*6.2. Proteomic Analysis of LOCBE*

Proteomic analyses of LOCBE are scarce, and studies are focused on specific bioactive molecules related to the hematological disturbances observed during envenomation rather than an in-depth description of the bristles extract protein content [50]. A protein profile of LOCBE analyzed through 2D electrophoresis revealed the presence of 159 to 129 spots under nonreducing or reducing conditions, respectively. Most of the spots were detected at acidic to neutral isoelectric point values (4 < pI > 7) distributed in a wide molecular mass range (<10 to 105 kDa). This complexity was predominantly diminished at low molecular mass range under reducing conditions, suggesting the presence of dimers or oligomers, and its monomers were not retained on the acrylamide gel mesh used. According to Coomassie blue staining and immunogenic potential, 25 spots were submitted to mass spectrometry analysis, and three protein categories were identified: lipocalins (eight spots), cuticle proteins (five spots), and serpin (one spot). Twelve spots were described as unknown proteins; some of them were immunodetected using ALS or anti-Lopap rabbit serum, suggesting the presence of interesting immunogenic molecules to be further investigated [50].

### *6.3. Procoagulant Toxins from LOCBE*

Losac is the first factor X activator purified as a monomer of 45 kDa from LOCBE [43,63]. The cloning, heterologous expression, and characterization of recombinant Losac (rLosac) was described by Alvarez-Flores and collaborators [43,48]. rLosac specifically activates factor X in the absence of calcium and phospholipids, although the presence of these cofactors accelerates its activity. Its enzymatic characterization was performed, revealing that this protein has no homology to known procoagulant proteases. Instead, Losac

belongs to the hemolin family of proteins, a group of multifunctional proteins exclusively expressed by Lepidoptera order insects involved in several cell interactions, but mainly in immunity [43,80]. The tertiary structure model of rLosac was built through homology modeling using the crystal structure of *H. crecopia* hemolin as a template, and it shares the multidomain structure D1–D4 and its conserved motifs. In addition, the multiple amino-acid sequence alignment of rLosac showed up to 76% identity with other hemolin protein families [43].

Studies were conducted to evaluate its effects on endothelium. rLosac inhibited apoptosis in serum-deprived human umbilical vein endothelial cells (HUVECs) and induced cell proliferation [63]. This reduction in cell death under nutrient deprivation conditions was also observed in mouse cortical neurons and human dermal fibroblasts, showing an effective prevention of reactive oxygen species generation and loss of mitochondrial membrane potential, suggesting an antioxidant activity [60–62]. An in vivo experimental model in rats demonstrated that rLosac improved wound healing by increasing the epidermal proliferation, as well as by preserving the extracellular matrix organization through collagen type I, fibronectin, and laminin expression. Thus, rLosac was indicated as a very promising molecule, potentially useful as a bioactive agent to develop new formulations for wound healing [59].

Lopap is a 69 kDa prothrombin activator that shares with LOCBE its role in inflammatory processes and belongs to the lipocalin protein family, being the most abundantly studied isolated toxin from the LOCBE [47,48,51,57]. The recombinant form of Lopap (rLopap) recognizes and hydrolyzes prothrombin, which in turn leads to an active thrombin generation, showing a proteolytic activity similar to native Lopap [47,57]. Lopap displayed a Ca2+-activating serine protease activity that was included into the group I of prothrombin activators [36,45,47]; the infusion of native Lopap produced intravascular coagulation and thrombosis in the post capillary vessels of mice [46].

Properties of rLopap were evaluated in an in vivo model of leukocyte–endothelial cell interaction, revealing that rLopap as the native Lopap induced NO production and ICAM-1 expression in both neutrophils and endothelial cells. In addition, it induced antiapoptotic effects mediated by NO production [81]. The study of its effects in human platelets showed that there is not a direct effect on platelet function, since Lopap showed no effect on platelet aggregation induced by collagen ADP or thrombin. On the other hand, Lopap induced the expression of adhesion molecules ICAM-1 and E-selectin of human endothelial cells [56]. In those cells, Lopap promotes survival mechanisms since it induces the release of nitric oxide and prostaglandin I2, along with the release of inflammatory cytokine IL-8 and t-PA. Moreover, synthetic peptides based on lipocalin motif 2, which is found in a primary sequence of Lopap, showed cytoprotective and antiapoptotic activity in vitro and in vivo approaches, suggesting the involvement of that lipocalin motif in cell protection [82–85]. This knowledge brought new perspectives on the use of these synthetic molecules since they do not exhibit hemostatic functions.

#### **7. Effect of LOCBE and Toxins in the Inflammatory Response**

Dermatitis and skin reactions such as urticaria are well-known signs after accidental contact with the spines and bristles of venomous lepidopteran caterpillars [1]. Generally, the consequences of these reactions are limited to local inflammation, with no systemic or tissue damage. In the case of the envenomation caused by *L. obliqua*, the process is characterized by triggering an intense inflammatory response in victims followed by coagulation, complement, and kallikrein–kinin systems [36,48,51]. In recent years, several studies have been carried out aiming to clarify and describe the role of the venom-induced inflammatory response in the clinical symptoms characteristic of lonomism.

