Endostatin- and interleukin-2-expressing retroviral bicistronic vector for gene therapy of metastatic renal cell carcinoma.

BACKGROUND: Metastatic renal cell carcinoma (mRCC) is one of the most treatment-resistant malignancies. Despite all new therapeutic advances, almost all patients develop resistance to treatment and cure is rarely seen. In the present study, we evaluated the antitumor effect of a bicistronic retrovirus vector encoding both endostatin (ES) and interleukin (IL)-2 using an orthotopic metastatic RCC mouse model. METHODS: Balb/C-bearing Renca cells were treated with NIH/3T3-LendIRES-IL-2-SN cells. In the survival studies, mice were monitored daily until they died. At the end of the in vivo experiment, serum levels of IL-2 and ES were measured, the lung was weighed, and the number of metastatic nodules, nodule area, tumor vessels and proliferation of tumor-infiltrating Renca cells were determined. RESULTS: Inoculation of NIH/3T3-LendIRES-IL-2-SN cells resulted in an increase in ES and IL-2 levels in the treated group (p < 0.05). There was a significant decrease in lung wet weight, lung nodule area and tumor vessels in the treated group compared to the control group (p < 0.001). The proliferation of Renca cells in the bicistronic-treated group was significantly reduced compared to the control group (p < 0.05). Kaplan-Meier survival curves showed that the probability of survival was significantly higher for mice submitted to bicistronic therapy (log-rank test, p = 0.0016). Bicistronic therapy caused an increase in the infiltration of CD4, CD4 interferon (IFN)γ-producing, CD8, CD8 IFNγ-producing and natural killer (CD49b) cells. CONCLUSIONS: Retroviral bicistronic gene transfer led to the secretion of functional ES and IL-2 that was sufficiently active to: (i) inhibit tumor angiogenesis and tumor cell proliferation and (ii) increase the infiltration of immune cells.

Endostatin gene therapy enhances the efficacy of IL-2 in suppressing metastatic renal cell carcinoma in mice.

We investigated whether the administration of IL-2 combined with endostatin gene therapy was able to produce additive or even synergistic immunomodulatory activity in a mouse model of metastatic renal carcinoma. Renca cells were injected into the tail vein of BALB/c mice. After 24 h, the animals were randomly divided into four groups (5 mice/group). One group of mice was the control, the second group received treatment with 100,000 UI of Recombinant IL-2 (Proleukin, Chiron) twice a day, 1 day per week during 2 weeks (IL-2), the third group received treatment with a subcutaneous inoculation of 3.6 x 10(6) endostatin-producing cells, and the fourth group received both therapies (IL-2 + ES). Mice were treated for 2 weeks. In the survival studies, 10 mice/group daily, mice were monitored daily until they died. The presence of metastases led to a twofold increase in endostatin levels. Subcutaneous inoculation of NIH/3T3-LendSN cells resulted in a 2.75 and 2.78-fold increase in endostatin levels in the ES and IL-2 + ES group, respectively. At the end of the study, there was a significant decrease in lung wet weight, lung nodules area, and microvascular area (MVA) in all treated groups compared with the control group (P < 0.001). The significant difference in lung wet weight and lung nodules area between groups IL-2 and IL-2 + ES revealed a synergistic antitumor effect of the combined treatment (P < 0.05). The IL-2 + ES therapy Kaplan-Meier survival curves showed that the probability of survival was significantly higher for mice treated with the combined therapy (log-rank test, P = 0.0028). Conjugated therapy caused an increase in the infiltration of CD4, CD8 and CD49b lymphocytes. An increase in the amount of CD8 cells (P < 0.01) was observed when animals received both ES and IL-2, suggesting an additive effect of ES over IL-2 treatment. A synergistic effect of ES on the infiltration of CD4 (P < 0.001) and CD49b cells (P < 0.01) was also observed over the effect of IL-2. Here, we show that ES led to an increase in CD4 T helper cells as well as cytotoxic lymphocytes, such as NK cells and CD8 cells, within tumors of IL-2 treated mice. This means that ES plays a role in supporting the actions of T cells.

Erythrocyte protoporphyrin fluorescence as a potential marker of diabetes.

Protoporphyrin (PpIX), a porphyrin derivative, is the intermediate metabolic precursor of the heme molecule. Abnormal metabolism of total erythrocyte PpIX has been observed in diseases such as cancer, lead poisoning, psoriasis, iron deficiency anemia and acute porphyries. Diabetes mellitus (DM) is a complex metabolic syndrome in which hyperglycemia is the primary clinical manifestation and contributes to the diabetic complications. The aim of this study was to evaluate the utility of fluorescence spectroscopy of erythrocyte PpIX for monitoring the early stages of diabetes. A total of 14 male C 57BL mice, 6 weeks old, were divided into two groups: diabetic and non-diabetic. Diabetes was induced by intraperitoneal injection of streptozotocin (SZT). Blood cells were cultured with standard and 50 mM supplemented RPMI medium. Blood smears were prepared and stained for qualitative morphology analysis under optical microscopy. Blood porphyrin autofluorescence was analyzed by fluorescence spectroscopy. Characteristic PpIX emission spectra were obtained by exciting the samples at 405 nm. Average blood glucose was lower in the control group than in the diabetic group (156.50 +/- 8.11 mg/dL vs. 371.10 +/- 14.43 mg/dL, P < 0.05). Both diabetic and glucose-cultured erythroblasts showed a significant decrease (around 30.5% and 40%, respectively) in the emission band intensity at 635 nm. Our results indicate that the erythrocyte PpIX profile could be used as a biological monitor for diabetes.