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HER-2/neu is the human epidermal growth factor receptor 2, which is associated with the progression of prostate cancer (PCa). HER-2/neu-specific T cell immunity has been shown to predict immunologic and clinical responses in PCa patients treated with HER-2/neu peptide vaccines. However, its prognostic role in PCa patients receiving conventional treatment is unknown, and this was addressed in this study. The densities of CD8+ T cells specific for the HER-2/neu(780-788) peptide in the peripheral blood of PCa patients under standard treatments were correlated with TGF-ß/IL-8 levels and clinical outcomes. We demonstrated that PCa patients with high frequencies of HER-2/neu(780-788)-specific CD8+ T lymphocytes had better progression-free survival (PFS) as compared with PCa patients with low frequencies. Increased frequencies of HER-2/neu(780-788)-specific CD8+ T lymphocytes were also associated with lower levels of TGF-ß and IL-8. Our data provide the first evidence of the predictive role of HER-2/neu-specific T cell immunity in PCa.
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Vacinas Anticâncer , Neoplasias da Próstata , Humanos , Masculino , Epitopos , Interleucina-8 , Linfócitos T CD8-Positivos , Receptor ErbB-2/metabolismoRESUMO
Breast cancer is a heterogeneous and complex disease. Although the use of tumor biomarkers has improved individualized breast cancer care, i.e., assessment of risk, diagnosis, prognosis, and prediction of treatment outcome, new markers are required to further improve patient clinical management. In the present study, a search for novel breast cancer-associated genes was performed by mining the UniGene database for expressed sequence tags (ESTs) originating from human normal breast, breast cancer tissue, or breast cancer cell lines. Two hundred and twenty-eight distinct breast-associated UniGene Clusters (BUC1-228) matched the search criteria. Four BUC ESTs (BUC6, BUC9, BUC10, and BUC11) were subsequently selected for extensive in silico database searches, and in vitro analyses through sequencing and RT-PCR based assays on well-characterized cell lines and tissues of normal and cancerous origin. BUC6, BUC9, BUC10, and BUC11 are clustered on 10p11.21-12.1 and showed no homology to any known RNAs. Overall, expression of the four BUC transcripts was high in normal breast and testis tissue, and in some breast cancers; in contrast, BUC was low in other normal tissues, peripheral blood mononuclear cells (PBMCs), and other cancer cell lines. Results to-date suggest that BUC11 and BUC9 translate to protein and BUC11 cytoplasmic and nuclear protein expression was detected in a large cohort of breast cancer samples using immunohistochemistry. This study demonstrates the discovery and expression analysis of a tissue-restricted novel transcript set which is strongly expressed in breast tissue and their application as clinical cancer biomarkers clearly warrants further investigation.
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Biomarcadores Tumorais , Neoplasias da Mama/genética , Mama/metabolismo , Perfilação da Expressão Gênica , Transcriptoma , Adulto , Idoso , Idoso de 80 Anos ou mais , Processamento Alternativo , Azacitidina/farmacologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Biologia Computacional , Mineração de Dados , Bases de Dados de Ácidos Nucleicos , Etiquetas de Sequências Expressas , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Transcrição GênicaRESUMO
OBJECTIVE: To identify changes in peripheral immune responses in patients with metastatic colorectal cancer (mCRC) treated with irinotecan/5-fluorouracil/leucovorin (IFL) alone or in combination with cetuximab (C-IFL). METHODS: Peripheral blood mononuclear cells (PBMCs) collected from healthy donors (n = 20) and patients with mCRC receiving treatment with either IFL (n = 30) or C-IFL (n = 30) were tested for cytokine production upon polyclonal stimulation with anti-CD3 monoclonal antibody, T cell proliferation in the autologous mixed lymphocyte reaction (auto-MLR) and T regulatory cell (Treg) frequency. The respective results were evaluated over two treatment cycles and further assessed in relation to response to treatment. RESULTS: PBMCs prior to treatment exhibited significantly lower production of IL-2, IFN-γ, IL-12 and IL-18 cytokines and lower auto-MLR responses, whereas Treg frequency, IL-4, IL-10 cytokines were increased compared to healthy donors. During treatment, IL-2, IFN-γ, IL-12, IL-18 and auto-MLR responses increased, while Treg frequency and IL-10 secretion decreased significantly compared to the baseline. Responders to treatment exhibited a significantly higher increase in IL-2, IFN-γ, IL-12 and IL-18 production and auto-MLR responses, and higher decrease in IL-4, IL-10 secretion and Treg frequency. Among all patient subgroups analysed, responders to C-IFL demonstrated significantly higher increase in auto-MLR responses, IL-12 and IL-18 secretion and higher decrease in Treg frequency. CONCLUSION: The disturbed immune parameters observed in patients with mCRC at presentation can be significantly improved during treatment with IFL and this effect can be potentiated by the addition of cetuximab. Monitoring of the peripheral immune system function could be used as surrogate marker in predicting treatment-related outcome.
