RESUMO
T-cell prolymphocytic leukemia (T-PLL) is a poor-prognostic neoplasm. Differentiation stage and immune-effector functions of the underlying tumor cell are insufficiently characterized. Constitutive activation of the T-cell leukemia 1A (TCL1A) oncogene distinguishes the (pre)leukemic cell from regular postthymic T cells. We assessed activation-response patterns of the T-PLL lymphocyte and interrogated the modulatory impact by TCL1A. Immunophenotypic and gene expression profiles revealed a unique spectrum of memory-type differentiation of T-PLL with predominant central-memory stages and frequent noncanonical patterns. Virtually all T-PLL expressed a T-cell receptor (TCR) and/or CD28-coreceptor without overrepresentation of specific TCR clonotypes. The highly activated leukemic cells also revealed losses of negative-regulatory TCR coreceptors (eg, CTLA4). TCR stimulation of T-PLL cells evoked higher-than-normal cell-cycle transition and profiles of cytokine release that resembled those of normal memory T cells. More activated phenotypes and higher TCL1A correlated with inferior clinical outcomes. TCL1A was linked to the marked resistance of T-PLL to activation- and FAS-induced cell death. Enforced TCL1A enhanced phospho-activation of TCR kinases, second-messenger generation, and JAK/STAT or NFAT transcriptional responses. This reduced the input thresholds for IL-2 secretion in a sensitizer-like fashion. Mice of TCL1A-initiated protracted T-PLL development resembled such features. When equipped with epitope-defined TCRs or chimeric antigen receptors, these Lckpr-hTCL1Atg T cells gained a leukemogenic growth advantage in scenarios of receptor stimulation. Overall, we propose a model of T-PLL pathogenesis in which TCL1A enhances TCR signals and drives the accumulation of death-resistant memory-type cells that use amplified low-level stimulatory input, and whose loss of negative coregulators additionally maintains their activated state. Treatment rationales are provided by combined interception in TCR and survival signaling.
Assuntos
Memória Imunológica , Leucemia Prolinfocítica de Células T/imunologia , Proteínas Proto-Oncogênicas/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Animais , Humanos , Leucemia Prolinfocítica de Células T/genética , Leucemia Prolinfocítica de Células T/patologia , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais/genética , Linfócitos T/patologiaRESUMO
BACKGROUND: Iron deficiency anemia (IDA) and anemia of chronic diseases (ACD) are common in the geriatric population. However, differentiation between IDA and ACD is still problematic. Hepcidin is a key regulator of iron homeostasis: downregulation in the presence of iron deficiency allows enteral iron resorption, while upregulation in case of chronic inflammation blocks it. We aimed at studying whether serum hepcidin levels might serve as diagnostic parameter to differentiate between IDA and ACD among elderly. PATIENTS AND METHODS: A total of 37 patients (age 69-97 years) were divided into 4 groups: group I (IDA), group II (ACD), group III (controls), and group IV (IDA/ACD). Serum hepcidin levels were analyzed using a commercially available ELISA kit (DRG Instruments, Marburg, Germany). Differences in hepcidin levels were tested with nonparametric methods. RESULTS: We could show a strong positive correlation between serum hepcidin and ferritin (Spearman rho 0.747) and a statistic significant difference of hepcidin levels among all groups (p = 0.034). Hepcidin levels between ACD and controls differed significantly (p = 0.003). CONCLUSION: Despite the small number of patients included in this study, which reduces the strength of the study's evidence, results conform with the current literature: it can be assumed that hepcidin will be used as a diagnostic parameter to differentiate between IDA and ACD in the future. However, more studies with larger patient groups are urgently needed to answer this question.
