , Feminino , Hemoglobinas Glicadas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/patologia , Triglicerídeos/sangueRESUMO
The aim of this experimental study was to estimate the effect of Simvastatin on glycemic variability-related insulin resistance in the course of diabetes mellitus (DM) in rats. Fifty seven male Wistar rats were divided into four groups: I - rats with diabetes mellitus and glycemic variability treated with Simvastatin (20 mg/kg body weight, intragastral during 8 weeks); II - placebo-treated rats with DM and glycemic variability; III - placebo treated rats with DM and IV - nondiabetic control rats. DM was induced by feeding rats with high-fat diet (61%) during five weeks and low-dose of Streptozotocin (30 mg/kg, intraperitoneally). Daily glucose excursions were stimulated by feeding animals twice a day. We measured fasting blood glucose, glycated hemoglobin (HbA1c), insulin and HOMAIR was calculated. Higher insulin resistance in diabetic rats is related to greater daily glycemic variability. In our study was installed significant increasing HOMAIR in diabetics rats with glycemic excursions comparison with the control. Our results showed that the simvastatin-treatment decreases the indices glycemic variability and HOMA in diabetic rats with glycemic excursions.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Resistência à Insulina , Sinvastatina/administração & dosagem , Animais , Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/patologia , Humanos , Insulina/sangue , RatosRESUMO
Many different cells produce and release membraneous microvesicles (MV) or exosomes into their microenvironment. Exosomes represent a specific subtype of secreted derived vesicles which are defined as homogenous vesicles of 30-100 nm lined by a lipid bilayer, which contain a specific set of proteins, lipids, and nucleic acids. There are clear evidences that they serve as important biological signals messengers and carriers in physiological as well as in pathological processes. Those derived from tumours (tumour-derived exosomes, TD-exosomes) function as protumourigenic factors that can mediate intercellular communication in the tumour microenvironment and also contribute to cancer progression. The main functions of exosomes in the cancer microenvironment include the following: promotion of primary cancer growth, stimulation of angiogenesis, activation of stromal fibroblasts, sculpting the cancer ECM, generation of a premetastatic niche and suppression of host immune response. Exosomes have recently emerged as potentially promising diagnostic and prognostic biomarkers in cancer and other diseases. This article is a summary of information about the structure and origin of exosomes and also indicates the importance of exosomes and microRNAs in lung cancer. The role of exosomes in NSCLC is little known, and its explanation requires thorough research.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Exossomos , Neoplasias Pulmonares/patologia , Microambiente Tumoral , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Comunicação Celular , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Exossomos/química , Exossomos/genética , Exossomos/metabolismo , Humanos , Lipídeos/análise , Neoplasias Pulmonares/metabolismo , MicroRNAs/genética , Corpos Multivesiculares/metabolismo , Neovascularização Patológica/patologiaRESUMO
The CD3+56+ cells are a small but significant population of T lymphocytes encompassing NKT-like and NKT cells, which may play the essential role at the very early stages of atherosclerotic plaque development. The frequency and activity of CD3+56+ cells in atherosclerosis-inducing dysglycaemic disease (diabetes type 2 or pre-diabetes) is largely unknown.We analysed CD3+56+ cell count, granzyme, perforin and annexin V profiles in the peripheral blood from a group of patients with pre-diabetes, with diabetes type 2 and from non-dysglycaemic controls. Measurements were made of fasting glucose levels, HbA1c, 1,5-anhydroglucitol and lipid profile.The mean counts of CD3+56+ cells were significantly higher in patients with pre-diabetes compared to both patients with diabetes and to control group. There was an increase in the number of CD3+56+ cells producing granzyme and perforin in pre-diabetic patients compared to other groups, while there were no difference in annexin V+ populations within examined groups. It was confirmed that CD3+56+ cells count is modified by metabolic factors and their parameters, namely HbA1c and 1,5-anhydroglucitol values.It could be stated that the alterations of CD3+ 56+ cells count in peripheral blood of pre-diabetic and type 2 diabetic patients are related to different grades of carbohydrate deteriorations - postprandial hyperglycaemia and chronic hyperglycaemia.
