RESUMO
We show that a lectin like protein from the mushroom Agaricus bisporus (LSMT) is capable to permeate the epithelial monolayer barrier of the intestine ex vivo. The protein is not toxic or immunogenic upon prolonged administration and elevated dose in mice. Thus, it could be a candidate as a drug carrier for oral administration. However, its permeability should be tested after the protein has been modified, mimicking the condition in which it is used as a drug carrier. The protein was conjugated to captopril, the selected model of a Biopharmaceutical Classification System (BCS) class III drug, with high solubility but poor permeability. The drug was conjugated to LSMT that had been modified with 4-succinimidyloxycarbonyl-alpha-methyl-2-pyridyldithiotoluene (SMPT) as a linker. The success of LSMT modification was confirmed with TLC and MS; the latter also indicated the amount of captopril molecule linked. The modified LSMT could permeate through the intestinal monolayer barrier, and thus could be absorbed in the intestine after modification. The modified protein appears to remain stable after incubation in simulated gastrointestinal fluids. This pioneering work provides an essential basis for further development of the protein as a drug carrier for oral administration.
Assuntos
Agaricus , Captopril/química , Portadores de Fármacos/química , Monofenol Mono-Oxigenase/química , Administração Oral , Agaricales/metabolismo , Agaricus/metabolismo , Células CACO-2 , Captopril/administração & dosagem , Captopril/metabolismo , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Ácido Gástrico/metabolismo , Humanos , Absorção Intestinal/efeitos dos fármacos , Absorção Intestinal/fisiologia , Monofenol Mono-Oxigenase/administração & dosagem , Monofenol Mono-Oxigenase/metabolismoRESUMO
Light subunit of mushroom tyrosinase (LSMT) is a protein of unknown function from mushroom Agaricus bisporus that has been demonstrated to permeate through rat intestine ex vivo. Thus, it can be absorbed in the intestine, thereby holding a promise as a drug carrier for oral administration, similar to HA-33 protein from botulinum, one of the closest structural homologs of LSMT. However, the safety of LSMT should be ensured prior to its use. Here, we described biological response of LSMT upon weekly intraperitoneal administration of 50 µg/day to the Balb/c mice for 12 weeks. Motoric and behavior profiles, as well as the index of main organs (liver, spleen, lung, heart, and kidney), and body weight, were not significantly changed as compared with the control group. Also, no IgG was detected in the serum. The results suggest that LSMT is safe for further development.
Assuntos
Agaricus/enzimologia , Comportamento Animal/efeitos dos fármacos , Monofenol Mono-Oxigenase/administração & dosagem , Animais , Peso Corporal/efeitos dos fármacos , Feminino , Sistema Imunitário/efeitos dos fármacos , Imunoglobulina G/sangue , Infusões Parenterais , Masculino , Camundongos Endogâmicos BALB C , Subunidades Proteicas/administração & dosagemRESUMO
BACKGROUND: Malignant melanoma (MM) often shows multiple chemo-resistance, leading to poor prognosis of the patients. Therapeutic anti-cancer vaccination may be a feasible way to prolong the survival of patients. We have demonstrated that application of antigenic peptides via the tape-stripped, horny layer-removed skin, known as percutaneous peptide immunization (PPI), induces tumor cell-specific cytotoxic T lymphocytes (CTLs) in rodents and humans. OBJECTIVE: To evaluate clinical significance of PPI in advanced MM patients. METHODS: We performed PPI in 59 patients undergoing advanced MM with Melan-A, tyrosinase, MAGE-2, MAGE-3 and gp-100 peptides based on HLA typing in individuals. The induction of CTLs was assessed by the tetramer or pentamer flow cytometry in 35 patients. Patients showing positive CTL responses to all antigens were defined as complete responder (n=18), and those showing negative responses to at least one applied antigen were classified as incomplete responder (n=17). The primary endpoint of the study was overall survival (OS). For statistical analysis, log-rank test, univariate and multivariate Cox proportional hazard model were used. RESULTS: OS of the complete responders was longer than that of the incomplete responders (median survival time: 55.8 vs 20.3 months, log rank P=0.089). A hazard ratio for the complete responders relative to the incomplete responders was 0.23 (95% confidence interval: 0.06-0.93, P=0.039) in a multivariate Cox proportional hazard model. CONCLUSION: The induction of CTLs was a novel independent survival factor, and the induction of peptide-specific CTLs by PPI contributes to the prolonged survival and represents an impact on therapeutic approaches in MM. Unique trial number: UMIN000005706.