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Anticorpos Monoclonais Humanizados/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Idoso , Anticorpos Monoclonais/imunologia , Complexo CD3/metabolismo , Camptotecina/análogos & derivados , Camptotecina/uso terapêutico , Cetuximab , Neoplasias Colorretais/imunologia , Citocinas/metabolismo , Feminino , Fluoruracila/uso terapêutico , Humanos , Leucovorina/uso terapêutico , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Fenótipo , Linfócitos T Reguladores/imunologiaRESUMO
Prostate cancer (PCa) is one of the most common male cancers worldwide and one of the deadliest if unsuccessfully treated. Τhe need for reliable, easily accessible immune-related molecular biomarkers that could be combined with clinically defined criteria, including PSA and Gleason score, to accurately predict PCa patients' clinical outcomes is emerging. Herein, we describe for the first time a blood-identified immune-related gene signature comprising eight upregulated multi-functional genes associated with poor prognosis. Next-generation sequencing (NGS) analysis of PCa patients' peripheral blood samples revealed a more than three-fold upregulation of each of the eight genes as compared to samples originating from healthy donors. The construction of gene and protein interaction networks revealed different extents of the functional implications of these genes in the regulation of cell proliferation and immune responses. Analysis of the available data from The Cancer Genome Atlas (TCGA) regarding gene expression and survival of prostate adenocarcinoma (PRAD) and pan-cancer (PANCAN) patients revealed that intra-tumoral upregulation of this eight-gene signature (8-GS) was associated with poor 5-year progression-free intervals in PCa patients, even in those with high Gleason scores, and also with an unfavorable prognosis for cancer patients irrespective of the cancer type and even in the early stages. These observations suggest that further investigation of the 8-GS prospectively in randomized clinical trials, in which clinical benefit in terms of evaluating time to disease progression can be assessed, is warranted.
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The authors wish to make the following corrections to this paper [...].
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Disrupting tumor-mediated mechanisms suppressing host immunity represents a novel approach to tumor immunotherapy. Depletion of regulatory T cells (Tregs) increases endogenous anti-tumor immunity and the efficacy of active immunotherapy in experimental tumor models. HLA-A2.1/HLA-DR1 (A2.1/DR1) × BALB- neuT+ (neuT+) triple transgenic mice represent an improvement over neuT+ mice for evaluating vaccination regimens to overcome tolerance against HER-2/neu. We questioned whether depletion of Tregs with Denileukin diftitox (Ontak) enhances the efficacy of a therapeutic vaccine consisting of HER-2(85-94) (p85) CTL and HER-2(776-790) (p776) Th peptides against the growth of TUBO.A2 transplantable tumor in male A2.1/DR1 × neuT+ Tg mice. While the therapeutic vaccine primed the tumor-reactive CD8+ CTLs and CD4+ effector T lymphocytes (Teffs) compartment, inducing activation, tumor infiltration, and tumor rejection or delay in tumor growth, treatment with Ontak 1 day prior to vaccination resulted in enhanced CD4+ and CD8+ T-cell-mediated vaccine-specific immune responses in the periphery. This was closely associated with greater infiltration and a striking change in the intratumor balance of Tregs and vaccine-specific CTLs/Teffs that directly correlated with markedly enhanced antitumor activity. The data suggest that Tregs control both CD4+ and CD8+ T-cell activity within the tumor, emphasize the importance of the intratumor ratio of vaccine-specific lymphocytes to Tregs, and demonstrate significant inversion of this ratio and correlation with tumor rejection during Ontak/vaccine immunotherapy.
