Unraveling the Reaction Mechanism of Russell's Viper Venom Factor X Activator: A Paradigm for the Reactivity of Zinc Metalloproteinases?

Snake venom metalloproteinases (SVMPs) are important drug targets against snakebite envenoming, the neglected tropical disease with the highest mortality worldwide. Here, we focus on Russell's viper (Daboia russelii), one of the "big four" snakes of the Indian subcontinent that, together, are responsible for ca. 50,000 fatalities annually. The "Russell's viper venom factor X activator" (RVV-X), a highly toxic metalloproteinase, activates the blood coagulation factor X (FX), leading to the prey's abnormal blood clotting and death. Given its tremendous public health impact, the WHO recognized an urgent need to develop efficient, heat-stable, and affordable-for-all small-molecule inhibitors, for which a deep understanding of the mechanisms of action of snake's principal toxins is fundamental. In this study, we determine the catalytic mechanism of RVV-X by using a density functional theory/molecular mechanics (DFT:MM) methodology to calculate its free energy profile. The results showed that the catalytic process takes place via two steps. The first step involves a nucleophilic attack by an in situ generated hydroxide ion on the substrate carbonyl, yielding an activation barrier of 17.7 kcal·mol-1, while the second step corresponds to protonation of the peptide nitrogen and peptide bond cleavage with an energy barrier of 23.1 kcal·mol-1. Our study shows a unique role played by Zn2+ in catalysis by lowering the pKa of the Zn2+-bound water molecule, enough to permit the swift formation of the hydroxide nucleophile through barrierless deprotonation by the formally much less basic Glu140. Without the Zn2+ cofactor, this step would be rate-limiting.

Efficacy of photobiomodulation therapy on healing of ionizing irradiated bone: a systematic review of in vivo animal studies.

This systematic review aimed to answer the research focused question: What are the effects of photobiomodulation (PBM) therapy on bone healing after ionizing irradiation in animal models? The EMBASE, LILACS, LIVIVO, PubMed, Scopus, and Web of Science databases, including gray literature, were searched using the following keywords: "Head and Neck Neoplasms"; "Ionizing Radiation"; "Low-Level Light Therapy"; and "Bone regeneration", focusing on the primary studies that assessed the effects of PBM therapy on animal models of irradiated bone. Six studies have met the eligibility criteria and presented an overall regular quality according to the risk of bias assessment tools. All the studies utilized rat animal model and near-infrared laser PBM at low power output setting. Most of the studies showed increased new bone formation, osteocytes, osteoblasts, and vascularization networking, as a result of PBM therapy. However, only one out of the six studies has not shown any differences in bone healing in both lased and non-lased animal groups. Nevertheless, PBM therapy is a potential tool to improve bone healing induced by ionizing radiation. However, due to the scarce number of studies and the great variability of laser parameters and treatment protocols, a clear conclusion cannot be drawn. Hence, extensive preclinical in vivo studies are warranted to ensure these beneficial effects have been addressed prior to translational clinical trials.

Changes in antiparasitical activity of gold nanorods according to the chosen synthesis.

Gold nanorods (GNRs) are increasingly being studied for diagnostic and therapeutic purposes. Green synthesis based methods with natural compounds as additives stand out as a hope in terms of better synthesis methodology, with advantages of producing potentially less toxic and, perhaps, biologically active GNRs due to influence of natural additives used during synthesis. Exploring green chemistry using different natural phenolic compounds, the present work reveals different in vitro activity of GNRs evaluated against different parasites that causes skin infectious diseases compared to GNRs produced by convencional seed mediated method. This approach brings advantages in producing active GNRs, with ease calling, less cytotoxic and with a better selectivity index (SI) than GNRs synthesized by conventional seed mediated synthesis, opening new possibilities for therapies. Natural compounds used in green syntheses were gallic acid (GA), resveratrol (RSV) and a purified fraction of the hydroalcoholic extract of Stryphnodendron obovatum. GNRs exhibited great activity against Leishmania braziliensis, and the dermatophytes Tricophyton rubrum, T. interdigitale and Microsporum gypseum. The anti-Leishmania and antidermatophytic activity of GNRs reinforce the applicability of GNRs in biomedical field and the influence of synthesis method in biological activity, showing benefits related to the seedless synthesis with natural compounds. In addition, these preliminary results indicate the possibility of exploring at maximum the physical and chemical properties of GNRs in addition to the biological activity itself, such as the development of topical antiparasitic formulations for association with phototherapy.