Assuntos
Vacinas Anticâncer/administração & dosagem , Melanoma/tratamento farmacológico , Fragmentos de Peptídeos/administração & dosagem , Neoplasias Cutâneas/tratamento farmacológico , Linfócitos T Citotóxicos/efeitos dos fármacos , Administração Cutânea , Adulto , Idoso , Vacinas Anticâncer/imunologia , Feminino , Antígenos HLA/imunologia , Humanos , Imunização , Antígeno MART-1/administração & dosagem , Masculino , Melanoma/imunologia , Melanoma/mortalidade , Melanoma/patologia , Antígenos Específicos de Melanoma/administração & dosagem , Pessoa de Meia-Idade , Monofenol Mono-Oxigenase/administração & dosagem , Análise Multivariada , Fragmentos de Peptídeos/imunologia , Modelos de Riscos Proporcionais , Estudos Prospectivos , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia , Linfócitos T Citotóxicos/imunologia , Fatores de Tempo , Resultado do Tratamento , Antígeno gp100 de Melanoma/administração & dosagemRESUMO
Enhancement of transdermal absorption through rat skin and stability of the human tyrosinase plasmid (P) using Tat (T) and an entrapment in elastic cationic niosomes (E) were described. E (Tween61:cholesterol:DDAB at 1:1:0.5 molar ratio) were prepared by the freeze-dried empty liposomes (FDELs) method using 25% ethanol. TP was prepared by a simple mixing method. TPE was prepared by loading T and P in E at the T:P:E ratio of 0.5:1:160 w/w/w. For gel formulations, P, TP, PE and TPE were incorporated into Carbopol 980 gel (30 µg of plasmid per 1 g of gel). For the transdermal absorption studies, the highest cumulative amounts and fluxes of the plasmid in viable epidermis and dermis (VED) were observed from the TPE of 0.31 ± 0.04 µg/cm(2) and 1.86 ± 0.24 µg/cm(2)/h (TPE solution); and 4.29 ± 0.40 µg/cm(2) and 25.73 ± 2.40 µg/cm(2)/h (TPE gel), respectively. Only plasmid from the PE and TPE could be found in the receiving solution with the cumulative amounts and fluxes at 6 h of 0.07 ± 0.01 µg/cm(2) and 0.40 ± 0.08 µg/cm(2)/h (PE solution); 0.10 ± 0.01 µg/cm(2) and 0.60 ± 0.06 µg/cm(2)/h (TPE solution); 0.88 ± 0.03 µg/cm(2) and 5.30 ± 0.15 µg/cm(2)/h (PE gel); and 1.02 ± 0.05 µg/cm(2) and 6.13 ± 0.28 µg/cm(2)/h (TPE gel), respectively. In stability studies, the plasmid still remained at 4 ± 2 °C and 25 ± 2 °C of about 48.00-65.20% and 27.40-51.10% (solution); and 12.34-38.31% and 8.63-36.10% (gel), respectively, whereas at 45 ± 2 °C, almost all the plasmid was degraded. These studies indicated the high potential application of Tat and an entrapment in elastic cationic niosomes for the development of transdermal gene delivery system.
Assuntos
Produtos do Gene tat/fisiologia , Monofenol Mono-Oxigenase/metabolismo , Fragmentos de Peptídeos/metabolismo , Plasmídeos/metabolismo , Absorção Cutânea/fisiologia , Regulação para Cima/efeitos dos fármacos , Administração Cutânea , Sequência de Aminoácidos , Animais , Cátions , Estabilidade de Medicamentos , Elasticidade , Produtos do Gene tat/administração & dosagem , Produtos do Gene tat/genética , Humanos , Lipossomos , Masculino , Dados de Sequência Molecular , Monofenol Mono-Oxigenase/administração & dosagem , Monofenol Mono-Oxigenase/genética , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/genética , Plasmídeos/administração & dosagem , Plasmídeos/genética , Ratos , Ratos Sprague-Dawley , Absorção Cutânea/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
PURPOSE: Electroporation of dendritic cells (DC) with mRNA encoding tumor-associated antigens (TAA) has multiple advantages compared to peptide loading. We investigated the immunologic and clinical responses to vaccination with mRNA-electroporated DC in stage III and IV melanoma patients. EXPERIMENTAL DESIGN: Twenty-six stage III HLA*02:01 melanoma patients scheduled for radical lymph node dissection (stage III) and 19 melanoma patients with irresectable locoregional or distant metastatic disease (referred to as stage IV) were included. Monocyte-derived DC, electroporated with mRNA encoding gp100 and tyrosinase, were pulsed with keyhole limpet hemocyanin and administered intranodally. TAA-specific T-cell responses were monitored in blood and skin-test infiltrating lymphocyte (SKIL) cultures. RESULTS: Comparable numbers of vaccine-induced CD8(+) and/or CD4(+) TAA-specific T-cell responses were detected in SKIL cultures; 17/26 stage III patients and 11/19 stage IV patients. Strikingly, in this population, TAA-specific CD8(+) T cells that recognize multiple epitopes and produce elevated levels of IFNγ upon antigenic challenge in vitro, were significantly more often observed in stage III patients; 15/17 versus 3/11 stage IV patients, P = 0.0033. In stage IV patients, one mixed and one partial response were documented. The presence or absence of IFNγ-producing TAA-specific CD8(+) T cells in stage IV patients was associated with marked difference in median overall survival of 24.1 months versus 11.0 months, respectively. CONCLUSION: Vaccination with mRNA-electroporated DC induces a broad repertoire of IFNγ producing TAA-specific CD8(+) and CD4(+) T-cell responses, particularly in stage III melanoma patients.