Dissertations / Theses on the topic 'Inflammation Phospholipase A2'

Pour obtenir de nouveaux antiinflammatoires dépourvus d'effets secondaires, nous développons des inhibiteurs spécifiques de la sPLA₂-II, enzyme impliquée dans l'inflammation en amont des COX. Nous avons montré, in vitro, que les molécules synthétisées, comme le PMS 1062 (leader), inhibent spécifiquement l'activité des sPLA₂-II, -V, et -X et qu'ils sont peu cytotoxiques. Dans diverses cellules stimulées par le LPS(ou LPS+cytokines), le PMS 1062 inhibe la production du NO, de PGE₂, de TNF-α et d'II-6 ainsi que l'activation du NF-кB, mais reste sans effet sur les activités COX₂ et COX₁. En outre, le PMS 1062 abolit la production de H₂O₂, la chute du potentiel transmembranaire et la déplétion en glutathion induites par le LPS ainsi que la formation de MDA induite par le fer/ascorbate. Ces résultats attestent que le PMS 1062 possède une activité antiinflammatoire importante liée, d'une part, à l'inhibition des activités sPLA₂-II et NOSi via NF-кB, et d'autre part, à son pouvoir antioxydant.

cPLA2α – a central regulator of inflammation Bioactive lipids are central in regulating the inflammatory process and imbalance in lipid mediator signaling contributes to progression of pathological conditions such as atherosclerosis, allergy, autoimmunity, degenerative diseases and cancer. Phospholipase A2 (PLA2) enzymes release fatty acids such as arachidonic acid (AA) and a lysophospholipid from cellular membranes. Lysophospholipids can me metabolized to biologically active lipid mediators including platelet-activating factor (PAF). PAF is a potent mediator of inflammation, but can also exert a range of other physiological and pathophysiological processes including apoptosis, proliferation and cancer development. AA is a precursor of many bioactive lipid including prostaglandins such as prostaglandin E2 (PGE2), a potent immunoregulator and inducer of inflammation, fever and pain. In particular cytosolic phosholipase A2 (cPLA2α) is associated with inflammation and inflammatory disease as a main enzyme mediating AA release and proinflammatory eicosanoid production, and is proposed as a future therapeutic target. However, lipid signaling is complex and sophisticatedly regulated, and the downstream consequences of cPLA2α inhibition are not fully understood. The overall objective of this thesis was to investigate the role of PLA2 enzymes, in particular cPLA2α, and downstream lipid messengers in cellular signaling mechanisms involved in chronic inflammatory disease. In Paper I, we investigated the role of PAF in differentiated keratinocytes, a cellular model system for psoriatic skin. We found that PAF did not primarily induce pro-inflammatory signaling, but rather proliferative responses possibly linking the inflammatory response to re-epithelialization and wound-healing. In the second part of this thesis comprising Papers II-IV, we focused on the role of cPLA2α in regulating pro-inflammatory signaling pathways central in the pathogenesis of rheumatoid arthritis (RA). In Paper II, we found cPLA2α to regulate joint-destructive and pro-inflammatory effectors induced by tumor necrosis factor (TNF), a “master” cytokine in RA. In Papers III and IV, we investigated the role of cPLA2α in modulating TLR-induced signaling. TLRs constitute a central part in the innate immune system sensing invading pathogens and tissue injury. However, TLRs can also induce “sterile” inflammation by recognizing molecules derived from the host itself, and increased TLR activation is believed to contribute to the pathogenesis of a range of inflammatory and autoimmune diseases including RA. We found that cPLA2α regulates TLR-induced activation of the transcription factor NF-κB and expression of several pro-inflammatory mediators. We furthermore identified PGE2 and possibly other related prostanoids as actors in this mechanism. Taken together, our findings expand the understanding of cPLA2α as a central regulator of molecular mechanisms in chronic inflammation, and enlighten the potential role of cPLA2α and PAF in linking the inflammatory and proliferatory processes
