RESUMEN

Rapid diagnosis and immediate infection control precautions are cornerstones in the prevention of norovirus outbreaks. However, faecal sampling for norovirus PCR--the standard of care--is time consuming. From 2009 to 2011, parallel faecal and rectal swab samples were consecutively obtained from patients with acute gastroenteritis presenting at our emergency department. In total, 109 complete sample pairs of 108 patients were analysed by specific norovirus real-time PCR. The sensitivity of real-time PCR was 97.3% (36/37) for both sampling methods. A rectal swab is a reasonable option for detection of norovirus by real-time PCR, if a stool specimen is not readily available.

Asunto(s)

Infecciones por Caliciviridae/diagnóstico , Norovirus/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Recto/virología , Manejo de Especímenes/métodos , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Sensibilidad y Especificidad

RESUMEN

OBJECTIVE: To determine the relative frequency of secondary hyperparathyroidism after 1 of 4 standard bariatric surgical procedures, with respect to vitamin D-receptor (VDR) Bsm1 genotype-polymorphism (VDRP). METHODS: Included were 141 obese men and women [aged 44.6±10.4 years, body mass index (BMI) 44.4±5.4 kg/m²], who had undergone either gastric banding (GB; n = 39), laparoscopic sleeve-gastrectomy (LSG; n = 31), Roux-en-Y-gastric-bypass (RYGB; n=43), or biliopancreatic-diversion with "duodenal switch" (BP-DS; n = 28)]. They were tested for VDR-genotype (Bsm1), vitamin D, and serum-PTH-levels postoperatively. RESULTS: Analysis of Covariance revealed a treatment effect, showing statistically significantly higher PTH-levels after BP-DS than after GB (mean difference = 32.14, p<0.001), after SG (mean difference = 25.18, p=0.001), or after RYGB (mean difference = 18.15, p=0.020). VDR-BSM1-genotype did not influence PTH-levels and vitamin-D postoperatively. Logistic regression indicated that the risk of developing SHPT after BP-DS was 12.5 times higher than after GB and 16.7 times higher than after SG. Beside other variables, VDR-genotype and the interaction between VDR-genotype and type of surgery did not attain statistical significance. CONCLUSIONS: In a comparison of the 4 most frequently performed bariatric operations vitamin-D-receptor polymorphism (VDRP) had no influence on the development of postoperative secondary hyperparathyroidism (SHPT) and is not useful as a predictor. SHPT occurs most often after BP-DS. Operation type, gender, VDRP, preoperative BMI, and relative postoperative BMI-loss, however, only explain 24% of the variance in postoperative PTH levels. Other gastral or intestinal factors physiologically promoting calcium-turnover and PTH regulation are postulated.

Asunto(s)

Cirugía Bariátrica/efectos adversos , Hiperparatiroidismo Secundario/etiología , Hiperparatiroidismo Secundario/genética , Mutación , Complicaciones Posoperatorias/etiología , Complicaciones Posoperatorias/genética , Receptores de Calcitriol/genética , Adulto , Cirugía Bariátrica/métodos , Desviación Biliopancreática/efectos adversos , Desviación Biliopancreática/métodos , Estudios de Cohortes , Estudios Transversales , Femenino , Estudios de Asociación Genética , Humanos , Hiperparatiroidismo Secundario/epidemiología , Hiperparatiroidismo Secundario/metabolismo , Incidencia , Masculino , Persona de Mediana Edad , Hormona Paratiroidea/sangre , Complicaciones Posoperatorias/epidemiología , Complicaciones Posoperatorias/metabolismo , Periodo Posoperatorio , Receptores de Calcitriol/metabolismo , Estudios Retrospectivos , Factores de Riesgo , Suiza/epidemiología , Vitamina D/sangre