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer , Imunoterapia , Melanoma , Adulto , Idoso , Antígenos de Neoplasias/administração & dosagem , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Eletroporação , Feminino , Humanos , Interferon gama/sangue , Masculino , Melanoma/tratamento farmacológico , Melanoma/imunologia , Melanoma/patologia , Pessoa de Meia-Idade , Monofenol Mono-Oxigenase/administração & dosagem , Monofenol Mono-Oxigenase/genética , Metástase Neoplásica , Estadiamento de Neoplasias , RNA Mensageiro/administração & dosagem , RNA Mensageiro/imunologia , Antígeno gp100 de Melanoma/administração & dosagem , Antígeno gp100 de Melanoma/genéticaRESUMO
Efforts to develop effective cancer vaccines often use combinations of immunogenic peptides to increase the applicability and effectiveness of the immunizations. The immunologic consequences of combining more than 1 self/tumor antigen in a single vaccine emulsion remain unclear, however. We performed 2 sequential clinical trials in patients at high risk for melanoma recurrence. Patients were given the highly immunogenic gp100:209-217(210M) peptide and the less immunogenic tyrosinase:368-376(370D) peptide once every 3 weeks for 4 weeks. This vaccination course was 12 weeks long, and patients were vaccinated for up to 4 courses (16 total vaccinations). In the first trial in 31 patients, the peptides were emulsified separately in incomplete Freund adjuvant and injected at 2 different sites. In the second trial in 33 patients, the peptides were emulsified together and injected at the same site. Cryopreserved lymphocytes were obtained by apheresis after each course and were evaluated for antipeptide activity using tetramer, enzyme-linked immunospot, and in vitro sensitization boost assays. When the peptides were injected at separate sites, robust specific reactivity to the native gp100:209-217 peptide was measured by each of the assays, whereas immunization with the tyrosinase:368-376(370D) peptide was far less effective. When the peptides were emulsified and injected together at the same site, immunization to the gp100:209-217(210M) epitope dropped precipitously, whereas reactivity to the tyrosinase:368-376(370D) peptide was enhanced. These cautionary data indicate that mixing peptides in the same emulsion can alter reactivity compared with peptides injected separately by mechanisms that may include the induction of localized nonspecific inflammation or competitive binding of peptides to major histocompatibility complex molecules.
Assuntos
Vacinas Anticâncer/imunologia , Melanoma/imunologia , Melanoma/terapia , Glicoproteínas de Membrana/imunologia , Adulto , Idoso , Antígenos de Neoplasias/administração & dosagem , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/administração & dosagem , Interações Medicamentosas/imunologia , Feminino , Humanos , Masculino , Melanoma/patologia , Glicoproteínas de Membrana/administração & dosagem , Pessoa de Meia-Idade , Monofenol Mono-Oxigenase/administração & dosagem , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/imunologia , Prevenção Secundária , Resultado do Tratamento , Antígeno gp100 de MelanomaRESUMO
Melanoma now represents the fifth most common cancer in North America and it has increased dramatically in the past decade. One of the approaches shows that lowering of tyrosine level can inhibit the growth of melanoma in cell culture and in mice bearing B16BL6 melanoma. However, human cannot tolerate the tyrosine restricted diets for lowering tyrosine due to nausea, vomiting, and severe body weight loss. We therefore prepare a novel soluble polyhemoglobin-tyrosinase complex. Our studies show that this preparation can lower systemic tyrosine level in normal animals. This preparation also prevents the native tyrosinase from having adverse effects and from rapid removal after injection. In cell culture study, we find that this preparation inhibits the growth of murine B16F10 melanoma culture. Furthermore, in animal studies we observe that daily intravenous injection of this polyhemoglobin-tyrosinase preparation significantly delays the growth of B16F10 melanoma in mice, without causing adverse effects or changes in the growth of the treated animals.
