Karine Bizzi Schlemmer

Cytoprotective and genoprotective effects of β-glucans against aflatoxin B1-induced DNA damage in broiler chicken lymphocytes

The polysaccharide β-glucan presents beneficial effects on the immune system, although the mechanisms of the immunomodulatory effect remain poorly understood. The potential cytoprotective and genoprotective effects of β-glucans were evaluated in broiler chicken lymphocytes exposed to increasing concentrations of aflatoxin B₁ (AFB₁) and/or β-glucans. AFB₁ significantly decreased cell viability at the concentrations of 10 and 20 μg/ml at 72 h of incubation (p<0.01 and p<0.001, respectively). Moreover, the AFB₁ concentrations of 1, 10 and 20 μg/ml increased DNA fragmentation levels at 24 h (p<0.001). Conversely, lymphocyte death was prevented by β-glucans at the concentrations of 1% and 10%, indicating a cytoprotective effect. Reactive oxygen species levels were increased in the cells treated with 20 μg/ml AFB₁ at 24 h (p<0.05) and 10% β-glucans with or without AFB₁ at 24, 48 and 72 h of incubation (p<0.001). DNA damage increased by more than 100% in AFB₁-treated lymphocytes when compared to control group. β-glucans at 1% was able to fully revert the AFB₁-induced lymphocyte DNA damage, indicating a genoprotective effect and maintaining DNA integrity. In conclusion, β-glucans showed in vitro dose-dependent cytoprotective and genoprotective effects in broiler chicken lymphocytes exposed to AFB₁.

Immunotherapy for pythiosis: Effect on NTPDase activity in lymphocytes of an experimental model.

NTPDase (EC 3.6.1.5) occurs in lymphocytes and plays an important role in immune function, in that hydrolyzes extracellular nucleoside tri- and/or diphosphates to form AMP. Pythium insidiosum causes the disease pythiosis, a pyogranulomatous disease of horses, dogs, cattle, cats and humans. Most antifungal drugs are ineffective against this pathogen, and immunotherapy, a treatment approach that relies on the injection of P. insidiosum antigen, has been successfully used in humans and horses to manage this disease. In this study, we investigated NTPDase activity in lymphocytes from rabbits inoculated with zoospores of P. insidiosum. After immunotherapy, we investigated the relationship between enzymatic activity and the pattern of the immune response. One milliliter of zoospores was inoculated subcutaneously into the coastal region of each rabbit. An average of 17,500 viable mobile zoospores/mL of induction medium was administered. Inoculated rabbits were checked weekly, and the subcutaneous nodular area (cm²) was measured 28 days after inoculation. Rabbits that developed lesions received four doses of immunotherapy at intervals of 14 days. Blood samples were collected by heart puncture twice a month for the determination of NTPDase activity. The results demonstrated that NTPDase activity in lymphocytes was increased in relation to ATP hydrolysis (by about 100%) in pythiosis and returned to normal values after immunotherapy. The data demonstrating NTPDase activity before and after immunotherapy reinforce the previously elaborated hypothesis that the change from a Th2 to a Th1 immune response is responsible for the curative properties of immunotherapy.

E‐ADA activity in lymphocytes of an experimental model of pythiosis treated with immunotherapy

Pythiosis is a life‐threatening disease caused by the oomycete Pythium insidiosum. Some authors have suggested the involvement of a Th2‐like immune response in the infected host, which leads to extensive tissue damage. The switch from a Th2 to a Th1 response pattern is one hypothesis to explain the curative properties of immunotherapy. Taking into account the importance of immunotherapy for pythiosis treatment and the contribution of adenine nucleotides in the immunoregulation of the host, we evaluated the ecto‐adenosine deaminase (E‐ADA; EC 3·5.4·4) activity in lymphocytes from rabbits inoculated with P. insidiosum. Rabbits were inoculated with 1 milliliter of zoospores subcutaneously injected into the lateral thorax; after developing lesions, the rabbits received eight doses of immunotherapy. E‐ADA activity was measured in lymphocytes and the adenine nucleotides and adenosine levels were quantitatively determined in serum. Rabbits with characteristic lesions of pythiosis showed a decreased E‐ADA activity (82·36%), a decreased adenosine triphosphate concentration (54·04%) and a higher adenosine concentration (2·51 fold), when compared with controls, after 28 days of inoculation. However, after the immunotherapy, the rabbits showed an increase in the E‐ADA activity when compared with control (78·62%), contributing for the change in the immune response. Our results reinforce the hypothesis that the change from a Th2 to a Th1 immune response with the participation of the purinergic system could be responsible for the curative properties of immunotherapy. Copyright

An experimental model of contact dermatitis: Evaluation of the oxidative profile of Wistar rats treated with free and nanoencapsulated clobetasol

