Immunotherapy against antigenic tumors: a game with a lot of players.

Our understanding of how immune responses are generated and regulated drives the design of possible immunotherapies for cancer patients. For that reason, we first describe briefly the actual immunological theories and their common perspectives about cancer vaccine development. Second, we describe cancer vaccines that are able to induce tumor-specific immune responses in cancer patients. However, these responses are not always followed by tumor rejection. At the end of the review, we discuss two possible reasons that might explain this dichotomy of cancer immunology. First, the immune response generated, although detectable, may not be quantitatively sufficient to reject the tumor. Second, the tumor microenvironment may modulate tumor cell susceptibility to the systemic immune response induced by the immunization. Finally, we discuss what, in our opinion, might be the best way to improve cancer vaccine strategies and how the relationship between the tumor and its surroundings might be studied in more details.

Kinetics of TCR use in response to repeated epitope-specific immunization.

Selection of T cell-directed immunization strategies is based extensively on discordant information derived from preclinical models. We characterized the kinetics of T cell selection in response to repeated antigenic challenge. By enumerating with epitope/HLA tetrameric complexes (tHLA) vaccine-elicited T cell precursor frequencies (Tc-pf) in melanoma patients exposed to the modified gp100 epitope gp100:209-217 (g209-2M) we observed in most patients that the Tc-pf increased with number of immunizations. One patient's kinetics were further characterized. Dissociation kinetics of g209-2M/tHLA suggested enrichment of T cell effector populations expressing TCR with progressively higher affinity. Furthermore, vaccine-elicited T cells maintained the ability to express IFN-gamma ex vivo and proliferate in vitro. Thus, repeated exposure to immunogenic peptides benefited immune competence. These results provide a rationale for immunization strategies.

Genetic heterogeneity in the toxicity to systemic adenoviral gene transfer of interleukin-12.

Despite the efficacy of IL-12 in cancer experimental models, clinical trials with systemic recombinant IL-12 showed unacceptable toxicity related to endogenous IFNgamma production. We report that systemic administration of a recombinant adenovirus encoding IL-12 (AdCMVmIL-12) has a dramatically different survival outcome in a number of mouse pure strains over a wide range of doses. For instance at 2.5 x 10(9) p.f.u., systemic AdCMVmIL-12 killed all C57BL/6 mice but spared all BALB/c mice. Much higher IFNgamma concentrations in serum samples of C57BL/6 than in those from identically treated BALB/c were found. Causes for heterogeneous toxicity can be traced to differences among murine strains in the levels of gene transduction achieved in the liver, as assessed with adenovirus coding for reporter genes. In accordance, IL-12 serum concentrations are higher in susceptible mice. In addition, sera from C57BL/6 mice treated with AdCMVmIL-12 showed higher levels of IL-18, a well-known IFNgamma inducer. Interestingly, lethal toxicity in C57BL/6 mice was abolished by administration of blocking anti-IFNgamma mAbs and also by simultaneous depletion of T cells, NK cells, and macrophages. These observations together with the great dispersion of IFNgamma produced by human PBMCs upon in vitro stimulation with IL-12, or infection with recombinant adenovirus encoding IL-12, suggest that patients might also show heterogeneous degrees of toxicity in response to IL-12 gene transfer.

Status of activation of circulating vaccine-elicited CD8+ T cells.

Selective blunting of the status of activation of circulating tumor-specific T cells was invoked to explain their paradoxical coexistence with unhampered tumor growth. By analogy, lack of tumor regression in the face of observable melanoma vaccine-induced T cell responses might be attributed to their status of activation. We enumerated with HLA-A*0201/peptide tetramers (tHLA) vaccine-elicited T cell precursor frequency directly in PBMC of patients with melanoma undergoing vaccination with the HLA-A*0201-associated gp100:209-217(210 M) epitope (g209-2 M). Furthermore, we tested by intracellular (IC)-FACS analysis and quantitative real-time PCR (qRT-PCR) the ability of postvaccination PBMC to produce cytokine in response to challenge with vaccine-related epitopes or vaccine-matched (HLA-A*0201) melanoma cells. Vaccine-induced enhancement of T cell precursor frequency could be detected with tHLA in PBMC from six of eight patients studied at frequencies ranging between 0.3 and 2.3% of the total CD8+ population. Stimulation with vaccine-related epitopes induced IFN-gamma expression detectable by IC-FACS or qRT-PCR, respectively, in five and six of these patients. Furthermore, down-regulation of tHLA staining was noted upon cognate stimulation that could be utilized as an additional marker of T cell responsiveness. Finally, we observed in six patients an enhancement of reactivity against vaccine-matched tumor targets that was partly independent of documented vaccine-specific immune responses. A strong correlation was noted between tHLA staining of postvaccination PBMC and IFN-gamma expression by the same samples upon vaccine-relevant stimulation and assessed either by IC-FACS or qRT-PCR. Thus, blunting of the status of T cell activation on itself cannot easily explain the lack of clinical responses observed with vaccination.