Assuntos
Antineoplásicos/uso terapêutico , Hemoglobinas/uso terapêutico , Melanoma Experimental/tratamento farmacológico , Monofenol Mono-Oxigenase/uso terapêutico , Aldeídos/química , Aldeídos/farmacologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Técnicas de Cultura de Células , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/farmacologia , Feminino , Hemoglobinas/administração & dosagem , Hemoglobinas/farmacologia , Melanoma Experimental/metabolismo , Camundongos , Monofenol Mono-Oxigenase/administração & dosagem , Monofenol Mono-Oxigenase/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Tirosina/metabolismoRESUMO
Melanoma is an increasingly common fatal skin cancer. Many groups are carrying out research on potential treatments for melanoma. One of these approaches has shown that lowering tyrosine can inhibit the growth of melanoma in cell cultures and of B16BL6 melanoma in mice. However, humans cannot tolerate tyrosine-restricted diets for lowering tyrosine because of nausea, vomiting and weight loss. We report here our preparation and characterization of a novel soluble polyhaemoglobin-tyrosinase complex. This preparation prevents native tyrosinase from having adverse effects and from rapid removal after injection. The preparation inhibited murine B16F10 melanoma cell growth in culture and delayed its growth in a mice model. Intravenous injection of the preparation lowers the systemic tyrosine level without causing adverse effects such as vomiting and weight loss in mice. It is therefore possible that this complex could be useful in the treatment of human melanoma.
Assuntos
Hemoglobinas/farmacologia , Melanoma/patologia , Monofenol Mono-Oxigenase/farmacologia , Animais , Bovinos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Hemoglobinas/administração & dosagem , Hemoglobinas/química , Injeções Intravenosas , Masculino , Melanoma/metabolismo , Camundongos , Peso Molecular , Monofenol Mono-Oxigenase/administração & dosagem , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/metabolismo , RatosRESUMO
Our previous studies have indicated that encapsulated tyrosinase and crosslinked hemoglobin with tyrosinase (polyhemoglobin-tyrosinase) decrease systemic tyrosine level significantly in rats. However, we need a few days of oral administration of encapsulated tyrosinase before the systemic tyrosine level starts to decrease. Although intravenous injection of polyhemoglobin tyrosinase can lower the systemic tyrosine to about 10% within an hour, the level increases towards normal after 24 h. We therefore investigate the effects of intravenous injection of polyhemoglobin-tyrosinase combined with oral administration of encapsulated tyrosinase on lowering the systemic tyrosine level. In addition, we further optimize this combined method for lowering systemic tyrosine in animal studies and have found out that two intravenous injections of polyhemoglobin-tyrosinase followed by three times a day oral administration of encapsulated tyrosinase could immediately lower the body tyrosine and maintain this low level as long as the oral administration is continued.
Assuntos
Monofenol Mono-Oxigenase/administração & dosagem , Tirosina/efeitos dos fármacos , Administração Oral , Animais , Cápsulas , Hemoglobinas/uso terapêutico , Injeções Intravenosas , Masculino , Melanoma/tratamento farmacológico , Oxigênio/metabolismo , Ratos , Ratos Sprague-Dawley , Tirosina/sangueRESUMO
There is no effective treatment for melanoma, a fatal skin cancer occurring with increasing frequency. Dietary tyrosine restriction lowers systemic tyrosine and suppresses the growth of melanoma in mice, but this is not tolerated by human resulting in nausea, vomiting, and weight loss. We report here the successful use of oral polymeric microcapsules containing tyrosinase to lower the systemic tyrosine level in the rats. We found that microencapsulated tyrosinase incubated with intestinal content of rats selectively lowered the tyrosine level. We then studied the daily oral administration of microencapsulated tyrosinase in rats of one dose a day, two doses a day, and three doses a day over a period of up to 22 days. With three doses a day, the tyrosine levels in the test group decreased to 68.8% of the control group by day 4 and then decreased to 52.6% after this and remained at this level throughout the 22 days test period. This is the level shown earlier by other workers using dietary restriction of tyrosine to result in suppression of growth of melanoma. However, unlike dietary tyrosine restriction, oral tyrosinase microcapsules did not result in adverse effects nor significant differences in growth (weight gain) when compared to the control group. This approach can also be used for the lowering of systemic tyrosine in hypertyrosinemia, an inborn error of metabolism.
