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El objetivo del presente estudio fue determinar, mediante la técnica de RT-PCR Tiem- po Real y cuantificación relativa según el método 2-ΔΔCt, los niveles relativos de expresión in vitro de ARN mensajero de IL-2 e IL-10 en leucocitos circulantes de alpacas en presencia de antígenos clostridiales, empleándose como control endógeno a GAPDH. Se colectó sangre entera de 10 alpacas adultas. Las muestras fueron centrifugadas para obtener la fracción leucocitaria en gradiente de Ficoll, colectándose la capa flogística. Se hizo un pool de leucocitos que fueron purificados con cloruro de amonio. Se centrifugó y la concentración se ajustó a 500 000 células vivas/ml de medio mínimo esencial (MEM). Fueron cultivados en placas para cultivo celular de 24 pocillos y enfrentados a suspensiones de extracto clostridial a las concentraciones de 400, 16, 0.8 y 0.2 µg/ml por 1, 12 y 24 h. Posteriormente, se realizó la RT-qPCR. La cinética de la expresión de IL-2 mostró una tendencia no significativa inversamente proporcional a la dosis de antígeno clostridial en los tres tiempos de incubación, a diferencia de IL-10 cuya expresión fue variable, alcan- zando el máximo a las 12 h de incubación (p, The aim of the study was to determine the relative in vitro ARNm expression levels of IL-2 and IL-10 in peripheral blood leukocytes by real time RT-PCR and relative quantification using the 2-ΔΔCt method in presence of clostridial antigens and GAPDH as an endogenous control. Whole blood was collected from 10 adult alpacas. Samples were centrifuged to obtain leukocytes using the Ficoll reagent, purified with ammonium chloride and cultured in 24-well plates at a concentration of 500 000 live cells/ml minimal essential medium (MEM) with clostridial extract suspensions at concentrations of 400, 16, 0.8 and 0.2 µg/ml. Subsequently, the RT-qPCR test was performed. IL-2 kinetic expression patterns were inversely proportional to the clostridial antigen dose in the three incubation times (p>0.05), unlike IL-10 which its kinetic expression patterns were variable, reaching its maximum at 12 h (p

The aim of the study was to determine the relative in vitro ARNm expression levels of IL-2 and IL-10 in peripheral blood leukocytes by real time RT-PCR and relative quantification using the 2-ΔΔCt method in presence of clostridial antigens and GAPDH as an endogenous control. Whole blood was collected from 10 adult alpacas. Samples were centrifuged to obtain leukocytes using the Ficoll reagent, purified with ammonium chloride and cultured in 24-well plates at a concentration of 500 000 live cells/ml minimal essential medium (MEM) with clostridial extract suspensions at concentrations of 400, 16, 0.8 and 0.2 µg/ml. Subsequently, the RT-qPCR test was performed. IL-2 kinetic expression patterns were inversely proportional to the clostridial antigen dose in the three incubation times (p>0.05), unlike IL-10 which its kinetic expression patterns were variable, reaching its maximum at 12 h (p

El cáncer renal presenta varias características que lo diferencian de otros tumores. El aumento de supervivencia observado en pacientes con carcinoma renal metastásico tras la realización de nefrectomía, rompe un concepto clásico de la oncología según el cual la extirpación del tumor primario en tumores avanzados carece de sentido. Junto con el melanoma, es el único tumor en el que tratamientos inmunomoduladores, como interleukina-2 producen un beneficio clínico al paciente. El tratamiento del cáncer renal metastásico con interleukina-2 intravenosa a dosis altas ha confirmado en estudios aleatorios que puede producir respuestas completas de muy larga duración, en la práctica equivalentes a la curación. Por último, el cáncer renal está siendo empleado como modelo clínico para demostrar la eficacia de múltiples tratamientos dirigidos frente a nuevas dianas. En la actualidad se están estudiando más de 30 nuevos fármacos como tratamiento del cáncer renal, habiendo sido el primer tumor en el que se ha demostrado el beneficio clínico del tratamiento con inhibidores de la angiogénesis. En este artículo se revisan los aspectos más relevantes sobre el cáncer renal, incluyendo epidemiología, factores pronósticos, presentación clínica; bases moleculares y el estado actual de desarrollo de varios de los múltiples fármacos que se están estudiando en esta enfermedad. Renal cell carcinoma presents several unique features, which distinguish it from other tumours. The increase in survival that has been described in patients with renal cell carcinoma following nephrectomy breaks a classical rule of oncology, which states that surgery of the primary tumour has no role in the treatment of patients with advanced disease. Together with melanoma, it is the only tumour in which immunomodulatory treatments with drugs such as interleukin-2 produces a clinical benefit to patients. In randomized trials treatment of metastatic renal cell carcinoma with high dose interleukin-2 has confirmed its ability to induce long-term complete responses, which in practice can be considered equivalent to cure. Lastly, renal cell carcinoma is being used as a clinical model to demonstrate the role of several targeted treatments, with over 30 novel agents under development. It has been the first tumour type in which treatment with angiogenesis inhibitors has shown a clinical benefit. This article reviews the most relevant aspects of renal cell carcinoma, including epidemiology, prognostic factors, clinical presentation, molecular bases and the current status of development of the most relevant novel treatments for this disease.

