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/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.

Usefulness of serum lipid peroxide as a diagnostic test for hypoxic ischemic encephalopathy in the full-term neonate.

OBJECTIVE: To evaluate the usefulness of serum lipid peroxide (LPO) for hypoxic ischemic encephalopathy (HIE) in full-term neonates. STUDY DESIGN: Diagnostic test evaluation forming three groups: (1) healthy full-term neonates (n=59), (2) at-risk full-term neonates without HIE (n=57) and (3) at-risk full-term neonates with HIE (n=57). HIE diagnosis was made using the Finer clinical classification at 48 h after birth. Serum LPO was taken at 4 h after birth and determined by spectrophotometry. RESULT: One hundred seventy-three full-term neonates were studied. Fifty-one of the at-risk full-term neonates with HIE (51/57) had high serum LPO and two of the at-risk full-term neonates without HIE (2/57) (P<0.001). Serum LPO level had 89% sensitivity, 96% specificity, 96% positive predictive value, 90% negative predictive value, 24 positive probability ratio, 0.11 negative probability ratio and 92% diagnostic usefulness. CONCLUSION: Serum LPO level could be a useful test for early diagnosis of HIE in full-term neonates.

[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.

Human papillomavirus in tonsillar and nasopharyngeal carcinoma: isolation of HPV subtype 31.

We retrospectively studied biopsy specimens obtained from 16 patients who had carcinoma of the tonsil or nasopharynx. Polymerase chain reaction testing detected the presence of human papillomavirus (HPV) in 13 samples (81.3%)--six tonsillar and seven nasopharyngeal. Eleven of the 13 positive samples (84.6%) contained HPV subtype 31. We believe that this is the first report of the presence of HPV subtype 31 in these carcinomas. In addition to the significant association between tonsillar and nasopharyngeal cancer and HPV, our analysis of descriptive variables confirmed the association between the incidence of these neoplasms and poor oral hygiene and low socioeconomic status in older adults.

Effects of melatonin on the Harderian gland of lipopolysaccharide-treated rats: morphological observations.

Melatonin is a free radical scavenger and antioxidant. This indol is reported to efficiently scavenge both hydroxyl and peroxyl radicals and it also reduces both in vitro and in vivo tissue damage due to oxidants which generate oxygen toxic radicals. Lipopolysaccharide (LPS) administration induces oxidative damage in various tissues mainly due to its ability to increase reactive oxygen species. In the present work, we studied the morphological changes and lipid peroxidation in the Harderian gland after LPS administration and the effects of melatonin in preventing the induced changes. Hyperchromasia, vesicular degeneration, necrosis and infiltration with macrophages, monocytes and neutrophils were observed in the LPS-treated group (10 mg/kg, intraperitonally [i.p.]). Also, a typical structure of the glandular acini of the gland exhibited diffuse damage. In the LPS rats treated with melatonin (10 mg/kg, i.p.), a diminished number of infiltrative cells was seen, and cloudy swelling was reduced, as was nuclear hyperchromasia. Neither necrosis nor vesicular degeneration were noted in the melatonin-treated rats, and in general, glandular structure was preserved. Lipid peroxidation products increased significantly within six hours after LPS administration, and melatonin treatment decreased the LPS-dependent lipid peroxidation products. These data together suggest that melatonin protects the Harderian gland against LPS toxicity in terms of morphological damage.

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.

Proteolytic activity in extracts of invasive cervical carcinoma and precursor lesions.

In this preliminary report, we showed that proteolytic activity of extracts from 85 cervical samples of patients with normal cervix, low and high-grade squamous intra-epithelial lesions and invasive carcinoma, increased according to the natural history of the cervical cancer when measured with three different substrates. Inhibitor assays for four different catalytic classes of endopeptidases indicated that the predominant catalytic class in extracts of all groups was that of metalloproteinases. Substrate gel electrophoresis revealed that invasive carcinoma extracts had two bands with proteolytic activity (with M(r) of 72 and 52 kDa) which were not present in normal tissue or biopsies with precursor lesions. Immunological and molecular characterization of these bands may provide information relevant to cervical cancer biology and clinical applications.

Hepatotoxicity induced by a single ip injection of ruthenium red.

Ruthenium red (RR) has been used as a marker in morphological observations of the glycocalix because it interacts with polyanionic mucopolysaccharides. This fact may explain its agglutinating effect on rat blood red cells following a single 20 mg/kg intraperitoneal injection, which increases with time post-injection. This study was performed to determine whether such an effect was due to a direct effect of the RR on the blood cells, to interference with coagulation, or to the non-specific general toxicity of this dye. Male rats were injected with 20 mg/kg RR ip and the enzymatic and coagulation parameters, plus the liver morphology were examined. Alanine aminotransferase (ALAT) activity was increased at 30, 60 and 120 min, and aspartic aminotransferase (ASAT) activity was increased 60, 120 and 480 min after RR injection. The prothrombin time (PT) and partially activated thromboplastin time (PTT) were significantly decreased, particularly after 60-120 min. The liver had an external granular appearance with clear signs of congestion and oedema, and showed degenerative changes very soon after RR injection. A single administration of RR induces serious functional and structural changes in the liver. Such a toxicity, and these changes must be taken into consideration, particularly with regard to neurological studies.

