Preactivation of neutrophils and systemic oxidative stress in dogs with hyperleptinemia.

High occurrence of obesity currently constitutes the main nutritional disease of the canine species. There is evidence that leptin increases during obesity in dogs. Hyperleptinemia is associated with increased neutrophil oxidative metabolism in obese humans and contributes to oxidative stress. However, in obese dogs, the probable relationship between this condition and the activation of the oxidative metabolism of neutrophils has yet to be established. Thus, we investigated the hypothesis that neutrophil activation and systemic oxidative stress occur in dogs with hyperleptinemia. A control group of 24 healthy dogs with a body condition score (BCS) of 4-5, an overweight group of 25 dogs with a BCS of 6-7, and 27 obese dogs with a BCS of 8-9, were composed. Two subgroups were formed composed of dogs with and without hyperleptinemia, grouped according to the 95% confidence interval obtained for plasma leptin values of the control group. Changes in obesity markers (body condition score, adiponectin and plasma leptin) and plasma oxidative stress (lipid peroxidation, total antioxidant and oxidant capacities and oxidative stress index) were measured in all the dogs selected. Neutrophil oxidative metabolism was evaluated in flow cytometry by superoxide production with the probe hydroethidine and by hydrogen peroxide production with the probe 2',7'-dichlorofluorescein diacetate, with or without phorbol myristate acetate (PMA) stimulation. Apoptosis and neutrophil viability were quantified in a capillary flow cytometer using Annexin VPE, with or without camptothecin apoptosis inducing effect. Obese dogs presented higher systemic oxidative stress, hyperleptinemia and preactivated neutrophils with accelerated apoptosis. Dogs with hyperleptinemia and obese dogs presented higher neutrophil superoxide production under PMA stimulation and the presence of systemic oxidative stress compared with control. To our knowledge, this is probably the first evidence that preactivation of the oxidative metabolism of circulating neutrophils occurs in dogs with hyperleptinemia, a condition that can induce systemic oxidative stress in the canine species.

Dog skin parasite load, TLR-2, IL-10 and TNF-α expression and infectiousness.

Visceral leishmaniosis is a zoonotic disease that is transmitted by Lutzomyia longipalpis sandflies. Dogs are the main peri-urban reservoir of the disease, and progression of canine leishmaniosis is dependent on the type of immune response elaborated against the parasite. Type 1 immunity is characterized by effective cellular response, with production of pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF-α). In contrast, Type 2 immunity is predominantly humoral, associated with progression of the disease and mediated by anti-inflammatory cytokines such as interleukin 10 (IL-10). Although seemly important in the dynamics of leishmaniosis, other gene products such as toll-like receptor 2 (TRL-2) and inducible nitric oxide synthase (iNOS) exert unclear roles in the determination of the type of immune response. Given that the dog skin serves as a micro-environment for the multiplication of Leishmania spp., we investigated the parasite load and the expression of TLR-2, iNOS, IL-10 and TNF-α in the skin of 29 infected and 8 control dogs. We found that increased parasite load leads to upregulation of TLR-2, IL-10 and TNF-α, indicating that abundance of these transcripts is associated with infection. We also performed a xenodiagnosis to demonstrate that increased parasitism is a risk factor for infectiousness to sandflies.

Induction of haem oxygenase-1 increases infection of dog macrophages by L. infantum.

We aimed to induce and inhibit HO-1, ascertaining its effect on infection rate, parasite load and the levels of superoxide, reactive oxygen species (ROS), nitric oxide (NO), TNF-alpha and IL-10 in cultured macrophages from healthy dogs infected by Leishmania infantum. Macrophages obtained from 15 healthy dogs were cultured alone or infected with L. infantum, with or without association of HO-1 inducer and inhibitor. The infection rate and the parasite load were determined by the number of infected macrophages and number of promastigotes per macrophage, respectively. HO-1 levels and gene expression, as well as IL-10 and TNF-alpha levels were also measured in these cultures. Superoxide, ROS and NO levels in macrophages were measured through flow cytometry. Induction of HO-1 increased the infection rate and parasite load, while its inhibition decreased the infection rate and IL-10 production. There was a positive correlation between HO-1 and infection rate or parasite load. Increased infection rate was associated with decreased superoxide, ROS and NO levels. Induction of HO-1 metabolism in dogs infected by L. infantum is possibly one of the mechanisms responsible for increasing the infection of macrophages, mainly through reduction in the oxidative and nitrosative metabolisms of these cells.