Assuntos
Monofenol Mono-Oxigenase/farmacologia , Tirosina/sangue , Administração Oral , Animais , Peso Corporal/efeitos dos fármacos , Cápsulas , Composição de Medicamentos , Técnicas In Vitro , Mucosa Intestinal/metabolismo , Cinética , Masculino , Monofenol Mono-Oxigenase/administração & dosagem , Ratos , Ratos Sprague-DawleyRESUMO
The recent identification and molecular characterization of tumor-associated antigens recognized by tumor-reactive CD8+ T lymphocytes has led to the development of antigen-specific immunotherapy of cancer. Among other approaches, clinical studies have been initiated to assess the in vivo immunogenicity of tumor antigen-derived peptides in cancer patients. In this study, we have analyzed the CD8+ T cell response of an ocular melanoma patient to a vaccine composed of four different tumor antigen-derived peptides administered simultaneously in incomplete Freund's adjuvant (IFA). Peptide NY-ESO-1(157-165) was remarkably immunogenic and induced a CD8+ T cell response detectable ex vivo at an early time point of the vaccination protocol. A CD8+ T cell response to the peptide analog Melan-A(26-35 A27L) was also detectable ex vivo at a later time point, whereas CD8+ T cells specific for peptide tyrosinase(368-376) were detected only after in vitro peptide stimulation. No detectable CD8+ T cell response to peptide gp100(457-466) was observed. Vaccine-induced CD8+ T cell responses declined rapidly after the initial response but increased again after further peptide injections. In addition, tumor antigen-specific CD8+ T cells were isolated from a vaccine injection site biopsy sample. Importantly, vaccine-induced CD8+ T cells specifically lysed tumor cells expressing the corresponding antigen. Together, these data demonstrate that simultaneous immunization with multiple tumor antigen-derived peptides can result in the elicitation of multiepitope-directed CD8+ T cell responses that are reactive against antigen-expressing tumors and able to infiltrate antigen-containing peripheral sites.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Epitopos de Linfócito T/imunologia , Ativação Linfocitária/imunologia , Melanoma/imunologia , Proteínas de Membrana , Neoplasias Uveais/imunologia , Antígenos de Neoplasias/administração & dosagem , Linfócitos T CD8-Positivos/química , Vacinas Anticâncer/administração & dosagem , Testes Imunológicos de Citotoxicidade/métodos , Esquema de Medicação , Epitopos de Linfócito T/administração & dosagem , Adjuvante de Freund/administração & dosagem , Adjuvante de Freund/imunologia , Antígeno HLA-A2/administração & dosagem , Antígeno HLA-A2/imunologia , Humanos , Injeções Subcutâneas , Lipídeos/administração & dosagem , Lipídeos/imunologia , Estudos Longitudinais , Antígeno MART-1 , Melanoma/prevenção & controle , Monofenol Mono-Oxigenase/administração & dosagem , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/imunologia , Proteínas de Neoplasias/imunologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/imunologia , Proteínas/administração & dosagem , Proteínas/imunologia , Linfócitos T Citotóxicos/química , Linfócitos T Citotóxicos/imunologia , Células Tumorais Cultivadas , Neoplasias Uveais/prevenção & controle , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
PURPOSE: To determine clinical and immunologic responses to a multipeptide melanoma vaccine regimen, a randomized phase II trial was performed. PATIENTS AND METHODS: Twenty-six patients with advanced melanoma were randomly assigned to vaccination with a mixture of four gp100 and tyrosinase peptides restricted by HLA-A1, HLA-A2, and HLA-A3, plus a tetanus helper peptide, either in an emulsion with granulocyte-macrophage colony-stimulating factor (GM-CSF) and Montanide ISA-51 adjuvant (Seppic Inc, Fairfield, NJ), or pulsed on monocyte-derived dendritic cells (DCs). Systemic low-dose interleukin-2 (Chiron, Emeryville, CA) was given to both groups. T-lymphocyte responses were assessed, by interferon gamma ELIspot assay (Chiron, Emeryville, CA), in peripheral-blood lymphocytes (PBLs) and in a lymph node draining a vaccine site (sentinel immunized node [SIN]). RESULTS: In patients vaccinated with GM-CSF in adjuvant, T-cell responses to melanoma peptides were observed in 42% of PBLs and 80% of SINs, but in patients vaccinated with DCs, they were observed in only 11% and 13%, respectively. The overall immune response was greater in the GM-CSF arm (P <.02). Vitiligo developed in two of 13 patients in the GM-CSF arm but in no patients in the DC arm. Helper T-cell responses to the tetanus peptide were detected in PBLs after vaccination and correlated with T-cell reactivity to the melanoma peptides. Objective clinical responses were observed in two patients in the GM-CSF arm and one patient in the DC arm. Stable disease was observed in two patients in the GM-CSF arm and one patient in the DC arm. CONCLUSION: The high frequency of cytotoxic T-lymphocyte responses and the occurrence of clinical tumor regressions support continued investigation of multipeptide vaccines administered with GM-CSF in adjuvant.