Assuntos
Complexo CD3/sangue , Antígeno CD56/sangue , Diabetes Mellitus Tipo 2/sangue , Células T Matadoras Naturais/metabolismo , Placa Aterosclerótica/sangue , Idoso , Glicemia/metabolismo , Desoxiglucose/sangue , Jejum/sangue , Hemoglobinas Glicadas/metabolismo , Humanos , Hiperglicemia/sangue , Lipídeos/sangue , Masculino , Pessoa de Meia-Idade , Células T Matadoras Naturais/patologiaRESUMO
There are differences in the clinical course of chronic viral hepatitis C between adults and children, but it is generally accepted that the disease has cell-mediated immune background. The aim of this study was to evaluate PBMC subsets in children with chronic hepatitis C before treatment in order to find some predictive factors, useful for patients management. Several PBMC subsets, in particular lymphoid and dendritic cell (DC) ones, were tested by flow cytometry in HCV(+) paediatric patients (n = 46) and in control children matched in terms of age and sex (n = 20). Data were subjected to extensive statistics. It was found that cells with cytotoxic potential such as CD8(+)CD28(-) T cells, NK and NKT cells as well as lineage(-)HLA-DR(+) DC were increased in per cent values, while CD4(+) T cells and CD4:CD8 ratio were decreased in hepatitis C group. In HCV(+) patients, CD4(+) T cells were inversely correlated with alanine aminotransferase (ALT) levels and with viraemia. DC subset of myeloid origin (CD11c(+)) assessed both in per cent values and as mean fluorescence intensity (MFI) of HLA-DR expression was shown to be downregulated in hepatitis patients, in spite of increased numbers. To conclude, PBMC subsets, and in particular DC, are affected by HCV chronic infection in children, reflected by the correlation with clinical parameters, such as ALT and viraemia.
Assuntos
Antígenos CD/análise , Células Dendríticas/imunologia , Hepatite C Crônica/imunologia , Leucócitos Mononucleares/imunologia , Adolescente , Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Estudos de Casos e Controles , Criança , Feminino , Citometria de Fluxo , Antígenos HLA-DR/metabolismo , Humanos , Imunofenotipagem , MasculinoRESUMO
The aim of this study was to assess alterations of zeta chain expression and their relation to apoptosis of T lymphocytes. T lymphocytes were obtained from malignant pleural effusions (MPE) of 15 patients. The work focused on TCR-zeta chain expression, apoptosis of T lymphocytes, and on the content of monocyte/macrophages in effusions. Analysis was performed using three color flow cytometry combining CD3, TCR-zeta and TUNEL reaction. The content of tumor and monocyte/macrophage cells has been determined using CD3, CD14, and cytokeratins, as markers of distinct cell subpopulations. Our findings strongly indicate that decreased zeta chain expression in T cells depends on the content of monocyte/macrophage cells, and that the range of the decrease is inversely proportional to the number of monocytes/macrophages in the effusion. Those having low zeta chain expression were the main subpopulation of T cells undergoing apoptosis. These data suggest that decreased zeta chain expression in T cells in MPE may be related to the abundance of monocyte/macrophages in effusions.