Effects of Orally Administered Resveratrol on TNF, IL-1ß, Leukocyte Phagocytic Activity and Oxidative Burst Function in Horses: A Prospective, Randomized, Double-Blinded, Placebo-Controlled Study.

Resveratrol, a phytophenol, is a commonly used equine nutraceutical supplement touted to exert anti-inflammatory effects. The effect of orally administered resveratrol on tumor necrosis factor (TNF), interleukin-1ß (IL-1ß), leukocyte phagocytic activity or oxidative burst function have not been reported in horses. The objective of this study was to determine the effects of a commercially available, orally administered resveratrol product on innate immune functions in healthy adult horses. Whole blood was collected from 12 horses prior to and following 3 weeks of treatment with either the manufacturer's recommended dose of resveratrol or placebo. Phagocytosis, oxidative burst and pathogen associated molecular pattern (PAMP) motif-stimulated leukocyte production of TNF and IL-1ß were compared pre- and post-treatment between treatment groups. Phagocytosis and oxidative burst capacity were evaluated via flow cytometry. Tumor necrosis factor and IL-1ß were measured using cytotoxicity and ELISA assays, respectively. There were no significant differences in phagocytosis, oxidative burst or stimulated TNF or IL-1ß production between resveratrol and placebo treatment groups. Orally administered resveratrol at a routinely recommended dose for a duration of 3 weeks did not significantly affect phagocytic activity, oxidative burst function or PAMP-stimulated leukocyte cytokine production.

Dogs with osteosarcoma have altered pro- and anti-inflammatory cytokine profiles.

BACKGROUND: Current advances in immunotherapy are an exciting area of study in canine osteosarcoma (OSA). The objective of this study was to determine the immune response in dogs with osteosarcoma by measuring stimulated leukocyte production of tumor necrosis factor (TNF), interleukin (IL)-6, IL-10 and TNF and IL-6 to IL-10 ratios. METHODS: Whole blood was collected from dogs with osteosarcoma receiving non-steroidal anti-inflammatory drugs (NSAIDs, n = 11), dogs with osteosarcoma not receiving NSAIDs (n = 14) and healthy dogs (n = 5). RESULTS: No difference in TNF production was found among healthy and OSA dogs regardless of NSAID administration following stimulation with lipopolysaccharide (LPS) (p = .410), lipoteichoic acid (LTA) (p = .693) or PBS (p = .120). Leukocyte IL-6 production was greater in all dogs with OSA after stimulation with LPS (p = .015), LTA (p = .014) and PBS (p = .034) with no difference between OSA dogs receiving NSAIDs and those not. No differences in IL-10 were found among healthy controls and dogs with OSA regardless of NSAID use. There was no difference among groups for LPS-stimulated TNF to IL-10 ratios (p = .407). For LTA-stimulated leukocytes, the TNF to IL-10 ratio was lower in dogs with OSA than in healthy dogs (p = .031) with no difference between OSA NSAID dogs compared to OSA non-NSAID dogs (p = .059). No differences were found in LPS (p = .310)- or LTA (p = .265)-stimulated leukocyte IL-6 to IL-10 production ratios among groups. CONCLUSIONS: Dogs with osteosarcoma have an altered pro- and anti-inflammatory immunologic profile compared to healthy dogs regardless of NSAID use. Further study is indicated to determine the potential prognostic and therapeutic implications of these findings.

Resveratrol administration increases phagocytosis, decreases oxidative burst, and promotes pro-inflammatory cytokine production in healthy dogs.