Abstract Objective An experimental animal model of contact dermatitis (CD) was used to investigate the effects of free and nanoencapsulated clobetasol propionate on the skin and on the oxidative profile of liver tissue. Methods Female Wistar rats were divided into six groups, each containing eight rats. The first group, control (C), was sensitized with solid vaseline. Group 2, (CD), was sensitized with 5% NiSO4. Groups 3 and 4 were sensitized with 5% NiSO4 and treated with free (FC) and nanoencapsulated (NC) clobetasol (0.42 mg/g), respectively, daily for 5 days. Group 5 was treated with nanoencapsulated clobetasol (0.42 mg/g) on days 1, 3, and 5 (C135) and group 6 received a hydrogel containing empty nanoparticles (NP) daily for 5 days. Thiobarbituric acid reactive substances (TBARS), carbonyl levels, non-protein sulfhydryl groups (NPSH) and catalase activity were measured in liver homogenates. Results A significant increase was observed in the levels of TBARS, NPSH, and catalase activity for the groups CD and NP. Discussion Our results suggest that both NiSO4 sensitization and NP administration induced oxidation of cellular lipids and activated the antioxidant enzyme catalase to protect from this damage. These results also indicated that daily treatment with the free and nanoencapsulated clobetasol, as well as treatment with the nanoencapsulated clobetasol every other day, were able to prevent these redox alterations and protect against histological damage.

In vitro effect of ochratoxin A and deoxynivalenol on the expression of interleukin 5 and interferon-gamma in broiler chicken lymphocytes

Cytokines are proteins secreted by cells of innate and acquired immunity, produced in response to various antigens and responsible for mediating several function of these cells. Our study evaluated the profile of cytokines interleukin 5 (IL-5) and interferon gamma (IFN-γ), induced in lymphocytes of broiler chickens in response to secondary fungal metabolites ochratoxin A (OTA) and deoxynivalenol (DON) at concentrations of 0.001, 0.01, 0.1 and 1 μg/ml. The quantification of the cytokines was analysed at 24, 48 and 72 h after incubation with mycotoxins, using real-time PCR (qRT-PCR). The results obtained showed that OTA induced mRNA synthesis of IL-5 at concentrations 0.001, 0.1 and 1 μg/ml after 24 h of lymphocyte incubation, while at 48 h only the expression of the IL-5 cytokine at a concentration of 1 μg/ml (P<0.05) was detected. DON in a concentration of 1 μg/ml induced the expression of IL-5 in the lymphocytes only at 48 h post-incubation period (P<0.05). Regarding IFN-γ, gene expression was not observ...

An experimental murine model of otitis and dermatitis caused by Malassezia pachydermatis

We report a malasseziosis model in immunocompromised Swiss mice. For this model, the mice were immunosuppressed with a combination of cyclophosphamide at 150 mg/kg and hydrocortisone acetate at 250 mg/kg. Two groups were formed according to the site of inoculation. Dermatitis group received an intradermal injection of 5 × 106 cell/mouse at a shaved dorsal region, while the otitis group received the same inoculum in the middle ear. Five animals/group were euthanised at different times, and the skin and ear were histopathologically analysed. During the first euthanasia, which occurred after inoculation, microscopic examination showed that all mice presented budding yeast‐like in a tissue sample. The presence of yeasts decreased over time being undetected on the 17th day (dermatitis group) and the 21st day (otitis group) after inoculation. This is the first murine model for malasseziosis that can be useful for evaluating new treatment approaches.

Embryonated chicken eggs: An experimental model for Pythium insidiosum infection

Pythiosis is a severe disease caused by Pythium insidiosum. Currently, the research on the treatment of pythiosis uses rabbits as an experimental infection model. To reduce the use of animals in scientific experimentation, alternative models are increasingly necessary options. The objective of this study was to establish a new experimental infection model for pythiosis using embryonated chicken eggs. First, we tested the inoculation of 4 zoospore concentrations into the egg allantoic cavity at 3 embryonic days. We observed that increased zoospore concentration causes a decrease in survival time, and at a later embryonic day (the 14th) of infection, embryos showed delayed mortality. To confirm the reproducibility of the model, we chose the 14th embryonic day for the inoculation of 50 zoospores/egg, and the experiment was repeated twice. Mortality began with 30% embryos 48 hours after inoculation, and 95% embryos died within 72 hours. There was no mortality in the uninfected control group. The infection was confirmed by culture, PCR and histopathology. Immunohistochemistry confirmed the presence of hyphae in blood vessels in the umbilical cords in 95% of embryos and only 1 liver (5%). Our results suggest that embryonated eggs can be a very useful alternative infection model to study pythiosis.

In vitro combination of antifungal agents against Malassezia pachydermatis

The yeast Malassezia pachydermatis is a common commensal and occasional opportunistic pathogen of theskin microbiota of animals and humans. In this study, the susceptibility of M. pachydermatis isolates to fluconazole (FLC), itraconazole (ITZ), ketoconazole (KTZ), clotrimazole (CLZ), and miconazole (MCZ) alone and in combination with terbinafine (TRB), nystatin (NYS), and caspofungin (CSP) was evaluated in vitro based on the M27-A3 technique and the checkerboard microdilution method using Sabouraud dextrose broth with 1% tween 80 (SDB). Based on the mean FICI values, the main synergies observed were combinations of ITZ+CSP and CLZ+CSP (55.17%). The most significant combinations deserve in vivo evaluations because might provide effective alternative treatments against M. pachydermatis due to their synergistic interactions.