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Toxina Diftérica/farmacologia , Interleucina-2/farmacologia , Neoplasias Mamárias Experimentais/terapia , Receptor ErbB-2/imunologia , Vacinação/métodos , Animais , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Toxina Diftérica/imunologia , Feminino , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Antígeno HLA-DR1/genética , Antígeno HLA-DR1/imunologia , Humanos , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/imunologia , Imunossupressores/imunologia , Imunossupressores/farmacologia , Interleucina-2/imunologia , Masculino , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Ratos , Receptor ErbB-2/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/imunologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
BACKGROUND: Bevacizumab, a monoclonal antibody (mAb) targeting vascular endothelial growth factor (VEGF), has produced promising results when combined with chemotherapy in the treatment of advanced colorectal cancer (CRC). The aim of the present study was to define the immunological profile of metastatic CRC patients at baseline and following chemotherapy with either irinotecan/5-fluorouracil/leucovorin (IFL) alone or IFL in combination with.bevacizumab (B-IFL). METHODS: Peripheral blood mononuclear cells (PBMCs) obtained from healthy donors (HD) (n = 20) and patients (n = 40) were tested for T-cell proliferation in the autologous mixed lymphocyte reaction (auto-MLR), and cytokine production following stimulation with anti-CD3 mAb. RESULTS: PBMCs obtained from CRC patients prior to treatment exhibited lower auto-MLR responses and low production of IL-2, IFN-γ, IL-12 and IL-18 cytokines, whereas IL-4 and IL-10 cytokines were increased as compared to HD (p < 0.001, for all parameters) following in vitro stimulation with anti-CD3 mAb. During treatment, and in particular in week 12 of evaluation, IL-2 (p < 0.001 for both IFL and B-IFL groups), IFN-γ (p < 0.001 for IFL and p = 0.001 for B-IFL), IL-12 (p < 0.001 for both IFL and B-IFL) and IL-18 (p < 0.001 for both IFL and B-IFL) production, as well as auto-MLR responses increased (p < 0.001 for both IFL and B-IFL), whereas IL-4 (p < 0.001 for IFL and p = 0.001 for B-IFL) and IL-10 [p < 0.001 for IFL and p = 0.067 (non-significant) for B-IFL] production decreased over baseline in the two treatment groups, yet their respective values never reached those of HD. Moreover, IL-2, IFN-γ production, and auto-MLR were higher in the B-IFL over the IFL treatment group (p < 0.001, p < 0.04, p < 0.001, respectively). CONCLUSION: Our study demonstrates that the abnormal immune parameters observed in metastatic CRC patients at presentation can substantially improve during treatment with either IFL or B-IFL. The immune parameters examined can provide a sensitive and valuable tool for monitoring immune function in CRC patients, and could be applied as surrogate markers predicting treatment-related outcome.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Idoso , Inibidores da Angiogênese/administração & dosagem , Anticorpos Monoclonais Humanizados/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Bevacizumab , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/secundário , Citocinas/metabolismo , Feminino , Fluoruracila/administração & dosagem , Grécia , Humanos , Irinotecano , Leucovorina/administração & dosagem , Teste de Cultura Mista de Linfócitos , Masculino , Pessoa de Meia-Idade , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fatores de Tempo , Resultado do TratamentoRESUMO
During the last decade, there has been significant progress in the field of prostate cancer therapeutic treatments based on androgen receptor-axis-targeted therapies, which resulted in improved clinical outcomes [...].
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Acquired immune resistance (AIR) describes a situation in which cancer patients who initially responded clinically to immunotherapies, after a certain period of time, progress with their disease. Considering that AIR represents a feedback response of the tumor against the immune attack generated during the course of immunotherapies, it is conceivable that AIR may also occur before treatment initiation as a mechanism to escape endogenous adaptive antitumor immunity (EAAI). In the present study, we assessed the EAAI in paraffin-embedded breast primary tumor tissue samples and drew correlations with the clinical outcomes. In particular, we analyzed densities of CD8+ cells as elements mediating antitumor cytotoxicity, and of CD163+ and FoxP3+ cells as suppressor elements. We found a direct correlation between the densities of CD8+ cells and of CD163+ and/or FoxP3+ cells in the vast majority of patients' tumors. Importantly, the vast majority of patients whose tumors were overpopulated by CD8+ cells developed AIR, which was characterized by high intratumoral CD163+ and/or FoxP3+ cell densities and reduced overall survival (OS). We also showed that AIR depends on the levels of CD8+ cell-ratios in the tumor center to the invasive margin. Our data suggest that tumors develop AIR only when under a robust endogenous immune pressure.