Assuntos
Vacinas Anticâncer/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Manitol/análogos & derivados , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Torácicas/tratamento farmacológico , Adulto , Idoso , Células Dendríticas , Esquema de Medicação , Feminino , Humanos , Interleucina-2/administração & dosagem , Linfonodos/imunologia , Masculino , Manitol/administração & dosagem , Melanoma/diagnóstico por imagem , Melanoma/imunologia , Melanoma/mortalidade , Melanoma/secundário , Glicoproteínas de Membrana/administração & dosagem , Pessoa de Meia-Idade , Monofenol Mono-Oxigenase/administração & dosagem , Proteínas de Neoplasias/administração & dosagem , Ácidos Oleicos/administração & dosagem , Fragmentos de Peptídeos/administração & dosagem , Radiografia , Neoplasias Cutâneas/diagnóstico por imagem , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia , Análise de Sobrevida , Linfócitos T/imunologia , Neoplasias Torácicas/diagnóstico por imagem , Neoplasias Torácicas/imunologia , Neoplasias Torácicas/mortalidade , Neoplasias Torácicas/secundário , Resultado do Tratamento , Antígeno gp100 de MelanomaRESUMO
We studied the size and avidity of primary and recall CD8(+) T cell responses in vivo in mice immunized with dendritic cells presenting different densities of a MHC class I-restricted peptide. Increasing the epitope density on a fixed number of dendritic cells increased the size of the primary response, yet had no influence on the avidity of the effector cells. However, epitope density-based selection of cells with different avidities was evident in the subsequent memory population, and in recall responses. Additionally, mice primed with different peptide densities had similarly sized quiescent memory and recall responses. Our findings provide evidence for an important role for epitope density in the selection of T cells in vivo.
Assuntos
Apresentação de Antígeno , Linfócitos T CD8-Positivos/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Epitopos de Linfócito T/metabolismo , Memória Imunológica , Subpopulações de Linfócitos T/citologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Adesão Celular/imunologia , Diferenciação Celular/imunologia , Células Cultivadas , Epitopos de Linfócito T/administração & dosagem , Epitopos de Linfócito T/biossíntese , Epitopos de Linfócito T/genética , Antígenos H-2/metabolismo , Antígeno HLA-A2/metabolismo , Antígeno de Histocompatibilidade H-2D , Humanos , Imunização , Ativação Linfocitária , Contagem de Linfócitos , Melanoma/enzimologia , Melanoma/imunologia , Camundongos , Camundongos Transgênicos , Monofenol Mono-Oxigenase/administração & dosagem , Monofenol Mono-Oxigenase/biossíntese , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/imunologia , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Tumorais Cultivadas , Vaccinia virus/genética , Vaccinia virus/imunologiaRESUMO
We conducted a randomized trial in HLA*A0201+ patientswith American Joint Committee on Cancer stage III or IV melanoma immunized with tyrosinase 368-376(370D) peptide and gp100 209-217(210M) peptide to compare the potency of three different adjuvants. Patients received 3 monthly immunizations with 500 microg of each peptide either with incomplete Freund's adjuvant (IFA), QS-21, or granulocyte macrophage colony-stimulating factor (GM-CSF). The primary end point was induction of IFN-gamma release by CD8+ T cells against tyrosinase and gp100 peptides measured by enzyme-linked immunospot assays without in vitro prestimulation measured pretreatment, 2 and 8 weeks after the third vaccination. Four of 9 and 4 of 8 patients immunized using QS-21 and GM-CSF, respectively, developed increased frequencies of CD8+ T cells against tyrosinase 370D peptide compared with 0 of 9 patients immunized using IFA (P = 0.045). T-cell responses against a gp100-related peptide showed similar results, but their relevance to T-cell reactivity against native gp100 209-217 is uncertain. These results show that: (a) QS-21 and GM-CSF are superior to IFA as immunological adjuvants for vaccination against tyrosinase 370D peptide; and (b) with appropriate adjuvants, increased frequencies of peptide-specific T cells after vaccination can be detected by enzyme-linked immunospot without prolonged prestimulation in vitro.
