Combination Blockade of the IL6R/STAT-3 Axis with TIGIT and Its Impact on the Functional Activity of NK Cells against Prostate Cancer Cells.

Methods: We analyzed the secretion of cytokines, chemokines, and growth factors in 22Rv1, LNCaP, and DU145 cells. In these cells, we also evaluated the expression of NK ligands, IL6R, STAT-3, and phosporylated STAT-3. In NK-92 cells, we evaluated the effects of Stattic (Stt) and tocilizumab (Tcz) on NK receptors. In addition, we assessed if the disruption of the IL6R/STAT-3 pathway and blockade of TIGIT potentiated the cytotoxicity of NK-92 cells versus DU145 cells. Results: DU145 abundantly secretes M-CSF, VEGF, IL-6, CXCL8, and TGF-ß. Furthermore, the expression of CD155 was found to increase in accordance with aggressiveness and metastatic status in the prostate cancer cells. Stt and Tcz induce a decrease in STAT-3 phosphorylation in the DU145 cells and, in turn, induce an increase of NKp46 and a decrease of TIGIT expression in NK-92 cells. Finally, the disruption of the IL6R/STAT-3 axis in prostate cancer cells and the blocking of TIGIT on NK-92 were observed to increase the cytotoxicity of NK-92 cells against DU145 cells through an increase in sFasL, granzyme A, granzyme B, and granulysin. Conclusions: Our results reveal that the combined use of inhibitors directed against the IL6R/STAT-3 axis and TIGIT enhances the functional activity of NK cells against castration-resistant prostate cancer cells.

Capsaicin attenuates immunosuppression induced by chronic stress in BALB/C mice.

Although acute stress generally exerts positive effects on the immune system, chronic stress typically causes immunosuppression via the hypothalamic-pituitary-adrenal (HPA) axis. In this study, the effects of capsaicin (1.28 mg/kg intraperitoneally [i.p.] for 7 days) on immune parameters were evaluated under conditions of chronic stress. Capsaicin treatment significantly increased the immune response as evaluated by the delayed-type hypersensitivity (DTH) reaction to dinitrofluorobenzene (DNFB) and splenocyte proliferation assays- It also is able to rescue the splenocytes of the apoptosis induced by stress. The capsaicin treatment increased the production of Th1 cytokines and decreased the production of Th2 cytokines and TGF-ß1 in the plasma and culture supernatants of immunosuppressed mice, which is associated with the modulation of Th2 induced by stress cells. Moreover, the production of corticosterone significantly decreased in capsaicin-treated animals as compared to control groups. The capsaicin treatment further attenuated the immunosuppression induced by the corticosterone treatment (40 mg/kg i.p. for 7 days), albeit less potently, as exhibited in the DTH response. Intriguingly, the capsaicin treatment decreased the induction of IL-10, IL-4, and TGF-ß1 through high doses of corticosterone, indicating direct cellular immunomodulation. These results show, that capsaicin is able to modulate chronic stress-induced immunosuppression, mediating corticosterone released inhibition, but also, that capsaicin significantly modulates the pharmacological action of corticosterone in vivo.

Regulation of immunophenotype modulation of monocytes-macrophages from M1 into M2 by prostate cancer cell-culture supernatant via transcription factor STAT3.

BACKGROUND: Transcription factor STAT3 has a prominent innate immunity effect on cancer progression. We determined the regulation of STAT3 in the immunophenotype modulation of macrophages from M1 into M2 induced by the cell-culture supernatant of the Prostate-Cancer line PC3. METHODS: Monocytes-macrophages from healthy donors were cultured in the supernatant of PC3 cells, membrane proteins, and intracytoplasmic and phosphorylated STAT3 were measured using flow cytometry, while cytokines and growth factors were studied using luminescence. Cytotoxicity and nitric oxide were evaluated via colorimetric assays. RESULTS: The supernatant of PC3 prostate-tumor cells effectively induced macrophages toward an M2 profile, and the expression of phosphorylated STAT3 in the monocytes-macrophages notably increased, and mainly related to IL-10. In the group of monocytes-macrophages treated with a STAT3 inhibitor, the macrophages were induced toward an M1 phenotype. CONCLUSIONS: In this study, we showed that the secretion profile of PC3 prostate-cancer cells induces a change in macrophage phenotype from M1 into M2, and that the phenomenon is related to phosphorylation of transcription factor STAT3 and IL-10.

