Scorpion Venom and the Inflammatory Response

Scorpion venoms consist of a complex of several toxins that exhibit a wide range of biological properties and actions, as well as chemical compositions, toxicity, and pharmacokinetic and pharmacodynamic characteristics. These venoms are associated with high morbility and mortality, especially among children. Victims of envenoming by a scorpion suffer a variety of pathologies, involving mainly both sympathetic and parasympathetic stimulation as well as central manifestations such as irritability, hyperthermia, vomiting, profuse salivation, tremor, and convulsion. The clinical signs and symptoms observed in humans and experimental animals are related with an excessive systemic host inflammatory response to stings and stings, respectively. Although the pathophysiology of envenomation is complex and not yet fully understood, venom and immune responses are known to trigger the release of inflammatory mediators that are largely mediated by cytokines. In models of severe systemic inflammation produced by injection of high doses of venom or venoms products, the increase in production of proinflammatory cytokines significantly contributes to immunological imbalance, multiple organ dysfunction and death. The cytokines initiate a cascade of events that lead to illness behaviors such as fever, anorexia, and also physiological events in the host such as activation of vasodilatation, hypotension, and increased of vessel permeability

Balance Between Pro- and Anti-Inflammatory Cytokines in Mice Treated With Centruroides noxius Scorpion Venom

CSV consists of a very complex of molecules and demonstrates significant cellular activities capable of stimulating immune functions in vivo. The purpose of this study was to analyze the effects of CSV on sex, weight, route of injection and the balance of pro- and anti-inflammatory cytokines in mice. The susceptibility and route of injection were analyzed by lethal (LD(50)) determination. The effects of CSV were also analyzed in blood from immunized mice using detection by means of antibodies and mediators production. Several functional bioassays were employed: TNF activity was assayed by measuring its cytotoxic activity in L929 cells, and other cytokines were assayed by enzyme-linked immunosorbent assay, whereas nitric oxide levels were detected by Griess colorimetric reactions in sera from BALB/c mice. After injecting subcutaneously, the LD(50) presented an increase of the CSV correlation and similar levels of susceptibility were obtained for female and male from BALB/c mice. Significant differences were observed in the time-course of cytokine levels. The balance of pro- and anti-inflammatory cytokines TNF/IL-10 and IL-6/IL-10 ratios were significantly higher in injected mice group when compared with those obtained for non-injected group. The CSV is poor in antigenic composition and it is difficult to get antibodies specific to neutralizing the lethal factor. The effect of immunization with 0.5 LD(50) of CSV on the balance of pro- and anti-inflammatory cytokines was measured. The maximum levels of TNF and IL-6, IFN-γ and NO were observed on days 7 and 21 after immunization, respectively. IL-10 levels peaked between days 21 and 28 after immunization with CSV. With respect to balance of pro- and anti-inflammatory cytokines it was possible to observe that negative correlation between serum levels of IL-6/IL-10 and TNF/IL-10 exists. These ratios may possibly reflect the balance of pro- and anti-inflammatory cytokines in serum, which may by manifested in the inflammatory status during the envenoming processes. In conclusion, an increase in the serum levels of TNF and IL-6 may be a useful marker for scorpion envenomation

The dynamics of cytokine d nitric oxide secretion in mice injected with Tityus serrulatus scorpion venom.

AIMS: The effects of Tityus serrulatus venom (TSV) were analysed with respect to the susceptibility of four isogenic mouse, the symptoms following injection of venom and the inflammatory mediators in an experimental model of severe envenomation induced in mice. METHODS: The susceptibility was analysed by lethal dose (LD50) determination, including the symptoms observed during envenomating and glucose levels. The detection of cytokines in serum from mice were analysed using enzyme-linked immunosorbent assay, and nitric oxide (NO) was analysed using nitrite determination. RESULTS: The estimated LD50 values were in micrograms per 100 microliters, and the susceptibility of mice to TSV varies with: (a) mouse strain and route of injection (A/J < BALB/c < C57Bl/6 = DBA); (b) mouse strain and sex (A/J female and male < BALB/c female and male); and (c) body weight (all groups of A/J < BALB/c groups). Among the mouse strains studied, BALB/c mice presented moderate sensibility to TSV, with changes in specific signs and serum levels of glucose, several cytokines and NO, when injected intraperitoneally (i.p.) with 1 LD50 of venom. Sweating, salivation and tremor were the specific signs that preceded death. The maximum levels of glucose in sera from mice injected i.p. with 1 LD50 of TSV were observed 60-90 min post-injection. Significant differences were observed in the time-course of cytokine levels, and the venom induced marked elevations of interleukin (IL)-1alpha, IL-1beta, IL-6, IL-10 and interferon gamma (IFN-gamma). The maximum levels of IL-1alpha and IL-1beta were observed 2 h post-injection. The more pronounced levels of IL-6 were observed 4 h post-injection. There was an early increase in IFN-gamma followed by an even higher level after 4 h. IL-10 levels peaked between 6 and 8 h, and this cytokine probably modulates the secretion of IFN-gamma. Tumor necrosis factor release was not detected in BALB/c mice injected with TSV. NO levels attained maximal release after 2 h, following venom injection, while a second peak for NO was at 6 h. CONCLUSIONS: These findings indicate that the susceptibility to the systemic effects of the venom varies among mice of different haplotypes, and that the cytokines such as IL-1, IL-6, IFN-gamma and NO are strongly involved in the pathogenesis caused by this venom and are correlated with the severity of envenomation

Role of cytokines and nitric oxide in the induction of tuberculostatic macrophage functions.