Resveratrol is a polyphenol that is safe to administer to dogs and has immunomodulating properties. Canine in vitro work indicated that resveratrol spared polymorphonuclear cell (PMN) phagocytosis but reduced the robustness of PMN oxidative burst and resulted in a pro-inflammatory leukocyte cytokine profile. The objective of this study was to determine the short-term effect of resveratrol on the healthy canine innate immune system in vivo. The hypothesis was that resveratrol would spare phagocytosis, depress the vigor of PMN oxidative burst, and result in a proinflammatory stimulated leukocyte cytokine profile in vivo. In an open-label study, whole blood was collected from 12 healthy, adult client-owned dogs on day 0 and 3. Six dogs received resveratrol, 200 mg kg-1, orally once daily for three days and six dogs served as controls with no supplement administered. Phagocytosis, oxidative burst and pathogen associated molecular pathogen stimulated leukocyte production of tumor necrosis factor (TNF), interleukin (IL)-6, and IL-10 were measured. Results between days 0 and 3 were compared using two way repeated measures analysis of variance and Fisher least significant difference method. A P -value of < 0.05 was considered statistically significant. Resveratrol administration resulted in an increased number of Escherichia coli phagocytized by PMNs and decreased robustness of the oxidative burst reaction. Resveratrol also increased stimulated TNF and IL-6 production with no effect on IL-10. Resveratrol had differential effects on peripheral innate immune system function in dogs. Studies of resveratrol including tissue compartments and the adaptive immune system are indicated to determine if these immunologic effects may be beneficial in disease states.

Effects of calcitriol on apoptosis, toll-like receptor 4 expression, and cytokine production of endotoxin-primed canine leukocytes.

OBJECTIVE To determine the in vitro effect of calcitriol on indicators of immune system function in endotoxin-primed blood samples from healthy dogs. SAMPLE Blood samples from 6 healthy adult dogs. PROCEDURES Leukocytes were primed by incubation of blood samples with lipopolysaccharide (LPS; endotoxin) or PBS solution (unprimed control group) for 1 hour. Following priming, blood samples were incubated with calcitriol (2 × 10-7M) or ethanol (control substance) for 24 hours. After sample incubation, LPS-stimulated leukocyte production of tumor necrosis factor (TNF) and interleukin-10 (IL10) was measured with a canine-specific multiplex assay, and apoptosis and toll-like receptor 4 (TLR4) expression were evaluated via flow cytometry. RESULTS LPS stimulation of unprimed leukocytes but not endotoxin-primed leukocytes resulted in a significant increase in TNF and IL10 production, confirming the presence of endotoxin tolerance in dogs in vitro. Endotoxin priming significantly increased neutrophil viability with no effect on lymphocyte viability or TLR4 expression by neutrophils and monocytes. Calcitriol exposure significantly decreased LPS-stimulated production of TNF by unprimed and endotoxin-primed leukocytes. Conversely, calcitriol exposure had no effect on IL10 production by unprimed leukocytes but did significantly increase IL10 production by endotoxin-primed leukocytes. Calcitriol had no significant effect on the degree of neutrophil or lymphocyte apoptosis, nor was neutrophil and monocyte TLR4 expression affected in unprimed or endotoxin-primed leukocytes. CONCLUSIONS AND CLINICAL RELEVANCE These data indicated that calcitriol induced an anti-inflammatory shift in unprimed and endotoxin-primed canine leukocytes in vitro, without compromising neutrophil and monocyte TLR4 expression or altering the viability of neutrophils and lymphocytes in canine blood samples.

Effects of calcitriol on phagocytic function, toll-like receptor 4 expression, and cytokine production of canine leukocytes.

OBJECTIVE To determine the in vitro effects of calcitriol on indicators of immune system function in blood samples collected from healthy dogs. SAMPLE Blood samples from 8 healthy adult dogs. PROCEDURES Blood samples were incubated with calcitriol (10-7M) or control substance for 24 hours. Afterward, lipopolysaccharide (LPS)-, lipoteichoic acid (LTA)-, and N-acetylmuramyl-l-alanyl-d-isoglutamine hydrate (MDP)-stimulated leukocyte production of tumor necrosis factor (TNF) and interleukin-10 (IL10) were measured with a canine-specific multiplex assay. Phagocytosis of opsonized Escherichia coli and leukocyte expression of constitutive toll-like receptor 4 (TLR4) were evaluated via flow cytometry. Blood samples from 3 dogs were used to create a concentration-response curve to evaluate whether the observed cytokine modulation was concentration dependent. RESULTS Incubation of canine blood samples with calcitriol resulted in significant decreases in LPS-, LTA-, and MDP-stimulated leukocyte production of TNF but not IL10. Blunting of TNF production was concentration dependent. Leukocyte calcitriol exposure had no significant effect on phagocytosis and TLR4 expression. CONCLUSIONS AND CLINICAL RELEVANCE These data indicated that calcitriol induced an anti-inflammatory shift in canine leukocytes exposed to LPS, LTA, and MDP in vitro, without altering phagocytosis or TLR4 expression. Thus, calcitriol could represent a novel candidate immunomodulatory treatment for dogs.

Atendimento das necessidades em saúde das travestis na atenção primária / Meeting the health needs of transvestites in primary care / El atendimiento de las necesidades en salud de las travestis en la atención primaria

A identidade trans está relacionada ao reconhecimento da identidade de gênero oposta ao sexo biológico. Essa característica faz com que essa população sofra diversos preconceitos e se torne vulnerável. Em nível programático, esse processo, quando ocorre nas unidades de saúde, faz com que as necessidades em saúde das travestis não sejam reconhecidas pelos profissionais da Atenção Primária. O presente trabalho tem como objeto de estudo as necessidades em saúde das travestis e objetivou discutir o atendimento das suas necessidades nos serviços de saúde da Atenção Primária. Trata-se de uma pesquisa de abordagem qualitativa do tipo descritiva. A coleta de dados ocorreu nos meses de abril e maio de 2013, quando foram realizadas entrevistas semiestruturadas com cinco travestis. A análise da entrevista foi realizada por meio da técnica de análise de conteúdo. Diante das falas das participantes emergiram três categorias temáticas: o conceito de saúde para travestis, a construção do corpo das travestis e o atendimento das travestis nas Unidades Básicas de Saúde. Foram identificadas como necessidades em saúde específicas: o acolhimento no serviço de saúde com a utilização do nome social da travesti, de modo a evitar a discriminação e constrangimentos na sala de espera; atendimento especializado por profissionais de saúde capacitados; proteção contra a violência desencadeada, principalmente em função da discriminação; orientações quanto à utilização de hormônios femininos; oferta de testes rápidos em toda Atenção Básica para diagnóstico de HIV, Hepatite B/C e Sífilis.

Trans identity is related to the recognition of gender identity as opposed to biological sex. This characteristic causes this population to suffer diverse prejudices and become vulnerable. At the programmatic level, this process, when it occurs in health facilities, makes the health needs of transvestites not to be recognized by Primary Care professionals. The objective of the present study is to study the health needs of the transvestites and aimed to discuss the meeting of their needs in Primary Health Care services. It is a qualitative research of descriptive type. Data collection took place in April and May 2013, when semi-structured interviews were carried out with five transvestites. The analysis of the interviews was performed by means of the technique of content analysis. Three themes emerged from the participants' speeches: the concept of health for transvestites, the construction of the body of transvestites and the treatment of transvestites in Basic Health Units. Specific health needs were identified: the reception in the health service using the social name of the transvestite, in order to avoid discrimination and constraints in the waiting room; specialized care by trained health professionals; protection against violence, mainly due to discrimination; guidelines on the use of female hormones; offer rapid testing throughout Basic Care for the diagnosis of HIV, Hepatitis B / C and Syphilis.