Immunoregulating properties of peptides related to tumor rejection antigens: effect on human monocytes and natural killer cells.

The authors analyzed the effect of several 15-amino acid peptides with sequences related to tumor-rejection antigens, tyrosinase, and the MAGE family on peripheral blood mononuclear cells from healthy donors cultured for periods of 1 to 7 days. Some of these peptides promoted stimulation of monocytes, manifested by phenotypic changes, release of interleukin (IL)-1a, IL-6, and tumor necrosis factor-alpha, and induction of nitric oxide synthase on differentiated CD14++/+ CD16+ DR++ monocytes. An increase in the percentage of cytotoxic monocytes (CD14+/- CD16+) containing granule-associated DNase activity was also observed. Active peptides induced the release of IL-2 and interferon-gamma. Nonspecific natural killer and lymphokine-activated killer cell-mediated cytotoxicity was also observed against classical target cell lines (K-562 and Daudi) and allogenic melanoma cell lines AC and BB, together with an increase in granule-associated DNase in the natural killer cell-enriched population. Monocytes were needed to enhance this innate response, because peptides failed to induce the release of IL-2 on monocyte-depleted peripheral blood mononuclear cells. Data show an enhancement of the rapid innate immune response by peptides related to tumor rejection antigens and suggest that they could also determine the nature of a slow and more definitive specific immune response against tumor cells.

[Benzodiazepine prescription at a health center: prevalence, its use and user characteristics]. / Prescripción de benzodiacepinas en un centro de salud: prevalencia, cómo es su consumo y características del consumidor.

OBJECTIVES: To find the prevalence of prescription of benzodiazepines (BDZ) in 1997 at a health centre (HC), and the characteristics of both their consumption and the takers. DESIGN: Crossover, observational study. SETTING: Primary care urban centre. PATIENTS: From a total of 7356 patients over 14 with clinical records and belonging to four lists, a random sample stratified by lists was selected. INTERVENTION: A form was used to gather social and demographic data, educational level, family context, linked pathologies, number of visits to HC per year, BDZ prescription and variables defining the kind of consumption. MAIN RESULTS: The prevalence of BDZ prescription was 7.7% (CI, 6-10%). Consumption profile: 33% long BDZ, 31% intermediate and 33% short. 44% consumed BDZ occasionally or for less than 2 weeks, and 42% had been taking it for over a year. For 56% (95% CI, 40-70) their G.P. was the origin of the prescription. The reason for the prescription was not specified in 42% of cases. The variables which defined the profile of the consumers, included in the logistic regression, were: sex, number of visits and linked pathologies, whose OR were: 1.57 (CI, 1.08-2.03), 1.11 (CI, 1.06-1.17) and 1.61 (CI, 1.04-2.05). CONCLUSIONS: The prevalence of BDZ prescription during 1997 in the population seen at our clinics was very similar to the figures found in other studies, with higher annual consumption averages and without the reason for taking it being specified in half the cases. The profile of BDZ takers was: mainly women, people with linked pathologies, and as a function of the number of attendances.

Prescripción de benzodiacepinas en un centro de salud: prevalencia, cómo es su consumo y características del consumidor / Prescription of benzodiazepines at a health centre: prevalence, how they are taken and characteristics of the taker