*L. obliqua* proinflammatory effects are first manifested by pain, burning sensation, edema, and erythema formation [8,19,28]. The first pharmacological studies showed that venom-induced nociception in animal models is largely facilitated by the production of prostaglandins, and later edematogenic symptoms are induced by prostanoids and

histamines [48,71]. The kallikrein–kinin system is also involved in the edematogenic and hypotensive responses triggered by the venom. Bohrer and collaborators demonstrated that administration, prior to treatment with LOCBE, of plasma kallikrein inhibitor reduces the volume of venom-induced edema in a mouse paw model [35].

Envenomed patients presented low levels of plasma prekallikrein [8,19], indicating that kallikrein was activated and released into the blood circulation. The kallikrein–kinin system is composed of proteolytic enzymes and their substrates, being able to generate potent vasoactive and proinflammatory molecules that are involved in the control of blood pressure, vascular permeability, vascular smooth muscle cell contraction or relaxation, and pain [86]. One of the common consequences of lonomism is the sudden loss of basic renal functions. Kidneys and urine from envenomed animals were enriched with proteins related to inflammatory stress, tissue damage, oxidative stress, coagulation, complement system activation, and kinin system [87,88]. When simultaneously treated with kallikrein inhibitors and antilonomic serum, envenomed rats showed improvements in renal and vascular function, reducing tubular necrosis and renal inflammation [32]. The mechanism underlying these effects was associated with lowering renal inflammation, with a decrease in proinflammatory cytokines and matrix metalloproteinase expression, reduced tubular degeneration, and protection against oxidative stress.

An increase in the permeability of the endothelium allows greater infiltration of cells of the immune system, effectors, and regulators of acute inflammation into tissues [89]. Increased vascular tissue permeability is a characteristic event of the inflammatory response and can be induced by several proinflammatory and vasoactive substances such as bradykinin, histamine, thrombin, cytokines, prostaglandins, and free radicals [74,90]. Activation of the vascular tissue was observed after a single subcutaneous injection of LOCBE in rats [91]. Envenomed animals demonstrated neutrophilic leukocytosis in several tissues, where their histological sections provided evidence of inflammatory cell infiltrates in the heart, lungs, and kidneys, characterizing a systemic acute inflammatory response induced by the venom [92]. Furthermore, an increase was observed in leukocyte rolling and adhesion of these circulating blood cells to the endothelium of hamster cheek pouch tissue that was previously incubated with low doses of LOCBE [72]. The ability of LOCBE to induce an increase in the permeability of the vasculature and immune cell infiltration may provide a favorable environment for hemorrhages, especially in microvessels in the brain.

Due to the important relationship between inflammation and vasculature, studies were carried out seeking to elucidate the direct effect of LOCBE on vascular tissue. In vitro studies showed that non-hemorrhagic concentrations of LOCBE modify the cytoskeleton dynamics and increase focal adhesion in endothelial cells [72]. Furthermore, low doses of the LOCBE can induce activation of the nuclear transcription factor κB (NF-κB) pathway in these cells [73]. The NF-κB pathway is a critical signaling in several events associated with triggering acute inflammation and immune system cell recruitment [93]. Consequently, LOCBE also induced significant increases in the expression of COX-2, NOS-2, HO-1, MMP-2, and MMP-9, enzymes related to prostaglandin production, oxidative stress, and extracellular matrix degradation [72,74].

Additionally, the LOCBE was also shown to be a potent activator of vascular smooth muscle cells, being able to induce cell chemotaxis, exacerbated proliferation, and production of reactive oxygen species. Smooth muscle cell dysfunction is characterized by increased cell migration and proliferation, events that are amplified by the release of inflammatory mediators [91]. Furthermore, researchers also carried out a broad analysis of the gene expression profile of fibroblasts treated with LOCBE. The results show an upregulation of several proinflammatory mediator genes, such as IL-8, IL-6, and CCL2, as well as the adhesion molecule ICAM-3 and COX-2 [74]. Recently, our group showed a direct effect of LOCBE upon macrophage activation. The LOCBE directly induces THP-1 macrophages to a proinflammatory phenotype by activating NF-κB pathway, leading the cells to release proinflammatory cytokines and chemokines such TNFα, IL-1β, IL-6, IL-8, and CXCL10 [73].

The role of isolated toxins present in the venom triggering inflammatory responses is still to be investigated. In addition to its procoagulant activity, in vivo studies show that injection of a high concentration of recombinant *L. obliqua* prothrombin activator protease (rLopap) in rats promotes neutrophil and monocyte infiltration in pulmonary microcirculation vessels. In HUVECs, rLopap stimulates the increase of IL-8, ICAM-1, and E-selectin, proteins involved in the recruitment of immune cells to the tissue [56,75]. In contrast, there is no evidence that Losac presents proinflammatory activity beyond its cytoprotective and proliferative effects.

Taken together, the evidence indicates that LOCBE can induce a local acute inflammatory response that can evolve into a systemic response. Many studies have characterized the role of the kinin–kallikrein system and the liberation of other proinflammatory mediators by the affected tissues, related to several clinical stomps. The isolated effect of the toxins present in the LOCBE, such as Lopap, and their roles in the activation of prekallikrein, the ability to directly induce cell responses, and the molecular mechanism underlying these effects still need to be clarified.

### **8. Innovation in Toxinology: Research Centers and New Molecule Development**