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Radiotherapy for localized prostate cancer has increased the cure and survival rates of patients. Besides its local tumoricidal effects, ionizing radiation has been linked to mechanisms leading to systemic immune activation, a phenomenon called the abscopal effect. In this study, we performed gene expression analysis on peripheral blood from prostate cancer patients obtained post- radiotherapy and showed that 6 genes, including CCR7, FCGR2B, BTLA, CD6, CD3D, and CD3E, were down-regulated by a range of 1.5-2.5-fold as compared to pre-radiotherapy samples. The expression of the signature consisting of these six genes was also significantly lower post- vs. pre-radiotherapy. These genes are involved in various tumor-promoting immune pathways and their down-regulation post-radiotherapy could be considered beneficial for patients. This is supported by the fact that low mRNA expression levels for the 6-gene signature in the prostate tumor tissue was linked to better survival. Importantly, we report that this 6-gene signature strongly correlated with a favorable prognosis regardless of poor standard clinicopathological parameters (i.e., Gleason score ≥ 8 and T3 (including T3a and T3b). Our pioneering data open the possibility that the 6-gene signature identified herein may have a predictive value, but this requires further long-term studies.
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Radiotherapy (RT) is a therapeutic modality that aims to eliminate malignant cells through the induction of DNA damage in the irradiated tumor site. In addition to its cytotoxic properties, RT also induces mechanisms that result in the promotion of antitumor immunity both locally within the irradiation field but also at distant tumor lesions, a phenomenon that is known as the "abscopal" effect. Because the immune system is capable of sensing the effects of RT, several treatment protocols have been assessing the synergistic role of radiotherapy combined with immunotherapy, collectively referred to as radioimmunotherapy. Herein, we discuss mechanistic insights underlying RT-based immunomodulation, which also enhance our understanding of how RT regulates antitumor T-cell-mediated immunity. Such knowledge is essential for the discovery of predictive biomarkers and for the improvement of clinical trials investigating the efficacy of radio-immunotherapeutic modalities in cancer patients.
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Radiation therapy (RT) is an essential component in the therapeutic treatment of patients with localized prostate cancer (LPCa). Besides its local effects, ionizing radiation has been linked to mechanisms leading to systemic immune activation. The present study explored the effect of RT on the Tcell receptor variable ß (TCR Vß) chain repertoire of peripheral blood T cells in patients with LPCa. Highthroughput TCR Vß sequencing was performed on 20 blood samples collected from patients with LPCa at baseline and 3 months postRT. The diversity index was altered, as were TCR Vß clonal evenness and convergence before and postRT; however, these findings were not significant. Notably, marked changes in the frequencies among the top 10 TCR Vß clonotypes were detected and some patients developed new clonotypes of high abundance. These data provided initial evidence that RT in patients with LPCa may induce systemic immune changes, which could be exploited by future therapies for improved clinical results.
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Neoplasias da Próstata , Receptores de Antígenos de Linfócitos T alfa-beta , Humanos , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/radioterapia , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
Our aim is to develop peptide vaccines that stimulate tumor antigen-specific T-lymphocyte responses against frequently detected cancers. We describe herein a novel HLA-A*0201-restricted epitope, encompassing amino acids 828-836 (residues QIAKGMSYL), which is naturally presented by various HER-2/neu (+) tumor cell lines. HER-2/neu(828-836), [HER-2(9(828))], possesses two anchor residues and stabilized HLA-A*0201 on T2 cells in a concentration-dependent Class I binding assay. This peptide was stable for 3.5 h in an off-kinetic assay. HER-2(9(828)) was found to be immunogenic in HLA-A*0201 transgenic (HHD) mice inducing peptide-specific and functionally potent CTL and long-lasting anti-tumor immunity. Most important, using HLA-A*0201 pentamer analysis we could detect increased ex vivo frequencies of CD8(+) T-lymphocytes specifically recognizing HER-2(9(828)) in 8 out of 20 HLA-A*0201(+) HER-2/neu (+) breast cancer patients. Moreover, HER-2(9(828))-specific human CTL recognized the tumor cell line SKOV3.A2 as well as the primary RS.A2.1.DR1 tumor cell line both expressing HER-2/neu and HLA-A*0201. Finally, therapeutic vaccination with HER-2(9(828)) in HHD mice was proven effective against established transplantable ALC.A2.1.HER tumors, inducing complete tumor regression in 50% of mice. Our data encourage further exploitation of HER-2(9(828)) as a promising candidate for peptide-based cancer vaccines.
