The Crotalus durissus terrificus rattlesnake venom, its main toxin, crotoxin (CTX), and its crotapotin (CA) and phospholipase A2 (CB) subunits modulate the immune system. Formyl peptide receptors (FPRs) and lipoxin A4 (LXA4) are involved in CTX's effect on macrophages and neutrophils. Dendritic cells (DCs) are plasticity cells involved in the induction of adaptive immunity and tolerance maintenance. Therefore, we evaluated the effect of CTX, CA or CB on the maturation of DCs derived from murine bone marrow (BM). According to data, CTX and CB—but not CA—induced an increase of MHC-II, but not costimulatory molecules on DCs. Furthermore, CTX and CB inhibited the expression of costimulatory and MHC-II molecules, secretion of proinflammatory cytokines and NF-κBp65 and p38/ERK1/2-MAPK signaling pathways by LPS-incubated DCs. Differently, CTX and CB induced IL-10, PGE2 and LXA4 secretion in LPS-incubated DCs. Lower proliferation and IL-2 secretion were verified in coculture of CD3+ cells and DCs incubated with LPS plus CTX or CB compared with LPS-incubated DCs. The effect of CTX and CB on DCs was abolished in cultures incubated with a FPRs antagonist. Hence, CTX and CB exert a modulation on functional activity of DCs; we also checked the involvement the FPR family on cell activities.

Acetylcholinesterase (AChE) is an important enzyme in the control of the neuronal action potential and sensitive to organophosphate inhibition. Brain fish AChE is less sensitive to organophosphate inhibition than AChE from terrestrial animals, although this sensitivity is variable among species and has not yet been fully evaluated in fish species. In this setting, inhibition kinetic constants for progressive irreversible inhibition of brain acetylcholinesterase due to methyl-paraoxon exposure were determined in three fish species (Mugil liza, Genidens genidens and Lagocephalus laevigatus) and hen (Gallus domesticus). Enzyme extraction using a detergent was shown to be adequate, and samples presented activity inhibition in high substrate concentrations and suppression of inhibition by methyl-paraoxon in the presence of the substrate, similar to kinetic patterns from purified enzyme preparations. Catfish (G. genidens) AChE presented the highest sensitivity among the evaluated fish species (IC50 = 1031.20 nM ± 63.17) in comparison to M. liza and L. laevigatus (IC50: 2878.83 ± 421.94 and 2842.5 ± 144.63 nM respectively). The lower dissociation constant (Kd = 20.3 ± 2.95 μM) of catfish AChE showed greater enzyme affinity for methyl-paraoxon, explaining this species higher sensitivity to organophosphates. Hen AChE presented higher ki (900.57 ± 65.3 mM-1min-1) and, consequently, greater sensitivity to methyl-paraoxon, explained by a lower Kd (0.6 ± 0.13 μM). Furthermore, hen AChE did not differentiate between the propionylthiocholine and acetylthiocholine substrates, indicating easier access of methyl-paraoxon to the hen enzyme activity site. The results obtained herein indicate a suitable extraction of AChE and, despite different inhibition kinetic constants, demonstrate that fish AChE is less sensitive to methyl-paraoxon, probably due to reduced access to the catalytic center which provides greater enzyme substrate selectivity.

BackgroundThe mosquito-borne alphaviruses chikungunya virus (CHIKV) and o'nyong-nyong virus (ONNV) are closely related Alphaviruses that belong to the Semliki forest virus serocomplex. The two viruses are associated with large outbreaks with significant morbidity. However, they are transmitted by different mosquito vectors and accordingly need different prevention strategies. The viruses are difficult to distinguish clinically and there is a lack of sensitive and specific assays that can discriminate between CHIKV and ONNV. Therefore, there is a need for new methods that may be able to determine the true burden of the diseases caused by these viruses, especially in resource-poor settings.MethodTo distinguish between CHIKV and ONNV, we designed and optimized two genetic methods, melt analysis of mismatch amplification mutation assay (Melt-MAMA) and agarose gel-based mismatch amplification mutation assay (Agarose-MAMA). The identification was based on single nucleotide polymorphisms using two competing forward primers and a common reverse primer that targeted selected sites in the envelope genes (E1 and E2). A specific shift in the melting point and mobility on agarose gels was obtained by tailing one of the two competing primers with a G/C-rich stretch of nucleotides.ResultsThe melting point analyses by real-time polymerase chain reaction (qPCR Melt-MAMA) or gel-shift assay (Agarose-MAMA assay) for CHIKV and ONNV were found to be reproducible and the sensitivity of the two assays was estimated at under 100 template copies/reaction. Furthermore, no cross-reactivity with related viruses of the same serocomplex such as Mayaro virus, Ross River virus or Semliki forest virus was detected, or with other viruses such as Sindbis virus (Alphavirus), West Nile virus, dengue virus (Flavivirus), Inkoo virus and Tahyna virus (Orthobunyavirus). The results from the two assays were comparable when the obtained amplicons were analyzed by Melt-MAMA or by Agarose-MAMA.ConclusionHerein we present reliable and robust methods that can discriminate between CHIKV and ONNV. These methods can be used in well-equipped laboratories and basic clinical settings (e.g., in developing countries), as well as in field situations. The approach may also be applicable in the distinction of other closely-related mosquito-borne viruses that belong to the same serogroup.