M1 polarization and the effect of PGE2 on TNF-α production by lymph node cells from dogs with visceral leishmaniasis.

Canine visceral leishmaniasis (CVL) is caused by the intracellular parasite Leishmania infantum. Increased levels of arginase, nitric oxide (NO2 ) and prostaglandin E2 (PGE2 ) can play a regulatory role regarding the immune response in CVL cases. This study aimed to evaluate the arginase activity in adherent macrophages cultured from the lymph nodes of healthy and naturally infected dogs and to examine the NO2 and PGE2 levels in the supernatant of these cultures. In addition, the regulatory effect of PGE2 on the production of tumour necrosis factor (TNF-α) and interleukin-10 (IL-10) in supernatants from the total lymph node was observed in leucocyte cultures. The arginase activity was lower in the adherent macrophages cultured from the lymph nodes of naturally infected dogs and there were higher concentrations of NO2 and PGE2 in the supernatants of these cultures. Higher TNF-α and IL-10 concentrations were observed in supernatants from total lymph node leucocytes cultures, from infected dogs, and the presence of indomethacin only decreased TNF-α in the supernatant of these cultures. We conclude that the low arginase activity in macrophages suggested that M1 polarization and PGE2 were participating in the immune response and were increasing TNF-α in CVL.

Blood-brain barrier disruption during spontaneous canine visceral leishmaniasis.

Visceral leishmaniasis is a complex disease caused by Leishmania infantum, and in dogs, besides the classical symptoms, there are descriptions of inflammatory alterations in the brain. Brain inflammation is a strictly controlled process, and as the brain counts on the efficiency of the blood-brain barrier (BBB), we aimed to assess BBB integrity in dogs with spontaneous visceral leishmaniasis. Therefore, we evaluated markers in the cerebrospinal fluid (CSF) and in brain tissue related to BBB disruption and brain inflammation. Elevated albumin quota revealed BBB breakdown, corroborated by increased concentrations of anti-Leishmania antibodies in the CSF. In the brain, albumin and IgG staining formed halos around blood vessels, a classical indicator of BBB leakage. Soluble IgG was also detected in the choroid plexus and ependyma, and in these structures, IgG stained random resident cells. IgG(+) cells and Fcγ-RI(+) cells were identified in the choroid plexus, ependyma and perivascular in the brain parenchyma. The data support the occurrence of BBB disruption in dogs with spontaneous visceral leishmaniasis, and IgG as a key molecule that is capable of initiating and/or maintaining the inflammatory stimuli in the nervous milieu and the CSF as an important disseminator of inflammatory stimuli within the CNS.

Recombinant K28 antigen in ELISA in the diagnosis of canine visceral leishmaniosis.

Crude total antigen (CTA) from Leishmania infantum and recombinant antigen K39 (rK39) and recombinant antigen K28 (rK28) were compared using an ELISA for the diagnosis of canine visceral leishmaniosis (CVL). Forty-two blood samples from healthy dogs from a nonendemic area and 80 blood samples from an endemic area for dogs with visceral leishmaniosis (VL), confirmed with positive parasitological tests for Leishmania spp., were used in an ELISA. The parasitological diagnosis was chosen as a gold standard. The ELISA with rK28 antigen showed sensitivity of 100% and specificity of 100%, high agreement with CTA and rK39, indicating that the rK28 antigen is useful for ELISA serological diagnosis of CVL.

Effects of P-MAPA immunomodulator on Toll-like receptor 2, ROS, nitric oxide, MAPKp38 and IKK in PBMC and macrophages from dogs with visceral leishmaniasis.