cPLA2α - en sentral regulator i kronisk inflammasjon Lipider spiller en viktig rolle som signalmolekyler i inflammatoriske sykdommer som aterosklerose (hjerte- og karsykdom), revmatoid artritt, psoriasis, multiple sklerose og også i kreft. I dette forskningsprosjektet har vi undersøkt rollen til et enzym, cPLA2α og ulike lipider i molekylære mekanismer i kronisk inflammasjon, med tanke på utvikling av framtidige medisiner mot kronisk inflammatoriske sykdommer. PLA2-enzymer klipper løs fettsyrer fra fosfolipider i cellemembranen, og regulerer dermed produksjonen av en rekke ulike bioaktive lipider som platelet-activating factor (PAF) og prostaglandin E2 (PGE2). Både PAF og PGE2 er kjent som potente pro-inflammatoriske signalmolekyler, med de er også involvert i en rekke andre prosesser. I Del I av prosjektet undersøkte vi rollen til PAF i hudceller, som modellsystem for psoriasis. Vi fant at PAF primært induserte proliferasjon og migrasjon, og ikke inflammasjon. Dette kan bety at PAF i hud produseres som et signal som forbinder den inflammatoriske prosessen og sårheling, og kan potensielt også være involvert i patologisk hyperproliferasjon, som hudkreft. I Del II undersøkte vi hvordan cPLA2α regulerer inflammatorisk signalisering i leddhinneceller, som et modellsystem for revmatoid artritt. Vi fant at cPLA2α regulerer genuttrykk og produksjon proteiner og lipider relatert til inflammasjon, dannelsen av nye blodårer og ledd-destruksjon. Ved å hemme cPLA2α ble disse faktorene redusert, noe som kan være gunstig med tanke på sykdomshemmende effekt. Sett i sammenheng viser våre resultater at cPLA2α og lipider dannet nedstrøms dens aktivitet regulerer viktige prosesser i kronisk inflammasjon, prosesser som også er relatert til kreft. Molekyler som hemmer cPLA2α eller PAF-signalisering kan dermed representere nye medisiner mot kronisk inflammatoriske sykdommer som revmatoid artritt og psoriasis, og potensielt også kreft.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on March 25, 2010). Vita. Thesis advisor: Grace Y. Sun. "December 2009" Includes bibliographical references

In the presented study, we demonstrate that the interaction of group IVA cytosolic phospholipase A2 and ceramide-1-phosphate is crucial for production of eicosanoid synthesis in inflammation. Inflammation is a critical component of many disease states including anaphylaxis, cancer, cardiovascular disease, rheumatoid arthritis, diabetes and asthma. Eicosanoids are well established mediators of inflammation, and the initial rate limiting step in the production of eicosanoids is the liberation of arachidonic acid (AA) from membrane phospholipids by a phospholipase A2 (PLA2). The major phospholipase involved in this liberation of AA during the inflammatory response is group IVA cytosolic phospholipase A2 (cPLA2α). Previous studies from our laboratory demonstrated that the bioactive sphingolipid, ceramide-1-phosphate (C1P), binds cPLA2α at a three amino acid sequence, which is located in the cationic β-groove of the C2 domain of cPLA2α. In this study we examined the effects of the genetic ablation of ceramide kinase (CERK) on eicosanoid synthesis, as CERK is the only known enzyme to produce C1P in mammalian systems. We utilized primary mouse fibroblasts (MEFs) and macrophages isolated from CERK-/- and +/+ mice. The ceramide-1-phosphate and eicosanoid profiles were investigated, and both ceramide-1-phosphate and eicosanoid levels in CERK-/- MEFs were found to be dysregulated. This study also presents the development of a global eicosanoid method to analyze eicosanoids via LC-ESI-MS/MS. Using this new analysis method, we demonstrated that there are significant differences in eicosanoid levels in ex vivo CERK-/- cells when compared to wild type counterparts, but the effect of the genetic ablation of CERK on eicosanoid synthesis and the serum levels of C1P was not apparent in vivo.