An alternative strategy for cancer treatment is the manipulation of the immune system, denominated cancer immunotherapy. The immunotherapeutical use of cells of the immune system, like dendritic cells (DC), is being explored in different clinical protocols. Recently, we finalized a clinical phase I protocol, for the treatment of malignant melanoma, using DCs loaded with tumor lysates. Our results indicate that the subcutaneous application of DCs do not produce adverse effects. We also observed an increase of tumor spe-cific T lymphocytes precursors in the blood, associated to hypersensitivity reactions (DTH) in 60% of the treated patients. In most cases, an stability in the disease was observed, although without a significant association between vaccination and survival. Additionally, therapies based on Interleukin-2 (IL-2) have been used with relative success in the treatment of some kind of tumors since 1985. However, problems associated to the toxicity of IL-2 still restrict its massive use. Our direct experience with the use of IL-2, indicates that low doses and its subcutaneous application, maintains the beneficial effects for patients, eliminating the adverse effects. Based on the accumulated evidence during last the five years, we decided to implement an optimized clinical protocol, which alternatively combines dendritic cells vaccines with the use of low doses of IL-2 for the reinforcement of the immunological system (Rev Méd Chile 2004; 132: 1115-26)

An alternative strategy for cancer treatment is the manipulation of the immune system, denominated cancer immunotherapy. The immunotherapeutical use of cells of the immune system, like dendritic cells (DC), is being explored in different clinical protocols. Recently, we finalized a clinical phase I protocol, for the treatment of malignant melanoma, using DCs loaded with tumor lysates. Our results indicate that the subcutaneous application of DCs do not produce adverse effects. We also observed an increase of tumor spe-cific T lymphocytes precursors in the blood, associated to hypersensitivity reactions (DTH) in 60% of the treated patients. In most cases, an stability in the disease was observed, although without a significant association between vaccination and survival. Additionally, therapies based on Interleukin-2 (IL-2) have been used with relative success in the treatment of some kind of tumors since 1985. However, problems associated to the toxicity of IL-2 still restrict its massive use. Our direct experience with the use of IL-2, indicates that low doses and its subcutaneous application, maintains the beneficial effects for patients, eliminating the adverse effects. Based on the accumulated evidence during last the five years, we decided to implement an optimized clinical protocol, which alternatively combines dendritic cells vaccines with the use of low doses of IL-2 for the reinforcement of the immunological system (Rev Méd Chile 2004; 132: 1115-26)

Background: Type 1 diabetes is an organ specifc autoimmune disease whose incidence is increasing worldwide. A functional imbalance in cytokine production resulting in dominance of T helper (Th1) over Th2-type response has been suggested to play a critical role in the pathogenesis of type 1 diabetes. Aim: To measure serum concentrations of interleukin (IL)-1ß, IL-2 and IL-4 in children with recently diagnosed type 1 diabetes and to evaluate the autoimmune response measuring glutamic acid decarboxylase (GAD65) and tyrosine phosphatase like (IA-2) autoantibodies. Patients and Methods: 120 diabetic children and 118 age and gender matched control children, were recruited for this study. Circulating levels of IL-1ß, IL-2 and IL-4 were measured by ELISA. GAD65 and IA-2 were measured by RIA. Results: Circulating levels of IL-1ß were elevated in type 1 diabetic children as compared to the control group (9.3±7.3 and 4.9±3.8 pg/ml respectively, p=0,01). Serum concentration of IL-2 was also higher in diabetic patients (19.8±13.1 and 11.3±9.1 pg/ml respectively, p=0,01). No differences in serum IL-4 were observed between diabetics and control. Diabetic children with one or two positive autoantibodies (IA-2 and/or GAD65) had significantly higher levels of IL-1ß and IL-2 and lower levels of IL-4 than diabetic children without positive autoantibodies. High concentrations of IL-1ß were associated with an early onset of the disease. Conclusions: High levels of IL-1ß and IL-2 were found in diabetic children with recent diagnosis of the disease. Diabetics with positive antibodies against GAD65 and IA-2 had higher levels of IL-1ß and IL-2 and lower levels of IL-4 than their counterparts without positive antibodies (Rev Méd Chile 2004; 132: 413-20)