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Vacinas Anticâncer/imunologia , Epitopos de Linfócito T/imunologia , Imunoterapia/métodos , Neoplasias/terapia , Receptor ErbB-2/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Neoplasias da Mama/imunologia , Separação Celular , Citometria de Fluxo , Antígenos HLA-A/imunologia , Antígeno HLA-A2 , Humanos , Camundongos , Neoplasias/imunologia , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/terapia , Fragmentos de Peptídeos/imunologiaRESUMO
Semen hyperviscosity impairs sperm motility and can lead to male infertility. This prospective study aimed at assessing the ability of exogenous DNase in improving sperm quality, taking into consideration that DNase has been found in the seminal plasma of several species and that neutrophils release chromatin in order to trap bacteria. A total of seventy-seven semen samples with high seminal viscosity (HSV) as the study group and sixty-two semen samples with normal seminal viscosity (NSV) as the control group were compared in this analysis. These semen samples were divided into three groups of receiving treatment (a) with DNase I at 37°C for 15 min, (b) by density gradient centrifugation, and (c) with a combination of the above two methods. Following a fifteen-minute treatment of hyperviscous semen, the motility of spermatozoa in 83% of semen samples increased to a statistically significant degree. On the contrary, DNase treatment of semen with normal viscosity had no such effects. The above treatment was also accompanied by a significant increase in the percentage of normal spermatozoa, resulting in a major decrease of the teratozoospermia index. Comparison between semen samples that underwent density gradient centrifugation following DNase I treatment, to those collected after density gradient treatment alone, showed that in the first case the results were more spectacular. The evaluation of each preparation in terms of yield (% total progressively motile sperm count after treatment in relation to the initial total sperm count) revealed that the combined approach resulted in 29.8% vs. 18.5% with density treatment alone (p=0.0121). DNase I treatment results in an improvement of sperm motility and morphology and could be beneficial to men with hyperviscous semen in assisted reproduction protocols.
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HER-2/neu is a self-antigen expressed by tumors and nonmalignant epithelial tissues. The possibility of self-tolerance to HER-2/neu-derived epitopes has raised questions concerning their utility in antitumor immunotherapy. Altered HER-2/neu peptide ligands capable of eliciting enhanced immunity to tumor-associated HER-2/neu epitopes may circumvent this problem. The human CTL peptide HER-2/neu (435-443) [hHER-2(9(435))] represents a xenogeneic altered peptide ligand of its mouse homologue, differing by one amino acid residue at position 4. In contrast to mHER-2(9(435)), vaccination of HLA-A*0201 transgenic (HHD) mice with hHER-2(9(435)) significantly increased the frequency of mHER-2(9(435))-specific CTL and also induced strong protective and therapeutic immunity against the transplantable ALC tumor cell line transfected to coexpress HLA-A*0201 and hHER-2/neu or rHER-2/neu. Similar results were also obtained with wild-type C57BL/6 mice inoculated with HER-2/neu transfectants of ALC. Adoptive transfer of CD8(+) CTL from mice immunized with hHER-2(9(435)) efficiently protected naive syngeneic mice inoculated with ALC tumors. In conclusion, our results show that HER-2(9(435)) serves as a tumor rejection molecule. They also propose a novel approach for generating enhanced immunity against a self-HER-2/neu CTL epitope by vaccinating with xenogeneic altered peptide ligands and provide useful insights for the design of improved peptide-based vaccines for the treatment of patients with HER-2/neu-overexpressing tumors.