RESUMEN

We performed a phase I/II clinical trial in metastatic melanoma patients with an ultraviolet (UV)-inactivated nonreplicating recombinant vaccinia virus enabling the expression, from a single construct, of endoplasmic reticulum-targeted HLA-A0201-restricted Melan-A/MART-1(27-35), gp100(280-288), and tyrosinase(1-9) epitopes, together with CD80 and CD86 costimulatory proteins. Corresponding soluble peptides were used to boost responses and granulocyte-macrophage colony-stimulating factor was used as systemic adjuvant. Safety and immunogenicity, as monitored with in vitro-restimulated peripheral blood mononuclear cells by cytotoxic T lymphocyte precursor (CTLp) frequency analysis and tetramer staining, were specifically addressed. Of 20 patients entering the protocol, 2 had to withdraw because of rapidly progressing disease. Immune responses were evaluated in 18 patients (stage III, n = 5; stage IV, n = 13) and increases in specific CTLp frequencies were observed in 15. In 16 patients responsiveness against all 3 antigens could be analyzed: 7 (43%), including all stage III cases, showed evidence of induction of CTLs specific for the three epitopes, and 2 (12%) and 4 (25%), respectively, showed reactivity against two or one tumor-associated antigen. In three stage IV patients no specific CTL reactivity could be induced. Increases in CTLp frequency were detected mostly after viral vaccine injections. However, in a majority of patients final CTLp levels were comparable to initial levels. Tetramer characterization of Melan-A/MART-1(27-35)-specific CTLs during the protocol also suggested preferential expansion after recombinant virus administration. Vector-specific humoral responses, frequently undetectable in stage IV patients, did not appear to prevent tumor-associated antigen-specific CTL induction. Aside from a single occurrence of transient grade 3 leukopenia, no major clinical toxicity was reported. Seventeen of 18 patients completed the 3-month trial (one patient died before the last delayed-type hypersensitivity test). Three displayed regression of individual metastases, seven had stable disease, and progressive disease was observed in seven patients. This is the first report on the administration of a UV-inactivated recombinant vaccinia virus coexpressing five transgenes in cancer patients. The results described here, in terms of safety and immunogenicity, support the use of this reagent in active specific immunotherapy.

Asunto(s)

Vacunas contra el Cáncer/uso terapéutico , Epítopos/inmunología , Antígenos HLA-A/inmunología , Melanoma/terapia , Linfocitos T Citotóxicos/inmunología , Virus Vaccinia/inmunología , Adulto , Anciano , Antígenos CD/inmunología , Antígenos de Neoplasias , Antígeno B7-1/inmunología , Antígeno B7-2 , Vacunas contra el Cáncer/administración & dosificación , Virus Defectuosos , Femenino , Estudios de Seguimiento , Vectores Genéticos , Humanos , Antígeno MART-1 , Masculino , Melanoma/inmunología , Glicoproteínas de Membrana/inmunología , Persona de Mediana Edad , Proteínas de Neoplasias/inmunología , Vacunas Sintéticas/uso terapéutico

RESUMEN

AIMS: The Mitotic Activity Index (MAI) is a strong prognostic factor for disease free survival in breast cancer. The MAI is lower in screen detected tumours, correlating with less aggressive biological behaviour in this group. In this study the MAI is compared between screen detected, interval and symptomatic breast cancers. METHODS: Between 1991 and 1999, the MAI was determined in 581 breast cancers, 160 were detected by screening, 66 were interval carcinomas, and 355 were symptomatic breast cancers. Other prognostic factors were also registered. RESULTS: The interval group had a significantly higher median MAI (17-18, range 1-134) than the screen detected group (7-8, range 0-94,P <0.0001). There was no difference with the symptomatic group (MAI 15, range 0-149,P =0.92). CONCLUSIONS: Interval cancers had an intermediate outcome when correlated with other prognostic factors, compared to screen detected and symptomatic cancers.

Asunto(s)

Neoplasias de la Mama/diagnóstico , Carcinoma Ductal de Mama/diagnóstico , Índice Mitótico , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Femenino , Humanos , Tamizaje Masivo , Persona de Mediana Edad , Invasividad Neoplásica , Estadificación de Neoplasias , Países Bajos , Pronóstico , Receptores de Estrógenos/metabolismo , Biopsia del Ganglio Linfático Centinela , Salud de la Mujer

RESUMEN

The effect on immunogenicity of different tumor T cell epitope formulations was evaluated in vitro using nonreplicating recombinant vaccinia vector expressing two forms of the melanoma-associated MART-1/Melan-A antigen. The first recombinant virus expressed a minigene encoding a fusion product between an endoplasmic reticulum (ER)-targeting signal and the HLA-A201 binding 27-35 peptide. The second viral construct encoded the complete MART-1/Melan-A protein. The capacity of HLA-A201 cells infected with either viral construct to generate and to stimulate MART-1/Melan-A 27-35 specific cytotoxic T-lymphocytes (CTL), was comparatively characterized. The results obtained here with a tumor antigen confirmed the capacity of vaccinia virus-encoded ER-minigene to generate a very strong antigenic signal. In cytotoxicity assays, recognition of target cells infected with high amounts of both recombinant viruses with activated specific CTL clones, resulted in similar lytic activity. With regard to calcium mobilization, TCR down-regulation, IFN-gamma release, and T cell proliferation assays, the targeted epitope elicited 10- to 1000-fold stronger responses. Remarkably, the immunogenic difference between the two formulations, in their respective capacity to generate CTL from naive HLA-A2 peripheral blood mononuclear cells in vitro as measured by tetramer detection, was lower (2- to 3-fold). Recombinant vectors expressing complete antigens have demonstrated their capacity to generate specific responses and such vaccines might take advantage of a broader potential of presentation. However, as demonstrated here for the HLA-A201-restricted MART-1/Melan-A immunodominant epitope, nonreplicative vaccinia virus expressing ER-targeted minigenes appear to represent a significantly more immunogenic epitope vaccine formulation.