The aim of this study was to determine phenotypic differences when BCG invades macrophages. Bacilli prepared from the same BCG primary seed, but produced in different culture media, were analysed with respect to the ability to stimulate macrophages and the susceptibility to treatment with cytokines and nitric oxide (NO). Tumour necrosis factor (TNF) activity was assayed by measuring its cytotoxic activity on L-929 cells, interleukin-6 (IL-6) and interferon-gamma (IFN-gamma) were assayed by enzyme-linked immunosorbent assay (ELISA), whereas NO levels were detected by Griess colorimetric reactions in the culture supernatant of macrophages incubated with IFN-gamma, TNF or NO and subsequently exposed to either BCG-I or BCG-S. We found that BCG-I and BCG-S bacilli showed different ability to simulate peritoneal macrophages. Similar levels of IL-6 were detected in stimulated macrophages with lysate from two BCG samples. The highest levels of TNF and IFN-gamma were observed in macrophages treated with BCG-S and BCG-I, respectively. The highest levels of NO were observed in cultures stimulated for 48 h with BCG-S. We also found a different susceptibility of the bacilli to exogenous treatment with IFN-gamma and TNF which were capable of killing 60 and 70% of both bacilli, whereas NO was capable of killing about 98 and 47% of BCG-I and BCG-S, respectively. The amount of bacilli proportionally decreased with IFN-gamma and TNF, suggesting a cytokine-related cytotoxic effect. Moreover, NO also decreased the viable number of bacilli. Interestingly, NO levels of peritoneal macrophages were significantly increased after cytokine treatment. This indicates that the treatment of macrophages with cytokines markedly reduced bacilli number and presented effects on NO production. The results obtained here emphasize the importance of adequate stimulation for guaranteeing efficient killing of bacilli. In this particular case, the IFN-gamma and TNF were involved in the activation of macrophage bactericidal activity

Effect of thymocytes on reproductive parameters of nude female mice

Heterozygous and homozygous nude female mice with the genetic background BALB/c were housed in a clean conventional colony at the Laboratory Animal Facilities of Instituto Butantan. Nude animals are known to have severe deficiencies in reproductive function. Nude female is usually sterile where only 8.8% of them present spontaneous fertility. In order to study the involvement of the thymus for these reproductive defects, athymic nude female at different stages of development were treated with thymocytes suspensions and mated with heterozygous males. The results obtained for nude females at 10 and 30 days of age were respectively: a) number of pregnant = 48% and 12%; b) number offspring per female = 5.0 and 4.0; c) natality rates were 2.40 and 0.48; and d) under conditions used in this study the life span for nude animals was prolonged until 7 months. The use of this treatment would increase the animal numbers, which are important research models for biomedical investigations. Considering that nude mice are difficult to raise, an obtainment of these animals in higher number by this method appear to be an essential event, and might offer to grow better for the animals, which are used for breeding.A colônia de camundongos da linhagem BALB/c, heterozigotos e homozigotos, foi mantida em condições convencionais no Biotério Experimental do Instituto Butantan. Os camundongos "nude" apresentam severa deficiência no sistema reprodutor. As fêmeas de camundongos "nude" são geralmente estéreis; apenas 8,8% apresentam fertilidade espontânea. Para estudar o envolvimento do timo sobre os defeitos na reprodução em diferentes estágios de desenvolvimento, as fêmeas de camundongos "nude" foram tratadas com suspensões de timócitos, e, em seguida, acasaladas com machos heterozigotos. Os resultados obtidos para fêmeas "nude" com 10 e 30 dias de idade foram respectivamente: a) número de fêmeas prenhes = 48% e 12%; b) número de filhotes por fêmea = 5,0 e 4,0; c) as taxas de natalidade foram 2,40 e 0,48; e d) nas condições utilizadas neste trabalho, a vida média dos camundongos "nude" foi prolongada até 7 meses. Este método descreve o aumento do número de animais "nude", importantes em estudos biológicos. Considerando a dificuldade de obtenção de camundongos "nude" em grande número, o uso deste método poderia contribuir para o melhoramento das condições de vida dos animais utilizados nos acasalamentos

EVALUATION OF ANTI-ARTHRITIC POTENTIAL OF PARTITIONED EXTRACTS OF BOUGAINVILLEA X BUTTIANA (VAR. ROSE) HOLTTUM AND STANDL

