Premolis semirufa (Walker, 1856) Envenomation, Disease Affecting Rubber Tappers of the Amazon: Searching for Caterpillar-Bristles Toxic Components

Pararama, the popular name of the larval form of the moth Premolis semirufa inhabits rubber plantations in the Amazon region and the accidental contact of the skin with the caterpillar's bristles or cocoons results in immediate and intense heat, pain, edema, and itching. In many cases a chronic inflammatory reaction with immobilization of the joints occurs. The current study has evaluated the biological and immunochemical characteristics of the Pararama caterpillar bristles extract. Electrophoretic analysis showed the presence of several components, including a very intense 82 kDa band. This latter component was endowed with intense gelatinolytic activity, as observed in zymography assays. Further analysis revealed that the extract also contained hyaluronidase activity but is devoid of phospholipase A2 activity. In vivo assays, using mice, showed that the extract was not lethal, but caused significant edema and induced intense infiltration of inflammatory cells to the envenomation site. The extract also induced high specific antibody titers, but no autoantibodies were detected. The data obtained, so far, demonstrate the existence of a mixture of different enzymes in the bristles of Premolis semirufa caterpillar, which can act together in the generation and development of the clinical manifestations of the Pararama envenomation

Immunochemical and biological characterization of the venom from caterpillar Premolis semirufa, etiological agent of pararamose, occupational disease of rubber tappers in the Amazon.

O contato com as cerdas da lagarta de Premolis semirufa (Pararama) desperta sintomas da inflamação aguda e nos indivíduos poliacidentados pode causar deformidades comuns às sinovites crônicas (pararamose). No presente estudo foi mostrado que o extrato das cerdas da lagarta apresenta intensa atividade proteolítica, sendo capaz de ativar o Sistema Complemento, promover hidrólise de C3, C4 e C5 e a geração de anafilatoxinas. Análises cromatográficas do extrato permitiram o isolamento de uma serinoprotease de 82 kDa capaz de promover tais atividades. Em modelo murino, foi verificado que o extrato é capaz de induzir altos títulos de anticorpos, pronunciada reação inflamatória, ativação de linfócitos T e APCs, bem como produção de citocinas pró-inflamatórias. Os dados obtidos demonstram a existência, no extrato das cerdas da pararama, de várias enzimas que podem atuar em conjunto na geração e desenvolvimento das manifestações clínicas da pararamose.The contact with the Premolis semirufas caterpillar bristles (Pararama) causes symptoms of the acute inflammation and, in the individuals after multiple accidents, joint deformities common to chronic synovitis (pararamose) can occur. In the current study it was shown that caterpillar bristles extract has intense proteolytic activity, being able to activate the Complement System, induce cleavage of C3, C4 and C5, and the generation of anaphylatoxins. Chromatographic analysis of the extract allowed the isolation of a serine protease with Mr of 82 kDa able to promote such activities. In murine model, it was demonstrated that the extract is able to induce high antibody titers, a pronounced inflammatory reaction, activation of T lymphocytes and APCs, as well as the generation of proinflammatory cytokines. The data obtained demonstrate the existence, in the pararama bristles extract, of numerous enzymes that can act together in the generation and development of clinical manifestations of pararamose

Enzymatic and Pro-Inflammatory Activities of Bothrops lanceolatus Venom: Relevance for Envenomation

Bothrops lanceolatus, commonly named ‘Fer-de-Lance’, is an endemic snake of the French Caribbean Island of Martinique. Envenomations by B. lanceolatus present clinical aspects characterized by systemic thrombotic syndrome and important local inflammation, involving edema and pain but limited hemorrhage. To investigate mechanisms of venom-induced inflammation, B. lanceolatus venom was characterized, its cross-reactivity with bothropic antivenom explored, its cytotoxicity on human keratinocytes and vascular cells, and the production of cytokines and chemokines were analyzed. We used electrophoretic separation, zymography, colorimetric or fluorimetric enzymatic assays, and immunochemical assays. Therapeutic South American bothropic antivenom cross-reacted with B. lanceolatus venom and completely or partially abolished its PLA2, hyaluronidase, and proteolytic activities, as well as its cytotoxicity for keratinocytes. The substrate specificity of B. lanceolatus venom proteases was emphasized. B. lanceolatus venom cytotoxicity was compared to the B. jararaca venom. Both venoms were highly cytotoxic for keratinocytes (HaCaT), whereas B. lanceolatus venom showed particularly low toxicity for endothelial cells (EAhy926). Patterns of cytokine and chemokine production by cells exposed to the venoms were highly pro-inflammatory. Thus, the results presented here show that B. lanceolatus venom toxins share important antigenic similarities with South American Bothrops species toxins, although their proteases have acquired particular substrate specificity. Moreover, the venom displays important cytotoxic and pro-inflammatory action on human cell types such as keratinocytes and endothelial cells, which are important players in the local and systemic compartments affected by the envenomation