Leishmania (L.) chagasi is the etiologic agent of visceral leishmaniasis (VL) that can be transmitted to humans and dogs. VL in Brazil represents a serious public health problem; therefore, it is important to study new alternatives to treat infected dogs. In dogs, the therapeutic arsenal against canine VL is limited. The immunomodulator protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride (P-MAPA) improves immunocompetence when the immune system is impaired, but its dependence on Toll-like receptors (TLRs) and the mechanisms involved in immune response remain unclear. The in vitro action of P-MAPA on the expression of TLR2 and TLR4, reactive oxygen species (ROS), nitric oxide (NO) and p38 mitogen-activated protein kinase (p38 MAPK) and IKK phosphorylation was studied in mononuclear cells from peripheral blood and macrophages from healthy and Leishmania-infected dogs. The PBMC or macrophages were isolated and cultured with different concentrations of P-MAPA (20,100 and 200 µg/ml) in a humid environment at 37°C with 5% CO(2). Observation revealed that Leishmania-infected dogs showed a decrease in TLR2 in macrophages compared with healthy dogs and in induction with P-MAPA. ROS were increased in PBMCs from Leishmania spp.-infected dogs compared with healthy dogs and P-MAPA improved ROS production. NO production was increased in culture supernatant from macrophages stimulated by P-MAPA in both healthy and Leishmania spp. infected dogs. Treatment of macrophages from healthy dogs with immunomodulatory P-MAPA induced p38 MAPK and IKK phosphorylation, suggesting signal transduction by this pathway. These findings suggest that P-MAPA has potential as a therapeutic drug in the treatment of canine visceral leishmaniasis.

Leishmaniasis causes oxidative stress and alteration of oxidative metabolism and viability of neutrophils in dogs.

The aim of the present study was to test the hypothesis that oxidative stress and alteration of oxidative metabolism and apoptosis of neutrophils in dogs vary with the stage of leishmaniasis and to determine the contribution of uremia to such alterations. Dogs with leishmaniasis were classified into two stages: moderate (Leish II, n=20) or very severe (i.e. with concurrent uremia; Leish IV, n=20) according to the LeishVet Consensus. The two leishmaniasis groups were compared with uremic dogs without leishmaniasis (Uremic, n=10) and to healthy dogs (Control, n=30). To determine oxidative stress, total antioxidant/oxidant capacity, lipid peroxidation, total glutathione and the plasma antioxidants albumin, uric acid and bilirubin were quantified. Superoxide production was determined using the hydroethidine probe and viability and apoptosis were measured using annexin V-PE by capillary flow cytometry. Oxidative stress was present in both uremia and leishmaniasis with reduced total antioxidant capacity and was associated with increased induced production of superoxide and apoptosis. The greatest amount of oxidants was observed in animals with moderate disease only. Neutrophils from uremic dogs with and without leishmaniasis had decreased viability and an increased apoptosis rate in addition to increased lipid peroxidation. In conclusion, oxidative stress occurs in both stages of leishmaniasis with differences in intensity and levels of plasma markers; however, uremia does contribute to the decreased spontaneous viability of neutrophils in dogs in the final stage of the disease.

Improvement in clinical signs and cellular immunity of dogs with visceral leishmaniasis using the immunomodulator P-MAPA.

This study investigated the immunotherapeutic potential of the protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride immuno-modulator (P-MAPA) on canine visceral leishmaniasis. Twenty mongrel dogs presenting clinical symptoms compatible with leishmaniasis and diagnosis confirmed by the detection of anti-leishmania antibodies were studied. Ten dogs received 15 doses of the immunomodulator (2.0 mg/kg) intramuscularly, and 10 received saline as a placebo. Skin and peripheral blood samples were collected following administration of the immunomodulator. The groups were followed to observe for clinical signals of remission; parasite load in the skin biopsies using real-time PCR, the cytokines IL-2, IL-10 and IFN-γ in the supernatant of peripheral blood mononuclear cells stimulated in vitro with either total promastigote antigen or phytohemagglutinin measured by capture ELISA, and changes in CD4⁺ and CD8⁺ T cell subpopulations evaluated by flow cytometry. Comparison between the groups showed that treatment with the immunomodulator promoted improvement in clinical signs and a significant reduction in parasite load in the skin. In peripheral blood mononuclear cell cultures, supernatants showed a decrease in IL-10 levels and an increase in IL-2 and IFN-γ. An increase in CD8⁺ T cells was observed in peripheral blood. In addition, the in vitro leishmanicidal action of P-MAPA was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and no leishmanicidal activity was detected. These findings suggest that P-MAPA has potential as an immunotherapeutic drug in canine visceral leishmaniasis, since it assists in reestablishing partial immunocompetence of infected dogs.