Objetivos.Prevalencia de prescripción de benzodiacepinas (BDZ) en 1997 en un centro de salud (CS), cómo es el consumo y características del consumidor. Diseño. Estudio observacional transversal. Emplazamiento.Centro urbano de atención primaria. Pacientes. Entre un total de 7.356 pacientes mayores de 14 años con historia clínica pertenecientes a 4 consultas, se seleccionó una muestra aleatoria estratificada por consultas de 667. Intervención. Hoja de recogida de datos sociodemográficos, nivel de instrucción, entorno familiar, patologías asociadas, n.º visitas/año, prescripción de BDZ y variables que definen el tipo de consumo. Resultados principales. Prevalencia de prescripción de BDZ: 7,7 por ciento (IC del 95 por ciento, 6-10 por ciento). Perfil de consumo: 33 por ciento BDZ de vida media larga, 31 por ciento de intermedia y 33 por ciento de corta.El 44 por ciento las consume de forma ocasional o menos de 2 semanas y un 42 por ciento lleva más de un año consumiéndolas; en un 56 por ciento (IC del 95 por ciento, 40-70) el origen de la prescripción es el médico de cabecera y no consta el motivo de la prescripción en el 42 por ciento de los casos. Las variables que definen el perfil del consumidor, incluidas al aplicar la regresión logística, fueron: sexo, n.º de visitas y patologías asociadas, cuyas OR fueron 1,57 (IC del 95 por ciento, 1,08-2,03), 1,11 (IC del 95 por ciento, 1,06-1,17) y 1,61 (IC del 95 por ciento, 1,04-2,05), respectivamente. Conclusiones. La prevalencia de prescripción de BDZ durante 1997 en la población atendida en nuestras consultas fue muy similar a la encontrada en otros estudios, con medias de consumo superiores al año y sin que conste el motivo de la prescripción en la mitad de los casos, recayendo la misma sobre todo en mujeres, con patología asociada y en función del n.° de visitas (AU)

Myeloablative chemotherapy with autologous peripheral blood stem cell transplantation for metastatic breast cancer: immunologic consequences affecting clinical outcome.

Autologous peripheral blood stem cell transplantation following myeloablative chemotherapy is being increasingly utilized in the treatment of a variety of malignancies. We administered busulfan 16 mg/kg orally, thiotepa 500-700 mg/m2 i.v., and carboplatin 800-1000 mg/m2 i.v. to 56 women with metastatic carcinoma of the breast. Autologous peripheral blood stem cells, which had been collected after a combination of chemotherapy and granulocyte colony-stimulating factor, were infused on day 0. The major toxicities of the conditioning regimen included severe pancytopenia, stomatitis, nausea, emesis, diarrhea, fever, and infection. Transplant-related mortality was 1.8%. The incidence of opportunistic viral infections was 42.9%. Fourteen individuals achieved a complete response. The actuarial survival at 1223 days was 13.7% for the entire group of patients; the actuarial survival at 1009 days was 39.3% among complete responders. The functional status of the immune system was determined following transplantation in a subset of patients. Peripheral blood mononuclear cells were obtained before and after stem cell infusion, and were analyzed phenotypically and functionally. Proliferative and interleukin-2 synthetic ability of these cells was assessed following stimulation with phytohemagglutinin and anti-CD3 antibody. The response to influenza peptides was also ascertained. Proliferative and interleukin-2 synthetic capacity was markedly impaired for over a year. Memory response was virtually absent for up to 2 years following transplantation. The prolonged and marked immunosuppression following this myeloablative regimen was associated with a high incidence of opportunistic viral infections, and may have contributed to disease relapse and progression especially in patients who failed to achieve a complete response following transplantation.

Generation of CD8+ and CD4+ T-cell response to dendritic cells genetically engineered to express the MART-1/Melan-A gene.

Both CD8+ and CD4+ T cells have demonstrated roles in antitumor immune response in many animal tumor systems. In many human tumor systems, although abundant literature exists on the evidence of tumor antigen-specific CD8+ CTL response, only limited information is available on tumor antigen-specific CD4+ T-cell response. Using the MART-1/Melan-A (MART-1) antigen system as a prototype human tumor-associated antigen (TAA)- and dendritic cell (DC)-based MART-1 antigen presentation system (i.e., DCs transduced with an adenoviral vector-based construct carrying the MART-1 gene), we explored, in vitro, the feasibility of generating both CD8+ and CD4+ T-cell responses in the same individual. Here, we show that autologous DCs from both HLA-A2-positive melanoma patients and normal healthy individuals that are transduced with an adenoviral vector containing the MART-1 antigen are capable of inducing both MART-1-specific CD8+ and CD4+ T cells in in vitro coculture. After several rounds of stimulation, both the CD4+ and CD8+ T cells synthesized IFN-gamma when they were specifically stimulated. The CD8+ T cells generated in such cocultures also recognized the MART-1(27-35) peptide, AAGIGILTV, in 4-h cytotoxicity assays. These observations, therefore, suggest that Th1-type responses can be generated, in vitro, by stimulation with DCs that are genetically modified to express a TAA. Although the outcome of this type of genetically engineered DC-based stimulation may vary from system to system, this type of in vitro antigen presentation may be very useful in more comprehensive analyses of CD4+ T-cell response to defined TAAs, and such genetically engineered autologous DCs might be better candidates to serve as surrogate cancer vaccines.