Assuntos
Apoptose , Regulação para Baixo , Macrófagos/metabolismo , Proteínas de Membrana/biossíntese , Monócitos/metabolismo , Receptores de Antígenos de Linfócitos T/biossíntese , Linfócitos T/metabolismo , Complexo CD3/biossíntese , Citometria de Fluxo , Humanos , Marcação In Situ das Extremidades Cortadas , Receptores de Lipopolissacarídeos/biossíntese , Neoplasias/metabolismo , Derrame Pleural/metabolismo , Derrame Pleural Maligno/metabolismoRESUMO
Prolactin (PRL) has been reported to inhibit apoptosis in various cell types and to serve as a cofactor in the upregulation of CD25 on T cells during activation. We investigated a possible relation between prolactin receptor (PRL-R) or IL-2 receptor alpha (IL-2Ralpha, CD25) expression on circulating T lymphocytes and their apoptosis in patients with breast cancer. Peripheral blood mononuclear cells obtained from 25 patients, 25 normal controls (NC) and three cord blood samples were evaluated for Annexin V binding and expression of CD95, CD25, and PRL-R on CD3(+) T cells by multicolour flow cytometry. Plasma levels of PRL, sCD95L, and sIL-2R were determined in patients and controls and related to T-cell apoptosis. The ability of PRL to protect T cells from apoptosis induced by various agents was also studied. Expression of PRL-R on the surface of T cells was comparable in patients with breast cancer and NC, but PRL plasma levels in patients were significantly lower (P<0.05). In patients, 18+/-11% (mean+/-s.d.) of CD3(+) cells bound Annexin V, compared to 9+/-6% in NC (P<0.0004). Percentages of CD3(+)Fas(+) and CD3(+)CD25(+) cells were higher in the peripheral circulation of patients than NC (P<0.0001 and <0.04, respectively). Levels of sFasL were lowest in plasma of the patients with the highest proportions of CD3(+)Fas(+) T cells. Most T cells undergoing apoptosis were CD3(+)CD25(-) in patients, and the proportion of CD3(+)CD25(-) Annexin V(+) cells was significantly increased in patients compared to NC (P<0.006). Ex vivo PRL protected T cells from starvation-induced or anti-CD3Ab-induced but not from Fas/FasL-dependent apoptosis. These results indicate that expression of CD25 but not of PRL-R on the surface of activated T lymphocytes appears to be involved in modulating Fas/Fas - ligand interactions, which are, in part, responsible for apoptosis of T lymphocytes and excessive turnover of immune cells in the circulation of patients with breast cancer.
Assuntos
Apoptose/imunologia , Neoplasias da Mama/imunologia , Receptores de Interleucina-2/imunologia , Receptores da Prolactina/fisiologia , Linfócitos T/imunologia , Idoso , Antígenos CD/imunologia , Neoplasias da Mama/patologia , Feminino , Sangue Fetal , Humanos , Células Jurkat , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Valores de Referência , Receptor fas/imunologiaAssuntos
Aneurisma da Aorta Abdominal/metabolismo , Glutamina/farmacologia , Nutrição Parenteral Total , Idoso , Aneurisma da Aorta Abdominal/imunologia , Aneurisma da Aorta Abdominal/cirurgia , Estudos de Casos e Controles , Glutamina/administração & dosagem , Hospitalização , Humanos , Tempo de Internação , Masculino , Nitrogênio/metabolismoRESUMO
BACKGROUND: Limited research has been performed concerning the effects of MR imaging on the immune system. In this study the influence of MR imaging exposure on polymorphonuclear neutrophil (PMN) adhesion was evaluated. MATERIAL AND METHODS: In vivo and in vitro studies were performed in 10 patients undergoing an MR imaging procedure, PMN adhesion to a plastic surface, as well as the expression of adhesion molecules beta 2-integrins CD11b, CD18, and L-selectin on the surface of PMN were estimated. RESULTS: Exposure to MR imaging significantly increased adhesion of isolated PMNs to plastic surfaces. PMNs from blood samples obtained from patients undergoing MR imaging as well as from blood samples placed beside patients during MR imaging did not differ from controls in adhesion to plastic surfaces. Similarly, plasma from three tested samples did not change control PMN adhesion to plastic surface. Expression of beta 2-integrins (CD11, CD18) was significantly increased in samples left beside patients during MR imaging, while significantly decreased in samples obtained from patients after MR imaging exposure when compared to control samples. Expression of the surface adhesion molecule L-selectin on the surface of PMN decreased significantly in blood samples left beside patients during MR imaging. CONCLUSION: The results indicate that the PMN adhesion properties increase under the influence of MR imaging exposure. This phenomenon may be the result of direct stimulation of polymorphonuclear neutrophils by the exposure to MR imaging.