Pentoxifylline during steroid window phase at induction to remission increases apoptosis in childhood with acute lymphoblastic leukemia

Purpose: Pentoxifylline (PTX) has been shown to increase chemotherapy-induced apoptosis. A clinical trial was developed to evaluate the effect of the addition of PTX to the induction steroid window phase in children with acute lymphoblastic leukemia (ALL). Methods: Thirty-two children were enrolled on this study. Children with a new diagnosis of ALL were randomly assigned to receive prednisone (PRD) 40 mg/m2/day only during the 7-day treatment pre-phase (PRD group, 11 patients) or to receive PRD with PTX (10 mg/kg/day) (PTX group, 11 patients); the control group included children with normal bone marrow (10 patients). Bone marrow aspiration (BMA) was performed at diagnosis (day -7) in all groups, and at day 0 (end of PRD window) for patients with ALL (PRD and PTX groups). Apoptosis was evaluated by flow cytometry (FC) using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) stains. Statistical analysis was performed using the Mann–Whitney U test. Results: Apoptotic index at day -7 was similar in all groups. However, at day 0 post-treatment, apoptosis was significantly higher in the PTX group than in the PRD group (p < 0.001). There were no serious adverse effects associated with PTX. Conclusions: PTX potentiates blast apoptosis induced by PRD in children with ALL during steroid window phase (AU)

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Pentoxifylline during steroid window phase at induction to remission increases apoptosis in childhood with acute lymphoblastic leukemia.

PURPOSE: Pentoxifylline (PTX) has been shown to increase chemotherapy-induced apoptosis. A clinical trial was developed to evaluate the effect of the addition of PTX to the induction steroid window phase in children with acute lymphoblastic leukemia (ALL). METHODS: Thirty-two children were enrolled on this study. Children with a new diagnosis of ALL were randomly assigned to receive prednisone (PRD) 40 mg/m(2)/day only during the 7-day treatment pre-phase (PRD group, 11 patients) or to receive PRD with PTX (10 mg/kg/day) (PTX group, 11 patients); the control group included children with normal bone marrow (10 patients). Bone marrow aspiration (BMA) was performed at diagnosis (day -7) in all groups, and at day 0 (end of PRD window) for patients with ALL (PRD and PTX groups). Apoptosis was evaluated by flow cytometry (FC) using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) stains. Statistical analysis was performed using the Mann-Whitney U test. RESULTS: Apoptotic index at day -7 was similar in all groups. However, at day 0 post-treatment, apoptosis was significantly higher in the PTX group than in the PRD group (p < 0.001). There were no serious adverse effects associated with PTX. CONCLUSIONS: PTX potentiates blast apoptosis induced by PRD in children with ALL during steroid window phase.

[Stevens-Johnson syndrome plus intrahepatic cholestasis caused by clindamycin or chlorpheniramine]. / Síndrome de Stevens Johnson más colestasis intrahepática inducido por clindamicina o clorfeniramina.

A 48-year-old woman was hospitalized with the diagnosis of hepatitis. She presented with symptoms of jaundice, headache, elevated bilirubin, and elevated hepatic enzymes. She related a recent episode of a bronchial infection that was treated during the previous eight days with paracetamol (500mg, 2 doses only), chlorpheniramine, betamethasone and clindamycin. After an initial clinical and laboratorial improvement, she began to complain of pruritus of the palms and soles. Thereafter, vesicles evolving to blisters developed and a deterioration of her general health ensued. Serologies for hepatitis A, B, and C viruses were negative. Intrahepatic cholestasis and Stevens Johnson Syndrome (SJS) were the final diagnosis. The association of the Stevens Johnson Syndrome and intrahepatic cholestasis simultaneously, related to adverse drug reactions, is very rare. The drugs reportedly involved are mainly antibiotics, such as ampicillin, vancomycin, amoxicillin/clavulinic acid and erythromycin. Other drugs involved are non-steroidal anti-inflamatory drugs, such as mefenamic acid, ibuprofen, and sulindac. The reactions can be minor or severe and can even cause death, an outcome that has been reported in patients of all races and ethnic groups, but appears to be more rare in patients of Latin origin. We present a discussion of this case and review the main characteristics of the Stevens Johnson Syndrome.

In vitro induction of apoptosis in U937 cells by perillyl alcohol with sensitization by pentoxifylline: increased BCL-2 and BAX protein expression.