Assuntos
Anemia Ferropriva/sangue , Anemia Ferropriva/diagnóstico , Ferritinas/sangue , Avaliação Geriátrica/métodos , Hepcidinas/sangue , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Doença Crônica , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A majority of cancer deaths are because of an uncontrolled relapse of the disease despite initial remission after therapy, asking for strategies to control tumour cells in the long term. Adoptive therapy with chimeric antigen receptor (CAR)-redirected T cells showed promising success in primary tumour elimination; the capacity of such engineered T cells to establish enduring tumour protection is currently a matter of discussion, in particular as most targeted 'tumour-associated antigens' are self-antigens. To address the issue in a clinically relevant model that closely mimics the human situation, we recorded rejection of carcinoembryonic antigen (CEA)-positive pancreatic tumours in the CEA transgenic mouse that expressed CEA as self-antigen in healthy cells of the gastrointestinal tract. Adoptive therapy with CD8(+) T cells, which were redirected by a CEA-specific, low-affinity CAR with CD3ζ endodomain, eliminated CEA(+) tumours in a primary response; cured mice produced an efficient recall response in the long term towards CEA(+) tumour cells upon rechallenge. Secondary tumour rejection was CEA specific, mediated by engineered T cells and did not require host T cells. No toxicity towards healthy tissues with CEA expression was recorded. Data indicate that adoptive therapy with engineered T cells can establish self-antigen-specific tumour protection in the long term without autoimmunity.
Assuntos
Carcinoma/prevenção & controle , Neoplasias Pancreáticas/prevenção & controle , Complexo Receptor-CD3 de Antígeno de Linfócitos T/genética , Linfócitos T/imunologia , Animais , Complexo CD3/química , Complexo CD3/genética , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/metabolismo , Carcinoma/terapia , Linhagem Celular Tumoral , Terapia Genética , Células HEK293 , Humanos , Imunoterapia Adotiva , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Pancreáticas/terapia , Estrutura Terciária de Proteína , Linfócitos T/transplanteRESUMO
Low-grade inflammation is important in the development of obesity related pathologies such as insulin resistance and type 2 diabetes, and also cardiovascular disease. Visfatin/PBEF/Nampt and resistin are proinflammatory adipokines secreted from adipocytes, monocytes, and macrophages, and have been linked to atherosclerotic plaque formation, recently. The aim of the present study was to investigate if the expression of these molecules in circulating blood monocytes is altered in obese and/or type 2 diabetic human subjects. Monocytes were isolated by CD14-antibody based magnetic cell sorting from blood samples of 17 lean controls, 20 obese nondiabetic subjects, and 19 obese patients with type 2 diabetes. FACS analysis was performed to test purity of the cell preparations. Expression of the different adipokines was measured by multiplex real-time PCR on RNA-level. Visfatin/PBEF/Nampt was found to be very strongly expressed in monocytes, whereas resistin levels were significantly lower. Furthermore, visfatin/PBEF/Nampt expression was significantly upregulated in obese type 2 diabetic patients, whereas obese nondiabetics exhibited similar levels compared to lean controls, indicating that visfatin/PBEF/Nampt levels are related to type 2 diabetes rather than to obesity. In contrast, resistin expression displayed a different pattern being significantly increased in obese subjects compared to controls but not related to type 2 diabetes. These data suggest a differential role for these two proinflammatory adipokines in linking metabolic diseases to atherosclerosis with visfatin/PBEF/Nampt being more important in patients with type 2 diabetes and resistin in obese but nondiabetic human subjects.
Assuntos
Citocinas/sangue , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/complicações , Monócitos/enzimologia , Nicotinamida Fosforribosiltransferase/sangue , Obesidade/sangue , Obesidade/complicações , Resistina/sangue , Antropometria , Citocinas/genética , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/genética , Feminino , Glucose/farmacologia , Humanos , Inflamação/sangue , Inflamação/complicações , Insulina/farmacologia , Receptores de Lipopolissacarídeos/metabolismo , Masculino , Pessoa de Meia-Idade , Nicotinamida Fosforribosiltransferase/genética , Obesidade/enzimologia , Obesidade/genética , Resistina/genéticaRESUMO
Recent insight into the balance of self-tolerance and auto-aggression has raised interest in using human regulatory T (Treg) cells for adoptive immunotherapy of unlimited autoimmune diseases including type-1 diabetes, rhematoid arthritis and multiple sclerosis. The therapeutic use of Treg cells, however, is so far hampered by the inefficiency of current protocols in making them accessible for genetic manipulations. We report here that TCR/CD3 stimulation that is accompanied by extensive CD28 costimulation makes human Treg cells susceptible to retroviral gene transfer ex vivo while preserving their properties in vitro and in vivo. To show the power of genetic manipulation of human Treg cells, we engineered 'designer Treg cells' by retroviral expression of a chimeric immunoreceptor with defined specificity, which activates Treg cells in a ligand-dependent manner to proliferate, to secrete high amounts of interleukin-10 and to repress an ongoing cytolytic T-cell response in vivo. The procedure in genetically modifying human Treg cells ex vivo will open a panel of applications for their use in the adoptive therapy of deregulated immune responses.
