Role of nitric oxide in the local and systemic pathophysiological effects induced by Bothrops asper snake venom in mice

Objective To assess the role of nitric oxide in the most relevant local and systemic manifestations in mice injected with the venom of the snake Bothrops asper. Mice were pretreated with nitric oxide synthase inhibitors, and the modifications of the pathological effects induced by the venom were tested. Results Inhibition of NO synthesis did not affect acute local myonecrosis and hemorrhage in muscle tissue upon intramuscular injection of venom. Local footpad edema was reduced in mice pretreated with the NO synthase inhibitor L-NAME, and a reduction in the extent of inflammatory infiltrate in muscle tissue was observed after envenomation in mice pretreated with L-NAME and aminoguanidine. The most pronounced effect of NOS inhibition by L-NAME was an increment in the lethal activity of the venom, when injected by the intraperitoneal route. Conclusion Nitric oxide does not seem to play a significant role in the local acute pathological alterations (hemorrhage and myonecrosis) induced by B. asper venom in mice, although it contributes to edema and inflammatory infiltrate. Nitric oxide exerts a protective role in the systemic pathophysiological manifestations leading to lethality.Universidad de Costa Rica/[2075-06-01]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Distinctive features of composts of different origin: a thorough examination of the characterization results

The potential of composts produced from different origin residues to be used in environmentally friendly agriculture is addressed in this work. Seven composts obtained from different raw materials and composting methodologies are compared using elemental, thermal and spectroscopic characterization data. Despite the stabilization of the organic matter in all composts being adequate for agricultural applications, they display distinct elemental and structural compositions. Likewise, the fertilisers have very different effects on lettuce growth. Despite the observed differences, some common features were found, namely a mass loss (TGA) of 25.2 g per mol C, association between groups of elements (Fe, Al, Ni, Co, Cr, Cu and S; Mg, Na, K and P, C, Coxi, N and Pb) and correlations between the amount of carbon nanostructures and the characteristic aromaticity parameters. These results suggest that the tuning of the compost features for specific cultures may be possible for sustainable food production.This work was financially supported by the Interreg VA Spain-Portugal Programme (EU) through the project Res2ValHum (0366_RES2VALHUM_1_P). A.C. Silva acknowledges receipt of a PhD grant (UMINHO/BD/40/2016) financed by the Operational Programme Norte 2020 (through the Project “NORTE-08-5369-FSE-000033”). A. Teixeira acknowledges the grant (Res2ValHum 01/2018) to develop experimental work for 11 months on the project. J. Antelo and S. Fiol are also grateful for the financial support provided by Xunta de Galicia—Consellería de Educación e Ordenación Universitaria de Galicia (Consolidation of Competitive Groups of Investigation; GI-1245, ED431C 2018/12 and CRETUS AGRUP2015/02, ref. 2018-PG10)

A snake venom secreted phospholipase A2 induces foam cell formation depending on the activation of factors involved in lipid homeostasis

MT-III, a snake venom GIIA sPLA2, which shares structural and functional features with mammalian GIIA sPLA2s, activates macrophage defense functions including lipid droplet (LDs) formation, organelle involved in both lipid metabolism and inflammatory processes. Macrophages (MΦs) loaded with LDs, termed foam cells, characterize early blood vessel fatty-streak lesions during atherosclerosis. However, the factors involved in foam cell formation induced by a GIIA sPLA2 are still unknown. Here, we investigated the participation of lipid homeostasis-related factors in LD formation induced by MT-III in macrophages. We found that MT-III activated PPAR-γ and PPAR-β/δ and increased the protein levels of both transcription factors and CD36 in macrophages. Pharmacological interventions evidenced that PPAR-γ, PPAR-β/δ, and CD36 as well as the endoplasmic reticulum enzymes ACAT and DGAT are essential for LD formation. Moreover, PPAR-β/δ, but not PPAR-γ, is involved in MT-III-induced PLIN2 protein expression, and both PPAR-β/δ and PPAR-γ upregulated CD36 protein expression, which contributes to MT-III-induced COX-2 expression. Furthermore, production of 15-d-PGJ2, an activator of PPARs, induced by MT-III, was dependent on COX-1 being LDs an important platform for generation of this mediator.Fundación de Apoyo a la Investigación del Estado de São Paulo/[00 / 11624-5]/FAPESP/BrasilFundación de Apoyo a la Investigación del Estado de São Paulo/[2011 / 21341-5]/FAPESP/BrasilFundación de Apoyo a la Investigación del Estado de São Paulo/[2014 / 18549-1]/FAPESP/BrasilFundación de Apoyo a la Investigación del Estado de São Paulo/[2010 / 06345-1]/FAPESP/BrasilFundación de Apoyo a la Investigación del Estado de São Paulo/[2015 / 24701-3]/FAPESP/BrasilFundación de Apoyo a la Investigación del Estado de São Paulo/[2013 / 22610-5]/FAPESP/BrasilFundación de Apoyo a la Investigación del Estado de São Paulo/[2010 / 08506-2]/FAPESP/BrasilFundación de Apoyo a la Investigación del Estado de São Paulo/[307379 / 2016-7]/FAPESP/BrasilUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