Assuntos
Adesão Celular/efeitos da radiação , Imageamento por Ressonância Magnética , Neutrófilos/citologia , Adulto , Idoso , Antígenos CD18/biossíntese , Feminino , Humanos , Selectina L/biossíntese , Antígeno de Macrófago 1/biossíntese , Masculino , Pessoa de Meia-Idade , Neutrófilos/efeitos da radiação , Plásticos/metabolismoRESUMO
Expression of T-cell receptor- or Fcgamma receptor III-associated signal-transducing zeta chain is important for the functional integrity of immune cells. We found that significantly higher proportions of circulating CD3+ T cells as well as natural killer cells had low or absent expression of the zeta chain in patients with advanced melanoma than in normal donors (P < 0.0005). Decreased zeta expression was always observed in a small subset of circulating CD3+ T cells that were in the process of apoptosis, i.e., bound Annexin V or were terminal deoxynucleotidyl transferase-mediated nick end labeling positive. Up to 80% of T cells in the peripheral blood of patients with melanoma were Fas+, with the mean percentage of Fas+CD3+ cells significantly higher in patients (P < 0.004) than normal controls. These Fas+CD3+ T cells were found to preferentially undergo apoptosis. Annexin V binding, the loss of Fas expression from the cell surface as well as zeta down-regulation, which are associated with early apoptosis, were detected in a proportion of circulating Fas+CD3+. In Jurkat cells incubated with agonistic anti-Fas antibody (CH-11), a rapid loss of Fas expression from the cell surface coincided with Annexin V binding and preceded the loss of zeta chain during early apoptosis. In a subset of Jurkat cells coincubated with human melanoma cells, Annexin V binding and zeta degradation as well as DNA fragmentation were observed, indicating that the tumor induced T-cell death. Triggering of death receptors expressed on activated T lymphocytes was accompanied by the loss of zeta expression. On the other hand, soluble factors secreted by melanoma cells induced down-regulation but no apoptosis in activated normal T cells. In the circulation of patients with melanoma, apoptosis of immune effector cells may be related to the state of chronic activation, resulting in the up-regulation of death receptors and increased susceptibility to apoptosis.
Assuntos
Apoptose , Complexo CD3/biossíntese , Melanoma/sangue , Melanoma/patologia , Proteínas de Membrana/biossíntese , Receptores de Antígenos de Linfócitos T/biossíntese , Linfócitos T/metabolismo , Anexina A5/metabolismo , Antígenos CD4/biossíntese , Antígeno CD56/biossíntese , Antígenos CD8/biossíntese , Técnicas de Cocultura , Fragmentação do DNA , Regulação para Baixo , Citometria de Fluxo , Humanos , Marcação In Situ das Extremidades Cortadas , Células Jurkat , Leucócitos Mononucleares/metabolismo , Melanoma/metabolismo , Transdução de Sinais , Fatores de Tempo , Células Tumorais Cultivadas , Regulação para Cima , Receptor fas/biossíntese , Receptor fas/imunologia , Receptor fas/metabolismoAssuntos
Linfócitos do Interstício Tumoral/imunologia , Proteínas de Membrana/metabolismo , Derrame Pleural Maligno/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Apoptose , Fragmentação do DNA , Humanos , Tolerância Imunológica , Marcação In Situ das Extremidades Cortadas , Ativação Linfocitária , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Derrame Pleural/imunologia , Derrame Pleural/metabolismo , Derrame Pleural/patologia , Derrame Pleural Maligno/metabolismo , Derrame Pleural Maligno/patologia , Transdução de Sinais , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
Thirty-five patients with dermatophyte onychomycosis caused by Trichophyton rubrum, T. mentagrophytes var. granulosum, T. tonsurans and Epidermophyton floccosum were examined before treatment and 27 of these patients were examined again when they came to the control check up 3 months after completion of treatment. The immunological investigations, including evaluation of immunological competence, were performed in vivo through the determination of lymphoid cell immunophenotype by a flow cytometry technique. The quantitative composition of basic lymphocyte subpopulations and natural killer cells in the peripheral blood of 35 patients before the treatment was compared with a control group of 20 individuals. Statistically significant differences in the percentages of CD3+ T lymphocytes (P<0.05), T helper lymphocytes (CD4+) (P<0.05) and activated T lymphocytes (CD3+/HLA-DR+) (P<0.05) were obtained. In the control check-up examinations of 27 patients 3 months after completion of treatment, in comparison with the control group of 20 healthy individuals, highly statistically significant differences in percehtages of T lymphocytes (CD3+) (P<0.001) and T helper lymphocytes (CD4+) (P<0.01) were obtained. In five of these 27 patients the treatment resulted in failure. Comparing the group of 22 recovered patients with these five patients in whom the treatment result was failure, the only statistically significant difference obtained before as well as after the treatment was in B lymphocytes (CD19+) percentage (P<0.05). The results obtained confirm that impairments of the patients' cellular immunity are crucial factors influencing the course and results of treatment in dermatophyte onychomycosis.