Assuntos
Engenharia Genética/métodos , Imunoterapia Adotiva/métodos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/transplante , Animais , Apoptose/imunologia , Linhagem Celular , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Humanos , Ativação Linfocitária/genética , Camundongos , Camundongos Nus , Receptores Imunológicos/metabolismo , Proteínas Recombinantes/metabolismo , Retroviridae/genética , Sarcoma Experimental/imunologia , Sarcoma Experimental/patologia , Sarcoma Experimental/terapiaRESUMO
Recently, Chlamydia pneumoniae-specific DNA and antigens were reported in the skin of patients with Mycosis fungoides (MF), the most common form of cutaneous T-cell lymphomas. In order to revalidate these data we analyzed skin sections of patients with MF for the expression of three different chlamydial antigens and C. pneumoniae DNA by immunohistochemistry and PCR according to previously described protocolls. Neither C. pneumoniae-specific DNA sequences nor antigens were detected in any of the skin biopses from 24 MF patients tested, suggesting that further studies are needed to establish any pathogenetic relevance of C. pneumoniae in MF.
Assuntos
Infecções por Chlamydophila/fisiopatologia , Chlamydophila pneumoniae , Micose Fungoide/fisiopatologia , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Micose Fungoide/microbiologiaRESUMO
In early stages of cutaneous T cell lymphoma (Sézary syndrome) both CD4+CD7- and CD4+CD7+ T cells clonally expand whereas in late stages of the disease CD7- cells are predominant in number, giving rise to the question whether CD7- T cells have a survival advantage in the skin. Galectin-1, a cell-bound lectin, was recently reported to trigger apoptosis in activated CD7+ T cells. Here, we demonstrate that in contrast to activated CD7(+) T cells, quiescent and activated CD69+ CD7- T cells from healthy donors and from Sézary patients are resistant to galectin-1-mediated cell death. CD7- T cells are apoptosis-resistant even during coculture with IFN-gamma-stimulated endothelial cells that constitutively express galectin-1 in high amounts. These data imply that resistance of CD7- T cells to galectin-1-induced apoptosis may contribute to the accumulation of CD7- Sézary T cells during progression of the disease.
Assuntos
Adjuvantes Imunológicos/farmacologia , Antígenos CD7/análise , Apoptose/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Hemaglutininas/farmacologia , Síndrome de Sézary/patologia , Adolescente , Adulto , Idoso , Antígenos CD7/efeitos dos fármacos , Antígenos CD4/análise , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Estudos de Casos e Controles , Criança , Feminino , Galectina 1 , Humanos , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Síndrome de Sézary/tratamento farmacológico , Síndrome de Sézary/imunologiaRESUMO
It has been suggested that extracellular mRNA might be released from tumor cells and might be protected from serum RNase by proteins or proteolipid complexes. Our group has recently assessed the potential value of extracellular RNA detection by RT-PCR in the peripheral blood of patients with metastatic melanoma.
