Mônica Sakai

Immunomodulatory effects of Pteridium aquilinum on natural killer cell activity and select aspects of the cellular immune response of mice

Pteridium aquilinum (bracken fern) is one of the most common plants. Epidemiological studies have revealed a higher risk of certain types of cancers (i.e., esophageal, gastric) in people who consume bracken fern directly (as crosiers or rhizomes) or indirectly through the consumption of milk from livestock that fed on the plant. In animals, evidence exists regarding the associations between chronic bracken fern intoxication, papilloma virus infection, and the development of carcinomas. While it is possible that some carcinogens in bracken fern could be responsible for these cancers in both humans and animals, it is equally plausible that the observed increases in cancers could be related to induction of an overall immunosuppression by the plant/its various constituents. Under the latter scenario, normal tumor surveillance responses against nascent (non-bracken-induced) cancers or responses against viral infections (specifically those linked to induction of cancers) might be adversely impacted by continuous dietary exposure to this plant. Therefore, the overall objective of this study was to evaluate the immunomodulatory effects of bracken fern following daily ingestion of its extract by a murine host over a period of 14 (or up to 30) days. In C57BL/6 mice administered (by gavage) the extract, histological analyses revealed a significant reduction in splenic white pulp area. Among a variety of immune response parameters/functions assessed in these hosts and isolated cells, both delayed-type hypersensitivity (DTH) analysis and evaluation of IFNγ production by NK cells during TH1 priming were also reduced. Lastly, the innate response in these hosts—assessed by analysis of NK cell cytotoxic functionality—was also diminished. The results here clearly showed the immunosuppressive effects of P. aquilinum and that many of the functions that were modulated could contribute to the increased risk of cancer formation in exposed hosts.

Anandamide prior to sensitization increases cell-mediated immunity in mice

The endocannabinoid system has become a topic of great interest in pharmacology due to its remarkable distribution in mammal organisms and capacity to play a modulatory role on several physiological systems, including modulation of immunity. Many studies have shown that administration of cannabinoids causes inhibitory effects on immune cells, including decreased proliferation and antigen-presenting cell (APC) co-stimulatory activity. In contrast, other groups have shown that some cannabinoids might present stimulatory actions on macrophage activity and T cell activation. Therefore, we aimed to investigate whether a treatment in vivo with a low dose of anandamide (0.1mg/kg) immediately prior to sensitization would have an immunosuppressive or immunostimulatory effect on cell-mediated immunity (Th1 response) in mice. We report here that anandamide, prior to sensitization, was able to increase the Th1 response to ovalbumin in vivo and ex vivo. Anandamide increased delayed type hypersensitivity (DTH), splenocyte proliferation, and IFN-gamma production in a co-culture of adherent and non-adherent splenocytes. Moreover, anandamide prior to sensitization increased both the expression of DC co-stimulatory molecules (CD80/CD86) and IL-12/IL23 (p40) production ex vivo. We have also assessed direct effects of anandamide in the IFN-gamma/IL-4 balance of ConA-stimulated splenocytes in vitro. Anandamide at nanomolar concentrations increased the production of IFN-gamma, while such production decreased at micromolar range. Thus, anandamide induced both the increment of DC activation and IFN-gamma production, which are likely the mechanisms involved in the increase of Th1 response reported here.

Translocator protein (18 kDa) mediates the pro-growth effects of diazepam on Ehrlich tumor cells in vivo

The Translocator Protein (TSPO), previously known as the peripheral-type benzodiazepine receptor, is a ubiquitous drug- and cholesterol-binding protein that is up regulated in several types of cancer cells. TSPO drug ligands (e.g., diazepam) induce or inhibit tumor cell proliferation, depending on the dose and tissue origin. We have previously shown that TSPO is expressed in Ehrlich tumor cells and that diazepam increases proliferation of these cells in vitro. Here, we investigated the in vivo effects of diazepam on Ehrlich tumor growth and the role of TSPO in mediating this process. Oral administration of diazepam to mice (3.0mg/kg/day for 7 days) produced plasma and ascitic fluid drug concentrations of 83.83 and 54.12 nM, respectively. Diazepam increased Ehrlich tumor growth, likely due to its ability to increase tumor cell proliferation and Reactive Oxygen Species production. Radioligand binding assays and nucleotide sequencing revealed that Ehrlich tumor cell TSPO had the same pharmacological and biochemical properties as TSPO described in other tumor cells. The estimated K(d) for PK 11195 in Ehrlich tumor cells was 0.44 nM and 8.70 nM (low and high binding site, respectively). Structurally diverse TSPO drug ligands with exclusive affinity for TSPO (i.e., 4-chlordiazepam, Ro5-4864, and isoquinoline-carboxamide PK 11195) also increased Ehrlich tumor growth. However, clonazepam, a GABA(A)-specific ligand with no affinity for TSPO, failed to do so. Taken together, these data suggest that diazepam induces in vivo Ehrlich tumor growth in a TSPO-dependent manner.