BACKGROUND: Chemotherapy is effective against a wide variety of tumor cells, although its use is limited by side effects. In vitro experiments and phase I and II trials have shown that phytochemicals such as perillyl alcohol (P-OH) have antitumor effects. Pentoxifylline (PTX), a synthetic methylxanthine used mainly to treat pathologies associated with hematological diseases, sensitizes tumor cells to chemotherapy. The aim of this study was to determine whether PTX amplifies the antitumor effects of P-OH in U937 human myelomonocytic leukemia cells. METHODS: Apoptosis was measured by the loss of mitochondrial membrane potential determined by flow cytometry using dihexyloxacarbocyanine iodide (DiOC6) and propidium iodide. Bcl-2 and Bax protein expression was also assessed by Western blot analysis. RESULTS: P-OH and PTX induced loss of the mitochondrial membrane potential in U937 cells in vitro. Culturing the cells in the presence of both compounds caused a significant increase (p < 0.001) in apoptosis and expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins. However, despite their coexistence, Bax expression prevailed in our experiments. These data suggest that the effects of PTX might be attributable to changes in the mitochondrial membrane potential. CONCLUSION: PTX sensitizes tumor cells to the anti-neoplastic action of P-OH. These observations may have clinical relevance in the treatment of cancer patients.

[Retinopathy of prematurity and oxidative stress]. / Retinopatía del prematuro y estrés oxidativo.

INTRODUCTION: There is some evidence that retinopathy of prematurity is due to excessive oxidative stress on the developing retina caused by high free radical production or reduced ability to eliminate these radicals. OBJECTIVE: To determine the relationship between high levels of oxidative stress and retinopathy of prematurity. MATERIAL AND METHODS: A prospective cohort study was designed. Fifty premature infants of less than 33 weeks' gestational age were included. Serum lipoperoxide levels were determined as a measure of oxidative stress. Samples were taken once a week for 1 month, starting from the first week of life. The results of all four samples were compared between infants who developed any degree of retinopathy of prematurity and those without it. Ophthalmological examinations were performed after the fourth week of life. RESULTS: The incidence of retinopathy of prematurity was 22 % (11/50). The mean values of all the samples showed a significant difference between infants who developed retinopathy of prematurity (5.44 +/- 1.30 nmol/ml) and those who did not (2.94 +/- 0.89 nmol/ml, p = 0.0001). The relative risk of developing retinopathy of prematurity with high serum lipoperoxide levels was 5.15, 5.63, 4.15 and 12.70 for each of the weekly samples. CONCLUSIONS: There is an association between high serum lipoperoxide levels, as a measure of oxidative stress, and the incidence of retinopathy of prematurity.

Retinopatía del prematuro y estrés oxidativo / Retinopathy of prematurity and oxidative stress

Introducción: Existen algunas evidencias de que la retinopatía del prematuro es consecuencia de un elevado estrés oxidativo sobre la retina en desarrollo, lo cual se debe a la generación exagerada de radicales libres o a una disminución en la capacidad para su eliminación. Objetivo: Determinar la asociación entre la concentración elevada de estrés oxidativo y la presencia de retinopatía del prematuro. Material y métodos: Se realizó un estudio de cohorte prospectiva. Se incluyeron 50 prematuros de menos de 33 semanas de gestación. El estrés oxidativo se midió con la concentración sérica de lipoperóxidos. Se tomaron muestras a partir de la primera semana de vida y después, cada semana hasta la cuarta. Se compararon los resultados de las 4 muestras juntas, entre los prematuros con retinopatía de cualquier grado con los que no la desarrollaron. Las evaluaciones oftalmológicas se realizaron a partir de la cuarta semana de vida. Resultados: La incidencia de retinopatía en el estudio fue del 22 % (11/50). Hay una diferencia significativa en los valores promedio de todas las muestras, entre aquellos que presentaron retinopatía del prematuro, 5,44 ± 1,30 nmol/ml, comparados con los que no presentaron la enfermedad, 2,94 ± 0,89 nmol/ml, con una p = 0,00001. El riesgo relativo para el desarrollo de retinopatía con concentraciones elevadas de lipoperóxidos fue de 5,15, 5,63, 4,15 y 12,70 para las muestras de cada una de las semanas, respectivamente. Conclusiones: Existe una asociación entre la concentración elevada de lipoperóxidos séricos, como una medida de estrés oxidativo y la incidencia de la retinopatía del prematuro