Assuntos
Melanoma/sangue , RNA Mensageiro/sangue , RNA Neoplásico/sangue , Antígenos de Neoplasias , Humanos , Antígeno MART-1 , Melanoma/genética , Melanoma/patologia , Monofenol Mono-Oxigenase/genética , Metástase Neoplásica , Proteínas de Neoplasias/genética , Células Neoplásicas Circulantes , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The CD7(-) subset of CD4(+) T cells reflects a stable differentiation state of post-thymic helper T cells with CD45R0(+)CD45RA(-) 'memory' phenotype. Here we report that CD4(+)CD7(-) T cells are prone to increased spontaneous apoptosis in vitro compared to CD4(+)CD7(+) T cells. Spontaneous apoptosis is prevented by IL-15, but not by IL-2. Moreover, IL-15 increases Bcl-2 and decreases CD95/Fas expression of CD7(-), but not of CD7(+) T cells. Because IL-15 is physiologically not secreted but expressed in a membrane-bound form, we cocultured T cells with TNF-alpha stimulated fibroblasts that expose membrane IL-15. TNF-alpha stimulated fibroblasts rescue CD4(+)CD7(-) T cells from apoptosis whereas unstimulated fibroblasts do not. Rescue from apoptosis requires cell-cell contact and is abolished by addition of neutralizing antibodies to IL-15. We conclude that membrane IL-15 prevents accelerated apoptosis of CD4(+)CD7(-) T cells. This mechanism may contribute to accumulation of CD7(-) T cells in chronic inflammatory skin lesions.
Assuntos
Antígenos CD7/análise , Apoptose , Linfócitos T CD4-Positivos/imunologia , Dermatite/imunologia , Interleucina-15/farmacologia , Subpopulações de Linfócitos T/imunologia , Apoptose/efeitos dos fármacos , Linfócitos T CD4-Positivos/citologia , Células Cultivadas , Derme/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , Memória Imunológica , Imunofenotipagem , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Interleucina-15 , Receptores de Interleucina-2/metabolismo , Subpopulações de Linfócitos T/classificação , Subpopulações de Linfócitos T/citologia , Fator de Necrose Tumoral alfa/farmacologia , Receptor fas/metabolismoRESUMO
Functional impairment of the Fas/CD95 receptor-ligand system is associated with the development and progression of malignancies. One possible cause might be the inhibition of the formation of a functional Fas/CD95-FasL complex by soluble Fas/CD95 molecules (sFas/CD95). In the present study we determined sFas/CD95 serum concentration in 125 melanoma patients of different clinical stages of disease compared with 30 healthy controls using an ELISA. sFas/CD95 serum level was significantly elevated (P < 0.0005) in melanoma patients (mean +/- SE = 8.60 +/- 0.26 ng/ml) compared with healthy controls (mean +/- SE = 6.27 +/- 0.25 ng/ml). Univariate analysis revealed a correlation of sFas/CD95 serum concentration with advanced stages of disease (P = 0.009). Only a slight increase in sFas/CD95 serum level (P = 0.057) could be observed in regard to the tumor burden. Patients undergoing current treatment with cytostatics (n = 18) revealed a strong increase in sFas/CD95 serum level (P < 0.0005), whereas treatment with IFN-alpha alone or combined with cytostatics (n = 19) showed no change in serum sFas/CD95 concentration. According to univariate analysis, elevated sFas/CD95 serum levels were associated with a poor overall (P < 0.005) and a progression-free (P < 0.0005) survival. Multivariate analysis revealed sFas/CD95 serum concentration as an independent predictive factor for progression-free (P = 0.011), but not overall (P = 0.078), survival. Our results show a prognostic relevance of serum sFas/CD95 in melanoma patients, indicating that the evaluation of sFas/CD95 serum level may be important for the selection of therapeutic strategies.
Assuntos
Biomarcadores Tumorais/sangue , Melanoma/diagnóstico , Receptor fas/sangue , Progressão da Doença , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Melanoma/sangue , Melanoma/mortalidade , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Taxa de SobrevidaRESUMO
PURPOSE: Reverse transcription-polymerase chain reaction (RT-PCR)-based detection of tyrosinase mRNA is the most frequently used laboratory method for the detection of circulating tumor cells in melanoma patients. However, previously published results showed considerable variability in the PCR positivity rates. MATERIALS AND METHODS: We designed a collaborative study to assess the sensitivity, specificity, and clinical relevance of a new standardized RT-PCR-based enzyme-linked immunosorbent assay (ELISA) for the detection of circulating melanoma cells. Blood samples of healthy donors mixed with cells of a melanoma cell line were prepared in a blinded fashion, and aliquots were sent to seven participating laboratories experienced in RT-PCR. RESULTS: The results demonstrate a high sensitivity (1 melanoma cell/mL blood) and specificity (no false-negatives and 7.4% [2 of 28] false-positives) of the assay and a satisfactory rate of interlaboratory reproducibility. The analysis of aliquots of blinded samples derived from 60 melanoma patients identified tyrosinase mRNA in 17 of 60 (28.3%): three (20%) of 15 stage I patients, two (13.3%) of 15 stage II patients, five (35.7%) of 14 stage III patients, and seven (43.8%) of 16 stage IV patients. The interlaboratory reproducibility of positive samples, however, was extremely low and indicates the presence of low amounts of target mRNA. CONCLUSION: Reverse transcriptase-PCR ELISA has a high sensitivity and specificity for the detection of tyrosinase mRNA in peripheral blood cells. The low interlaboratory reproducibility for the detection of tumor cells in blood samples of melanoma patients, however, raises the question of relevance of this assay for clinical use.