Assuntos
Antígeno HLA-A2/imunologia , Melanoma/imunologia , Monofenol Mono-Oxigenase/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Feminino , Adjuvante de Freund/administração & dosagem , Adjuvante de Freund/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Contagem de Linfócitos , Masculino , Melanoma/tratamento farmacológico , Pessoa de Meia-Idade , Monofenol Mono-Oxigenase/administração & dosagem , Monofenol Mono-Oxigenase/química , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/imunologia , Saponinas/administração & dosagem , Saponinas/imunologia , Resultado do Tratamento , VacinaçãoRESUMO
Experimental autoimmune vitiligo was induced by intradermal injection of mushroom tyrosinase emulsified in complete Freund's adjuvant in female C57BL/6 mice. The onset of vitiligo was characterized by hair hypopigmentation and total melanocyte depletion in the basal layer of the epidermis. Oral administration of mushroom tyrosinase prevented the expression of mushroom tyrosinase induced experimental autoimmune vitiligo. Based on the results it is likely that oral administration of mushroom tyrosinase may have practical implications in vitiligo.
Assuntos
Agaricus/enzimologia , Doenças Autoimunes/tratamento farmacológico , Monofenol Mono-Oxigenase/imunologia , Monofenol Mono-Oxigenase/uso terapêutico , Vitiligo/tratamento farmacológico , Administração Oral , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Modelos Animais de Doenças , Feminino , Hipersensibilidade Tardia , Camundongos , Camundongos Endogâmicos C57BL , Monofenol Mono-Oxigenase/administração & dosagem , Monofenol Mono-Oxigenase/metabolismo , Pele/patologia , Vitiligo/imunologia , Vitiligo/patologiaRESUMO
Lymphocytes expanded from excised specimens can be used to characterize intratumoral T cell responses. These analyses, however, are limited to one time point in the natural history of the removed tumor. The expansion of autologous tumor cells and tumor-infiltrating lymphocytes (TIL) from fine needle aspirates (FNA) of tumors potentially allows a dynamic evaluation of T cell responses within the same lesion at moments relevant to the disease course or response to therapy. Fourteen TIL cultures and 8 tumor cell lines were generated from 18 FNA (12 patients). Five of six TIL that could be tested against autologous tumor demonstrated specific reactivity. Two additional TIL for which no autologous tumor was available demonstrated recognition of HLA-matched melanoma cell lines. Serial FNA of the same lesions were performed in five HLA-A*0201 patients vaccinated with the emulsified melanoma Ag (MA) epitopes: MART-1:27-35; tyrosinase:368-376(370D); gp100:280-288(288V); and gp100:209-217 (210M). FNA material was separately cultured for a short time in IL-2 (300 IU/ml) after stimulation with irradiated autologous PBMC pulsed with each peptide or FluM1:58-66 (1 micromol/ml). No peptide-specific TIL could be expanded from prevaccination FNA. However, after vaccination, TIL specific for gp100:280(g280), gp100:209 (g209), and MART-1:27-35 (MART-1)-related epitopes were identified in three, three, and two patients, respectively. No Flu reactivity could be elicited in TIL, whereas it was consistently present in parallel PBMC cultures. This excluded PBMC contamination of the FNA material. This analysis suggests the feasibility of TIL expansion from minimal FNA material and localization of vaccine-specific T cells at the tumor site.