Background: Type 1 diabetes is an organ specifc autoimmune disease whose incidence is increasing worldwide. A functional imbalance in cytokine production resulting in dominance of T helper (Th1) over Th2-type response has been suggested to play a critical role in the pathogenesis of type 1 diabetes. Aim: To measure serum concentrations of interleukin (IL)-1ß, IL-2 and IL-4 in children with recently diagnosed type 1 diabetes and to evaluate the autoimmune response measuring glutamic acid decarboxylase (GAD65) and tyrosine phosphatase like (IA-2) autoantibodies. Patients and Methods: 120 diabetic children and 118 age and gender matched control children, were recruited for this study. Circulating levels of IL-1ß, IL-2 and IL-4 were measured by ELISA. GAD65 and IA-2 were measured by RIA. Results: Circulating levels of IL-1ß were elevated in type 1 diabetic children as compared to the control group (9.3±7.3 and 4.9±3.8 pg/ml respectively, p=0,01). Serum concentration of IL-2 was also higher in diabetic patients (19.8±13.1 and 11.3±9.1 pg/ml respectively, p=0,01). No differences in serum IL-4 were observed between diabetics and control. Diabetic children with one or two positive autoantibodies (IA-2 and/or GAD65) had significantly higher levels of IL-1ß and IL-2 and lower levels of IL-4 than diabetic children without positive autoantibodies. High concentrations of IL-1ß were associated with an early onset of the disease. Conclusions: High levels of IL-1ß and IL-2 were found in diabetic children with recent diagnosis of the disease. Diabetics with positive antibodies against GAD65 and IA-2 had higher levels of IL-1ß and IL-2 and lower levels of IL-4 than their counterparts without positive antibodies (Rev Méd Chile 2004; 132: 413-20)

Resumen La interleucina-2 es una glicoproteína humana de 133 aminoácidos que resulta vital para obtener una respuesta inmunológica efectiva. En el Centro de Ingeniería Genética y Biotecnología ha podido obtenerse por vía recombinante a partir de una cepa de Escherichia coli. Para el control de la calidad de cada lote producido se hizo necesaria la elaboración de un material de referencia de trabajo. En esta publicación se describen los pasos seguidos en la obtención del lote candidato y la preparación del material de referencia como tal. Se demostró que el patrón de trabajo preparado es homogéneo para el uso en las técnicas analíticas de control de calidad de la interleucina-2 y se predijo una pérdida de actividad inferior al 5 % anual mediante un estudio de estabilidad acelerada. Se obtuvieron valores adecuados para la pureza del material de referencia, evaluada por electroforesis y para la actividad biológica, que se estableció con trazabilidad al material de referencia internacional para este producto.Establishment of a working reference material for recombinant interleukin-2 Interleukin-2 is a human glycoprotein with 133 aminoacids that is crucial for attaining an effective immunological reaction. In the Center of Genetic Engineering and Biotechnology this protein has been obtained as a recombinant product from Eschericia coli bacteria. For the quality control of every batch produced, it was necessary to establish a working reference material. In this paper we describe the steps taken to obtain a candidate batch and the preparation of the working material as such. It was proved that the working pattern obtained.is homogenous for its use in the analytical techniques of quality control of interleukin-2. A loss of activity lower than an annual 5 % was predicted by an accelerated stability study. Adequate values were attained for the purity of the reference material, evaluated by electrophoresis, and for the biological activity that was established with traceability to the international reference material for this product.