Assuntos
Ensaio de Imunoadsorção Enzimática/normas , Melanoma/diagnóstico , Células Neoplásicas Circulantes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Neoplasias Cutâneas/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , DNA de Neoplasias/análise , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Melanoma/patologia , Pessoa de Meia-Idade , Variações Dependentes do Observador , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Neoplasias Cutâneas/patologiaRESUMO
BACKGROUND: Absence of CD7 antigen expression in T cells defines a subset of normal CD4(+) CD45RO(+) CD45RA(-) memory cells and is furthermore observed in Sézary syndrome (SS). OBJECTIVE: Our purpose was to identify circulating T-cell clones in patients with SS and to elucidate whether the dominant T-cell clones express the CD7 antigen. METHODS: Peripheral blood lymphocytes of patients with SS were analyzed by two-color flow cytometry using antibodies to the V beta region of the T cell receptor (TCR) in combination with an antibody to CD7. In addition, T cells were analyzed for TCR-gamma gene rearrangement by polymerase chain reaction (PCR) techniques. RESULTS: Clonal T-cell expansion was detected in 7 patients with SS by immunostaining of the TCR V beta regions. PCR analysis confirmed the presence of dominant T cell clones. Double-immunostaining revealed that in each case cells of the clonal V beta TCR rearrangement homogeneously express the CD4(+)CD7(-) phenotype. Furthermore, CD4(+)CD7(-) cells express the CD15s antigen but lack expression of CD26 and CD49d. CONCLUSION: Expansion of clonal T cells strongly correlates with the expansion of CD4(+)CD7(-) T cells in 7 tested patients with SS. This supports our model that a subset of late differentiated, normal CD4(+)CD7(-) memory T cells may represent the physiologic counterpart of Sézary cells. Monitoring of circulating T cells with the CD4(+)CD7(-)CD15s(+)CD26(-)CD49d(-) phenotype proved to be useful for the identification of clonal T cells in patients with SS.
Assuntos
Antígenos CD7/análise , Antígenos CD4/análise , Síndrome de Sézary/imunologia , Linfócitos T/imunologia , Idoso , Diferenciação Celular , Divisão Celular , Células Clonais , Feminino , Citometria de Fluxo , Genes Codificadores da Cadeia gama de Receptores de Linfócitos T , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da PolimeraseRESUMO
PURPOSE: To determine the predictive value of the angiogenic serum factors angiogenin, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and interleukin-8 (IL-8) for the prognosis of patients with malignant melanoma. PATIENTS AND METHODS: Angiogenin, VEGF, bFGF, and IL-8 were measured in sera of 125 melanoma patients with different stages of disease and with or without current therapy including interferon alfa and different cytostatics in comparison with 30 healthy controls using enzyme-linked immunosorbent assay. RESULTS: Serum levels of angiogenin, VEGF, bFGF, and IL-8 were significantly increased in melanoma patients compared with healthy controls. Elevated serum concentrations of VEGF, bFGF, and IL-8 were associated with advanced disease stages and tumor burden. Cytostatic therapy of patients was accompanied by increased serum levels of angiogenin, bFGF, and IL-8. As shown by univariate analysis, elevated serum levels of VEGF (P = .0001 and .0036), bFGF (P < .00005 and < .00005), and IL-8 (P < .00005 and < .00005) were strongly correlated with a poor overall and progression-free survival, respectively. Multivariate analysis revealed stage of disease (P = .0238), tumor burden (P = .0347), VEGF (P = .0036), bFGF (P = .0252), and IL-8 (P = .0447) as independent predictive factors of overall survival. Tumor burden (P = .0081), VEGF (P = .0245), and IL-8 (P = .0089) were found as independent predictive factors of progression-free survival. CONCLUSION: Our data suggest that the angiogenic serum factors VEGF, bFGF, and IL-8 are useful predictive markers for overall and progression-free survival in melanoma patients.