Naloxone treatment prevents prenatal stress effects on peritoneal macrophage activity in mice offspring.

The present study analyzed the effects of maternal stress (PS) and/or naloxone treatment on the activity of peritoneal macrophage in male and female Swiss mice offspring. Pregnant female rats received a daily footshock (0.2 mA) and/or a naloxone injection from gestational day 15 to 19. Experiments were performed on postnatal day 30 on male and female pups. The following results were obtained in male offspring: (1) PS decreased both the index and the percentage of phagocytosis, this decrement being reversed by naloxone treatment, and (2) naloxone alone decreased the percentage of phagocytosis. The following results were obtained in female offspring: (1) PS decreased spontaneous and phorbol myristate acetate-induced macrophage oxidative burst, this decrement being reversed by naloxone pretreatment, and (2) PS decreased both the index and percentage of the phagocytosis, this effect was prevented by naloxone treatment. These data are discussed focussing on a putative neuroimmune interaction involving opioidergic systems during the ontogeny of the central nervous and immune systems.

Effects of different doses and schedules of diazepam treatment on lymphocyte parameters in rats

Benzodiazepines (BZD) are widely used for the treatment of anxiety. They enhance GABA-ergic neurotransmission through the binding on specific BDZ recognition sites, within the GABA(A) receptor-ion channel complex. However, recent studies showed that BZD also act on peripheral benzodiazepine receptor sites (PBR) or translocator protein 18 kDa (TSPO). Evidence for a direct immunomodulatory action for BZD emerged from studies that demonstrated the presence of TSPO on immune/inflammatory cells. The present study was designed to analyze the effects of diazepam on rat lymphocyte parameters, specifically on phenotype, cell proliferation and cell death. The effects of both acute and long-term (21 days) diazepam (1 and 10 mg/kg/day) administrations were evaluated. Results showed that diazepam (1 mg/kg) treatment did not change the immune parameters analyzed. However, both diazepam (10 mg/kg) acute and long-term treatments decreased the number of apoptotic cells; they also increased the percentage of T cytotoxic cells; decreased the percentage of B cells and increased the corticosterone serum levels. The induction of functional tolerance was suggested for the highest dose of diazepam (10 mg/kg), but not for the smaller dose (1 mg/kg) used, at least for diazepam effects on corticosterone serum levels. Diazepam effects were discussed as being related to the number of TSPO sites present on immune cells and/or to the increased levels of serum corticosterone observed after the treatments used.

Methylenedioxymethamphetamine (Ecstasy) Decreases Neutrophil Activity and Alters Leukocyte Distribution in Bone Marrow, Spleen and Blood

Objective: Looking for possible neuroimmune relationships, we analyzed the effects of methylenedioxymethamphetamine (MDMA) administration on neuroendocrine, neutrophil activity and leukocyte distribution in mice. Methods: Five experiments were performed. In the first, mice were treated with MDMA (10 mg/kg) 30, 60 min and 24 h prior to blood sample collection for neutrophil activity analysis. In the second experiment, the blood of naïve mice was collected and incubated with MDMA for neutrophil activity in vitro analysis. In the third and fourth experiments, mice were injected with MDMA (10 mg/kg) and 60 min later, blood and brain were collected to analyze corticosterone serum levels and hypothalamic noradrenaline (NA) levels and turnover. In the last experiment, mice were injected with MDMA 10 mg/kg and 60 min later, blood, bone marrow and spleen were collected for leukocyte distribution analysis. Results: Results showed an increase in hypothalamic NA turnover and corticosterone serum levels 60 min after MDMA (10 mg/kg) administration, a decrease in peripheral blood neutrophil oxidative burst and a decrease in the percentage and intensity of neutrophil phagocytosis. It was further found that MDMA (10 mg/kg) treatment also altered leukocyte distribution in blood, bone marrow and spleen. In addition, no effects were observed for MDMA after in vitro exposure both in neutrophil oxidative burst and phagocytosis. Conclusion: The effects of MDMA administration (10 mg/kg) on neutrophil activity and leukocyte distribution might have been induced indirectly through noradrenergic neurons and/or hypothalamic-pituitary-adrenal axis activations.