Asunto(s)

Antígenos Virales/inmunología , Epítopos/inmunología , Antígeno HLA-A2/inmunología , Melanoma/inmunología , Proteínas de Neoplasias/inmunología , Linfocitos T Citotóxicos/inmunología , Virus Vaccinia/inmunología , Presentación de Antígeno , Antígenos de Neoplasias , Calcio/metabolismo , Citotoxicidad Inmunológica , Regulación hacia Abajo , Humanos , Inmunización , Interferón gamma/metabolismo , Activación de Linfocitos/inmunología , Antígeno MART-1 , Melanoma/patología , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes , Linfocitos T/inmunología , Transfección , Células Tumorales Cultivadas , Vacunas Virales , Replicación Viral

RESUMEN

To gain new insights into the functional interaction between DC and neoplastic cells, we have analyzed the effects of melanoma and colorectal cancer lines on the chemotaxis and the phenotype of monocyte-derived DC in vitro. Both types of tumor cells displayed effective chemoattractive capacity towards immature, but not mature DC. Furthermore, conditioned medium of discrete melanoma lines induced upregulation of CD80, CD86, MHC class I, and MHC class II molecules on immature DC. However, de novo expression of E-cadherin and strong upregulation of CD15 could also be detected in the absence of CD83 expression. Melanoma-conditioned DC exhibited an increased adhesion capacity to a melanoma cell line in vitro and did not migrate in response to SLC chemokine. Tumor-infiltrating CD15(+) cells displaying DC morphology could also be detected by immunohistochemistry in the original tumor specimens from which discrete melanoma cell lines under investigation were derived. Colorectal cancer cell lines, although able to chemoattract immature DC, were apparently unable to modulate their phenotype. Altogether our results suggest that tumor cells can attract immature DC in vitro and, eventually, modulate their phenotype. As a result, DC mobility could be severely impaired.

Asunto(s)

Quimiotaxis de Leucocito/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Inmunofenotipificación , Células Tumorales Cultivadas/inmunología , Animales , Cadherinas/biosíntesis , Cadherinas/genética , Quimiocinas/biosíntesis , Quimiocinas/genética , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Medios de Cultivo Condicionados/farmacología , Células Dendríticas/patología , Regulación Neoplásica de la Expresión Génica/inmunología , Humanos , Inmunohistoquímica , Antígeno Lewis X/análisis , Antígeno Lewis X/biosíntesis , Melanoma/inmunología , Melanoma/patología , Ratones , Regulación hacia Arriba/inmunología

RESUMEN

NY-ESO-1 gene encodes a novel member of the cancer/testis (CT) family of human tumour-associated antigens (TAA). Specific monoclonal antibodies (mAb) have identified the corresponding gene product in lysates of tumour cell lines as a 22 kDa protein but no data are available concerning its intracellular location or distribution within neoplastic tissues. We have generated NY-ESO-1 specific mAbs recognizing the target molecule in cytospin preparations and in sections from clinical tumour specimens. These reagents identify NY-ESO-1 TAA in melanoma cell lines expressing the specific gene as a cytoplasmic protein, sharing the intracellular location of most MAGE TAA. In a series of 12 melanoma specimens, specific staining, limited to neoplastic cells, was detectable in the five cases where NY-ESO-1 gene expression was observed. In two of them over 90% of tumour cells showed evidence of positive staining. Lower percentages of positive neoplastic cells ranging between single cells and 50% were observed in the remaining tumours. These data suggest that active specific immunotherapies targeting NY-ESO-1, alone or in combination with other TAA could be of high clinical relevance in sizeable subgroups of melanoma patients.

Asunto(s)

Anticuerpos Monoclonales , Antígenos de Neoplasias/análisis , Melanoma/metabolismo , Proteínas de la Membrana , Proteínas/análisis , Línea Celular , Humanos , Inmunohistoquímica , Proteínas/inmunología , Proteínas Recombinantes/inmunología , Células Tumorales Cultivadas

