Ts6 and Ts2 from Tityus serrulatus venom induce inflammation by mechanisms dependent on lipid mediators and cytokine production

AbstractInflammatory mediators are thought to be involved in the systemic and local immune response induced by the Tityus serrulatus scorpion envenomation. New functional aspects of lipid mediators have recently been described. Here, we examine the unreported role of lipid mediators in cell recruitment to the peritoneal cavity after an injection with Ts2 or Ts6 toxins isolated from the T. serrulatus scorpion venom. In this report, we demonstrate that following a single intraperitoneal (i.p.) injection of Ts2 or Ts6 (250 μg/kg) in mice, there was an induction of leukocytosis with a predominance of neutrophils observed at 4, 24, 48 and 96 h. Moreover, total protein, leukotriene (LT)B4, prostaglandin (PG)E2 and pro-inflammatory cytokine levels were increased. We also observed an increase of regulatory cytokines, including interleukin (IL)-10, after the Ts2 injection. Finally, we observed that Ts2 or Ts6 injection in 5-lipoxygenase (LO) deficient mice and in wild type (WT) 129sv mice pre-treated with LTs and PGs inhibitors (MK-886 and celecoxib, respectively) a reduction the influx of leukocytes occurs in comparison to WT. The recruitment of these cells demonstrated a phenotype characteristic of neutrophils, macrophages, CD4 and CD8 lymphocytes expressing GR1+, F4/80+, CD3+/CD4+ and CD3+/CD8+, respectively. In conclusion, our data demonstrate that Ts2 and Ts6 induce inflammation by mechanisms dependent on lipid mediators and cytokine production. Ts2 may play a regulatory role whereas Ts6 exhibits pro-inflammatory activity exclusively

Successful and failed mini-implants: microbiological evaluation and quantification of bacterial endotoxin

Objectives: Using two groups of mini-implants (successful and failed) the objectives of this in vivo study were: to evaluate the microbial contamination by the checkerboard DNA-DNA hybridization technique and to quantify the bacterial endotoxin by the limulus amebocyte lysate assay. Material and Methods:The 15 successful and 10 failed mini-implants (1.6 mm diameter × 7.0 or 9.0 mm long), placed in the maxilla and/or mandible, were obtained from 15 patients undergoing orthodontic treatment. Data were analyzed statistically by the Wilcoxon rank-sum test using the SAS software (a=0.05). Results: All 40 microbial species were detected in both groups of mini-implants, with different frequencies. No differences were observed between the groups with respect to microbial complexes (blue, purple, yellow, green, orange, red and other species) and endotoxin quantification (p>0.05). Conclusion: Neither microbial contamination nor endotoxin quantification was determinant for the early loss of stability of the mini-implant

Impairment of neutrophil migration to remote inflammatory site during lung histoplasmosis

Histoplasma capsulatum (Hc) induces a pulmonary disease in which leukotrienes promote activation and recruitment of effectors cells. It is also well-recognized that leukotriene B 4 (LTB 4) and platelet-activating factor (PAF) induce leukocyte recruitment to inflammatory sites. We investigated the impact of pulmonary Hc infection on PMN migration to a remote inflammatory site. Our results show that pulmonary Hc infection impairs LTB4- or PAF-stimulated PMN recruitment to air pouch. Yet, remote inflammation did not modify PMN numbers in the bronchoalveolar lavage fluid (BALF) of Hc-infected mice. Interestingly, the concomitant administration of PAF and LTB 4 receptor antagonists inhibited PMN recruitment to both BALF and the remote site, demonstrating cooperation between both mediators. Along that line, our results show that PAF-elicited PMN chemotaxis was abrogated in 5-lipoxygenase-deficient animals. These results suggest caution in the indiscriminate use of anti-inflammatory drugs during infectious diseases.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

TLR2, TLR4 and CD14 Recognize Venom-Associated Molecular Patterns from <i>Tityus serrulatus</i> to Induce Macrophage-Derived Inflammatory Mediators

<div><p>Scorpion sting-induced human envenomation provokes an intense inflammatory reaction. However, the mechanisms behind the recognition of scorpion venom and the induction of mediator release in mammalian cells are unknown. We demonstrated that TLR2, TLR4 and CD14 receptors sense <i>Tityus serrulatus</i> venom (TsV) and its major component, toxin 1 (Ts1), to mediate cytokine and lipid mediator production. Additionally, we demonstrated that TsV induces TLR2- and TLR4/MyD88-dependent NF-κB activation and TLR4-dependent and TLR2/MyD88-independent c-Jun activation. Similar to TsV, Ts1 induces MyD88-dependent NF-κB phosphorylation via TLR2 and TLR4 receptors, while c-Jun activation is dependent on neither TLR2 nor TLR4/MyD88. Therefore, we propose the term venom-associated molecular pattern (VAMP) to refer to molecules that are introduced into the host by stings and are recognized by PRRs, resulting in inflammation.</p></div

TsV and Ts1 stimulation increases the mRNA expression of <i>Tlr2, Cd14, Myd88</i> and <i>Ptgs2</i> in peritoneal macrophages.

<p>Adherent macrophages from C57Bl/6 (WT) mice were treated with TsV or Ts1 (50 µg/ml) for 4 h. Unstimulated macrophages were used as the negative control. The cells were lysed, and total RNA was extracted. qRT-PCR was performed to determine the relative expression levels of transcripts encoding lipid metabolism enzymes, TLRs and adaptor proteins. The results were normalized to the expression levels of the endogenous internal controls <i>Actb</i>, <i>Gapdh</i> and <i>Tbp</i>. The 2^–ΔΔCt method was used for the analysis of the qRT-PCR data. *<i>p</i><0.05 (one-way ANOVA followed by Dunnett’s post-test) compared to medium alone. Statistically significant changes were considered when <i>p</i><0.05 and any gene presented a fold-change >2.0. The results are presented as the fold-change measured from 2 independent experiments.</p

Activation of NF-κB/AP-1 in RAW-Blue™ cells.

<p>These cells were derived from RAW 264.7 macrophages and contain a secreted embryonic alkaline phosphatase (SEAP) reporter construct that is integrated into the cellular DNA and that can be induced by NF-κB. The cells were incubated with either (A) anti-mTLR2-IgG (100 ng/ml) or (B) LPS-RS (10 ng/ml) for 30 min, with or without LPS (0.5 µg/ml), and TsV or Ts1 (50 µg/ml) for 24 h. The QUANTI-Blue™ substrate was used to measure the SEAP at 650 nm with an ELISA reader. The measurements were performed in triplicate, and a representative experiment is shown. *<i>p</i><0.05 (one-way ANOVA) compared to medium alone (dashed line). The values represent the means ± SD (<i>n</i> = 8), and the data are from 2 independent experiments.</p