Estresse oxidativo e aumento da apoptose em neutrófilos de cães com azotemia pré-renal / Oxidative stress and increase in apoptosis index of neutrophils from dogs with prerenal azotemia

O presente trabalho tem como objetivo testar a hipótese de que, à semelhança do que ocorre na uremia, cães com azotemia pré-renal sofrem estresse oxidativo, o qual está relacionado com alterações do metabolismo oxidativo e apoptose dos neutrófilos. Para tal, foi determinada a peroxidação lipídica pela quantificação do malondialdeído (MDA) e o status antioxidante total do plasma de 15 cães normais e 10 com azotemia pré-renal, correlacionando-os com a produção de superóxido e o índice apoptótico dos neutrófilos. As determinações do MDA e do status antioxidante total foram estabelecidas empregando-se um conjunto de reagentes comerciais. Por meio de citometria de fluxo capilar, a produção de superóxido e a apoptose de neutrófilos isolados de sangue periférico foram determinadas utilizando-se a sonda hidroetidina e o sistema anexina V-PE, respectivamente. Cães azotêmicos (26,29±5,32g/L) apresentaram menor concentração (p=0,0264) do antioxidante albumina em relação ao grupo-controle (30,36±3,29g/L) e também uma menor (p=0,0027) capacidade antioxidante total (2,36±0,32 versus 2,73±0,24mmol/L), enquanto não houve alteração da peroxidação lipídica plasmática e da produção de superóxido neutrofílica. Concluiu-se que, à semelhança do que ocorre na uremia, condições azotêmicas pré-renais no cão causam estresse oxidativo e aceleração da apoptose dos neutrófilos.

This study aims to test the hypothesis that, similarly to what occurs in uremia, dogs with prerenal azotemia suffer oxidative stress associated with changes in oxidative metabolism and apoptosis in neutrophils. For this purpose, fifteen normal dogs and ten with prerenal azotemia had lipid peroxidation determined by quantifying the malondialdehyde (MDA) and had plasma total antioxidant status evaluated, correlating them with the superoxide production and apoptotic index of neutrophils. MDA and plasma total antioxidant status were determined using commercial reagents. Using capillary flow cytometry, superoxide production and apoptosis were determined from isolated neutrophils of peripheral blood using the hydrithidine and Annexin V-PE probe system, respectively. Azotemic dogs (26.29±5.32g/L) had a lower concentration (p=0.0264) of the plasma antioxidant albumin than the control group (30.36±3.29g/L) and also had lower (p=0.0027) total antioxidant status (2.36±0.32 versus 2.73±0.24mmol/L), while no alterations were observed in plasma lipid peroxidation and superoxide production. It was concluded that, similarly to what occurs in uremia, prerenal azotemia causes oxidative stress and acceleration of neutrophil apoptosis in dogs.

Detection of cross infections by Leishmania spp. and Trypanosoma spp. in dogs using indirect immunoenzyme assay, indirect fluorescent antibody test and polymerase chain reaction.

The aim of this study was to detect cross infections by Leishmania spp. and Trypanosoma spp. using enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody test (IFAT) and polymerase chain reaction (PCR). Thus, 408 blood samples were collected from dogs domiciled in Araçatuba Municipality, São Paulo State, Brazil; the dogs were of both sexes, of several breeds and aged 6 months. For Leishmania spp., 14.95% (61 out of 408) of dogs were reactive using IFAT. Positivity was 20.10% (82 out of 408) using ELISA and 29.66% (121 out of 408) using PCR, with significant differences for the sex and age of these animals (p < 0.05). For Trypanosoma spp., antibody occurrence using ELISA was 10.54% (43 out of 408), while PCR indicated 2.45% (10 out of 408) positive dogs. Using IFAT, 10.29% (42 out of 408) of animals were considered positive and only sex showed a significant difference (p < 0.05). In this study, 10.54% (43 out of 408) of animals were seropositive according to ELISA for Trypanosoma spp., of which 79.07% (34 out of 43) showed positive results in the molecular diagnosis for Leishmania spp., while of the 10.29% (42 out of 408) positive dogs according to IFAT, 95.24 % (40 out of 42) had confirmed infection by this parasite. The obtained results demonstrate evidence of cross infections by both protozoa in the animals analysed in this study.