RESUMEN

In this study, a computer-assisted reverse immunology approach was utilized in order to identify potentially antigenic peptides derived from the differentiation antigen TRP-2, a melanosomal protein frequently expressed in melanoma. Among the seven peptides complying with HLA-A2.1-binding motifs, two induced specific CD8(+) cytotoxic T lymphocytes. HLA-A2.1(+) melanoma cells expressing TRP-2 were lysed by clones specific for TRP-2(360-368) (TLDSQVMSL) peptide, thus identifying it as a naturally processed epitope. Other T-cell clones directed against TRP-2(476-484) (VMGTLVALV) were unable to lyse HLA-matched TRP-2(+) cell lines. The role of intracellular proteolytic processing in the generation of this epitope was investigated by transfecting mini-genes encoding the TRP-2(476-484) peptide alone or carrying N- or C-terminal extensions. Specific T-cell clones recognized target cells expressing the cytotoxic T-lymphocyte (CTL)-defined epitope or its C-terminally extended precursor, but failed to recognize cells expressing the N-terminally extended TRP-2(476-484) peptide, suggesting the presence of a negative processing signal (NPS). Regarding C-terminus-flanking regions, mutational analysis indicates that the GLY485 residue plays a key role in the processing of the TRP-2(476-484) epitope. Interestingly, proteasome inhibitors preventing the generation of the MART-1/Melan-A(27-35) immunodominant melanoma tumor-associated antigen (TAA) promoted detectable presentation of TRP-2(476-484) epitope in HLA-A2.1(+) and TRP-2(+) tumor lines, as witnessed by cytokine release by specific T-cell clones.

Asunto(s)

Epítopos/inmunología , Antígeno HLA-A2/inmunología , Oxidorreductasas Intramoleculares/inmunología , Melanoma/inmunología , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular Transformada , Técnicas de Cocultivo , Inhibidores de Cisteína Proteinasa/farmacología , Análisis Mutacional de ADN , Ensayo de Inmunoadsorción Enzimática , Epítopos/efectos de los fármacos , Inhibidores de la Proteasa del VIH/farmacología , Antígeno HLA-A2/genética , Humanos , Oxidorreductasas Intramoleculares/química , Oxidorreductasas Intramoleculares/genética , Leucocitos Mononucleares/inmunología , Melanoma/genética , Melanoma/metabolismo , Péptidos/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ritonavir/farmacología , Linfocitos T Citotóxicos/metabolismo , Células Tumorales Cultivadas

RESUMEN

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that can be used for vaccination purposes, to induce a specific T-cell response in vivo against melanoma-associated antigens. We have shown that the sequential use of early-acting hematopoietic growth factors, stem cell factor, IL-3 and IL-6, followed by differentiation with IL-4 and granulocyte-macrophage colony-stimulating factor allows the in vitro generation of large numbers of immature DCs from CD34(+) peripheral blood progenitor cells. Maturation to interdigitating DCs could specifically be induced within 24 hr by addition of TNF-alpha. Here, we report on a phase I clinical vaccination trial in melanoma patients using peptide-pulsed DCs. Fourteen HLA-A1(+) or HLA-A2(+) patients received at least 4 i.v. infusions of 5 x 10(6) to 5 x 10(7) DCs pulsed with a pool of peptides including either MAGE-1, MAGE-3 (HLA-A1) or Melan-A, gp100, tyrosinase (HLA-A2), depending on the HLA haplotype. A total of 83 vaccinations were performed. Clinical side effects were mild and consisted of low-grade fever (WHO grade I-II). Clinical and immunological responses consisted of anti-tumor responses in 2 patients, increased melanoma peptide-specific delayed-type hypersensitivity reactions in 4 patients, significant expansion of Melan-A- and gp100-specific cytotoxic T lymphocytes in the peripheral blood lymphocytes of 1 patient after vaccination and development of vitiligo in another HLA-A2(+) patient. Our data indicate that the vaccination of peptide-pulsed DCs is capable of inducing clinical and systemic tumor-specific immune responses without provoking major side effects.

Asunto(s)

Presentación de Antígeno , Vacunas contra el Cáncer/administración & dosificación , Células Dendríticas/inmunología , Inmunoterapia Adoptiva , Melanoma/inmunología , Melanoma/terapia , Adolescente , Adulto , Anciano , Antígenos CD34 , Antígenos de Neoplasias , Vacunas contra el Cáncer/inmunología , Diferenciación Celular/inmunología , Citotoxicidad Inmunológica , Células Dendríticas/patología , Células Dendríticas/trasplante , Femenino , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/patología , Humanos , Masculino , Persona de Mediana Edad , Péptidos/inmunología , Proyectos Piloto