Objective: Bougainvillea is a natural source with potential for clinical use, and this plant is routinely employed in traditional Medicine in Mexico. This study planned to evaluate the effect of ethanolic extract partitioned of Bougainvillea x buttiana on acute and chronic inflammation.Methods: The extract from Bougainvillea x buttiana partitioned originated two phases the aqueous (BxbREaq) and organic (BxbREop) phases were employed in anti-inflammatory activity. Acute inflammation was evaluated using the carrageenan model, whereas the chronic inflammation with anti-arthritic potential was explored with complete Freund´s adjuvant (CFA). Arthritis was caused by intradermal inoculation of CFA, and the extract was administered orally at different doses for 21 d. Paw oedema was determined at 7, 14 and 21 d, and serum from the mice were obtained to detect cytokine levels by ELISA and for biological assays.Results: Phytochemistry studies revealed that these extracts contain alkaloids, carbohydrates, fatty acids, and tannins. The results demonstrated that these extracts significantly inhibited mouse paw oedema for acute and chronic inflammation in a dose-dependent manner. Additionally, BxbREop extracts markedly inhibited the production of TNF-α, IL-1β, and IL-6 and remarkably increased IL-10 in serum from mice with control or arthritic groups.Conclusion: The combined results suggest that BxbREop extract shows a potent effect in mice against CFA-induced arthritis for its ability to inhibit paw oedema and arthritic symptoms

Local inflammation, lethality and cytokine release in mice injected with Bothrops atrox venom.

We have provided evidence that: (a) lethality of mice to crude Bothrops venom varies according the isogenic strain (A/J > C57Bl/6 > A/Sn > BALB/c > C3H/HePas > DBA/2 > C3H/He); (b)BALB/c mice (LD50=100.0 microg) were injected i.p. with 50 microg of venom produced IL-6, IL-10, INF-gamma, TNF-alpha and NO in the serum. In vitro the cells from the mice injected and challenged with the venom only released IL-10 while peritoneal macrophages released IL-10, INF-gamma and less amounts of IL-6; (c) establishment of local inflammation and necrosis induced by the venom, coincides with the peaks of TNF-alpha, IFN-gamma and NO and the damage was neutralized when the venom was incubated with a monoclonal antibody against a 60 kDa haemorrhagic factor. These results suggest that susceptibility to Bothrops atrox venom is genetically dependent but MHC independent; that IL-6, IL-10, TNF-alpha, IFN-gamma and NO can be involved in the mediation of tissue damage; and that the major venom component inducers of the lesions are haemorrhagins

Inflammatory Mediators Release in Urine from Mice Injected with Crotalus durissus terrificus Venom

In this study, we investigated in groups of female BALB/c mice injected with Crotalus durissus terrificus venom (Cdt) the renal function based on creatinine clearance, percentage of fractional excretion cytokines and histological examination of renal tissue. Cdt caused renal alterations that induced proteinuria during the initial hours post-venom and reduced creatinine clearance 15 min. up to 2 hours post-venom administration. In urine from mice injected with Cdt induced a decrease in IL-4 levels. More pronounced increments of IL-5, IL-6 and IFN-γ were observed after 15 and 30 min, respectively. The highest levels of TNF and IL-10 were observed at 1 and 4 hs, respectively. The ratios of pro- and anti-inflammatory cytokines in animals injected with Cdt, which may be manifested in the inflammatory status during the envenoming. In groups of animals treated with Cdt were observed a decreasing in creatinine clearance and its effect on glomerular filtration rate was accompanied by decreased fractional excretion of cytokines and morphologic disturbances. This loss of change selectively in envenomation could thus explain why the relatively excretion of cytokines is reduced while of total proteins increases. In conclusion the fractional excretion of cytokines is significantly reduced in mice injected with Cdt, despite proteinuria

Pro-and Anti-Inflammatory Cytokines Release in Mice Injected with Crotalus durissus terrificus Venom

The effects of Crotalus durissus terrificus venom (Cdt) were analyzed with respect to the susceptibility and the inflammatory mediators in an experimental model of severe envenomation. BALB/c female mice injected intraperitoneally presented sensibility to Cdt, with changes in specific signs, blood biochemical and inflammatory mediators. The venom induced reduction of glucose and urea levels and an increment of creatinine levels in serum from mice. Significant differences were observed in the time-course of mediator levels in sera from mice injected with Cdt. The maximum levels of IL-6, NO, IL-5, TNF, IL-4 and IL-10 were observed 15 min, 30 min, 1, 2 and 4 hours post-injection, respectively. No difference was observed for levels of IFN-γ. Taken together, these data indicate that the envenomation by Cdt is regulated both pro-and anti-inflammatory cytokine responses at time-dependent manner. In serum from mice injected with Cdt at the two first hours revealed of pro-inflammatory dominance. However, with an increment of time an increase of anti-inflammatory cytokines was observed and the balance toward to anti-inflammatory dominance. In conclusion, the observation that Cdt affects the production of pro-and anti-inflammatory cytokines provides further evidence for the role played by Cdt in modulating pro/anti-inflammatory cytokine balance