Gyroxin and its biological activity: effects on CNS basement membranes and endothelium and protease-activated receptors.

Gyroxin is a glycoprotein isolated from rattlesnake venom, with known thrombin-like serine protease properties and behavioral action in the CNS. The mechanism of the latter has eluded experimenters for three decades. In this paper about the in vitro chick retina we demonstrate an excitotoxic CNS action of Gyroxin by observing retinal Intrinsic Optical Signals (IOS). These show sudden dynamic changes in the intact tissue due to gyroxin action. The very fast kinetics of this response precludes deep tissue penetration by the protein, a mechanism of tissue response described here for the first time. At nanomolar concentrations, Gyroxin alters profoundly the optical profiles of retinal spreading depression waves (RSDs), suggesting modulation of ionic transport and metabolism. This effect is reversible in contrast with the acute cell lysis induced with gyroxin pulses at higher concentration. Because there may be more than one target of Gyroxine at the retinal inner limiting membrane, additional biochemical assays were performed to study a possible Na/K-ATPase blockade and PAR receptor activation. We conclude that the Gyroxin interaction with basement membranes of CNS and endothelium triggers conformational phase transitions at basement membranes, with multiple functional consequences.

Disseminated transmissible venereal tumour associated with Leishmaniasis in a dog.

This report addresses an atypical transmissible venereal tumour in an 8-year-old bitch that was pluriparous and seropositive for leishmaniasis. There were ascites and a serosanguineous discharge from the vulva, but no lesions on the external genital mucosa. An aspirate of the peritoneal fluid showed mononuclear round cells characteristic of transmissible venereal tumour (TVT). Exploratory laparotomy revealed light red, granulomatous structures in the peritoneum, omentum, spleen, liver and uterine horns. Cytological and histopathological tests confirmed the diagnosis of intra-abdominal TVT. Dissemination of the TVT to several organs inside the abdominal cavity probably resulted from immunosuppression caused by leishmaniasis, which favoured the presence and aggressiveness of TVT.

Evaluation of TNF-α, IL-4, and IL-10 and parasite density in spleen and liver of L. (L.) chagasi naturally infected dogs.

Dogs are the main domestic reservoirs of L. (L.) chagasi. Once in the vertebrate host, the parasite can cause visceral leishmaniasis, which can also be transmitted to humans. Cytokines are key elements of the host immune response against Leishmania spp. To investigate whether tumor necrosis factor (TNF)-α, interleukin (IL)-4 and IL-10 are associated with pattern infection in dogs, these cytokines were quantified in the spleen and liver of dogs naturally infected with L. (L.) chagasi, with or without clinical manifestations, and their levels were correlated with the parasite load verified in these organs. A total of 40 adult dogs naturally infected with L. (L.) chagasi were assessed, together with 12 uninfected control dogs. Samples from spleen and liver were used to determine the cytokine levels by capture ELISA and for quantifying parasite load by real-time PCR. Statistical analysis was performed using the minimum Chi square method and group means were compared using the Tukey test. TNF-α, IL-4 and IL-10 levels in infected dogs were higher than in control groups; the liver was the main cytokine-producing organ during infection. The level of splenic TNF-α showed correlation with parasite load and may represent an important marker for infection process evolution, with the participation of IL-10. These results may contribute to a clearer understanding of the immune response in dogs infected with L. (L.) chagasi, which may lead to the development of prophylactic or preventive measures for these animals.

Effects of lidocaine infusion during experimental endotoxemia in horses.

