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.

[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.

Enhanced activity of murine peritoneal cells after aclacinomycin injection: characteristics of the enhanced respiratory burst.

After the i.p. injection into normal mice, of 4 mg/kg of aclacinomycin (ACM), a dose which prolongs the survival of tumor-bearing mice, the zymosan-elicited chemoluminescence (CL) of the peritoneal cells (PC) is greater than that of control cells. The volume in which the drug is administered plays an important role in the intensity of the response. ACM also stimulated the CL of PC from tumor-bearing mice. It is known that CL can also be elicited by soluble stimuli such as 4 beta-phorbol-12-myristate-13 alpha-acetate or Ca2+ ionophore A 23187, which, however, act in different ways. The response of ACM cells to these stimuli is also greater than in control cells. The enhanced CL of ACM-treated cells can be inhibited by incubating in vitro the zymosan-triggered PC with superoxide dismutase (300 units/ml) and catalase (2750 units/ml), but not with ethanol (20 microM) or potassium cyanide (100 microM). This indicates the participation of O2- and H2O2 in the CL of ACM-treated cells, whereas mitochondrial respiration does not appear to be involved. Furthermore, the following facts suggest the participation of arachidonic acid metabolism in the control of CL: (a) the in vitro addition of nordihydroguaiaretic acid (7 x 10(-6) M) and indomethacin (10(-3) M) inhibits the CL, while indomethacin (10(-6) M) has the opposite effect; (b) the PC from normal or ACM-treated mice when stimulated with zymosan secrete high amounts of prostaglandin (PG); (c) treated cells secrete the same amounts of PGE2 and 6-keto-PGF1 alpha but the secretion of PGF2 alpha and particularly of thromboxane B2 is greater in treated cells than in control cells and indomethacin (10(-6) M) strongly inhibits PG secretion in all groups; (d) in vitro addition of PGE2 at a concentration of 10(-6) M has an inhibitory effect on the CL emission of control and of treated cells, but it does not have this effect at lower concentrations (10(-8) M). These data suggest that the lipoxygenase pathway of arachidonic acid metabolism may be involved in the triggering of CL of ACM-treated cells, as well as that of normal cells, whereas products of the cyclooxygenase pathway may act as feedback inhibitors.

Enhanced activity of peritoneal cells after aclacinomycin injection: effect of pretreatment with superoxide dismutase on aclacinomycin-induced cytological alterations and antitumoral activity.

Peritoneal macrophages from mice injected with aclacinomycin (ACM) (4 mg/kg, i.p.) showed increased functional activity, as assessed by increased antitumoral activity in vitro and in vivo and zymosan-triggered chemoluminescence. They also showed ultrastructural signs of activation (increased number of cytoplasmic organelles), and atypical alterations (giant vacuoles and giant lysosomes containing heterogenous myelinoid bodies, lipofuscine-like substance, cytoplasmic debris, and a fine granular material). As these atypical alterations could be due to the generation of superoxide following ACM injection, superoxide dismutase (SOD) was injected 1 h prior to ACM administration. Neither the morphological characteristics of activation, nor the enhanced metabolic and antitumoral activities induced by ACM were affected by SOD pretreatment, but the atypical alterations were inhibited in a dose-dependent manner. Heat-inactivated SOD did not prevent their appearance. The atypical alterations were not found in peritoneal macrophages from talc or lipopolysaccharide-injected mice, but they were present in Adriamycin-treated mice and were also prevented by SOD pretreatment, indicating that the alterations are due to anthracycline treatment. Finally, [125I]SOD was phagocytized by peritoneal macrophages in vitro and in vivo and not by L1210 tumoral cells, explaining why the atypical alterations induced by ACM were no longer seen after SOD pretreatment. The unchanged direct oncostatic activity of ACM following SOD pretreatment suggests that this combination may have some wider perhaps clinical, potential.

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.

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.

Urokinase-type plasminogen activator and plasminogen activator inhibitors (PAI-1 and PAI-2) in extracts of invasive cervical carcinoma and precursor lesions.