Le travail présenté décrit certains aspects du fonctionnement de la phospholipase A2 (PLA2), enzyme clé du métabolisme de l'acide arachidonique, et donc de la libération des médiateurs lipidiques. Nous avons d'abord montré (BBRC, 1990, 168: 644) que la PLA2 peut catalyser la synthèse de phospholipides en milieu non aqueux. Cela indique que les conditions physico-chimiques du micro-environnement de l'enzyme peuvent participer à sa régulation. La PLA2 ne peut cependant pas catalyser de réaction de transacylation dans les conditions utilisées. Nous avons ensuite cloné la partie codante du gène d'une PLA2 secrétée non digestive à partir de placenta humain (BBRC, 1991, 178: 1298). Ce gène est identique à celui isolé à partir de tissus inflammatoires. La surexpression de ce gène par transfection dans des cellules eucaryotes y entraîne une augmentation de la libération stimulée d'acide arachidonique. La PLA2 clonée peut donc interférer avec les mécanismes intracellulaires de transduction du signal. Enfin, nous proposons d'évaluer un mécanisme de régulation traductionnelle de l'adressage de la PLA2 pour rendre compte de sa présence dans la cellule.

L'athérosclérose est caractérisée par l'accumulation de lipides et la formation de cellules spumeuses par absorption de lipoprotéines de faible densité (LDL) modifiées dans la paroi vasculaire. La phospholipase A2 humaine sécrétée de groupe X (PLA2-GX) a la plus grande affinité hydrolytique pour la phosphatidylcholine. Notre équipe a montré qu’elle est exprimée dans les lésions humaines d’athérosclérose et que l’hydrolyse des LDL par PLA2-GX, (LDL-X) induit la formation de particules proathérogenes. Nous avons montré que la PLA2-GX hydrolyse efficacement le Platelet-Activating-Factor (PAF), un puissant médiateur proinflammatoire. Ces résultats suggèrent que le PAF peut être un substrat pour cette enzyme dans des conditions physiologiques ou physiopathologiques. Afin de déterminer si la PLA2-GX joue un rôle étiologique dans l’athérosclérose, nous avons genotypé des polymorphismes de la PLA2-GX dans l’étude cardiovasculaire ATHEROGENE. Nos résultats montrent qu’un polymorphisme est lié avec les événements cardiovasculaires et nous montrons qu’un polymorphisme non synonyme, est responsable d’une forte diminution de l’expression et de l’activité de PLA2-GX. De plus, l’analyse transcriptomique (microarray) de cellules endothéliales en présence de LDL-X montre la régulation d’un nombre important de gènes impliqués dans l’inflammation. D’autre part, les LDL-X ont un effet important sur la réponse calcique des cellules et l’expression de gènes cibles de la voie UPR (Unfolded Protein Response). L’ensemble de ces résultats contribue à mieux définir le rôle de la PLA2-GX dans la phase inflammatoire de l’athérosclérose et identifie de nouvelles cibles moléculaires.