A secreted phospholipase A2 induces formation of smooth muscle foam cells which trans-differentiate to macrophage-like state

Vascular smooth muscle cells (VSMCs) loaded with lipid droplets (LDs) are markers of atherosclerosis. In this disease, inflammatory Group IIA-secreted phospholipase A2s (GIIA sPLA2s) are highly expressed in VSMCs, but their actions in these cells are unknown. Here, we investigated the ability of myotoxin III (MT-III), an ophidian GIIA sPLA2 sharing structural and functional features with mammalian GIIA sPLA2s, to induce LD formation and lipid metabolism factors involved in this e ect. Modulation of VSMC phenotypes by this sPLA2 was also evaluated. Incubation of VSMCs with MT-III significantly increased the number of LDs. MT-III upregulated scavenger receptor type 1 (SR-A1) and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) protein expression and enhanced acetylated-low density lipoprotein (acLDL) uptake by VSMCs, revealing the ability of a GIIA PLA2 to modulate scavenger receptor activities. MT-III induced translocation and protein expression of PPAR- and - / . Inhibition of peroxisome proliferator-activated receptors (PPARs) and diacylglycerol O-acyltransferase (DGAT) and acyl-CoA:cholesterolacyltransferase (ACAT) enzymes abrogatedMT-III-induced LD formation. Moreover, in response toMT-III, VSMCs acquired phagocytic activity and expressed macrophage markers CD68 and MAC-2. In conclusion, MT-III is able to stimulate VSMCs and recruit factors involved in lipid uptake and metabolism, leading to the formation of VSMC-derived foam cells with acquisition of macrophage-like markers and functions.Butantan Institute/[FAPESP 00/11624-5]//BrasilUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Role of TNF-α, IL-1β and IL-6 in the local tissue damage induced by Bothrops asper snake venom: an experimental assessment in mice

The role of the cytokines TNF-α, IL-1β and IL-6 in the acute local pathological effects induced by Bothrops asper snake venom was assessed in mice. Intramuscular injection of this venom induced increments in IL-1β and IL-6 in muscle, but no elevations of TNF-α were detected. Pentoxifylline (PTX), a methylxanthine derivative that inhibits the synthesis of TNF-α, and antibodies against these three cytokines were used to assess the role of these cytokines in venom-induced effects. As a control, PTX pretreatment was effective at abrogating lethality and serum TNF-α increments in mice subjected to endotoxemia induced by injection of Escherichia coli lipopolysaccharide, although it did not affect the increment in IL-1β and IL-6 in such endotoxic model. PTX failed to reduce lethality, hemorrhage, myonecrosis, dermonecrosis and edema induced by B. asper venom. Moreover, pretreatment with anti-cytokine antibodies was also ineffective at reducing venom-induced myonecrosis and hemorrhage. It is concluded that TNF-α, IL-1β and IL-6 do not have a significant role in the pathogenesis of the acute local pathological effects induced by B. asper venom in mice, although this does not exclude the possibility that these cytokines play a role in other aspects of venom-induced local pathology, as well as in the reparative and regenerative responses that take place after the onset of tissue damage.Universidad de Costa Rica/[741-A3-025]/UCR/Costa RicaUnited Nations Educational, Scientific and Cultural Organization/[883.701-3]/UNESCO/UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Bothrops asper and Bothrops jararaca snake venoms trigger microbicidal functions of peritoneal leukocytes in vivo