RESUMEN

Dendritic cells (DC) are professional antigen presenting cells (APC) whose proliferation and functional differentiation can be induced by hematopoietic growth factors including GM-CSF and FLT3 ligand (FL). Colorectal cancers are known to be infiltrated by dendritic cells (DC) and neoplastic cells have been shown to produce GM-CSF. In this work we investigated FLT3 ligand (FL) gene expression and protein production in human colorectal cancer cell lines and clinical tumor specimens. Using reverse transcription polymerase chain reaction (RT-PCR), 6 out of 6 established tumor lines were found to express to variable extents FL gene. In 1 of them, SW480, FL immunoreactivity could be observed by taking advantage of specific antibodies. In contrast, soluble FL could not be detected in any culture supernatant. FLT3 receptor (FR) gene was not expressed and exogenous addition to the cultures of recombinant FL (rFL) did not affect the proliferation of the tumor lines. FL gene expression was investigated using a densitometry-assisted, semiquantitative RT-PCR in clinical tumor specimens. Specific FL gene transcripts were amplified from 12 of 12 surgical samples. In these cases, FL gene expression of significantly lower intensity was also detected in healthy mucosa sampled in the vicinity (2 cm) or at a distance (10 cm) from neoplastic outgrowth. Immunohistochemical studies identified FL-positive cancer cells in 5 of 5 cases tested. No positivity was detected in healthy mucosa epithelia at a distance from the tumor or in stromal cells. FL content in preoperative sera from colorectal cancer patients (n = 13) did not exceed the levels detected in healthy donors (

Asunto(s)

Neoplasias Colorrectales/metabolismo , Expresión Génica , Proteínas de la Membrana/genética , Neoplasias Colorrectales/química , Neoplasias Colorrectales/genética , Técnica del Anticuerpo Fluorescente , Hematopoyesis , Humanos , Inmunohistoquímica , Proteínas de la Membrana/análisis , Proteínas de la Membrana/biosíntesis , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas

RESUMEN

Human melanoma cell lines may induce a specific T cell response against tumor cells in vitro. However, after repeated restimulation with autologous tumor cells, expansion of CTL is limited and often apoptosis of the T cells occurs. In order to improve conditions inducing primary T cell responses and thus allowing further expansion of tumor specific T cells for an adoptive transfer, we transfected human melanoma cells with the B7.1 gene (CD80), known to be a potent costimulatory molecule for T cell activation. CD80 expression on melanoma cells resulted in improved primary T cell activation, especially of CD8+ T cells. Furthermore, restimulation with CD80+ tumor cells gave rise to long term proliferating CD8+ T cell lines demonstrating an 100-fold expansion of T cells compared to the 20-30-fold increased numbers obtained with the controls (parental tumor cells +/- anti-CD28). T cells stimulated with CD80+ melanoma cells were found to display a MHC class I-restricted cytotoxic activity against the autologous tumor cells. In conclusion, these studies demonstrate the requirement of costimulation in generating large numbers of tumor specific T cells in vitro that may be used for an adoptive transfer in tumor immunotherapy.

Asunto(s)

Antígeno B7-1/genética , División Celular/inmunología , Melanoma/inmunología , Linfocitos T Citotóxicos/citología , Traslado Adoptivo , Secuencia de Bases , Citotoxicidad Inmunológica , Cartilla de ADN , Humanos , Interleucina-12/farmacología , Activación de Linfocitos/efectos de los fármacos , Melanoma/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T Citotóxicos/inmunología , Transfección

RESUMEN

In this study we used TEPITOPE, a new epitope prediction software, to identify sequence segments on the MAGE-3 protein with promiscuous binding to histocompatibility leukocyte antigen (HLA)-DR molecules. Synthetic peptides corresponding to the identified sequences were synthesized and used to propagate CD4(+) T cells from the blood of a healthy donor. CD4(+) T cells strongly recognized MAGE-3281-295 and, to a lesser extent, MAGE-3141-155 and MAGE-3146-160. Moreover, CD4(+) T cells proliferated in the presence of recombinant MAGE-3 after processing and presentation by autologous antigen presenting cells, demonstrating that the MAGE-3 epitopes recognized are naturally processed. CD4(+) T cells, mostly of the T helper 1 type, showed specific lytic activity against HLA-DR11/MAGE-3-positive melanoma cells. Cold target inhibition experiments demonstrated indeed that the CD4(+) T cells recognized MAGE-3281-295 in association with HLA-DR11 on melanoma cells. This is the first evidence that a tumor-specific shared antigen forms CD4(+) T cell epitopes. Furthermore, we validated the use of algorithms for the prediction of promiscuous CD4(+) T cell epitopes, thus opening the possibility of wide application to other tumor-associated antigens. These results have direct implications for cancer immunotherapy in the design of peptide-based vaccines with tumor-specific CD4(+) T cell epitopes.