La identidad trans está relacionada al reconocimiento de la identidad de género opuesta al sexo biológico. Esta característica hace que esta población sufra diversos prejuicios y vuelvase vulnerable. A nivel programático, ese proceso, cuando ocurre en las unidades de salud, hace que las necesidades en salud de las travestis no sean reconocidas por los profesionales de la Atención Primaria. El presente trabajo tiene como objeto de estudio las necesidades en salud de las travestis y objetivó discutir la atención de sus necesidades en los servicios de salud de la Primaria Atención. Tratase de una investigación de abordaje cualitativo de tipo descriptivo. La recolección de datos ocurrió en los meses de abril y mayo de 2013, cuando se realizaron entrevistas semiestructuradas con cinco travestis. El análisis de la entrevista fue realizado por medio de la técnica del análisis de contenido. Ante las declaraciones de las participantes surgieron tres categorías temáticas: el concepto de salud para travestis, la construcción del cuerpo de las travestis y la atención a las travestis en las Unidades Básicas de Salud. Fueron identificadas como necesidades en salud específicas: la acogida en el servicio de salud con la utilización del nombre social de la travesti, para evitar la discriminación y las constreñimiento en la sala de espera; atención especializada por profesionales de la salud capacitados; protección contra la violencia desencadenada, principalmente en función de la discriminación; orientaciones sobre la utilización de hormonas femeninas; oferta de pruebas rápidas en toda Atención Básica para diagnóstico de VIH, Hepatitis B / C y Sífilis.

Modified pectin from Theobroma cacao induces potent pro-inflammatory activity in murine peritoneal macrophage.

In vitro effects of acetylated pectin (OP) isolated from cacao pod husks (Theobroma cacao L.), its partially deacetylated and de-esterified form (MOP), and a commercial homogalacturonan (PG) were investigated on murine peritoneal macrophages. MOP stood out among the studied pectins. After 48h of incubation, compared with the control group, it was able to promote significant macrophage morphological differentiation from resident to activated stage and also stimulated nitric oxide production, which reached a level of 85% of that of LPS stimulus. In the presence of the highest tested concentration of MOP (200µg·mL-1), the levels of the cytokines TNF-α (6h) and IL-12 and IL-10 (48h) increased substantially in relation to untreated cells. Our results show that the partial deacetylation and de-esterification of pectin extracted from cacao pod husks (T. cacao L.) produced a polymer with greater ability than its native form to activate macrophages to a cytotoxic phenotype. Like this, they provide the possibility of a therapeutic application to MOP, which could lead to a decreased susceptibility to microbial infection besides antitumor activity. Additionally, the present results also corroborate with the proposition of that the chemical modifications of the biopolymers can result in an improved molecule with new possibilities of application.

Investigation of the potential immunomodulatory effects of resveratrol on equine whole blood: An in vitro investigation.

Horses affected with gastrointestinal conditions such as colic or colitis are at substantial risk for translocation of bacterial components such as lipopolysaccharide (LPS, endotoxin) from the gastrointestinal tract into circulation resulting in systemic inflammation and subsequent morbidity and mortality. Therefore, there is a need for effective preventive and treatment strategies aimed at minimizing the host's inflammatory reaction to these pathogen-associated molecular patterns (PAMPs) from gastrointestinal disease. Resveratrol (RES, trans-3,5,4'-trihydroxystilbene) is a phytoalexin commonly found in fruits and beverages, including red wine. Health benefits associated with the consumption of red wine have been attributed to RES. Resveratrol has been significantly shown to exert a powerful anti-inflammatory effect in laboratory animals subjected to experimental endotoxemia/sepsis. Therefore, the objective of this study was to determine in vitro whether RES had an inhibitory effect on the production of tumor necrosis factor (TNF) in cultivated whole blood (Cwb) following stimulation by PAMPs. We hypothesized that RES would inhibit TNF production in Cwb following stimulation by LPS or lipoteichoic acid (LTA). Production of TNF bioactivity in Cwb was measured in the presence of phosphate buffered saline (control), ethanol (solvent control), dexamethasone (anti-inflammatory control), LPS, LTA, and three different concentrations of RES. Both LPS and LTA stimulated TNF production, and addition of dexamethasone was inhibitory to this effect. An anti-inflammatory effect for RES was not demonstrated under the current experimental conditions. Further studies are required to characterize the effect of RES on the equine innate immune system during systemic inflammation.