Complement System Inhibition Modulates the Inflammation Induced by the Venom of Premolis semirufa, an Amazon Rainforest Moth Caterpillar

The caterpillar of the Premolis semirufa moth, commonly called Pararama, is found in the Brazilian Amazon region. Contact with the hairs can cause a chronic inflammatory reaction, termed “pararamosis”. To date, there is still no specific treatment for pararamosis. In this study, we used a whole human blood model to evaluate the involvement of the complement in the proinflammatory effects of P. semirufa hair extract, as well as the anti-inflammatory potential of complement inhibitors in this process. After treatment of blood samples with the P. semirufa hair extract, there was a significant increase in the generation of soluble terminal complement complex (sTCC) and anaphylatoxins (C3a, C4a, and C5a), as well as the production of the cytokines TNF-α and IL-17 and the chemokines IL-8, RANTES, MIG, MCP-1, and IP-10. The inhibition of C3 with compstatin significantly decreased IL-17, IL-8, RANTES, and MCP-1 production. However, the use of the C5aR1 antagonist PMX205 promoted a reduction in the production of IL-8 and RANTES. Moreover, compstatin decreased CD11b, C5aR1, and TLR2 expression induced by P. semirufa hair extract in granulocytes and CD11b, TLR4, and TLR2 in monocytes. When we incubated vascular endothelial cells with extract-treated human plasma, there was an increase in IL-8 and MCP-1 production, and compstatin was able to decrease the production of these chemokines. C5aR1 antagonism also decreased the production of MCP-1 in endothelial cells. Thus, these results indicate that the extract of the Pararama bristles activates the complement system and that this action contributes to the production of cytokines and chemokines, modulation of the expression of surface markers in leukocytes, and activation of endothelial cells

Venom from Bothrops lanceolatus, a Snake Species Native to Martinique, Potently Activates the Complement System

Bothrops lanceolatus snake venom causes systemic thrombotic syndrome but also local inflammation involving extensive oedema, pain, and haemorrhage. Systemic thrombotic syndrome may lead to fatal pulmonary embolism and myocardial and cerebral infarction. Here, we investigated the ability of B. lanceolatus venom to activate the Complement system (C) in order to improve the understanding of venom-induced local inflammation. Data presented show that B. lanceolatus venom is able to activate all C-pathways. In human serum, the venom strongly induced the generation of anaphylatoxins, such as C5a and C4a, and the Terminal Complement complex. The venom also induced cleavage of purified human components C3, C4, and C5, with the production of biologically active C5a. Furthermore, the venom enzymatically inactivated the soluble C-regulator and the C1-inhibitor (C1-INH), and significantly increased the expression of bound C-regulators, such as MCP and CD59, on the endothelial cell membrane. Our observations that B. lanceolatus venom activates the three Complement activation pathways, resulting in anaphylatoxins generation, may suggest that this could play an important role in local inflammatory reaction and systemic thrombosis caused by the venom. Inactivation of C1-INH, which is also an important inhibitor of several coagulation proteins, may also contribute to inflammation and thrombosis. Thus, further in vivo studies may support the idea that therapeutic management of systemic B. lanceolatus envenomation could include the use of Complement inhibitors as adjunct therapy

Characterization of Phenotypes of Immune Cells and Cytokines Associated with Chronic Exposure to <i>Premolis semirufa</i> Caterpillar Bristles Extract

<div><p>The Brazilian moth <i>Premolis semirufa</i> (Walker, 1856), usually called pararama, is a parasite of the rubber <i>Hevea</i> genus. Contact with the bristles causes symptoms of acute inflammation. A chronic inflammatory reaction frequently occurs in individuals after multiple contacts, and this reaction is characterised by articular synovial membrane thickening with joint deformities, common characteristics of chronic synovitis. Extract from the bristles has been shown to induce an intense inflammatory response in a murine model, and this reaction was characterised by the presence of neutrophils in the paw tissues of injected mice and a strong, specific antibody response. There is not yet an effective treatment for incidents involving contact with pararama. In this study, we evaluated the phenotype of the immunological response and cytokine production in BALB/c mice subcutaneously injected in the footpad with <i>P</i>. <i>semirufa</i> bristle extract or sterile saline (control) seven times at 15 day intervals. An analysis of cells from the draining lymph node by flow cytometry showed that the absolute numbers of TCD4, TCD8 and B lymphocytes, as well as the expression of activation molecules, were higher in the extract-treated group. Furthermore, immunohistochemistry and immunofluorescence analyses showed a mixed inflammatory infiltrate composed of neutrophils and macrophages at the inoculation site. In addition, an analysis of paw cytokines showed elevated levels of IL-6, IL-12, IL-10, IL-17 and IL-23 after the 7^th inoculation. In conclusion, these data provide evidence of pro-inflammatory changes in the phenotypes of immune cells and cytokine production in animals subjected to injections with an extract from <i>Premolis semirufa</i> bristles, which may explain the intense and prolonged inflammatory response that characterises this disorder.</p></div