BACKGROUND: The clinical efficacy of IV infusion of lidocaine for treatment of equine endotoxemia has not been studied. HYPOTHESIS: Lidocaine infusion after exposure to lipopolysaccharide (LPS) will inhibit the inflammatory response and have inhibitory effects on the hemodynamic and cytokine responses to endotoxemia. ANIMALS: Twelve horses. METHODS: Two equal groups (n=6): saline (GI) and lidocaine (GII). In all animals, endotoxin (500 ng/kg body weight [BW]) was injected intraperitoneally over 5 minutes. Twenty minutes later, animals received a bolus of GI or GII (1.3 mg/kg BW) over 5 minutes, followed by a 6-hour continuous rate infusion of GI or GII (0.05 mg/kg BW/min). Treatment efficacy was judged from change in arterial blood pressure, peripheral blood and peritoneal fluid (PF) variables (total and differential cell counts, enzyme activities, and cytokine concentrations), and clinical scores (CS) for behavioral evidence of abdominal pain or discomfort during the study. RESULTS: Compared with the control group, horses treated with lidocaine had significantly lower CS and serum and PF tumor necrosis factor-alpha (TNF-alpha) activity. At several time points in both groups, total and differential cell counts, glucose, total protein and fibrinogen concentrations, and alkaline phosphatase, creatine kinase, and TNF-alpha activities were significantly different from baseline values both in peripheral blood and in PF. CONCLUSIONS AND CLINICAL IMPORTANCE: Lidocaine significantly decreased severity of CS and inhibited TNF-alpha activity in PF.

Qualidade espermática de sêmen de cães naturalmente infectados por leishmania sp / Semen quality of dogs naturally infected by leishmania sp

Avaliaram-se alterações espermáticas associadas à infecção por leishmaniose no sêmen de cães naturalmente infectados, utilizando-se, durante oito semanas consecutivas, ejaculados de seis cães soronegativos e seis cães soropositivos. As amostras foram colhidas uma vez por semana e avaliadas quanto ao volume, concentração, motilidade, vigor, morfologia espermática, integridade da cromatina, avaliação simultânea da integridade da membrana plasmática, acrossoma e potencial mitocondrial. Concomitantemente foram dosadas a proteína total do plasma seminal e sanguíneo. A leishmaniose visceral causou aumento dos defeitos maiores e menores nos espermatozoides dos animais acometidos pelo estágio moderado a severo da doença. Em estágios mais avançados da enfermidade, a integridade das membranas acrossomal e plasmática foi afetada negativamente. Não foi possível estabelecer um critério quanto à avaliação do potencial mitocondrial. A incidência de alterações morfológicas nos animais acometidos não promoveu aumento de injurias à cromatina. Todos os animais com leishmaniose apresentaram hiperproteinemia do sêmen.

The spermatic changes associated with the natural infection in dogs by Leishmania sp was evaluated during eight consecutive weeks, using ejaculates of six seronegative and six seropositive dogs. The samples were collected once a week and evaluated for volume, concentration, motility, vigor, sperm morphology, chromatin integrity, simultaneous evaluation of the plasmatic membrane integrity, acrosome, and mitochondrial potential. The total proteins of the seminal plasma and blood were measured. The visceral leishmaniasis caused increase of major and minor defects in spermatozoa of animals attacked by moderate to severe stages of the disease. In more advanced stages of the illness, the acrosomal and plasmatic membranes integrity was adversely affected. It was not possible to establish a pattern refering the evaluation of the mitochondrial potential. The incidence of morphological changes in the seropositive animals did not promote an increase of injuries to the chromatin. All animals with leishmaniasis presented hyperproteinemia of the semen.

Evaluation of renal and hepatic functions in dogs naturally infected by visceral leishmaniasis submitted to treatment with meglumine antimoniate.

The present study aimed to evaluate the renal and hepatic responses in eight dogs with visceral leishmaniasis submitted to treatment with meglumine antimoniate and to verify the occurrence of possible side effects. Urinalysis, hepatic and renal function tests were carried out in all animals at up to seven moments. After the end of a six-month observation period, all dogs were euthanized. Before the beginning of the experiment urinary and biochemical alterations were observed in four dogs due to the changes caused by the parasite itself. These alterations included the presence of renal cells, cylindruria, proteinuria, azotemia, hyperproteinemia and hypoalbuminemia. One dog died on the third day after treatment because an aggravation of the clinical picture, probably due to the medication. During the course of the study, an increase in hepatic enzymes was verified in two animals. Sixty days after the beginning of the treatment four dogs showed remission of clinical signs. The other three were asymptomatic with persistent biochemical alterations. From these, two presented recurrence of clinical signs about 150 days after the beginning of the treatment while in the other, hyperproteinemia persisted. Meglumine antimoniate was not efficient to treat dogs with severe renal dysfunction and the side effects observed were pain at the site of injection and the probable transient hepatotoxicity, evidenced by biochemical examinations, but without the presence of clinical signs.