Perezone, from the gorgonian Pseudopterogorgia rigida, induces oxidative stress in human leukemia cells

Abstract Four bisabolanes 1–4, including perezone (1) and triacetyl perezone (2), were isolated through a bioassay-guided fractionation of the extract obtained from the Caribbean gorgonian coral Pseudopterogorgia rigida collected during an expedition cruise to the Bahamas. All isolated compounds showed to be cytotoxic toward panel of four human tumor cell lines, as quantified by the MTT assay after 72 h incubation. Perezone (1), the most active one, was further analyzed, showing to be cytotoxic, but not selective, in a 12-cell line panel comprising tumor and non-tumor, as well as human and murine cells. Additionally, 1 was assayed for cytotoxicity against HL-60 leukemic cells. Pre-treatment with an acute free radical scavenger (L-NAC) before exposure of cells to perezone virtually eliminated the generation of intracellular ROS and lessened its severe cytotoxicity. The protective effect delivered by L-NAC evidences that the mechanism of perezone-induced cytotoxicity is partially associated to production of ROS and a consequent induction of oxidative stress.

Morphine and buprenorphine do not alter leukocyte cytokine production capacity, early apoptosis, or neutrophil phagocytic function in healthy dogs.

Opioids have immunomodulatory properties in many species, but there is little information pertaining to these properties in dogs. Our objective was to compare the in vivo effects of morphine, buprenorphine, and control solution on innate immune system function and apoptosis in healthy dogs. Six adult dogs received a 24-hour infusion of morphine, buprenorphine, or control solution (saline) in a randomized, controlled, crossover block design. Leukocyte apoptosis, phagocytosis, and oxidative burst were evaluated using flow cytometry. Lipopolysaccharide, lipoteichoic acid, and peptidoglycan-stimulated leukocyte production of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 were determined using canine specific multiplex assays. No significant treatment effects were detected among groups. These data suggest that healthy dogs could be less sensitive to the immunomodulatory effects of acute opioid administration compared with other species. Larger investigations in healthy and immunologically challenged dogs are recommended prior to application of these results in clinical patients.

Effects of tramadol and o-desmethyltramadol on canine innate immune system function.

OBJECTIVE: Tramadol is a commonly used opioid analgesic in dogs, particularly in dogs with a compromised immune system. An opioid may be selected for its immunomodulatory effects. Consequently, the objective of this study was to investigate the effects of tramadol on immune system function by evaluating the effect of tramadol and o-desmethyltramadol (M1) on the function of canine leukocytes in vitro. The hypothesis was that tramadol and M1 would not alter polymorphonuclear leukocyte (PMN) phagocytosis, PMN oxidative burst, or stimulated leukocyte cytokine production capacity of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10. STUDY DESIGN: In vitro pharmacodynamic study. ANIMALS: Six healthy dogs. METHODS: Blood from six dogs was obtained and incubated with various concentrations of tramadol and M1. Phagocytosis and oxidative burst were assessed using flow cytometry, and lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PG)-stimulated leukocyte production of TNF, IL-6, and IL-10 were measured using a canine specific multiplex assay. RESULTS: No differences were detected in phagocytosis or oxidative burst with any drug concentration. Tramadol did not alter leukocyte cytokine production, however, M1 significantly blunted IL-10 production. CONCLUSIONS: Tramadol and its metabolite M1 were sparing to PMN phagocytosis and oxidative burst in dogs in vitro. Tramadol did not alter leukocyte cytokine production, however, M1 blunted IL-10 production at clinically achievable concentrations suggesting that M1 may promote a proinflammatory shift. CLINICAL RELEVANCE: These data suggest that tramadol has minimal effect on phagocytosis and oxidative burst, and may promote a proinflammatory shift. Therefore, tramadol may be an ideal opioid analgesic in dogs at high risk of infection. Further investigation in vivo is warranted.