Assuntos
Antifúngicos/uso terapêutico , Epidermophyton/isolamento & purificação , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos/imunologia , Onicomicose/imunologia , Trichophyton/isolamento & purificação , Antígenos CD19/análise , Complexo CD3/análise , Antígenos CD4/análise , Dermatoses do Pé/tratamento farmacológico , Dermatoses do Pé/imunologia , Dermatoses do Pé/microbiologia , Antígenos HLA-DR/análise , Dermatoses da Mão/tratamento farmacológico , Dermatoses da Mão/imunologia , Dermatoses da Mão/microbiologia , Humanos , Itraconazol/uso terapêutico , Contagem de Linfócitos , Naftalenos/uso terapêutico , Onicomicose/tratamento farmacológico , Onicomicose/microbiologia , Salicilatos/uso terapêutico , TerbinafinaRESUMO
In tumor tissue obtained from a group of 21 patients treated surgically for laryngeal carcinoma, the expression of Fas, Fas-L, CD3, TCR zeta chain and Bcl-2 were estimated. It has been shown that tumor often express: Fas-L, Fas, Bcl-2, the molecules which may influence increased tumor survival. On the other hand lack of MHC class I antigen expression on tumor cells frequently has been observed. As it is known, such tumor cells can not be recognized by cytotoxic T lymphocytes. Frequently observed decreased expression of TCR zeta chain and high level of apoptosis among T cells present in tumor microenvironment seems to depend on direct interactions with molecules expressed by tumor. Understanding of these interactions may be of great importance in evaluation of tumor aggressiveness, patients follow up and the approach to treatment.
Assuntos
Neoplasias Laríngeas/metabolismo , Linfócitos T/metabolismo , Adulto , Apoptose , Complexo CD3/metabolismo , Regulação para Baixo , Proteína Ligante Fas , Feminino , Regulação Neoplásica da Expressão Gênica , Genes MHC Classe I/genética , Humanos , Neoplasias Laríngeas/cirurgia , Masculino , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Regulação para Cima , Receptor fas/metabolismoRESUMO
Alterations in the p53 gene occur frequently and can lead to accumulation of p53 protein in squamous cell carcinomas of the head and neck (SCCHN). Since accumulation of p53 is associated with enhanced presentation of wild-type sequence (wt) p53 peptides to immune cells, the development of pan vaccines against SCCHN has focused on wt p53 epitopes. We used the HLA-A2.1-restricted wt p53(264-272) epitope to generate CTL from circulating precursor T cells of HLA-A2.1(+) healthy donors and patients with SCCHN. Autologous peptide-pulsed dendritic cells were used for in vitro sensitization. CTL specific for the wt p53(264-272) peptide were generated from PBMC obtained from two of seven normal donors and three of seven patients with SCCHN. These CTL were HLA class I restricted and responded to T2 cells pulsed with p53(264-272) peptide as well as HLA-A2-matched SCCHN cell lines naturally presenting the epitope. Paradoxically, none of the tumors in the three patients who generated CTL could adequately present the epitope; two had a wt p53 genotype and no p53 protein accumulation, while the third tumor expressed a point mutation (R to H) in codon 273 that prevents presentation of the p53(264-272) epitope. In contrast, patients who did not generate CTL had tumors that accumulated altered p53 and potentially could present the p53(264-272) epitope. These findings suggest that in vivo, CTL specific for the wt p53(264-272) peptide might play a role in the elimination of tumor cells expressing this epitope and in immunoselection of epitope-loss tumor cells. Immunoselection of tumors that become resistant to anti-p53 immune responses has important implications for future p53-based vaccination strategies.