Venoms from snakes of the genus Bothrops cause pronounced local effects in the victims. These alterations result not only from the direct toxic action of venom components, but also from the prominent inflammatory reaction associated with these envenomations. In this study we investigated the ability of Bothrops asper (BaV) and Bothrops jararaca (BjV) venoms to induce cellular influx and microbicidal functions in leukocytes. BaV and BjV (5 μg/animal) caused a long lasting infiltration of leukocytes (3–48 h) when injected into mouse peritoneal cavity. Both venoms increased phagocytosis and production of hydrogen peroxide (H2O2) by polymorphonuclear (PMN) and mononuclear (MN) peritoneal leukocytes. In addition, nitric oxide (NO) production by macrophages was also enhanced after the venom injections. This effect was inhibited by treating animals with L-NAME and aminoguanidine, thus suggesting the induction of iNOS synthesis by the venoms. Western blot analysis confirmed the expression of iNOS in macrophages. BaV and BjV injection led to increased levels of IFN-γ at the site of inflammation. Since IFN-γ is an effective inducer of iNOS expression, an indirect action of the venoms on iNOS expression can be proposed. A marked formation of nitrotyrosine-containing proteins was also observed in macrophage homogenates. Based on these results, we suggest that reactive oxygen and nitrogen-derived species are involved in the pathogenesis of the local tissue damage characteristic of Bothrops sp envenomations.Fundação de Amparo à Pesquisa do Estado de São Paulo/[98/00162-9]/FAPESP/BrasilFundação de Amparo à Pesquisa do Estado de São Paulo/[97/13089-5]/FAPESP/BrasilFundação de Amparo à Pesquisa do Estado de São Paulo/[95/9699-7]/FAPESP/BrasilConselho Nacional de Desenvolvimento Científico e Tecnológico/[301199/91-4)]/CNPq/BrasilUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Pharmacological modulation of hyperalgesia induced by Bothrops asper (terciopelo) snake venom

The ability of Bothrops asper snake venom to cause hyperalgesia was investigated in rats, using the paw pressure test. Intraplantar injection of the venom (5–15 μg/paw) caused a dose and time-related hyperalgesia, which peaked 2 h after venom injection. Bothrops asper venom-induced hyperalgesia was blocked by the bradykinin B2 receptor antagonist HOE 140 and attenuated by dexamethasone, an inhibitor of phospholipase A2. Inhibition of the lipoxygenase pathway by NDGA abrogated the algogenic phenomenon. The hyperalgesic response was not modified by pretreatment with indomethacin, an inhibitor of the cyclo-oxygenase pathway, by meloxicam, an inhibitor of the type 2 cyclo-oxygenase pathway, by the PAF receptor antagonist BN52021 or by anti-TNF-α or anti-interleukin 1 antibodies. Intraplantar injection of the venom also caused an oedematogenic response which was not modified by any of these pharmacological treatments. These results suggest that hyperalgesia induced by Bothrops asper venom is, at least partially, mediated by bradykinin, phospholipase A2 activity and leukotrienes. Distinct mechanisms are involved in the development of hyperalgesia and oedema induced by the venom.Fundação de Amparo à Pesquisa do Estado de São Paulo/[98/00257-0]/FAPESP/BrasilInstituto Butantan///BrasilUniversidad de Costa Rica//UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Increments in serum cytokine and nitric oxide levels in mice injected with Bothrops asper and Bothrops jararaca snake venoms