Increased phagocytic activity of peripheral blood monocytes after intravenous injection of phospholipase A2 to monkeys.

One of the expressions of the activity of phagocytic cells such as monocytes or macrophages is a burst of increased oxidative activity on stimulation. The free oxygen radicals liberated, mainly O2- and H2O2, lead to chemoluminescence, which is thus a measure of activation. Chemoluminescence also depends on arachidonic acid metabolism, and this depends on phospholipase A2 (PLA2). We modified monocyte activity in monkeys by injecting them i.v. with this enzyme and observed that 30 min after injection, the phagocytic activity of peripheral blood monocytes and the chemoluminescence they emitted was greater than that of controls. We suggest that PLA2 may act as an in vivo immunomodulator in mammals.

[Increase of proliferative and cytotoxic responses to alloantigenic stimulation of mouse spleen cells treated with aclacinomycin]. / Incremento a las respuestas proliferativa y citotóxica a la estimulación aloantigénica de células esplénicas de ratones tratados con aclacinomicina.

Aclacinomycin (ACM) is an oncostatic substance on the family of the Anthracyclines, with a proven activity in human and rodents. Splenic cells from C57BL/6 ACM injected mice by intraperitoneal or intravenous route four days before their sacrifice showed a significant increase in the proliferative and cytotoxic response respectively measured by incorporation of 3H-TdR and by the liberation of 51Cr when stimulated in vitro with irradiated mouse DBA/2 splenic cells. This response is doses dependent, and one can clearly observe different effects on the proliferative and cytotoxic responses at high doses. The cultures supernatants of splenic cells from mice treated with ACM during allogeneic stimulation showed a greater activity to induce the proliferation of a line of T cytotoxic cells dependent on Interleukin-2. Finally, the cytotoxic activity of splenic cells induced by the allogeneic stimulation in vitro, of mice treated with ACM, was found in a subpopulation of cells non adherent to plastic, mainly made up of lymphocytes.

[Phagocytic activity in PMN leukocytes and macrophages of athymic nu/nu mice]. / Actividad fagocítica en leucocitos PMN y macrófagos de ratones atímicos nu/nu.

The athymic nu/nu mice represent a natural model for studying defense mechanisms. This study had the object of evaluating the phagocytic activity of polymorphonuclear leucocytes (PMN) and peritoneal macrophages (PM) in congenitally athymic homozygotic nu/nu mice. The results showed that the chemotaxis, random mobility, ingestion of latex particles and reduction of nitroblue tetrazolium were all normal. Nevertheless, the enzyme myeloperoxidase in the PMN was significantly diminished when compared with Balb/c mice. These results suggest a partial decrease in the oxidative metabolism of the PMN in nu/nu mice.

Pulmonary toxicity of oxygen.

Prolonged exposure to hyperoxia in order to compensate for inefficient ventilation can lead to progressive pulmonary damage and death. Since pulmonary macrophages control bacterial and viral penetration, we studied the effect of hyperoxia on pulmonary alveolar macrophages from newborn and adult rats, kept in air or in a 95% normobaric oxygen atmosphere. The viability of pulmonary alveolar macrophages in bronchoalveolar lavages of newborn rats after 3 d in oxygen was significantly lower than that of newborn rats after 3 days in air. The functional capacity, as measured by the phagocytosis of Candida was similarly affected and these observations mays explain why infections develop. Adult rat macrophages were not sensitive to hyperoxia.

Actividad fagocitica de los macrofagos pulmonares de los ratones atimicos desnudos nu/nu. / Phagocytic activity of lung macrophages of nu/nu athymic nude mice.

La finalidad de este trabajo fue investigar la actividad fagocitica de los macrofagos pulmonares de los ratones atimicos desnudos nu/nu y compararla con la de los ratones normales BALB/C de los que se originaron. Se encontro que el indice fagocitico de los macrofagos pulmonares de 10 ratones nu/nu frente a particulas de latex fue de 4.23 +/- 1.15, y que el de un numero igual de ratones comparables BALB/C fue 1.71 +/- 0.43 (p < 0.001).Estos resultados parecen indicar que la actividad fagocitica de los macrofagos pulmonares de los ratones nu/nu no es timodependiente, y que probablemente se encuentra aumentada como mecanismo compensatorio de resistencia a las infecciones.}