Assuntos
Carcinoma de Células Escamosas/imunologia , Epitopos de Linfócito T/metabolismo , Neoplasias de Cabeça e Pescoço/imunologia , Epitopos Imunodominantes/metabolismo , Ativação Linfocitária/genética , Fragmentos de Peptídeos/metabolismo , Subpopulações de Linfócitos T/imunologia , Proteína Supressora de Tumor p53/metabolismo , Autoanticorpos/sangue , Carcinoma de Células Escamosas/genética , Citotoxicidade Imunológica/genética , Análise Mutacional de DNA , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Citometria de Fluxo , Variação Genética/imunologia , Antígeno HLA-A2/imunologia , Antígeno HLA-A2/metabolismo , Neoplasias de Cabeça e Pescoço/genética , Humanos , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Coloração e Rotulagem , Subpopulações de Linfócitos T/química , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/química , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologiaRESUMO
It has been suggested that dendritic cells (DCs) are capable of ingesting apoptotic tumor cells (ATCs) and presenting tumor-associated antigens to immune cells. We evaluated the potential of human DCs, which have ingested ATCs, to serve as a source of antigenic epitopes for presentation to T cells specific for PCI-13, a squamous cell carcinoma of the head and neck cell line. Immature DCs (DCimm) generated in the presence of interleukin 4 and granulocyte machrophage colony-stimulating factor from peripheral blood monocytes of HLA-A2+ healthy donors were incubated in the presence of ATCs. Uptake of ATCs by DCs was monitored by flow cytometry and confocal microscopy after 2-18 h of coincubation. When DCs were matured (DCmat) in the presence of proinflammatory cytokines, their capacity to uptake ATCs was reduced. Responses of PCI-13-specific CD8+ T cells to unmodified PCI-13 cells and to DCimm or DCmat +/- ATCs or +/- tumor lysates were tested in gamma-IFN enzyme-linked immunospot and cytotoxicity assays. Unmodified tumor cells were found to be the best stimulators of antitumor activity of the established T-cell line, and ATCs alone were minimally stimulatory. However, DCs that ingested ATCs were able to present tumor antigens to CTLs, and DCimm and DCmat were almost equally stimulatory. When DCs plus various tumor-derived preparations were used as antigen-presenting cells with autologous HLA-A2+ T cells obtained from normal donors, DCs that had ingested ATCs were more effective in generating CD8+ CTLs than tumor cells alone or DCs pulsed with tumor lysates. The results indicate that human DCs fed with ATCs and then matured effectively generated T cell-mediated antitumor responses in vitro.
Assuntos
Apoptose , Linfócitos T CD8-Positivos/imunologia , Citocinas/farmacologia , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Linfócitos T Citotóxicos/imunologia , Antígenos CD/análise , Carcinoma de Células Escamosas , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Antígeno HLA-A2/imunologia , Neoplasias de Cabeça e Pescoço , Humanos , Interferon gama/análise , Interferon gama/biossíntese , Interleucina-4/farmacologia , Células K562 , Proteínas Recombinantes/farmacologia , Células Tumorais CultivadasRESUMO
Peripheral blood mononuclear cells (PBMCs) obtained from patients with advanced melanoma but not from healthy individuals were found to undergo spontaneous ex vivo apoptosis upon incubation in medium. PBMCs were evaluated for evidence of apoptosis using Annexin V binding, caspase-3 activation, and DNA fragmentation (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). PBMCs of patients with melanoma contained a significantly higher proportion (P = 0.0027) of spontaneously apoptotic cells than PBMCs of controls after 24-h incubation in medium alone. The relative proportion of activated Fas+ and tumor necrosis factor receptor 1-positive (TNFR1+) PBMCs was significantly higher in patients with melanoma than that observed in controls. To demonstrate that the TNF family of receptors and ligands was involved in this type of apoptosis, PBMCs were incubated in the presence of agonistic anti-Fas antibody (CH-11) or TNF-alpha. The proportion of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive PBMCs undergoing spontaneous apoptosis was found to be comparable with that induced by CH-11 antibody or TNF-alpha. Three-color flow cytometry revealed that CD3+ Fas+ T cells were especially sensitive to apoptosis and were preprogrammed in vivo to die. Apoptosis occurred in all subsets of PBMCs but was significantly higher (P = 0.01) in the CD3+ CD8+ T-cell subset in patients relative to controls. In two patients with melanoma, who responded clinically to dendritic cell-based peptide vaccines, the proportion of apoptotic T cells was decreased by half after therapy. In patients who were treated previously with vaccination-based therapies, levels of T-cell apoptosis were lower than in the other melanoma patients. The observed accelerated death of T cells, which are activated and susceptible to apoptosis in patients with melanoma, may contribute to a rapid turnover of immune cells, resulting in a decreased immunocompetence.