Changes in serum levels of several cytokines and nitric oxide were studied in BALB/c mice injected intraperitoneally with one median lethal dose (LD(50)) of the venoms of Bothrops asper and Bothrops jararaca, two of the medically most important poisonous snakes of Latin America. Despite differences observed in the time-course of cytokine increments and in serum cytokine levels, both venoms induced prominent elevations of TNF-alpha, IL-1, IL-6, IL-10 and IFN-gamma. There was an early increase in TNF-alpha and IL-1, followed by a more pronounced increment by 18 h. IL-6 levels peaked between 4 and 6 h, and this cytokine probably modulates the secretion of TNF-alpha and IL-1 and the synthesis of acute-phase proteins. Both venoms induced an early increment in serum IL-10, whereas IFN-gamma levels reached higher values in mice injected with B. jararaca venom than in those receiving B. asper venom. Serum nitric oxide concentration increased in mice injected with both venoms rapidly after envenomation, remaining elevated for 24 h. It is concluded that a complex pattern of cytokine and nitric oxide synthesis and secretion occurs in severe experimental envenomation by B. asper and B. jararaca venoms. Furthermore, it is suggested that some of these mediators, particularly TNF-alpha, IL-1 and nitric oxide, might play a relevant role in the pathophysiology of systemic alterations induced by these venoms.Fundação de Amparo à Pesquisa do Estado de São Paulo//FAPESP/BrasilEl Consejo Nacional para Investigaciones Científicas y Tecnológicas//CONICIT/Costa RicaUniversidad de Costa Rica//UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Inflammatory effects of snake venom metalloproteinases

Metalloproteinases are abundant enzymes in crotaline and viperine snake venoms. They are relevant in the pathophysiology of envenomation, being responsible for local and systemic hemorrhage frequently observed in the victims. Snake venom metalloproteinases (SVMP) are zinc-dependent enzymes of varying molecular weights having multidomain organization. Some SVMP comprise only the proteinase domain, whereas others also contain a disintegrin-like domain, cysteine-rich, and lectin domains. They have strong structural similarities with both mammalian matrix metalloproteinases (MMP) and members of ADAMs (a disintegrin and metalloproteinase) group. Besides hemorrhage, snake venom metalloproteinase induce local myonecrosis, skin damage, and inflammatory reaction in experimental models. Local inflammation is an important characteristic of snakebite envenomations inflicted by viperine and crotaline snake species. Thus, in the recent years there is a growing effort to understand the mechanisms responsible for SVMP-induced inflammatory reaction and the structural determinants of this effect. This short review focuses the inflammatory effects evoked by SVMP

Preadipocytes are target cells for the actions of a representative gIIA phospholipase A2 which triggers proinflammatory pathways related to obesity

Adipose tissue secretes proinflammatory mediators which promote systemic and adipose tissue inflammation seen in obesity. Group IIA (GIIA)-secreted phospholipase A2 (sPLA2) enzymes are found to be elevated in plasma and adipose tissue from obese patients and are active during inflammation, generating proinflammatory mediators, including prostaglandin E2 (PGE2). PGE2 exerts anti-lipolytic actions and increases triacylglycerol levels in adipose tissue. However, the inflammatory actions of GIIA sPLA2s in adipose tissue cells and mechanisms leading to increased PGE2 levels in these cells are unclear. This study investigates the ability of a representative GIIA sPLA2, MT-III, to activate proinflammatory responses in preadipocytes, focusing on the biosynthesis of prostaglandins, adipocytokines and mechanisms involved in these effects. Our results showed that MT-III induced biosynthesis of PGE2, PGI2, MCP-1, IL-6 and gene expression of leptin and adiponectin in preadipocytes. The MT-III-induced PGE2 biosynthesis was dependent on cytosolic PLA2 (cPLA2)-α, cyclooxygenases (COX)-1 and COX-2 pathways and regulated by a positive loop via the EP4 receptor. Moreover, MT-III upregulated COX-2 and microsomal prostaglandin synthase (mPGES)-1 protein expression. MCP-1 biosynthesis induced by MT-III was dependent on the EP4 receptor, while IL-6 biosynthesis was dependent on EP3 receptor engagement by PGE2. These data highlight preadipocytes as targets for GIIA sPLA2s and provide insight into the roles played by this group of sPLA2s in obesityUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP