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Copyright of Universitas Médica is the property of Pontificia Universidad Javeriana and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Envenomation caused by contact with Lonomia obliqua bristles is characterized by pain, an intense systemic proinflammatory reaction and disturbances in the coagulation cascade that can cause severe clinical manifestations and death. However, the role of immune system components in these effects is still poorly understood. In this study, we evaluated the cytotoxic effect of L. obliqua venom on THP-1-derived macrophages and its ability to modulate inflammatory markers, as well as the cytokine and chemokine release profile. Our results show that L. obliqua venom is able to directly exert a potent pro-inflammatory reaction in macrophages, characterized by the activation of the NF-κB transcription factor pathway, the expression of CD80 and CD83, and the release of pro-inflammatory mediators such as TNF-α, IL-1β, IL-6, IL-8 and CXCL10. These results suggest that macrophages can play an important role during the orchestration of the inflammatory response present in envenomation caused by Lonomia obliqua caterpillars.

The presence of specialized cells for venom production in the Lonomia obliqua caterpillar has long been a controversial topic. In this study, we identify a cell inside the spine that specializes in the production of toxins. Our histological study showed that this glandular cell was inserted at the subapical region of the spine, in a constricted region like a ring. This cell type was not observed in all spines of the scolus. The constricted region of the spine observed by scanning electron microscopy displayed a circular groove in which the apical portion of the spine fits perfectly; however, some spines in the same scolus lacked this groove. After breaking off the spine at the most apical region, a small drop of orange or green liquid was observed to flow from its tip. These secretions were analysed by MALDI-ToF and found to possess biochemically different compositions. The green secretion demonstrated greater similarity to the haemolymph of the caterpillar than the orange secretion. Based on our findings, the spines with a groove probably contain the venom glands and produce an orange secretion. However, it is also possible that both secretions play an important role in envenoming because all spines in contact with the skin of the accidental victim should break regardless of whether they are present in a groove. [ABSTRACT FROM AUTHOR]

Lonomia obliqua caterpillar is frequently seen in accidents with humans especially in the south of Brazil. Patients develop a hemorrhagic syndrome that can be treated with specific antilonomic serum. A consumptive coagulopathy was found to be the main cause of bleeding complications observed in patients after contact with L. obliqua. Studies revealed that L. obliqua caterpillar bristle extract (LOCBE) displays a procoagulant activity that leads to intravascular thrombin formation, resulting in a special form of disseminated intravascular coagulation (DIC). Fibrinolysis seems to be secondary to the fibrin production, since no direct fibrinolytic activity was found in LOCBE. Two procoagulant toxins, a factor X activator (Losac) and a prothrombin activator (Lopap), were isolated from LOCBE and characterized. Infusion of Lopap into experimental animals triggered a condition similar to that observed in human envenomation.

This study presents a new recombinant protein that acts as a powerful antiviral (rAVLO-recombinant Antiviral protein of Lonomia obliqua). It was able to reduce the replication by 10 fold for herpes virus and by 10 fold for rubella virus. RT-PCR of viral RNA rAVLO treated infected cells also showed similar rate of inhibition in replication. The analysis of this protein by bioinformatics suggests that this protein is globular, secreted with a signal peptide and has the ability to bind to MHC class I. It was found that there are several protein binding sites with various HLA and a prevalence of α-helices in the N-terminal region (overall classified as a α/β protein type). BLAST similarity sequence search for corresponding cDNA did not reveal a similar sequence in Genbank, suggesting that it is from a novel protein family. In this study we have observed that this recombinant protein and hemolymph has a potent antiviral action. This protein was produced in a baculovirus/Sf-9 system. Therefore, these analyses suggest that this novel polypeptide is a candidate as a broad spectrum antiviral. [ABSTRACT FROM AUTHOR]