Assuntos
Substâncias de Crescimento/sangue , Melanoma/sangue , Proteínas de Neoplasias/sangue , Análise de Variância , Indutores da Angiogênese/sangue , Biomarcadores/sangue , Fatores de Crescimento Endotelial/sangue , Feminino , Fator 2 de Crescimento de Fibroblastos/sangue , Humanos , Interleucina-8/sangue , Linfocinas/sangue , Masculino , Melanoma/mortalidade , Melanoma/patologia , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Valor Preditivo dos Testes , Prognóstico , Modelos de Riscos Proporcionais , Ribonuclease Pancreático/sangue , Análise de Sobrevida , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
We have previously described the human homolog of a rat metastasis-associated molecule, hC4.4 A, with a weak homology to the urokinase-type plasminogen activator receptor. By the restricted expression in nontransformed tissues as opposed to expression in roughly 50% of a variety of carcinoma lines of different origins, a possible correlation between hC4.4 A and tumor progression emerged. This was explored in more detail in melanoma by quantitative polymerase chain reaction and in situ hybridization. As shown before, normal human skin weakly expresses hC4.4 A. Melanocytes and nevi are negative, but up to 60% of primary malignant melanoma and 100% of lymph node and skin metastases of melanoma are hC4.4 A positive. Signal intensity in both polymerase chain reaction and in situ hybridization varied considerably between individual samples, which is indicative for regulated expression of hC4.4 A. To test the hypothesis, melanoma lines were incubated with human serum. Whereas expression of hC4.4 was not influenced by heat-inactivated human serum, all melanoma lines responded to noninactivated human serum with upregulation of hC4.4 A expression. Regulated expression with highest level expression on metastases is a feature that hC4.4 A shares with the urokinase-type plasminogen activator receptor. This feature points towards functional activity of hC4.4 A in tumor progression.
Assuntos
Melanoma/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/fisiologia , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/secundário , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Regulação para CimaRESUMO
In chronic inflammatory conditions, mononuclear cells infiltrate the connective tissue attracted by fibroblast-secreted chemokines. The role of fibroblasts in sustaining the lymphocyte immune response upon cellular infiltration is so far unresolved. We here report that, upon prolonged stimulation with tumor necrosis factor-alpha, dermal fibroblasts enhance proliferation of activated T cells whereas unstimulated fibroblasts do not. T cell growth stimulation requires cell contact of tumor necrosis factor-alpha stimulated fibroblasts to T cells and is not due to soluble factors. Growth stimulation is substantially blocked by neutralizing antibodies to interleukin-15. Fluorescence-activated cell sorter analyses revealed that tumor necrosis factor alpha stimulated fibroblasts expose interleukin-15 in a membrane-bound form on the cell surface whereas nonstimulated fibroblasts and interferon-gamma treated fibroblasts do not. The amount of membrane interleukin-15 increases with the duration of tumor necrosis factor-alpha stimulation for at least 3 d. Unstimulated fibroblasts, however, accumulate interleukin-15 in the cytoplasm. No interleukin-15 could be detected in the culture supernatant. Immunohistochemical analyses confirmed membrane interleukin-15 on dermal fibroblasts in discoid lupus erythematosus skin lesions whereas no membrane interleukin-15 was found on the surface of fibroblasts in healthy skin. We conclude that dermal fibroblasts upon long-term tumor necrosis factor-alpha stimulation during chronic inflammation are involved via membrane-bound interleukin-15 in stimulating proliferation of accumulated, activated T cells.