Total number of CD3⁺CD4⁺CD44⁺ T cells and CD44 expression from <i>P. semirufa</i> group.

<p>BALB/c mice were repeatedly injected with 50 µL of pyrogen-free saline (□) or 10 μg (protein) of the extract (▪) into the footpad and, after the 1^st, 4^th and 7^th inoculations, the popliteal lymph nodes were collected and processed for flow cytometry analysis. (A) Total number of CD3⁺CD4⁺CD44⁺ T lymphocytes and (B) Median Fluorescence Intensity (MFI) of the expression of this molecule. All graphs show mean values ± SD. *<i>p</i><0.05, ** <i>p</i><0.01 and ***<i>p</i><0.001: significant differences between the mean values obtained with the saline group and the mean values of the <i>P</i>. <i>semirufa</i> group. The symbols indicate significant differences between the inoculations: 1^st×4^th (#), 1^st×7^th (&) and 4^th×7^th ($).</p

Determination of the total number of leukocytes from <i>P. semirufa</i> bristle extract-treated group.

<p>BALB/c mice were repeatedly injected with 50 µL of pyrogen-free saline with or without 10 μg (protein) of the extract in the footpad and, after the 1^st, 4^th and 7^th inoculations, the popliteal lymph nodes were collected and processed for flow cytometry analysis. (A) Total number of cells, (B) Total number of TCD4 lymphocytes, (C) Total number of TCD8 lymphocytes, (D) Total number of B lymphocytes and (E) Total number of other leukocytes. All graphs show mean values ± SD. *<i>p</i><0.05, ** <i>p</i><0.01 and ***<i>p</i><0.001: significant differences between the mean values obtained with the saline group and the mean values of the <i>P</i>. <i>semirufa</i> group. The symbols indicate significant differences between the inoculations: 1^st×4^th (#), 1^st×7^th (&) and 4^th×7^th ($).</p

Total number of CD40⁺, CD80⁺, CD86⁺ and MHC II⁺ B cells and their expression from <i>P. semirufa</i> group.

<p>BALB/c mice were repeatedly injected with 50 µL of pyrogen-free saline (□) or 10 μg (protein) of the extract (▪) in the footpad and, after the 1^st, 4^th and 7^th inoculations, the popliteal lymph nodes were collected and processed for flow cytometry analysis. Total number of (A) CD19⁺CD40⁺ B lymphocytes, (B) CD19⁺CD80⁺ B lymphocytes, (C) CD19⁺CD86⁺ B lymphocytes and (D) CD19⁺MHC II⁺ B lymphocytes. Expression of these molecules (MFI) is shown in panels E to H. All graphs show mean values ± SD. *<i>p</i><0.05, ** <i>p</i><0.01 and ***<i>p</i><0.0001: significant differences between the mean values obtained with the saline group and the mean values of the <i>P</i>. <i>semirufa</i> group. The symbols indicate significant differences between the inoculations: 1^st×4^th (#), 1^st×7^th (&) and 4^th×7^th ($).</p

Total number of CD3⁺CD4⁺IL-17⁺ T lymphocytes, IL-17⁺ cells and IL-17R⁺ cells from <i>P. semirufa</i> group.

<p>BALB/c mice were repeatedly injected with 50 µL of pyrogen-free saline (□) or 10 μg (protein) of the extract (▪) in the footpad and, after the 1^st, 4^th and 7^th inoculations, the popliteal lymph nodes were collected and processed for flow cytometry analysis. Total number of (A) CD3⁺CD4⁺IL-17⁺ T lymphocytes, (B) IL-17⁺ cells and (C) IL-17R⁺ cells. All graphs show mean values ± SD. *<i>p</i><0.05 and ***<i>p</i><0.0001: significant differences between the mean values obtained with the saline group and the mean values of the <i>P</i>. <i>semirufa</i> group. The symbols indicate significant differences between the inoculations: 1^st×4^th (#), 1^st×7^th (&) and 4^th×7^th ($).</p