Asunto(s)

Presentación de Antígeno , Antígenos de Neoplasias , Linfocitos T CD4-Positivos/inmunología , Citotoxicidad Inmunológica , Antígenos HLA-DR/inmunología , Melanoma/inmunología , Proteínas de Neoplasias/inmunología , Secuencia de Aminoácidos , Vacunas contra el Cáncer , Diseño de Fármacos , Epítopos , Predicción , Subtipos Serológicos HLA-DR , Humanos , Activación de Linfocitos , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Unión Proteica , Procesamiento Proteico-Postraduccional , Programas Informáticos , Subgrupos de Linfocitos T

RESUMEN

In this work, we addressed the possibility to enhance the "in vitro" generation of CTLs recognizing tumor-associated antigens (TAAs) by using an inactivated recombinant vaccinia virus encoding B7.1 and B7.2 costimulatory molecules (rVV-B7.1/2). Antigen presenting cells (APCs) infected by rVV-B7.1/2 and pulsed with MART-1/Melan-A27-35 HLA-A2.1-restricted peptide induced significantly higher specific cytotoxic activity than peptide-loaded APCs infected by wild-type VV, both in VV-sensitized and naive donors. When APCs were infected with a rVV encoding both MART-1/Melan-A27-35 and B7-1/2 (rVV-B7.1/2-M), a significantly more effective CTL generation was observed as compared with cultures stimulated by APCs infected with a rVV encoding the TAA epitope only (rVV-M). These enhancing effects were detectable irrespective of a previous VV-specific sensitization. Most importantly, fibroblasts, devoid of antigen-presenting capacity upon peptide pulsing or infection with rVV-M, could be turned into effective APCs after infection by rVV encoding TAA epitopes and costimulatory molecules. In these experiments, by using separate recombinant viral constructs, we observed a predominant role of B7-1 as compared with B7-2 in the induction of TAA-specific CTLs. Taken together, our data indicate that replication-incompetent rVV encoding TAA epitopes and costimulatory molecules are able to induce highly effective generation of tumor-specific CTLs. Therefore, these vectors could represent valuable clinical tools for immunotherapy of melanoma patients.

Asunto(s)

Antígenos de Neoplasias/inmunología , Antígeno B7-1/inmunología , Linfocitos T Citotóxicos/fisiología , Virus Vaccinia/inmunología , Células Presentadoras de Antígenos/fisiología , Antígenos de Neoplasias/genética , Antígeno B7-1/genética , Epítopos , Humanos , Inmunoterapia , Proteínas Recombinantes/inmunología , Rayos Ultravioleta , Virus Vaccinia/genética

RESUMEN

Granulocyte-macrophage colony stimulating factor (GM-CSF) gene expression and protein production were investigated in colorectal cancer cell lines and surgical specimens. In 6 of 6 established tumor lines, expression of the GM-CSF gene was observed by reverse transcription polymerase chain reaction (RT-PCR). Furthermore, for 2 of the lines, the cytokine was detectable in > or = 100 pg/ml amounts in culture supernatants by enzyme-linked immunosorbent assay tests. Addition of recombinant GM-CSF at doses ranging between 30 pg and 30 ng/ml did not appear to affect the proliferation of colorectal cancer cell lines as measured by 3H-thymidine incorporation. GM-CSF gene expression was then examined in surgical specimens by a densitometry-assisted, semiquantitative RT-PCR technique. In the 10 samples analyzed, significantly higher expression was detectable in tumors, as compared with autologous healthy mucosa sampled in the vicinity (2 cm) or at distance (10 cm) from the neoplastic focus. Immunohistochemistry studies performed on 13 specimens led to the identification of intracytoplasmic GM-CSF in tumor cells in 9 samples. In 6 of these, positivity of stromal fibroblasts and lymphocytes adjacent to the tumor was also observed. In contrast, intracellular GM-CSF was only detectable in 2 cases in untransformed epithelial cells, close to the neoplasm, but never in healthy mucosa at distance from the tumor. Infiltration by dendritic cells (DC) was also investigated. In 5 of 8 colorectal cancers tested, DC aggregates accounted for more than 10% of stromal cells. Lower numbers were detectable in healthy mucosa. However, DC infiltration could not be correlated with the presence of GM-CSF-positive neoplastic cells in the tumor specimens. Remarkably, cultured DC were unable to exert significant cytotoxic activity against colorectal cancer cells in vitro.

Asunto(s)

Neoplasias Colorrectales/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Biosíntesis de Proteínas , Transcripción Genética , Anciano , División Celular/efectos de los fármacos , Línea Celular , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/cirugía , Ensayo de Inmunoadsorción Enzimática , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Cinética , Masculino , Reacción en Cadena de la Polimerasa , Células Tumorales Cultivadas