The baculovirus Anticarsia gemmatalis nucleopolyhedrovirus ( AgMNPV), a member of the family Baculoviridae, has been widely applied as a biopesticide for the control of the velvetbean caterpillar, a pest of soybean crop field. Baculoviruses are considered safe and efficient agents for this purpose, because they do not infect vertebrates, being safe for the health of humans and animals, as well as to the environment. The objective of this work was to identify proteins obtained from Lonomia obliqua hemolymph with potential application in the optimization of baculovirus AgMNPV replication in Sf9 insect cell culture. In this work the improvement of the cell culture and viral replication of the AgMNPV baculovirus was observed when Grace medium was supplemented with 10 % (v/v) Fetal Bovine Serum (FBS), 1 % (v/v) hemolymph extract, or 3 % (v/v) of hemolymph fractions or hemolymph sub-fractions obtained by purifying hemolymph through High Performance Liquid Chromatography. Hemolymph presented a positive effect on the synthesis of polyhedra and enhanced baculovirus replication in Spodoptera frugiperda (Sf9) cells (TCID/mL), and led to Sf9 cell culture improvement. Grace medium supplemented with 10 % (v/v) FBS and 1 % (v/v) hemolymph provided an increase of baculovirus replication, when the cells were infected with multiplicity of infection of 1. In this case, the baculovirus replication was 6,443.91 times greater than that obtained with the control: Grace medium supplemented with 10 % (v/v) FBS. In addition, this work suggests that hemolymph from L. obliqua could have an interesting application in biotechnology, due to an increase in the viability of the cells and virus replication. [ABSTRACT FROM AUTHOR]

The case of a 70 year-old, previously healthy woman who developed a severe bleeding diathesis shortly after touching a Lonomia obliqua caterpillar and finally died from multiple intracerebral hemorrhages is described. Brain hemorrhages are the leading cause of death in patients envenomed by the Lonomia species. The pertinent literature is reviewed and the most relevant clinical features highlighted, with emphasis on diagnosis. The use of new therapeutic options such as anti-Lonomia serum is discussed.O caso de uma mulher de 70 anos, previamente hígida, que desenvolveu diátese hemorrágica grave após contato com uma lagarta Lonomia obliqua, resultando em óbito por hemorragia intracerebral é relatado. Hemorragias cerebrais são uma das causas de morte em acidentes por Lonomia. A literatura pertinente é revisada, sendo as características clínicas e laboratoriais mais relevantes discutidas com ênfase para o diagnóstico, e o uso de novas abordagens terapêuticas como o soro anti-Lonomia.

Lonomia obliqua is an insect that has urticant spines in the larval stage. This species may cause death as a result of haemorrhages caused by a toxin released from the caterpillar's spines onto the skin of the victim. Since 1989 when this species was identified in Rio Grande do Sul, Brazil, many accidents have happened. The aim of this study was to verify the oviposition, fecundity, fertility, viability of eggs and incubation period of the species. Adults were left in a moth's cage to mate and lay eggs. Thirteen couples were observed daily, and the number of eggs and the caterpillars emerged were recorded. The results showed a mean of 2.8 (± 1.3) ovipositions, a mean fecundity of 135.3 (± 54.4) eggs/female, a mean fertility of 111.9 (± 55.4) eggs/female, a mean egg viability of 80.9 (± 20.97) % and a mean incubation period of 31.8 (± 5.8) days.O objetivo desse trabalho foi estudar os parâmetros biológicos de oviposição, fecundidade, fertilidade, viabilidade dos ovos e período de incubação de Lonomia obliqua Walker, 1855, em laboratório. As lagartas foram coletadas no campo nos municípios de Santa Cruz do Sul, Erechim, Guaporé, Passo Fundo, Não-Me-Toque, São Jorge, Farroupilha e Vila Lângaro, no Rio Grande do Sul, e Concórdia em Santa Catarina. As lagartas foram mantidas em sala de criação, com a leitura diária da temperatura e umidade relativa do ar, até a emergência dos adultos. Após a sexagem, cada casal foi colocado em uma gaiola para acasalamento e postura. Acompanhou-se, diariamente, as posturas das fêmeas e a eclosão das lagartas, determinando-se o número de posturas por fêmea, a fecundidade, a fertilidade, a viabilidade e o período de incubação dos ovos. O número médio de oviposições foi de 2,8 (± 1,3), fecundidade média de 135,30 (± 54,4) ovos por fêmea, fertilidade média de 111,9 (± 55,4) ovos por fêmea. A viabilidade média dos ovos foi de 80,9 (± 21,0) % e obteve-se um período médio de incubação de 31,8 (± 5,8) dias.

Many active principles produced by animals, plants and microorganisms have been employed in the development of new drugs for the treatment of human diseases. Among animals known to produce pharmacologically active molecules that interfere in human cell physiology, the caterpillar Lonomia obliqua has become the focus of toxicological studies due to recent findings about its venom constituents. The objective of this study was to investigate the effects of L. obliqua venom upon the viability and the proliferation of different cell lineages and to propose mechanisms for the herein observed induction of cell proliferation in glioma cell lines. MTT analyses indicate that L. obliqua venom increases the viability of tumor cell lines U138-MG and HT-29; on the other hand, it inhibits the viability of V-79 nontumor cells. Cell count based on the trypan blue exclusion method suggests a proliferating activity of the venom upon U138-MG cells. Exposure of U138-MG to crude venom extract led to a decrease in the production of nitric oxide, and activation of the cAMP signaling pathway inhibited the effects of the venom, indicating that these mechanisms may influence cell proliferation triggered by the venom. Despite the proliferative effects of crude venom on U138-MG and HT-29 cell cultures, a protein purified from L. obliqua hemolymph previously shown to have cytoprotective activity had no effect on U138-MG and HT-29; however, this same protein increased the viability of V-79 cells that had previously been exposed to the cytotoxic activity of the crude venom extract. This study indicates that the venom and the antiapoptotic protein act differently and have different effects on cell cultures, depending on the cell line analyzed. Biomolecules displaying either mitogenic or cytotoxic activities are of great biotechnological interest. Further studies encompassing the purification of active principles from L. obliqua venom are necessary to further elucidate its effects on different cell types. [ABSTRACT FROM AUTHOR]

Abstract: Animal venoms and secretions have been screened, in our research group, to discover, identify and isolate peptide molecules active in the mammalian haemostatic system. As result, this kind of research has provided a portfolio of promising drug candidates. These novel recombinant proteins have turned out to be multifunctional molecules, and are currently under different development phases. Lopap from bristles of the Lonomia obliqua moth caterpillar, for instance, is a prothrombin activator which belongs to the lipocalin family. It displays serine protease-like activity with procoagulant effect, and also induces cytokine secretion and antiapoptotic pathways in human cultured endothelial cells. Furthermore, a Lopap-derived peptide has showed to induce collagen synthesis in fibroblast culture and in animal dermis. Here, the molecular properties (steric, electronic, hydrophobic, geometric), which are strongly dependent on chemical structure, were investigated by applying chemometric and computational chemistry methods. It was considered different patterns of amino acid substitution related to the lipocalins' motif 2, which was recently shown to modulate cell survival. The calculated molecular properties were generally maintained in all investigated peptides extracted from three-dimensional structures of Protein Data Bank (1t0v, 1bbp, 1kxo, 2hzr, 1iiu, 1jyj, 1gka, 1s44, 3ebw) when compared to Lopap-derived peptide, specially the molecular shape and electronic density distribution, validating the lipocalin sequence signature previously reported. Indeed, those two properties are quite important for the molecular recognition process. [Copyright &y& Elsevier]

Abstract: The control of viral infections, mainly those caused by influenza viruses, is of great interest in Public Health. Several studies have shown the presence of active properties in the hemolymph of arthropods, some of which are of interest for the development of new pharmacological drugs. Recently, we have demonstrated the existence of a potent antiviral property in the hemolymph of Lonomia obliqua caterpillars. The aim of this study was to produce an antiviral protein in a baculovirus/Sf9 cell system. The resulting bacmid contains the sequence coding for the antiviral protein previously described by our group. Total RNA from L. obliqua caterpillars was extracted with Trizol and used in the reverse transcription assay with oligo(d)T primer followed by polymerase chain reactions (RT-PCR) with specific primers for the cDNA coding for the antiviral protein, based on the sequence deposited in the GenBank database. Restriction sites were inserted in the cDNA for ligation in the donor plasmid pFastBac1™. The recombinant plasmid was selected in Escherichia coli DH5α and subsequently used in the transformation of E. coli DH10Bac for the construction of the recombinant bacmid. This bacmid was used for the expression of the antiviral protein in the baculovirus/Sf9 cell system. After identifying the protein by western blot, activity tests were performed, showing that the purified recombinant protein was able to significantly reduce viral replication (about 4 logs). Studies on the optimization of the expression system for the production of this antiviral protein in insect cells are in progress. [Copyright &y& Elsevier]

Abstract: Caterpillar envenomation has been an emergent health issue. Lonomia obliqua is a medically important animal that causes a hemorrhagic syndrome that can progress to acute renal failure, intracranial hemorrhage and death. In the past few years the molecular characterization of L. obliqua venom in addition to experimental models has provided fundamental information to the understanding of the envenomation syndrome. Herein studies from several authors which characterized the complex toxic-pharmacological actions of whole venom are reviewed. [ABSTRACT FROM AUTHOR]

Abstract: Potent antiviral activity against measles, influenza and polio viruses was observed in the hemolymph of Lonomia obliqua. The antiviral protein responsible for this activity was isolated, purified by gel filtration chromatography using a gel filtration column system (Superdex 75) and further fractionated using a Resource-Q ion exchange column system. Experiments with the purified protein led to a 157-fold reduction (from 3.3±1.25×107 to 2.1±1.5×105 TCID50 mL−1) in measles virus production and a 61-fold reduction (from 2.8±1.08×109 to 4.58±1.42×107 mL−1) in polio virus production. Heating and freezing seem to have no influence over its antiviral activity. Also, the protein does not display virucidal activity and does not act on receptors on the cell membrane. The observations suggest an intracellular mechanism of action and that the protein may act as a constitutive agent that affects the innate antiviral immune response. [Copyright &y& Elsevier]

Abstract: Lopap (Lonomia obliqua prothrombin activator protease) is a member of the lipocalin family isolated from the extract of L. obliqua bristles. Lopap displays serine protease-like activities, including coagulation disturbance, cytokine secretion and antiapoptotic activity in human cultured endothelial cells. Here, we have investigated the effects of the recombinant protein (rLopap) on the inflammatory and apoptotic processes of neutrophils and endothelial cells from male Wistar rats. We found that rLopap did not induce in vivo leukocyte–endothelial interactions in the microvasculature, initial steps of leukocyte recruitment during inflammation. Incubation of rLopap with neutrophils or endothelial cells prevented apoptosis evoked by serum deprivation and induced nitric oxide (NO) production in both cell types, and increased the expression of ICAM-1 by endothelial cells. Simultaneous incubation of endothelial cells or neutrophils with rLopap and Nω-nitro-l-arginine methyl ester (L-NAME), a non-specific inhibitor of NO synthases, inhibited NO production and impaired the protection on apoptosis. Differently, incubation of endothelial cells with monoclonal antibody anti ICAM-1 did not change the protection on apoptosis evoked by rLopap. Together, these results indicate that rLopap does not display inflammatory properties in vivo but inhibits apoptosis of neutrophils and endothelial cells depending, at least in part, on NO production. [Copyright &y& Elsevier]

Abstract: The accidental contact with Lonomia obliqua caterpillar causes local and systemic symptoms (such as fibrinogen depletion), leading, in some cases, to serious clinical complications (acute renal failure and intracranial haemorrhage). Fortunately, a successful therapeutical approach using anti-Lonomic serum, produced in horses against L. obliqua''s bristle extract, has already been put in place. However, a global view of immunogenic toxins involved in the coagulation disorders could help to elucidate the envenoming process. In the present study, our aim was to identify bristle extract''s immunogenic components, especially those related to the haemostasis, coupling proteomics and immunochemical approaches (bidimensional electrophoresis, mass spectrometry and immunoblotting). The bidimensional map of bristle extract showed a broad profile of 157 silver-stained spots, where at least 153 spots were immunochemically revealed. Twenty-four of these spots were submitted to sequencing by mass spectrometry and three different categories of proteins were identified: lipocalins, cuticle proteins and serpins. From these protein families, it was observed that the most abundant was the lipocalin family, specifically represented by different isoforms of Lopap (a prothrombin activator protein), reinforcing its relevance during envenoming. Peptide sequences of several other immunochemically revealed spots showed no correspondence to any known sequence and were classified as unknown proteins. These proteins could represent new immunogenic molecules and/or toxins. The sequences presented in this article can be used for oligonucleotide design aiming the amplification of cDNAs coding for new molecules using L. obliqua bristles’ cDNA libraries or isolated RNAs as template. [Copyright &y& Elsevier]

Abstract: Large number of accidents caused by contact with Lonomia obliqua caterpillars, with hemorrhagic complications, have occurred in southern Brazil. Based on Venezuelan expertise to treat Lonomia achelous envenomation, the use of the antifibrinolytic drug epsilon-aminocaproic acid (EACA) has been indicated to treat L. obliqua envenomation, although no evidence has been presented to justify its use. Specific antivenom (antilonomic serum (ALS)) that neutralizes toxins that cause envenomation was developed. To compare the effectiveness of such treatments, rats were injected i.d. with the bristle extract of L. obliqua caterpillars and treated 15min, 1 and 6h after with saline, ALS, EACA, or with both ALS and EACA. ALS elicited fibrinogen recovery and normalization of thrombin time (TT), prothrombin time (PT) and activated partial thromboplastin time (APTT), independent of when it was administered; however, hematocrit was decreased in the group treated later. Saline or EACA-treated groups presented neither fibrinogen recovery nor normalization of hemostatic parameters. A high death rate was observed in the group treated with EACA 15min after the envenomation. Prolongation of TT and APTT observed in the group treated with EACA and ALS indicated that this association gave no benefit in relation to the group treated solely with ALS. The results presented herein suggest that ALS is the only effective treatment for envenomation caused by contact with Lonomia obliqua caterpillars and indicate that EACA should not be administered in the initial phase of envenomation. [Copyright &y& Elsevier]

Abstract: Lonomia obliqua envenomation induces an intense burning sensation at the site of contact and severe hemorrhage followed by edema and hypotension, and after few days death can occur usually due to acute renal failure. In order to understand more about the envenomation syndrome, the present study investigates the role played by kallikrein–kinin system (KKS) in edematogenic and hypotensive responses to the envenomation by L. obliqua. The incubation of L. obliqua caterpillar bristles extract (LOCBE) with plasma results in kallikrein activation, measured by cromogenic assay using the kallikrein synthetic substrate S-2302 (H-D-Pro-Phe-Arg-pNA). It was also showed that LOCBE was able to release kinins from low-molecular weight kininogen (LMWK). Moreover, it was demonstrated that previous administration of a kallikrein inhibitor (aprotinin) or bradykinin B2 receptor antagonist (HOE-140) significantly reduces the edema and hypotension in response to LOCBE, using mouse paw edema bioassay and mean arterial blood pressure analysis, respectively. The results demonstrate a direct involvement of the KKS in the edema formation and in the fall of arterial pressure that occur in the L. obliqua envenomation syndrome. [Copyright &y& Elsevier]

Abstract: Lonomia obliqua caterpillar bristle extract induces both direct and indirect hemolytic activity on human and rat washed erythrocytes, and provokes intravascular hemolysis in Wistar rats. Indirect hemolytic activity is assumed to be caused by a phospholipase A2 (PLA2) present in this extract, and this investigation was initiated in order to characterize this enzyme. Phospholipase A2 activity of crude extract was inhibited by both a PLA2-specific inhibitor (pBpb) and the metal ion chelator EDTA. L. obliqua PLA2 was purified by liquid chromatography from the crude bristle extract and had a molecular mass of 15kDa and a pI of 5.9; its N-terminal sequence showed high homology to a sequence of a putative PLA2 obtained from a cDNA library of L. obliqua bristles, and it is tentatively placed among Group III phospholipases A2. This enzyme was stable at 4°C, sensitive to higher temperatures, and its maximum catalytic activity was at pH 8.0. L. obliqua PLA2 induced hemolysis only when incubated with exogenous lecithin. Thus, the PLA2 purified herein appears to be responsible for the indirect hemolytic activity of the crude bristle extract. [Copyright &y& Elsevier]

Abstract: The hemorrhagic syndrome caused by Lonomia obliqua caterpillars is an increasing problem in Southern Brazil. The clinical profile is characterized by both hemorrhagic and pro-coagulant symptoms, constituting a paradoxical action of the venom. The effects upon blood coagulation and fibrin(ogen)olysis have been shown to result from the combined action of several active principles found mostly in the bristle extract. The present study reports quali-quantitative differences among L. obliqua secretions: Cryosecretion, hemolymph, bristle extract and tegument extract. Cryosecretion and hemolymph displayed strong amidolytic activity upon several substrates, presented moderated procoagulant activity and high fibrinogen degrading ability. Bristle and tegument extracts presented low amidolytic activity, but bristle extract showed the most potent procoagulant activity and both extracts presented low fibrinogen degrading ability. The differential involvement of these secretions during the accidents with L. obliqua can elucidate the different symptoms presented after envenomation. [Copyright &y& Elsevier]

Abstract: Acute renal failure (ARF) is a complication of envenoming by contact with caterpillars of the species Lonomia obliqua. A cohort study was conducted to evaluate development of acute and chronic renal failure (CRF) in patients who been in contact with L. obliqua during the period from 1989 to 2003 in the State of Santa Catarina, southern Brazil. Patients were evaluated in two distinct groups: those prior to 1995, who did not receive specific treatment of any type, and those after this date who were treated with antilonomic serum (SALon). The presence of creatinine ≥1.5mg/dl in patients who had no history of previous renal illness was considered to be indicative of acute kidney failure. Of the 2067 patients evaluated, 39 (1.9%) developed ARF. Eleven (32%) of these patients were treated with dialysis and four (10.3%) developed CRF. The seven deaths (4%) occurred in the period before treatment with SALon. Blood coagulation measures (TC, TP, and TTPA), were significantly longer in the group with ARF. All patients with ARF and 67% of the control group presented hematuria. The majority of the patients recovered prior renal function, however, four (10.3%) needed chronic dialysis. Envenoming by L. obliqua can be considered an important risk factor for the development of potentially fatal ARF, as well as for developing CRF. [Copyright &y& Elsevier]

Abstract: A severe hemorrhagic syndrome produced by contact with Lonomia obliqua caterpillars has become epidemic in southern Brazil. A significant thrombin production with intense consumption of fibrinogen and high D-dimer production indicates a consumption coagulopathy and secondary fibrinolysis in patients. Lopap is a single-chain 69kDa serine protease isolated from the crude extract of L. obliqua bristles. Experiments in mice showed that the purified protein, similar to the crude extract, causes uncoagulable blood by fibrinogen depletion. In order to characterize the effects of Lopap on cells involved with hemostatic system, we performed experiments using human umbilical vein endothelial cells (HUVECs). Our results show that Lopap exerts a direct effect on endothelial cells by increasing the liberation of molecules involved in the regulation of vascular tone, inhibiting platelet activation and chemotaxis, apart from inducing the expression of cell adhesion molecules which participate in inflammatory responses. The release or new synthesis of mediators involved in coagulation as von Willebrand factor and tissue factor, or in fibrinolysis as tissue plasminogen activator, was not affected by Lopap. Also our results demonstrated that Lopap acts on cell survival of HUVECs, regulating the expression of molecules as NO and avoiding cell death. [Copyright &y& Elsevier]

Skin contact with Lonomia caterpillar bristles causes a consumptive coagulopathy. From a cDNA library we cloned and expressed a prothrombin activator (rLopap) in active form, and from the bristles extract we characterized a FX activator (Losac). Several clones were sequenced and analyzed by expressed sequence tags. A database of about 1,270 sequences was constructed and deposited in NCBI. Both the native protein from the venom (Lopap) and the recombinant form (r-Lopap) promoted prothrombin hydrolysis, generating prethrombin-2, F1.2 and thrombin. Losac is a single-chain (43 kDa) protein that cleaves the FX heavy chain producing FXaα. In HUVECs rLopap and Losac are able to modulate cell survival by preventing apoptosis. rLopap increases NO and PGI2 concentration and Losac induces t-PA expression. Finally, to identify the venom proteins related to human envenomation, a 2D electrophoresis map is being performed as an attempt to find the major toxins recognized by the anti-lonomia venom. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: By studying Lonomia obliqua (caterpillar) venom we were able to detect a lytic activity on purified hyaluronic acid. The venom hydrolyses purified chondroitin sulphate, but was unable to degrade either heparan sulphate or dermatan sulphate. Moreover, through purified hyaluronic acid-degrading kinetic assays, we observed that this lytic activity was caused by a hydrolase rather than lyase enzyme. In addition, by using the Reissig colorimetric reaction, we detected this hyaluronic acid hydrolase action as a β-endohexosaminidase enzyme originating terminal N-acetylglucosamine residues rather than β-endoglucuronidase, which may originate glucuronic acid residues. Zymogram analysis of the venom detected 49 and 53kDa molecules with hyaluronic acid lytic activity. An examination of these hyaluronic acid degrading activities as a function of pH showed that these hydrolases had no apparent activities at a pH below 5.0 and higher than 8.0 and displayed their optimal activities at pH ranging from 6.0 to 7.0. Finally, through a fluorescence reaction to hyaluronic acid and confocal microscopy, we confirmed this cleaving action upon hyaluronic acid organised on the extracellular matrix of the dermis of rabbit. The data provide experimental evidence of the presence of hyaluronidases in the L. obliqua venom, probably involved in the harmful effects of the venom. [Copyright &y& Elsevier]

Hemostatic disturbances are frequent findings in human accidents caused by Lonomia obliqua caterpillars in the southern region of Brazil. In severe envenomation, patients may present life-threatening bleedings. Such disturbances may be mimicked in rats, which also develop intravascular hemolysis. The scope of this study was to investigate the time-course and intensity of intravascular hemolysis induced by i.d. injection of 750μg/kg crude L. obliqua bristle extract in rats. Total blood cell count, reticulocyte count, plasma hemoglobin and haptoglobin assays were performed in control and envenomed rats at 1, 6, 24 and 48h after envenomation. Rats presented a drastic drop of haptoglobin levels at 1 and 6h with increased plasma hemoglobin levels, a decrease in packed cell volume values at 6, 24 and 48h, and increased reticulocyte counts throughout after envenomation. Such observations indicated that intravascular hemolysis occurred as early as 1h following envenomation, and lasted for more than 6h. Intravascular hemolysis is probably induced by phospholipase A2 and other proteins with direct hemolytic activity present in crude caterpillar bristle extract. [Copyright &y& Elsevier]

Human envenoming by caterpillars of the saturniid moth Lonomia obliqua in southern Brazil produces a mild local response (erythema, some edema, and pain) and systemic effects that include incoagulable blood, renal failure and in severe accidents intracerebral hemorrhage. In this work, we used light and electron microscopy to investigate the morphological alterations in the brain and blood–brain barrier of rats injected intravenously with venom from L. obliqua spicules (200μg/kg). Five semi-purified fractions of venom (200μg/kg each) were also assayed. Quantitative morphological and ultrastructural analyses were done 6, 18, 24 and 72h after the i.v. injection of venom and its fractions. Light microscopy showed that 6h after envenoming there was cerebellar edema, which decreased by 72h. Intracerebral hemorrhage occurred in only one rat 24h after the injection of venom. Blood–brain barrier (BBB) breakdown, assessed by transmission electron microscopy based on the passage of an extracellular tracer (lanthanum nitrate) between brain capillary endothelial cells, was observed in the cerebellum and hippocampus 18h after venom injection. At this time, the cerebellum was more sensitive to the venom than the hippocampus, as shown by the greater number of leaky vessels. The number of capillaries showing breakdown was lower after 72h than after 18h. None of the semi-purified fractions significantly increased the number of leaky vessels. These results indicate that L. obliqua caterpillar venom has a deleterious action on the rat BBB. The lack of effect of the venom fractions when administered alone suggested that a synergistic action of venom components may be responsible for the damage seen in the central nervous system, but this was not confirmed when three combinations of the fractions were tested. [Copyright &y& Elsevier]

The hemorrhagic syndrome caused by contact with Lonomia obliqua caterpillars has reached epidemic proportions in southern Brazil. This study aimed at assessing the efficacy of two different dosages of an antilonomic serum (SALon) in the treatment of patients who had accidental contact with L. obliqua caterpillars. A randomized, prospective controlled trial was conducted at São Vicente de Paulo Hospital, a tertiary hospital in Passo Fundo, Brazil. From January 2000 to April 2002, 44 patients with grade I or II hemorrhagic syndrome were randomly assigned to either group A: 22 patients treated with 10.5 mg total dose (three vials of SALon) or group B: 22 patients treated with 17.5 mg total dose (five vials of SALon). Treatment efficacy was assessed according to time necessary for blood coagulation to go back to normal, incidence of adverse reactions, and hospitalization time. There were no differences in clinical picture and laboratory findings between the two groups. Mean time for coagulation to go back to normal was 15.3±6.6 and 19.1±8 h in groups A and B, respectively. Adverse reactions and hospitalization time were similar in both groups. SALon was effective and well tolerated. This study demonstrated that three vials are as effective as five vials of SALon for the treatment of envenoming by Lonomia caterpillars. [Copyright &y& Elsevier]

Envenomation caused by Lonomia obliqua caterpillars is an increasing problem in Southern Brazil. The clinical profile is characterized by a profound hemorrhagic disorder. In the present study, we describe the characterization of a fibrin(ogen)olytic factor (lonofibrase) isolated from a venomous secretion of the caterpillars. The crude extract showed a dose-dependent inhibitory effect in the rate of thrombin-induced fibrinogen clotting and produced fragmentation of fibrinogen. Isolation of the fibrin(ogen)olytic enzyme was achieved by combining ion exchange chromatography followed by gel filtration in a fast protein liquid chromatography (FPLC) system. A single 35-kDa band was identified and the isolated enzyme named lonofibrase. Lonofibrase rapidly degrades Aα and Bβ chains of fibrinogen, also being able to cleave fibrin in a distinct way from that observed with plasmin. The presence of lonofibrase with both fibrinogenolytic and fibrinolytic activities in L. obliqua secretion is coherent with the severe hemorrhagic clinical profile resulting from envenomation caused by these insects. [Copyright &y& Elsevier]