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Epitopos de Linfócito T/imunologia , Linfoma/prevenção & controle , Neoplasias Ovarianas/prevenção & controle , Fragmentos de Peptídeos/imunologia , Receptor ErbB-2/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Antígenos HLA-A/genética , Antígenos HLA-A/imunologia , Antígeno HLA-A2 , Humanos , Imunoterapia Ativa/métodos , Linfoma/genética , Linfoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/imunologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Receptor ErbB-2/genética , TransfecçãoRESUMO
Currently, a time consuming biochemical method is used for GR quantification. Here we compare the biochemical approach with a newly developed flow cytometric method of measuring GR in cell lines, which is less time consuming and does not requires the use of radioactive materials. The biochemical assay is easy to apply but the cells need to be grown in media free of endogenous glucocorticoids, in order to prevent them from interfering with radiolabelled hormone binding to the receptor. The presence of endogenous GR ligands is known to reduce receptor levels and to often produce false negative results. The immunofluorescent method is free of such limitations, as it depends entirely on detecting the receptor using a highly specific monoclonal antibody. Additionally, the biochemical assay cannot measure heterogeneity in individual cells, in contrast the flow cytometric one enables the enumeration of the receptor on a per cell basis, allowing exact description of differences in receptor levels amongst intact cells. Our results demonstrate that the flow cytometric method is of similar accuracy but of higher precision compared to the biochemical one. Also, the data we obtained using the immunofluorescent method correlated well with the biochemical one (R(2)=0.9712). In conclusion, flow cytometric method requires small cell numbers, is more accurate and lesser time consuming than the biochemical one. Thus, it could be useful for the quantification of GR in lymphocyte subpopulations, in lymphoproliferative disorders and in tumor cells from cancer patients, in order to design more efficient clinical treatment protocols.
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Citometria de Fluxo/métodos , Receptores de Glucocorticoides/análise , Receptores de Glucocorticoides/química , Anticorpos Monoclonais/metabolismo , Linhagem Celular Tumoral , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Células Jurkat , Células K562 , Receptores de Glucocorticoides/imunologia , Receptores de Glucocorticoides/metabolismoRESUMO
BACKGROUND: CD4+ cells expressing Interferon-γ (IFN-γ), following stimulation with specific mycobacterial antigens, identified with flow cytometry (FCM-CD4+IFN-γ+), is a new method for the diagnosis of Mycobacterium tuberculosis (MTB) infection. The aim of this study is to investigate the performance of FCM-CD4+IFN-γ+ in comparison with tuberculin skin test (TST) and Quantiferon TB Gold In-Tube (QFT-G-IT) in the diagnosis of latent MTB infection (LTBI), in close contacts and in patients with rheumatic diseases under treatment with anti-TNFa and other biologic agents. METHODS: TST, QFT-G-IT, and FCM-CD4+IFN-γ+ were performed in 56 immunocompetent close contacts and in 65 medically immunosuppressed patients under biologic treatment. RESULTS: In close contacts, 63% were FCM-CD4+IFN-γ+ ESAT-6(+), 70% FCM-CD4+IFN-γ+ PPD(+), 41% QFT-G-IT(+) and 57% TST(+). FCM-CD4+IFN-γ+ ESAT-6 was the only test that was strongly correlated to the exposure time to infection. In the immunosuppressed group, 49% were FCM-CD4+IFN-γ+ ESAT-6(+), 62% FCM-CD4+IFN-γ+ PPD(+), 4.6% QFT-G-IT(+), and 18% TST(+). CONCLUSION: FCM-CD4+IFN-γ+ assays are more sensitive than QFT-G-IT and TST for the diagnosis of LTBI in close contacts and in immunosuppressed patients under anti-TNF-a treatment. FCM-CD4+IFN-γ+ is not affected by the chronic use of biologic agents. © 2015 International Clinical Cytometry Society.
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Linfócitos T CD4-Positivos/imunologia , Citometria de Fluxo , Mycobacterium tuberculosis/citologia , Tuberculose/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Citometria de Fluxo/métodos , Humanos , Interferon gama/imunologia , Masculino , Pessoa de Meia-Idade , Tuberculose/metabolismo , Adulto JovemRESUMO
BACKGROUND: Early secreted antigenic target 6 (ESAT-6) is a virulent factor of Mycobacterium tuberculosis (MTB). The identification of intracellular (i/c) ESAT-6 in host cells would be a direct marker of MTB infection. We developed a method to detect i/cESAT-6 by flow cytometry. The aim of this study is to investigate the expression of i/cESAT-6 in the host cells of individuals with MTB infection. METHODS: The expression of i/cESAT-6 was examined in the blood of 58 active TB patients, in 10 naïve to TB infection controls, in 17 patients who completed anti-TB treatment, and in 56 close contacts with an index TB case. Additionally, it was examined in the sputum of 12 active TB patients. RESULTS: The i/cESAT-6 was positively detected in the blood of 52 out of 58 (90%) active TB patients. All naïve to TB infection controls were negative. Three out of 17 (18%) patients at the end of anti-TB treatment were positive. Twenty-six out of 56 (46%) close contacts tested positive. The i/cESAT-6 was detected in all culture positive TB sputum specimens. CONCLUSIONS: The i/cESAT-6 is a promising biomarker of MTB infection that could be used in the evaluation of active TB patients and in the diagnosis of latent TB infection. Further studies are needed to validate its potential diagnostic role. © 2014 International Clinical Cytometry Society.
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Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Citometria de Fluxo , Mycobacterium tuberculosis/citologia , Escarro/metabolismo , Biomarcadores/metabolismo , Citoplasma/metabolismo , Citometria de Fluxo/métodos , Humanos , Interferon gama/biossíntese , Mycobacterium tuberculosis/imunologiaRESUMO
PURPOSE: Overexpression of human epidermal growth factor receptor-2 (HER-2/neu) in breast cancer patients is a prerequisite for treatment with trastuzumab. In the present study, we demonstrate by fluorescence in situ hybridization (FISH) analysis that HER-2/neu gene amplification and chromosome 17 (CEP17) polysomy can be induced by irradiation in human breast cancer cell lines with low basal level of HER-2/neu. MATERIALS AND METHODS: The irradiation-induced HER-2/neu gene amplification and CEP17 polysomy enhanced HER-2/neu at the protein level in both human MDA-MB-231 and MDA-MB-435 breast cancer cell lines which was determined by immunohistochemistry and fluorescence analysis and was correlated with mRNA levels. RESULTS: Irradiation affected to a high degree the responsiveness of both cell lines to in vitro treatment with trastuzumab. The direct antiproliferative effect of trastuzumab, as well as its capacity to induce natural killer (NK) cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC), was considerably higher in the irradiated tumor cells compared to their non-irradiated counterparts. CONCLUSION: Our data demonstrate that irradiation induces HER-2/neu gene amplification and CEP17 polysomy thereby enhancing expression of this protein in breast cancer cell lines rendering them susceptible to treatment with trastuzumab. They also suggest that patients with HER-2/neu negative inoperable tumors undergoing local radiation therapy may benefit from treatment with trastuzumab.
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Anticorpos Monoclonais Humanizados/farmacologia , Neoplasias da Mama/patologia , Terapia Combinada/métodos , Raios gama/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Receptor ErbB-2/metabolismo , Anticorpos Monoclonais Humanizados/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Aberrações Cromossômicas/efeitos dos fármacos , Aberrações Cromossômicas/efeitos da radiação , Cromossomos Humanos Par 17/efeitos dos fármacos , Cromossomos Humanos Par 17/genética , Cromossomos Humanos Par 17/efeitos da radiação , Resistencia a Medicamentos Antineoplásicos/efeitos da radiação , Humanos , Hibridização in Situ Fluorescente , TrastuzumabRESUMO
A novel modality toward the treatment of HER-2/neu-positive malignancies, mostly including breast and, more recently prostate carcinomas, has been the use of vaccines targeting HER-2/neu extracellular and intracellular domains. HER-2/neu-specific vaccines have been demonstrated to generate durable T-cell anti-HER-2/neu immunity when tested in Phase I and II clinical trials with no significant toxicity or autoimmunity directed against normal tissues. Targeting of HER-2/neu in active immunotherapy may involve peptide and DNA vaccines. Moreover, active anti-HER-2/neu immunization could facilitate the ex vivo expansion of HER-2/neu-specific T cells for use in adoptive immunotherapy for the treatment of established metastatic disease. In addition, early data from trials examining the potential use of HER-2/neu-based vaccines in the adjuvant setting to prevent the relapse of breast cancer in high-risk patients have shown promising results. Future approaches include multiepitope preventive vaccines and combinatorial treatments for generating the most efficient protective anti-tumor immunity.