Assuntos
Melanoma/patologia , Melanoma/secundário , Linfócitos T/imunologia , Linfócitos T/patologia , Antígenos de Neoplasias/administração & dosagem , Antígenos de Neoplasias/imunologia , Biópsia por Agulha , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Técnicas de Cultura de Células , Divisão Celular/imunologia , Epitopos de Linfócito T/imunologia , Humanos , Epitopos Imunodominantes/administração & dosagem , Epitopos Imunodominantes/imunologia , Ativação Linfocitária , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologia , Antígeno MART-1 , Melanoma/imunologia , Melanoma/terapia , Glicoproteínas de Membrana/administração & dosagem , Glicoproteínas de Membrana/imunologia , Monofenol Mono-Oxigenase/administração & dosagem , Monofenol Mono-Oxigenase/imunologia , Proteínas de Neoplasias/administração & dosagem , Proteínas de Neoplasias/imunologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/imunologia , Peptídeos , Células Tumorais Cultivadas , Antígeno gp100 de MelanomaRESUMO
The human tyrosinase gene codes for two distinct antigens that are recognized by HLA-A*0201-restricted CTLs. For one of them, tyrosinase peptide 368-376, the sequence identified by mass spectrometry in melanoma cell eluates differs from the gene-encoded sequence as a result of posttranslational modification of amino acid residue 370 (asparagine to aspartic acid). Here, we used fluorescent tetrameric complexes ("tetramers") of HLA-A*0201 and tyrosinase peptide 368-376 (YMDGTMSQV) to characterize the CD8+ T-cell response to this antigen in lymphoid cell populations from HLA-A2 melanoma patients. Taking advantage of the presence of significant numbers of tetramer-positive CD8+ T cells in tumor-infiltrated lymph node cells from a melanoma patient, we derived polyclonal and monoclonal tyrosinase peptide 368-376-specific CTLs by tetramer-guided flow cytometric sorting. These CTLs efficiently and specifically lysed HLA-A*0201- and tyrosinase-positive melanoma cells. As assessed with tyrosinase peptide variants, the fine antigen specificity of the CTLs was quite diverse at the clonal level. Flow cytometric analysis of PBMCs stained with tetramers showed that tyrosinase peptide 368-376-specific CD8+ T cells were hardly detectable in peripheral blood of melanoma patients. However, significant numbers of such cells were detected after short-term stimulation of CD8+ lymphocytes with tyrosinase peptide 368-376 in 6 of 10 HLA-A2 melanoma patients. Taken together, these findings emphasize the significant contribution of the natural tyrosinase peptide 368-376 to the antigenic specificities recognized by the tumor-reactive CTLs that may develop in HLA-A2 melanoma patients.
Assuntos
Citotoxicidade Imunológica , Antígeno HLA-A2/imunologia , Melanoma/tratamento farmacológico , Melanoma/imunologia , Monofenol Mono-Oxigenase/imunologia , Linfócitos T Citotóxicos/imunologia , Humanos , Imunoterapia , Monofenol Mono-Oxigenase/administração & dosagem , Peptídeos/administração & dosagem , Peptídeos/imunologia , Células Tumorais CultivadasRESUMO
Parkinson's disease is a neurodegenerative disorder which is mainly characterized by degeneration of the dopaminergic cells in the nigro-striatal system. Due to a lowered L-tyrosine 3-monooxygenase activity, L-tyrosine is not sufficiently transformed to L-DOPA. To date the most common therapy is the administration of the dopamine precursor L-DOPA, with severe collateral effects. Therefore, the substitution of the lacking tyrosine hydroxylase with tyrosinase might be a novel therapeutical approach that would generate specifically L-DOPA from L-tyrosine. We present here evidence that stereotaxic injection of liposome-entrapped tyrosinase is able to significatively increase the levels of dopamine in the rat brain. The catecholamines L-DOPA, dopamine, L-epinephrine, L-norepinephrine were extracted by acid treatment from the brains and detected by HPLC.
Assuntos
Corpo Estriado/efeitos dos fármacos , Dopamina/biossíntese , Monofenol Mono-Oxigenase/farmacologia , Animais , Cromatografia Líquida de Alta Pressão , Corpo Estriado/metabolismo , Di-Hidroxifenilalanina/biossíntese , Portadores de Fármacos , Avaliação Pré-Clínica de Medicamentos , Epinefrina/biossíntese , Lipossomos , Masculino , Monofenol Mono-Oxigenase/administração & dosagem , Norepinefrina/biossíntese , Doença de Parkinson/metabolismo , Ratos , Ratos Wistar , Técnicas EstereotáxicasRESUMO
1. The present paper reports the effects of liposome-entrapped tyrosinase (EC 1.14.18.1. L-Tyrosine, L-3,4-dihydroxyphenylalanine: oxygen oxidoreductase) infusion on the catecholamine contents of rat plasma. The actions of liposomes and free tyrosinase have also been investigated. 2. From the experiments, evidence has been obtained that liposome-entrapped tyrosinase is able to affect specifically L-3,4-dihydroxyphenylalanine (L-DOPA) levels which increase dramatically. 3. The possible use of liposome-entrapped tyrosinase to raise L-DOPA levels in catecholamine related disorders is discussed. 4. Liposomes without tyrosinase provoke no significant changes of catecholamine or L-DOPA levels while free tyrosinase does induce a change but in a less constant fashion than the liposome-entrapped enzyme.