RESUMEN

C-type lectin-like inhibitory receptors are heterodimers consisting of CD94 and NKG2-A-B molecules expressed on NK cells and on a subset of activated T lymphocytes. Their inhibitory effects on NK cytotoxicity and on the NK-like activity of T cell clones have been demonstrated, but no data are currently available on antigen-specific class I-restricted cytotoxic T lymphocytes (CTL). We have generated a panel of HLA-A2.1-restricted CTL clones directed against a nonapeptide derived from a melanoma-associated antigen, dopachrome tautomerase (TRP-2). All clones were CD8+ and TCR alphabeta+. About half of them expressed a CD94bright phenotype, whereas the remaining were CD94dim. Only the CD94bright CTL expressed the NKG2-A-B gene, consistent with the expression of a C-type, lectin-like, inhibitory CD94/NKG2-A-B heterodimer. Both CD94bright and CD94dim clones appeared to require similar amounts of synthetic epitope sensitizing target cells. Addition of anti-CD94 mAb resulted in a significant increase of specific killing by CD94bright, but not by CD94dim clones in the presence of suboptimal concentrations of peptide, whereas, when optimal amounts were used, the mAb did not induce a significant modulation of the cytotoxicity. Antigen-induced inward [Ca2+]i fluxes were unaffected, but an enhancement of TCR down-modulation could be observed in the presence of anti-CD94 mAb at high concentration of antigenic peptide. The analysis of the TCR-Vbeta repertoire of the CTL clones by RT-PCR and immunofluorescence revealed that all clones regardless of CD94 phenotype shared Vbeta22 expression. Most importantly, sequence analysis showed that they all expressed identical Vbeta22 TCR rearranged with Jbeta2.1 and Cbeta2. Taken together, these data indicate that different expression of functionally active lectin-like inhibitory receptors can be detected in CTL clones sharing identical TCR sequence and peptide specificity.

Asunto(s)

Antígenos CD/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Lectinas Tipo C , Glicoproteínas de Membrana/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Secuencia de Bases , Células Clonales , Epítopos/inmunología , Antígenos de Histocompatibilidad Clase I/biosíntesis , Humanos , Datos de Secuencia Molecular , Subfamília D de Receptores Similares a Lectina de las Células NK

RESUMEN

Monoclonal antibody 57B specifically detects MAGE-3 gene protein expression. MAGE-derived peptides are recognized by CD8+ T cells and applied in immunotherapy. We examined formalin-fixed, paraffin-embedded tissue of 61 melanoma (primary, n = 40; metastatic, n = 21) and 46 control cases (junctional, dermal, compound, Spitz, Reed, and balloon-cell nevi) by immunohistochemistry using the alkaline phosphatase anti-alkaline phosphatase method after antigen retrieval. Immunoreactivity was rated positive at 20 positive cells per tumor or more. Staining pattern was homogeneous, scattered, or focal. All control samples and internal controls were immunonegative. Staining with monoclonal antibody 57B showed a specificity of 100% with a sensitivity of 44%. Immunopositivity (overall, 44% of melanomas) increased along with tumor, node, and metastasis stage; pT1 showed 13%, pT2 22%, pT3a 29%, pT3b 45%, pT4 100%, pTxN1 60%, and pTxNxM1a 63% of samples positive. The staining pattern was homogeneous on pT1 to pT3a tumors, homogeneous or focal in pT3b and pT4a, and homogeneous, focal, or scattered in pTxN1 and pTxNxM1a. The frequency of immunopositivity relates well to data on mRNA expression using reverse transcriptase polymerase chain reaction in a subgroup analyzed by both methods. Monoclonal antibody 57B can be used to allow profiling of melanomas using routine archival tissue, when considering immunotherapeutic approaches involving MAGE-3-derived epitopes.

Asunto(s)

Antígenos de Neoplasias/metabolismo , Melanoma/metabolismo , Melanoma/secundario , Proteínas de Neoplasias/metabolismo , Neoplasias Cutáneas/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Células COS , Cartilla de ADN/química , Técnica del Anticuerpo Fluorescente Indirecta , Formaldehído , Humanos , Técnicas para Inmunoenzimas , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Adhesión en Parafina , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad , Neoplasias Cutáneas/patología , Fijación del Tejido/métodos , Transfección

Asunto(s)

Antígenos de Neoplasias , Carcinoma de Células Escamosas/genética , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad

RESUMEN

Active specific immunotherapy targeting tumor-associated antigens (TAA) requires reagents of high immunogenicity and safety. To address this issue, we constructed a recombinant vaccinia virus carrying a minigene insert encoding the HLA-A2.1-restricted MART-1/Melan-A27-35 melanoma TAA (rVV-M). To facilitate the entry of the antigenic epitope into the endoplasmic reticulum, a sequence coding for adenovirus E3/19K leader peptide was added. This rVV-M was made replication-incompetent by treatment with psoralen and UV light. Infection with rVV-M rendered HLA-A2.1 EBV-transformed lymphoblastoid cells sensitive to the cytotoxic effects of HLA-class-1-restricted, MART-1/Melan-A27-35-specific cytotoxic T lymphocytes (CTL). The capacity of rVV-M to generate HLA-A2.1-restricted MART-1/Melan A-specific CTL was demonstrated from tumor-infiltrating-lymphocyte (TIL) cultures and from healthy donors' peripheral-blood mononuclear cells (PBMC). MART-1/Melan-A27-35-specific CTL were generated from TIL after 2 weekly stimulation courses. Infection with rVV-M elicited a higher CTL response than addition of exogenous peptide, whereas, when a similar protocol was used to stimulate PBMC of healthy donors, significant and specific cytotoxic activity could be observed only upon rVV-M infection but not upon exogenous peptide addition. All CTL generated upon rVV-M stimulation were also able to efficiently kill melanoma cell lines expressing both MART-1/Melan-A and HLA-A2.1. In addition, TNF-alpha production could be induced in rVV-M-stimulated CTL upon co-culture with COS-7 cells transiently transfected with MART-1/Melan-A and HLA-A2.1 genes. This safe and highly immunogenic reagent could be of use in TAA-targeted clinical immunotherapy.