Assuntos
Fibroblastos/citologia , Interleucina-15/farmacologia , Pele/citologia , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Anticorpos , Divisão Celular/fisiologia , Membrana Celular/química , Humanos , Técnicas Imunoenzimáticas , Interleucina-15/genética , Interleucina-15/imunologia , Lúpus Eritematoso Discoide/patologia , Ativação Linfocitária/efeitos dos fármacos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Reverse transcription polymerase chain reaction (RT-PCR)-based detection of tyrosinase mRNA is a frequently used method for the identification of circulating tumor cells in melanoma patients. The significance and practical value of this procedure for the diagnosis of tumor dissemination in melanoma patients are unclear. The conflicting results may at least partially be related to very low amounts of circulating tumor cells and to our observation that melanoma cells only transiently persist in the peripheral blood. The purpose of the present study was to evaluate the relevance of detection of extracellular melanoma-specific mRNA in serum and plasma samples in comparison to blood cell samples from patients with disseminated disease (stage IV). We therefore compared the presence of specific mRNA for tyrosinase, gp100, and MART-1 by RT-PCR amplification of specific cDNA from serum, plasma, and whole blood samples of 10 melanoma patients. Melanoma-specific mRNA was detectable in whole blood samples of all ten patients tested indicating the presence of circulating melanoma cells. In addition, tyrosinase mRNA could be detected in the serum and/or plasma of 6 of 10 melanoma patients whereas gp100 and MART-1 specific transcripts were not detectable in any of the samples tested. The presence and integrity of amplifiable RNA was shown in all serum and plasma samples of patients and controls by RT-PCR-specific amplification of porphobilinogen deaminase (PBDG) mRNA. We conclude that tyrosinase mRNA but not gp100 and MART-1 mRNA can be amplified from serum and/or plasma in a subset of melanoma patients showing circulating melanoma cells. Therefore, extracellular-directed assays appear to be less sensitive and efficacious in detecting melanoma-specific transcripts compared to cellular-based assays.
Assuntos
Biomarcadores Tumorais/sangue , Melanoma/sangue , Proteínas de Neoplasias/genética , Células Neoplásicas Circulantes , RNA Mensageiro/sangue , RNA Neoplásico/sangue , Adulto , Idoso , Antígenos de Neoplasias , Biomarcadores Tumorais/genética , Células Sanguíneas/química , Feminino , Humanos , Hidroximetilbilano Sintase/genética , Antígeno MART-1 , Masculino , Melanoma/genética , Melanoma/patologia , Glicoproteínas de Membrana/genética , Pessoa de Meia-Idade , Monofenol Mono-Oxigenase/genética , Metástase Neoplásica , Plasma , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Antígeno gp100 de MelanomaRESUMO
Reverse transcription (RT) of tyrosinase mRNA and specific cDNA amplification to facilitate the early detection of circulating tumor cells in melanoma patients have been reported. The significance and practical value of these procedures for the diagnosis of tumor dissemination in melanoma patients is, however, still unclear. We analyzed peripheral blood samples of melanoma patients of different clinical stages for the presence of tyrosinase mRNA by reverse transcriptase polymerase chain reaction (RT-PCR). In addition to a nested RT-PCR-based system, we evaluated the new PCR enzyme-linked immunosorbent assay tyrosinase system for sensitivity and specificity in detecting circulating melanoma cells. Our results showed a high sensitivity and specificity for this system in detecting one melanoma cell in 1 ml of whole blood. Using different methods of detection, no tyrosinase mRNA was detectable in blood samples of patients with primary melanoma and regional lymph node metastases. In a small number of patients with visceral metastases (10-30%), we found tyrosinase mRNA-positive results. Analyses of different blood samples taken at 2-h intervals indicate that tumor cells persist only transiently in the peripheral blood. Successful establishment of melanoma cell growth from tyrosinase mRNA-positive samples indicates that viable tumor cells exist in melanoma patients' peripheral blood. Our results indicate a low amount of tyrosinase-specific transcripts in a small subset of stage IV patients and suggest that the analysis of tyrosinase mRNA in peripheral blood samples is not helpful as a prognostic marker or monitoring tool in melanoma patients.