Introduction: There is some evidence that retinopathy of prematurity is due to excessive oxidative stress on the developing retina caused by high free radical production or reduced ability to eliminate these radicals. Objective: To determine the relationship between high levels of oxidative stress and retinopathy of prematurity. Material and methods: A prospective cohort study was designed. Fifty premature infants of less than 33 weeks' gestational age were included. Serum lipoperoxide levels were determined as a measure of oxidative stress. Samples were taken once a week for 1 month, starting from the first week of life. The results of all four samples were compared between infants who developed any degree of retinopathy of prematurity and those without it. Ophthalmological examinations were performed after the fourth week of life. Results: The incidence of retinopathy of prematurity was 22 % (11/50). The mean values of all the samples showed a significant difference between infants who developed retinopathy of prematurity (5.44 ± 1.30 nmol/ml) and those who did not (2.94 ± 0.89 nmol/ml, p = 0.0001). The relative risk of developing retinopathy of prematurity with high serum lipoperoxide levels was 5.15, 5.63, 4.15 and 12.70 for each of the weekly samples. Conclusions: There is an association between high serum lipoperoxide levels, as a measure of oxidative stress, and the incidence of retinopathy of prematurity

In vivo modification of adriamycin-induced apoptosis in L-5178Y lymphoma cell-bearing mice by (+)-alpha-tocopherol and superoxide dismutase.

Apoptosis was followed in L5178Y lymphoma cell-bearing mice at different times after intraperitoneal injections of adriamycin (ADM). Apoptosis was determined morphologically and confirmed by DNA laddering on electrophoresis. Apoptosis was observed 36h after injection of 5mg/kg ADM (apoptotic cell index 64.2+/-5.6 vs. 1.5+/-2.1 from the untreated group) and confirmed by DNA electrophoresis. However, when the animals were pretreated with (+)-alpha-tocopherol acid succinate or superoxide dismutase before ADM administration apoptotic index significantly diminished (P<0.05) and the DNA electrophoresis did not show fragmentations. We conclude that in ADM-treated mice, tumour cell death occurs in two ways: first by necrosis, then later by apoptosis. These observations are likely to be associated with or caused by the generation of reactive oxygen species.

In vivo inhibition by antioxidants of adriamycin-induced apoptosis in murine peritoneal macrophages.

Adriamycin (ADM) is an oncostatic of the anthracycline family with confirmed experimental and clinical efficiency. This antitumoral drug has been reported to stimulate macrophage activity and is able to induce apoptosis (AP) in some tumour cells. The objective of the present work was to investigate if in vivo administration of ADM to mice induces AP in their peritoneal macrophages (PM). AP was expressed by the apoptotic index (AI) of peritoneal macrophages observed under fluorescence microscope after ethidium bromide and acridine orange staining and confirmed by detection of the ladder pattern on DNA electrophoresis, indicates DNA fragmentation in 80-120 bp characteristic of apoptotic state. 24 hours after i.p. ADM administration, AP was observed in PM. The effect was best visible after the injection of 5 mg/kg ADM. (Al: 76.3+/-8.9 vs untreated control group AI: 2.8+/-1.1). In the ADM treated group a DNA ladder electrophoretic pattern was observed while DNA from normal PM was genomic. Since ADM toxicity has been attributed to reactive oxygen species generation, we investigated its possible participation in AP induction by pretreating mice with antioxidants: (+)-alpha-tocopherol acid succinate (30 IU/mouse per os) for 3 days before ADM administration with E. coli lipopolysacharide (0.15 microg/mouse i.p.) 24 hours before ADM administration or with superoxide dismutase (10,000 IU/mouse i.p.) 1 hour before ADM administration. AI was significantly decreased, with values close to those of the untreated control group (AI: 15+/-5.7, 9.6+/-8.0 and 32.9+/-6.9, respectively). Antioxidants given before ADM treatment significantly increased the live cell index (p < or = 0.001) in PM the groups while inactivated antioxidants no longer protect PM against the ADM AP induction. DNA analysis confirmed the effect: in the untreated control and in the antioxidant protected groups DNA was genomic while in either ADM or inactivated-antioxidants + ADM treated groups, DNA presented the ladder pattern. AP can thus be induced in PM by ADM and inhibited by antioxidants. These observations may have clinical applications.

Enhanced functional capacity of the peritoneal macrophages as antigen presenting cells after aclacinomycin treatment in mice.

Aclacinomycin (ACM) is an oncostatic of the anthracycline family, largely used in patients and experimentally in mice. ACM has been reported to enhance phagocytosis, secretion of free oxygen radicals and of interleukin 1. Its injection is also followed by an increase of the cytotoxic and cytostatic activity of murine peritoneal macrophages. In the present work we investigated whether ACM modifies the antigen-presenting cell capacity of murine peritoneal macrophages. Purified T lymphocytes were cultured with peritoneal macrophages from either normal or ACM treated mice (4 mg/kg day -4) which were previously incubated with phytohemagglutinin. The T cell proliferative response was greater in cultures with normal macrophages, indicating that macrophages from ACM-treated mice had a better antigen presenting activity than normal untreated macrophages.