Assuntos
Apoptose , Linfócitos T CD8-Positivos/citologia , Melanoma/patologia , Adulto , Idoso , Anexina A5/metabolismo , Antígenos CD/análise , Antígenos CD19/análise , Complexo CD3/análise , Antígenos CD4/análise , Antígeno CD56/análise , Antígenos CD8/análise , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Caspase 3 , Caspases/metabolismo , Estudos de Coortes , Fragmentação do DNA , Feminino , Citometria de Fluxo , Humanos , Marcação In Situ das Extremidades Cortadas , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Receptores de Lipopolissacarídeos/análise , Masculino , Melanoma/imunologia , Melanoma/metabolismo , Pessoa de Meia-Idade , Ligação Proteica , Receptores de IgG/análise , Receptores do Fator de Necrose Tumoral/análise , Receptores Tipo I de Fatores de Necrose Tumoral , Receptor fas/análiseRESUMO
Enhanced green fluorescent protein (EGFP) is a novel marker gene product, which is readily detectable using techniques of fluorescence microscopy, flow cytometry, or macroscopic imaging. In the present studies, we have examined the immunogenicity of EGFP in murine models. A stable transfectant of the transplantable CMS4 sarcoma of BALB/c origin expressing EGFP, CMS4-EGFP-Zeo, was generated. Splenocytes harvested from mice immunized with a recombinant adenovirus expressing EGFP (Ad-EGFP) were restimulated in vitro with CMS4-EGFP-Zeo. Effector lymphocytes displayed strong cytotoxicity against CMS4-EGFP-Zeo, but not against mock-transfected CMS4-Zeo tumor cells. A number of candidate H2-Kd-binding peptides derived from the EGFP protein were chosen according to an epitope prediction program and synthesized. These peptides were tested for their ability to bind to H2-Kd molecules and stimulate IFNgamma-production by splenocytes harvested from Ad-EGFP-immunized mice. Using this methodology, the peptide, HYLSTQSAL (corresponding to EGFP200-208) which strongly binds to H2-Kd molecules, was identified as a naturally occurring epitope of EGFP. These results should facilitate the use of EGFP as a model tumor antigen in BALB/c mice.
Assuntos
Antígenos de Neoplasias/análise , Vacinas Anticâncer , Antígenos H-2/imunologia , Proteínas Luminescentes/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Proteínas de Fluorescência Verde , Camundongos , Camundongos Endogâmicos BALB C , Modelos AnimaisRESUMO
In search of factors governing the accumulation of tumor infiltrating lymphocytes (TIL), frozen sections from fresh surgical specimens of laryngeal carcinoma (n = 36) were tested by alkaline phosphatase-anti-alkaline phosphatase (APAAP) immunohistochemistry for monomorphic determinants of HLA class I and class II expression on tumor cells and for the distribution of lymphoid cells bearing CD differentiation antigens. Cell subsets were quantitated in two tumor compartments, tumor mass and tumor stroma, by computer-assisted image analysis. In a portion of examined samples lymphoid cell suspension was isolated from cancerous tissues and assessed by flow cytometry. It has been found that T cells, localized mostly in tumor stroma, were predominant cell population in the tumor microenvironment. Their ability to penetrate tumor mass but not tumor stroma, by CD8+ T cells in particular, but also by natural killer (NK) cells, was associated with HLA class I antigen expression on tumor cells. In flow cytometric analysis activated T lymphocytes (CD3+DR+) were abundant in HLA+ tumors as compared to HLA- ones. In 4 year follow up of 20 patients the mortality was higher in HLA- group but the data were not statistically significant. These results show that HLA class I expression on tumor cells favor penetration of cytotoxic lymphoid cells into tumor mass, at least in the laryngeal cancer.