Assuntos
Melanoma/sangue , Monofenol Mono-Oxigenase/genética , Células Neoplásicas Circulantes/patologia , RNA Mensageiro/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/sangue , Humanos , Melanoma/enzimologia , Metástase Neoplásica , Estadiamento de Neoplasias , Controle de Qualidade , Sensibilidade e Especificidade , Neoplasias Cutâneas/enzimologiaRESUMO
The CD7- subset of CD4+ memory T cells reflects a stable differentiation state of post-thymic helper T cells and represents a small subpopulation in circulating blood. We here demonstrate that CD7- T cells preferentially accumulate in skin lesions under chronic inflammatory conditions irrespective of the particular disease. As adhesion to vascular endothelial cells (EC) is required for migration of circulating lymphocytes into tissues, we analysed the adherence of purified subsets of CD4+ memory T cells to endothelial cells in vitro. Compared with CD4+CD7+ T cells, cells of the CD4+CD7- subset preferentially adhere to EC, which is moreover increased after prestimulation of EC with tumour necrosis factor-alpha (TNF-alpha). Stimulated EC increase expression of intercellular adhesion molecule-1 (CD54) and E-selectin (CD62E), the ligand of which, cutaneous lymphocyte-related antigen (CLA), is highly expressed in CD4+CD7- T cells but not in CD4+CD7+ T cells. LFA-1 is expressed in a bimodal distribution on CD4+CD7- T cells in contrast to CD4+CD7+ cells, whereas VLA-1, VLA-3, and VLA-5 are nearly similarly expressed in both T cell subsets. Our results imply that the preferred adherence of CD4+CD7- memory T cells to vascular EC, which is increased after long-term EC stimulation with TNF-alpha, is likely to facilitate their accumulation in various inflammatory skin lesions.
Assuntos
Antígenos CD7/imunologia , Linfócitos T CD4-Positivos/imunologia , Endotélio Vascular/citologia , Pele/imunologia , Linfócitos T CD4-Positivos/fisiologia , Adesão Celular , Eczema/imunologia , Eczema/patologia , Endotélio Vascular/imunologia , Células Epidérmicas , Humanos , Memória Imunológica/imunologia , Queratinócitos/imunologia , Psoríase/imunologia , Psoríase/patologia , Pele/citologia , Pele/patologia , Dermatopatias/imunologia , Dermatopatias/patologia , Subpopulações de Linfócitos T/fisiologiaRESUMO
The expression and functionality of the Fas receptor (CD95/APO-1) play an important role for the maintenance of tissue homeostasis. Various types of tumor cells have been shown to escape immune recognition by constitutive resistance to CD95-mediated apoptosis. Furthermore, several apoptosis-related proteins have been reported to influence CD95 sensitivity. We tested an unselected panel of 11 melanoma cell lines for sensitivity to CD95 and the corresponding expression of CD95, CD95L, bcl-2, bcl-x, bcl-xS, bax and FLIP proteins. Despite detection of CD95 cell-surface expression in 9 out of the 11 cell lines tested, only 3 melanoma cell lines were sensitive to anti-CD95-MAb-induced cell death. Apoptosis-related proteins CD95L, bcl-2, bcl-x, bcl-xS and bax were found to be heterogenously expressed in different melanoma cell lines tested. The susceptibility of melanoma cells to anti-CD95-MAb-mediated apoptosis was associated with low protein expression of both bcl-2 and bcl-x. The level of CD95 cell-surface expression in melanoma cells was no indicator for CD95 sensitivity. Furthermore, FLIP protein was detectable in 7 out of the 11 cell lines, but showed no correlation to CD95 sensitivity. Certain cytokines have been described as modulating the susceptibility of tumor cells to CD95-induced cell death. Since IFN-alpha was proved to be clinically efficient in melanoma therapy, we tested whether interferons have the ability to induce sensitivity to CD95 in primarily resistant melanoma cell lines. Here we show that IFN-gamma, but not IFN-alpha, is able to increase the susceptibility of sensitive cell lines and to induce CD95 sensitivity in resistant melanoma cell lines, accompanied by up-regulation of the protein expression level of CD95 and/or bcl-xS.