Asunto(s)

Antígenos Virales/inmunología , Citotoxicidad Inmunológica , Epítopos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/inmunología , Proteínas de Neoplasias , Linfocitos T Citotóxicos/inmunología , Vacunas Sintéticas , Virus Vaccinia/inmunología , Vacunas Virales , Secuencia de Aminoácidos , Animales , Antígenos Virales/biosíntesis , Antígenos Virales/química , Secuencia de Bases , Donantes de Sangre , Células COS , Línea Celular , Epítopos/biosíntesis , Epítopos/química , Humanos , Linfocitos Infiltrantes de Tumor/patología , Melanoma/patología , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Valores de Referencia , Transfección , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/biosíntesis , Virus Vaccinia/fisiología , Replicación Viral

RESUMEN

OBJECTIVE: The authors' objective is to identify MAGE-1 tumor antigen in clinical melanoma specimens and to verify the extent of its expression in tumors where evidence of specific gene transcripts can be obtained. BACKGROUND DATA: The MAGE-1 gene encodes a tumor-associated antigen that can be recognized by specific cytotoxic T lymphocytes. Transcription of the MAGE-1 gene has previously been demonstrated in various malignancies, but the production of the specific gene product and its distribution in neoplastic tissues have not yet been addressed. METHODS: Total cellular mRNA was extracted from six melanoma biopsies, reverse-transcribed and tested in 25-45 cycles of reverse polymerase chain reaction (rtPCR) in the presence of primers' pairs specific for the beta-actin-positive control gene and for the MAGE-1-encoding gene. Concurrently, portions of these specimens were lysed and probed for MAGE-1 protein by immunoblotting. Additional material from the same biopsies was analyzed following immunohistological staining with MAGE-1-specific monoclonal antibodies. RESULTS: MAGE-1 gene transcription could be demonstrated following 25 cycles of rtPCR in one out of six biopsies and in three more following 35 cycles of rtPCR. 2/6 samples were negative even after 45 cycles of rtPCR. MAGE-1 protein production could be detected by immunoblotting in the lysates from biopsies showing evidence of specific gene transcription. Cells positive for MAGE-1 protein expression could be identified by immunohistochemistry on snap-frozen sections in three of the four tumors displaying specific transcripts. Distribution of positivity ranged between focal cellular areas and single positive cells in the different tumors. CONCLUSIONS: The MAGE-1 tumor antigen can be detected by specific monoclonal antibodies in clinical tumor specimens. The pattern of positivity observed in samples showing evidence of MAGE-1 gene expression suggests a relevant heterogeneity regarding MAGE-1 antigen production within individual tumor specimens.

Asunto(s)

Variación Genética , Melanoma/genética , Melanoma/inmunología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Anticuerpos Monoclonales , Antígenos de Neoplasias , Humanos , Immunoblotting , Inmunohistoquímica , Melanoma/metabolismo , Antígenos Específicos del Melanoma , Reacción en Cadena de la Polimerasa , Transcripción Genética

RESUMEN

Genes of the MAGE family encode tumor-specific antigens recognized by cytotoxic T-lymphocytes in a variety of neoplasms. We investigated the protein expression of these antigens as related to the gene expression, in esophageal squamous cell carcinoma by using monoclonal antibodies recognizing MAGE gene products. Esophageal squamous cell carcinomas were found to express both MAGE-1 (4 out of 15 samples) and MAGE-3 (7 out of 15 samples) genes, by RT-PCR. Immunoblotting revealed MAGE-1 and MAGE-3 gene products in 2 and 6 out of 15 samples, respectively. Immunohistochemistry performed on 12 samples showed MAGE-1 protein expression, limited to single tumor cells, in 2 cases. MAGE-3 gene product was detectable in 7 cases: in 5 of them over 50% of neoplastic cells were positive. Considering the high percentages of tumor cells expressing MAGE-3 antigen, the use of epitope-based vaccines could be envisaged in patients displaying appropriate HLA-class I phenotype.