Immunization with P10 peptide increases specific immunity and protects immunosuppressed BALB/c mice infected with virulent yeasts of Paracoccidioides brasiliensis.

Paracoccidioidomycosis is a systemic granulomatous disease caused by Paracoccidioides spp. A peptide from the major diagnostic antigen gp43, named P10, induces a T-CD4(+) helper-1 immune response in mice and protects against intratracheal challenge with virulent P. brasiliensis. Previously, we evaluated the efficacy of the P10 peptide alone or combined with antifungal drugs in mice immunosuppressed and infected with virulent isolate of P. brasiliensis. In the present work, our data suggest that P10 immunization leads to an effective cellular immune response associated with an enhanced T cell proliferative response. P10-stimulated splenocytes increased nitric oxide (NO) production and induced high levels of IFN-γ, IL-1ß and IL-12. Furthermore, significantly increased concentrations of pro-inflammatory cytokines were also observed in lung homogenates of immunized mice. P10 immunization was followed by minimal fibrosis in response to infection. Combined with antifungal drugs, P10 immunization most significantly improved survival of anergic infected mice. Administration of either itraconazole or sulfamethoxazole/trimethoprim together with P10 immunization resulted in 100 % survival up to 200 days post-infection, whereas untreated mice died within 80 days. Hence, our data show that P10 immunization promotes a strong specific immune response even in immunocompromised hosts and thus P10 treatment represents a powerful adjuvant therapy to chemotherapy.

Effects of opioids on phagocytic function, oxidative burst capacity, cytokine production and apoptosis in canine leukocytes.

Opioids alter immune and apoptotic pathways in several species. They are commonly used in companion animals affected with infectious and/or inflammatory disease, but the immunomodulatory and apoptotic effects of these drugs in dogs are relatively unknown. The aim of the present study was to evaluate the effects of morphine, buprenorphine and fentanyl on canine phagocyte function, oxidative burst capacity, leukocyte cytokine production, and lymphocyte apoptosis. Blood from six healthy adult dogs was incubated in the presence or absence of morphine (200 ng/mL), buprenorphine (10 ng/mL) or fentanyl (10 ng/mL) for 3 h (phagocytic function; cytokine production) or 8 h (apoptosis). Neutrophil phagocytosis of opsonized Escherichia coli, respiratory burst capacity after stimulation with opsonized E. coli or phorbol 12-myristate 13-acetate (PMA), and Annexin V-FITC staining of apoptotic lymphocytes were evaluated using flow cytometry. Leukocyte production of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-10 was assessed after incubation with lipopolysaccharide (LPS), lipoteichoic acid (LTA) or peptidoglycan. Morphine promoted a more intense respiratory burst. Morphine, buprenorphine and fentanyl all promoted LPS- or LTA-induced TNF-α and IL-10 production. However, the opioids tested did not alter TNF-α:IL-10 ratios. Morphine, buprenorphine and fentanyl all inhibited neutrophil apoptosis, an effect that was not concentration dependent in nature. These data indicate that opioids alter immune and apoptotic pathways in dogs. The possible effects of opioids on immune and cellular responses should be considered when designing studies and interpreting outcomes of studies involving administration of opioids in dogs.

Macrophage activation and leishmanicidal activity by galactomannan and its oxovanadium (IV/V) complex in vitro.

Compounds that activate macrophage antimicrobial activity are potential targets for treatment of leishmaniasis. The present study investigated the in vitro immunomodulatory effects of a galactomannan (GALMAN-A) isolated from seeds of Mimosa scabrella and its oxovanadium (IV/V) complex (GALMAN-A:VO(2+)/VO(3+)) on macrophage activity. GALMAN-A increased nitric oxide levels by ~33% at a concentration of 250µg/ml, while GALMAN-A:VO(2+)/VO(3+) decreased nitric oxide levels by ~33% at a concentration of 50µg/ml. Furthermore, GALMAN-A increased interleukin-1 beta (IL-1ß) and interleukin-6 (IL-6) levels by 5.5 and 2.3 times, respectively, at a concentration of 25µg/ml; at the same concentration, GALMAN-A:VO(2+)/VO(3+) promoted an increase in IL-1ß and IL-6 production by 8 and 5.5 times, respectively. However, neither GALMAN-A nor GALMAN-A:VO(2+)/VO(3+) affected tumor necrosis factor alpha (TNF-α) or interleukin-10 (IL-10) levels. Importantly, both GALMAN-A and GALMAN-A:VO(2+)/VO(3+) exhibited leishmanicidal activity on amastigotes of Leishmania (L.) amazonensis, reaching ~60% activity at concentrations of 100 and 25µg/ml, respectively. These results indicate that GALMAN-A is three times more potent and its oxovanadium complex is twelve times more potent than Glucantime (300µg/ml), which is the drug of choice in leishmaniasis treatment. The IC50 value for GALMAN-A:VO(2+)/VO(3+) was 74.4µg/ml (0.58µg/ml of vanadium). Thus, the significant activation of macrophages and the noted leishmanicidal effect demonstrate the need for further studies to clarify the mechanisms of action of these compounds.

DNA vaccine encoding peptide P10 against experimental paracoccidioidomycosis induces long-term protection in presence of regulatory T cells.

Paracoccidioidomycosis is a granulomatous systemic mycosis endemic in Brazil and other Latin America countries. A DNA vaccine encoding the immunoprotective peptide 10 (P10) significantly reduced the fungal burden in mice when given prior to or after intratracheal challenge with Paracoccidioides brasiliensis. Presently, the generation/expansion of CD4+ CD44hi memory T cells as well as Foxp3+ Treg cells in mice immunized with the DNA vaccine (pcDNA3-P10) before and after infection with P. brasiliensis was investigated. Memory CD4+ CD44hi T cells simultaneously with Foxp3+ Treg cells increased in the spleens and lungs of pcDNA3-P10 immunized mice on day 0, 30, 60 and 120 postinfection. Histopathology of the lung tissue showed minimal inflammation in immunized mice compared with the unimmunized group, suggesting a role for regulatory T cells in controlling the immunopathology. The DNA vaccine shows that the repeated immunization generates memory cells and regulatory T cells that replace the initially protective pro-inflammatory T cells conferring a long term protection while preserving the integrity of the infected tissue.

Metabolism of the mesoionic compound (MI-D) by mouse liver microsome, detection of its metabolite in vivo, and acute toxicity in mice.

The mesoionic derivative 4-phenyl-5-[4-nitrocinnamoyl]-1,3,4-thiadiazolyl-2-phenylamine chloride (MI-D) has antitumoral and anti-inflammatory effects. In this study, we present aspects of its metabolism and toxicity in mice. MI-D was metabolized in vitro by liver microsome, generating a main product with a much shorter retention time than MI-D in high-performance liquid chromatography (HPLC) analysis but with a spectrum similar to that of the original molecule. Mass spectrometry with electrospray ionization in positive mode analysis of the purified compound by HPLC indicated that the product of metabolism has four additional hydroxyl groups (m/z = 465) compared with MI-D (m/z = 401). The HPLC analyses of plasma and urine samples from mice treated with MI-D showed the presence of the metabolite product. The kinetic parameters K(m) (19.5 +/- 4.5 microM) and V(max) [1.5 +/- 0.4 units of fluorescence/(100 microg of microsomal protein/mL/s)] were estimated, confirming the metabolism of MI-D and indicating that the reaction follows Michaelis-Menten kinetics. Acute toxicity was established on the basis of an estimation of mean lethal dose (LD-50; 181.2 mg/kg) and histopathological analysis of animals that survived the LD-50 test. Abdominal adhesions, inflammatory foci, and formation of granulomas were observed. Altogether, the results contribute to the advancement of research in support of MI-D as a future chemotherapeutic drug.