In a previous study we reported a direct correlation between the degree of total proteolytic activity and the natural history of cervical carcinoma. The present work examined whether an increase in the urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitors (PAIs) is correlated with the natural history of cervical carcinoma. We measured uPA and PAI-1 activities and uPA, PAI-1 and PAI-2 antigen concentrations in cervical extracts from normal, squamous intraepithelial lesions (SIL) or invasive carcinoma patients. The uPA activity in invasive carcinoma extracts was 8.46 times that of normal extracts and 4.9 times that of SIL extracts. The PAI-1 activity in invasive carcinoma extracts was 1.3 times that of normal extracts and 1.24 times that of SIL extracts. uPA, PAI-1 and PAI-2 amounts were 25.7-, 12.1- and 7.9-fold higher, respectively, in invasive carcinoma than in SIL, and 39.1-, 21.38- and 27.3-fold higher, respectively, than in normal extracts. uPA and PAI-1 activities were 2.02- and 1.42-fold higher in extracts from patients with stages II-IV than those from stage I extracts, respectively. uPA, PAI-1 and PAI-2 amounts were 3.06-, 4.2- and 1.4-fold higher in extracts from patients with stages II-IV than those from stage I extracts, respectively. The increase in uPA activity and the antigen levels of uPA and PAIs (PAI-1 and PAI-2) in stages II-IV of invasive carcinoma of the cervix suggests that these components play an important role in invasion and metastasis in advanced stages of this tumour.

Inhibition of hybrid resistance by 5-fluorouracil. Destruction of a Thy-1+ Lyt-1+2- spleen cell implicated in the expression of hybrid resistance.

Hybrid resistance designates the poor proliferation of parent-strain bone marrow cells injected into lethally irradiated hybrids. While testing a variety of oncostatic drugs for their capacity to inhibit hybrid resistance, we found that 5-fluorouracil (5-FU) injected into B6D2F1 hybrids three days before grafting enhances the growth of parent-strain B6 bone marrow cells. Hybrid resistance can be restored in these 5-FU-treated B6D2F1 recipients by the injection of spleen cells from untreated (normal) B6D2F1 mice. Treatment of these normal B6D2F1 spleen cells with anti-Thy-1 or anti-Lyt-1 antibodies plus complement abolishes their capacity to restore resistance, whereas treatment with anti Lyt-2 antibody plus complement has no effect. It is known that the rejection of parent-strain bone marrow cells by the F1 hybrid is a multistep process in which more than one cell type is involved, and that the final effector cell is asialo-GMI+, cyclophosphamide-sensitive, and responsive to IFN (possibly the natural killer cell). The Thy-1+Lyt-1+2- spleen cell we describe here, which is eliminated by the 5-FU, doesn't seem to be the final effector cell for hybrid resistance since transfer of spleen cells from cyclophosphamide-treated (final-effector-cell-depleted) B6D2F1 hybrids into 5-FU treated B6D2F1 mice restores hybrid resistance just as well as do normal B6D2F1 spleen cells; and, when injected into 5-FU-treated B6D2F1 hybrids, the IFN inducer pI:pC also restores resistance. The cell we now describe would be a third cell type, probably responsible for the initial recognition of grafted parent-strain bone marrow cells, which triggers the mechanism of hybrid resistance. This hypothesis could explain the observed specificity of parent-strain bone marrow graft rejection by F1 hybrids.

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.

The bactericidal capacity of peripheral blood monocytes from HIV positive patients may collapse very soon after the infection.

The activity of peripheral blood monocytes from HIV positive patients was measured by the intensity of the chemoluminescence those cells emit when they ingest a foreign particle. In most patients (84%) that value was impaired. Such an alteration may occur very early in the course of the disease. The anti-p24 antibody titer was correlated with monocyte phagocytic potential as measured by the chemoluminescent value thus indicating the need for adequate monocyte activity in order to obtain antibody formation. The severity of opportunistic infections that HIV positive subjects may develop is a clear indication that their immune systems are abnormal. The most frequently affected cells are those which bear the viral receptor, the CD4 antigen. Those cells are mainly the helper T cells and monocytes. The monocytes are the immune system phagocytic cells which actively control infections, in part by the release oxidative radicals. Those radicals can easily be measured by the chemoluminescence (CL) the cells emit when they ingest a foreign particle. This study examines the CL emitted by peripheral blood monocytes from HIV-positive and control subjects while they phagocytose opsonized zymosan in vitro and correlates these values with other laboratory parameters.

Enhanced production of interleukin 1 by mouse peritoneal macrophages after aclacinomycin administration.