Long-term maternal separation differentially alters serum corticosterone levels and blood neutrophil activity in A/J and C57BL/6 mouse offspring.

Objectives: In this work, we searched for maternal separation effects on serum corticosterone levels and blood neutrophil activity in adult male A/J and C57BL/6 mouse offspring. Methods: 40 male A/J mice and 40 male C57BL/6 mice were divided within each strain into two groups. Mice in the maternal separation group were separated from their mothers (1 h/day) on postnatal days 0–13. Mice in the control group were left undisturbed. On postnatal day 45, blood was drawn from all mice and used to assess neutrophil activity by flow cytometry and serum corticosterone levels by radioimmunoassay. Results: The results showed that each mouse strain responded differently to maternal separation, but in both cases, serum corticosterone levels were affected. In both strains, adult mice that experienced maternal separation showed lower serum corticosterone levels than control mice. In relation to control mice kept together with their mothers, the levels of serum corticosterone were 72.7 and 36.36% lower in A/J and C57BL/6 mice submitted to maternal separation, respectively. The current findings showed that maternal separation increased neutrophil activity in mice after reaching adulthood. The observed effects, although in the same direction, differed between A/J and C57BL/6 mice. Maternal separation increased both the percentage and intensity of phagocytosis in C57BL/6 mice, but had no effects on A/J mice. Furthermore, maternal separation increased basal and propidium iodide-labeled Staphylococcus aureus-induced oxidative burst in A/J mice but did not affect oxidative burst in C57BL/6 mice. Finally, phorbol myristate acetate-induced oxidative burst increased in both strains. Conclusion: These results indicate that early maternal separation increases innate immunity, most likely by modifying hypothalamus-pituitary-adrenal axis activity. This suggests that maternal separation is a good model for stress which produces long-term neuroimmune changes whatever the animal species and strain used.

Diazepam decreases leukocyte–endothelium interactions in situ

High doses of diazepam reduce the inflammatory paw edema in rats. This effect was attributed to an action of diazepam on the Translocator Protein (TSPO). We evaluated the effects of diazepam (10 mg/kg, intraperitoneally) on leukocyte rolling and migration. In carrageenan-induced acute inflammation, diazepam decreased the interaction of leukocytes with endothelial cells (rolling) and the number of leukocytes in the mesentery (migration). RU486 (antagonist of glucocorticoid receptors) reduced the effects of diazepam on leukocyte rolling and migration, suggesting a participation of endogenous corticosteroids. We also showed that the effects of diazepam on leukocyte–endothelium interactions are mediated by nitric oxide (NO), since prior treatment with l-arginine (precursor of NO) partially precludes the inhibitory effects of diazepam; conversely, pretreatment with L-NAME (false substrate of the NO synthase) somewhat potentiates the effects of diazepam. The pathways that underlie the effects of diazepam remain to be further elucidated, but we believe that both local and systemic mechanisms may overlap to explain the influence of diazepam on leukocyte–endothelium interactions.

Proliferação de linfócitos e apoptose de células CD5 + de bovinos infectados pelo vírus da leucose enzoótica bovina

The purpose of the present trail was to evaluate the lymphocyte proliferation and the apoptosis rates of CD5+ cells in dairy cows infected with bovine leukemia virus (BLV) with distinct lymphocyte profiles in infected animals known as alymphocytotic (AL) and persistent lymphocytosis (PL). A total of 100 Holstein cows were sera tested for bovine leukemia virus through agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent-assay (ELISA). From these animals, 15 cows were selected and divided uniformly in 3 groups (negative, AL, LP). The lymphocyte proliferation was performed using flow cytometric measurement of CFSE-DA dye, where 2x106/mL lymphocytes were plated per well. The apoptosis of CD5+ cells from peripheral blood was performed using the annexin V-FITC to measure the apoptosis rates and the identification of CD5+ was accessed using monoclonal antibodies. Animals from the LP group showed lower lymphocyte proliferation and also lower apoptosis rates of CD5+ cells compared with negative and AL animals. The development of PL which resulted from an increase in B cell count, is due to the decrease in the apoptosis rates of CD5+ cells, and the higher lymphocyte proliferation appears to be limited only in the initial stages of development of LP.