The hemorrhagic syndrome caused by accidents with caterpillars of the genus Lonomia has been the focus of several clinical and biochemical studies, since its venom is composed of many active principles that interfere with the hemostatic system. Whereas a fibrinolytic agent has been characterized in the venom of Lonomia achelous, in Lonomia obliqua, only a prothrombin activator activity has been reported so far, even though both species cause similar bleeding disorders, characterized by hemorrhage, disseminated intravascular coagulation (DIC), and acute renal failure. Considering the possibility that the hemorrhagic syndrome resulting from envenoming by L. obliqua may be due to fibrinolytic and procoagulant activities acting together, we decided to investigate the effects of bristle extract (BE) of this species upon blood coagulation and fibrin(ogen)olysis. This study shows that besides a procoagulant activity related to the activation of prothrombin, the venom contains at least one fibrin(ogen)olytic activity, as shown by fibrinolysis in a fibrin (F) plate assay, by interference in thrombin-catalyzed fibrinocoagulation, and by polyacrylamide gel electrophoresis profile of fibrin and fibrinogen (Fg) degradation. Considering that a recombinant prothrombin activator from L. obliqua has been suggested in other studies to be used as an anti-thrombotic agent, it is important in the first place to better characterize the different active principles of this venom. [Copyright &y& Elsevier]

Human accidental envenomation caused by skin contact with the bristles of Lonomia obliqua caterpillar causes coagulation and fibrinolysis disorders. Alterations of hematologic parameters are observed only in severe cases of envenomation, but with no clinical evidence of intravascular hemolysis. However, since we have observed intravascular hemolysis in preliminary studies using Wistar rats as an experimental model for investigating L. obliqua envenomation, the objective of the present study was to investigate the in vitro hemolytic activity of the bristle extract of L. obliqua caterpillars on human and rat erythrocytes.Our results showed that the bristle extract has indirect and direct hemolytic activity on human and rat erythrocytes, although direct hemolytic activity was only observed at higher bristle extract concentrations. We also observed that the bristle extract has a proteolytic activity on band 3 of human and rat erythrocyte membranes. Thus, crude L. obliqua bristle extract was found to contain at least two components with hemolytic activity on erythrocytes, a phospholipase enzyme and another protein with a direct activity on the erythrocyte membrane. [Copyright &y& Elsevier]

Bauhinia ungulata factor Xa inhibitor (BuXI) inactivates factor Xa and LOPAP, a prothrombin activator proteinase isolated from the venom of Lonomia obliqua caterpillar bristles. The reactive site of the enzyme-inhibitor interaction was explored to design specific substrates for both enzymes. Methionine is crucial for LOPAP and factor Xa substrate interaction, since the change of both Met residues in the substrates abolished the hydrolysis. Synthetic sub-strates containing the sequence around the reactive site of BbKI, a plasma kallikrein inhibitor, were shown to be specific for trypsin hydrolysis. Therefore, these substrates may be an alternative in studies aiming at a characterization of trypsin-like enzyme activities, especially non-mammalian enzymes. [ABSTRACT FROM AUTHOR]

Envenomation caused by contact with Lonomia obliqua bristles is characterized by pain, an intense systemic proinflammatory reaction and disturbances in the coagulation cascade that can cause severe clinical manifestations and death. However, the role of immune system components in these effects is still poorly understood. In this study, we evaluated the cytotoxic effect of L. obliqua venom on THP-1-derived macrophages and its ability to modulate inflammatory markers, as well as the cytokine and chemokine release profile. Our results show that L. obliqua venom is able to directly exert a potent pro-inflammatory reaction in macrophages, characterized by the activation of the NF-κB transcription factor pathway, the expression of CD80 and CD83, and the release of pro-inflammatory mediators such as TNF-α, IL-1β, IL-6, IL-8 and CXCL10. These results suggest that macrophages can play an important role during the orchestration of the inflammatory response present in envenomation caused by Lonomia obliqua caterpillars. [ABSTRACT FROM AUTHOR]