Cytokine and antibody production during murine leptospirosis

The aim of the present study was to investigate the kinetics of humoral and cellular responses during leptospirosis. We observed that the presence of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) was associated with antibody production and bacterial recovery, and the compromising of both TNF-alpha and IL-6 in the immunopathogenesis of leptospirosis during an experimental infection of BALB/c mice inoculated with Leptospira interrogans serovar Canicola was verified. Results showed higher levels of TNF-alpha and IL-6 in the initial phase of infection, in which the greatest bacterial clearance was observed. However, when the bacterial recovery was compared with the kinetics of the production of antibodies, the results revealed a kinetics proportionally inverted to antibody production. This fact may be related to some inhibitory factor which could be responsible for the selective suppression of the cellular immune response. We concluded that during leptospirosis there was a greater mobilization of the cellular immune response activity, mainly in the initial phase of the infectious process, for posterior involvement of the humoral response, and that both TNF-alpha and IL-6 could be associated with the immunopathogenesis of the disease.

Role of cytokines, NO, and H2O2 on the immunopathology of leptospirosis in genetically selected mice

Immune response to leptospirosis is mainly humorally mediated, and involves opsonization of leptospires for phagocytosis by macrophages and neutrophils. However, some aspects are still unknown. For a more detailed analysis of the cellular immune response to leptospirosis infection, trials were carried out in order to determine the hydrogen peroxide and nitric oxide (H2O2 and NO)production stimulated or not by Interferon-gamma. The participation of some specific cytokines, such as Tumor Necrosis Factor-alfa (TNF-alfa); Interferon-gamma (IFN-y); Interleukin-6 (IL-6); and Interleukin-4 (IL-4), in the immunopathology of this infection was also investigated. For this purpose, we analyzed the supernatant from peritonealmacrophage cell culture and the splenic cells of mice genetically selected as High (H) and Low (L) antibody producers, and inbred Balb/c mice infected with Leptospira interrogans serovar icterohaemorrhagiae. The IL-6 production varied from release speaks to inhibition in H, L, and Balb/c mice. The three strains presented constant and elevated production of TNF-alfa until day 14, suggesting its effective participation in the initial phase of the infection. Meanwhile, all the three strains presented a constant and irregular IFN-y production, with release peaks between the 7th and 14th days in L mice. The H and Balb/c mice strains presented a higher tendency to Th2 response pattern, whereas L mice tendend towards Th1 response

Single dose of a vaccine based on DNA encoding mycobacterial hsp65 protein plus TDM-loaded PLGA microspheres protects mice against a virulent strain of Mycobacterium tuberculosis.

The high incidence of tuberculosis around the world and the inability of BCG to protect certain populations clearly indicate that an improved vaccine against tuberculosis is needed. A single antigen, the mycobacterial heat shock protein hsp65, is sufficient to protect BALB/c mice against challenge infection when administered as DNA vaccine in a three-dose-based schedule. In order to simplify the vaccination schedule, we coencapsulated hsp65-DNA and trehalose dimicolate (TDM) into biodegradable poly(DL-lactide-co-glycolide) (PLGA) microspheres. BALB/c mice immunized with a single dose of DNA-hsp65/TDM-loaded microspheres produced high levels of IgG2a subtype antibody and high amounts of IFN-gamma in the supernatant of spleen cell cultures. DNA-hsp65/TDM-loaded microspheres were also able to induce high IFN-gamma production in bulk lung cells from challenged mice and confer protection as effective as that attained after three doses of naked DNA administration. This new formulation also allowed a ten-fold reduction in the DNA dose when compared to naked DNA. Thus, this combination of DNA vaccine and adjuvants with immunomodulatory and carrier properties holds the potential for an improved vaccine against tuberculosis.