The peritoneal cells of mice injected with aclacinomycin (ACM), an oncostatic drug of the anthracyclin family, were found to secrete more interleukin (IL-1), after two successive 24-h periods of in vitro LPS stimulation than those of control mice. This measured IL-1 production is one of the signs of enhanced macrophage activity. The cells of ACM-injected mice also contained more intracellular IL-1 than those of controls. In contrast, macrophages from ACM-injected mice only increased their IL-1 production after the first 24-h incubation with PMA, and not after the second 24-h incubation. The response to ACM was dose- and time-dependent. We have also compared the IL-1 production by macrophages from mice injected with other anthracyclins, at doses equimolar to that of 4 mg/kg ACM and we have observed that adriamycin, 4'-epiadriamycin and aclacinomycin had similar activity, while THP-adriamycin an daunorubicine were slightly more active. Exploitation of this increased IL-1 production by macrophages could be beneficial in the design of tumor treatment protocols.

Decreased natural killer cell activity after prolonged administration of interferon in cancer patients.

Two patients with metastatic neoplastic disease received 2-3 X 10(6) IU alpha recombinant interferon (IFN) 3 times/wk, every other week, for 3-6 mth. The natural killer (NK) activity of their peripheral blood leukocytes, was followed during the course of the treatment. A significant decrease was observed in the NK activity, which returned to normal values at the end of IFN administration. The treatment did not modify the evolution of metastasis.

Phospholipase A2, an in vivo immunomodulator.

Arachidonic acid (AA) can be released from membrane phospholipids by the action of phospholipase A2 (PLA2). There is evidence that unsaturated fatty acids, particularly AA, released from membrane phospholipids are required to activate the respiratory burst of macrophages. The data reported here indicate that peritoneal macrophages harvested 30 min after i.p. injection of PLA2 can phagocytose Candida albicans more efficiently and emit more chemoluminescence (CL) than normal cells when stimulated by zymosan. PLA2 injection also enhances the CL of peritoneal cells from mice already stimulated by immunomodulators such as trehalose dimycolate (TDM), bestatin, or oncostatic drugs such as aclacinomycin (ACM). CL is not sensitive to potassium cyanide (KCN), but is inhibited by catalase, superoxide dismutase (SOD), nordihydroguaiaretic acid (NDGA) and high doses of indomethacin (10(-3) M). In vivo PLA2 treatment stimulates the synthesis of both cyclooxygenase and lipoxygenase derivatives of AA metabolism (PGE2, 6-keto, PGF1 alpha TXB2 and LTC4). Inhibitors of AA metabolism (NDGA, indomethacin) modulate the production of free oxidizing radicals in this experimental model, partly because of their effect on AA metabolism, as determined by the measuring immunoreactive products. However, this work indicates that the effects of these inhibitors, which have been extensively used in CL studies, should be interpreted with caution, since their specificity for AA metabolism is relative.

Kinetics of in vivo tumor cell destruction after specific immunization.

The natural cytotoxic activity in vivo can be evaluated by measuring in vivo tumor cell destruction after injecting mice with 125IUdR-labeled tumor cells, measuring their total body radioactivity and calculating the % radioactivity lost. We have studied the in vivo destruction of 125IUdR-labeled L1210 leukemic cells by B10.D2 mice previously immunized 4 times with heavily irradiated L1210 leukemic cells. Mathematical analysis of our results indicates that the radiolabel loss on day 1 is similar in normal and immunized animals, but that it stays greater over the following days in immunized animals, indicating that the difference between the two groups is not the extent of the initial cell destruction but the durability of the response. There is a good correlation between the eventual survival, the % of 125IUdR lost and the number of tumor cells present in the peritoneal cavity three hours after their injection. Such a methodology provides a very early prediction of the survival of each mouse, thus identifying animals with a poor prognosis.

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.

L-5178-Y lymphoma associated immunosuppression in Balb/c mice.

Lymphoma L-5178-Y bearing Balb/c mice were unable to mount a delayed-type hypersensitivity reaction to dinitrofluorobenzene (DNFB). A suppressor factor present in the ascitis of these mice inhibited the response if given during DNFB sensitization, but not during challenge. The factor was not present in lymphoma-bearing Balb/c nu/nu mice. It appeared to be a 35-66 kDa protein. Non-specific esterase staining indicated that the spleens of tumor-bearing and ascitis-injected mice contained a large excess of macrophages. Our observation that the suppressor factor prevented the very start of the immune response may indicate why immunostimulation is difficult to obtain in cancer bearing hosts.

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.