Orientadores: Sergio Marangoni, Luis Alberto Ponce Soto
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Uma grande variedade de toxinas provenientes de venenos animais tem sido largamente utilizada no estudo de mecanismos de ação e processos fisiológicos, sendo consideradas valiosas ferramentas moleculares. As serpentes peçonhentas expressam no veneno diversas proteínas muito estudadas e utilizadas clinicamente, apresentando diferentes graus de variabilidade inter e intraespecífica em suas composições e nos seus efeitos biológicos. O conhecimento obtido com os estudos destas moléculas tem grande importância clínica na compreensão dos processos fisiopatológicos envolvidos nos envenenamentos ofidicos e também acadêmico-tecnológica, devido à possibilidade do desenvolvimento de novos instrumentos moleculares utilizados na pesquisa e também de novos modelos moleculares para princípios ativos de drogas. Neste trabalho pesquisamos as atividades neurotóxica, miotóxica e inflamatória de uma PLA2 básica, D49, purificada do veneno da serpente Bothrops roedingeri após duas etapas cromatográficas, exclusão molecular em Sephadex G-75 e hidrofobicidade em HPLC de fase reversa em coluna 'mi'-Bondapak C-18. A BrTX-I apresentou massa molecular relativa em tomo de ~14 kDa (SDS-PAGE) e confirmada por espectrometria de massas (ESI-MS), em 14.358,69 Da. A análise da composição de aminoácidos da BrTX-I, revelou que esta é constituída aproximadamente por 120 resíduos aminoacídicos, com alto conteúdo de aminoácidos básicos e hidrofóbicos, resultando em um valor calculado de pI de 8,63. A presença de 14 resíduos de cisteína sugere a formação de sete pontes dissulfeto. A análise estrutural da BrTX-I foi realizada por ESI-MS e as regiões analisadas mostraram semelhança com outras PLA2 miotóxicas isoladas de venenos botrópicos. A BrTX-I apresentou alta atividade PLA2 e um comportamento tipo sigmoidal em baixas concentrações do substrato. Atividade PLA2 ótima da BrTX-I foi em pH 8,0 e temperatura de 37°C. A BrTX-I mostrou-se dependente de Ca2+ (mM) e na sua substituição por zn+2, Mn+2, Mg+2 e Cd+2 a atividade foi reduzida. O estudo da homologia sequencial da BrTX-I mostrou posições extremamente conservadas na molécula. Nas posições 1 e 2 há predominância da sequência de aminoácidos (DL), na posição 4 (Q). Uma das regiões altamente conservadas na sequência de aminoácidos das PLA2 é a alça de ligação ao cálcio, segmento ...YGCYCGXGG. Resíduos formando a alça de ligação ao cálcio e a rede catalítica da BrTX-I mostraram um alto grau de conservação, refletindo na manutenção da atividade. A região relacionada à atividade neurotóxica pré-sináptica (80-11 O) apresentou principalmente resíduos hidrofóbicos. Em preparações ex vivo, o veneno e a BrTX-I causaram rápido bloqueio da neurotransmissão na preparação biventer cervicis de pintainho de modo similar a outras Bothrops, sem alterar significativamente as respostas contraturantes à adição de AChe de KCl (5 e 20 'mi'g/mL), indicando atividade neurotóxica pré-sináptica. Em camundongos, a BrTX-I induziu miotoxicidade local, determinada pelo aumento nos níveis plasmáticos de CK e mostrou efeito pró-inflamatório analisado através da formação do edema de pata e liberação das citocinas IL-1 , IL-6 e TNF-'alfa'. Como BrTX-I produz um efeito inflamatório, a hidrólise de fosfolipídios pode ser relevante na fisiopatología do envenenamento
Abstract: A great variety of animal venom toxins has been widely used in the study of action mechanisms and metabolic processes, thus considered valuable molecular tools. Poisonous snakes contains in their venom several well studied and clinically used proteins, showing these venoms different intra or interspecific variability degrees in their composition and biological effects. The knowledge obtained with these molecules study, has a great clinical relevancy understanding pathophysiological process regarding snake envenomations, and also academic technological, due to the possibility to develop new molecular tools and new molecular models to study active principies of some drugs. ln this work, we study neurotoxic, myotoxic and inflammatory activities of BrTX-I, a basic PLA2, purified from Bothrops roendigeri snake venom after two chromatographic steps, using molecular exclusion chromatography (Sephadex G-75) and reverse phase HPLC on 'mi'-Bondapak C-18 column. BrTX-I showed relative molecular mass around 14 kDa (PAGE) and specific molecular mass of 14,358.69 Da was determined by ESl-MS mass spectrometry. The amino acid composition analysis showed that BrTX-I contains 120 aminoacidic residues with high content of basic and hydrophobic amino acids, resulting in a calculated pi value of 8. 63. The presence of 14 Cysteine residues, suggests the formation of seven dissulfide bonds. Structural analysis of BrTX-I PLA2, performed by ESI-MS showed high identity values when compared to other myotoxic PLA2, isolated from Bothrops snakes venoms. BrTX-I presented high PLA2 activity and showed a sigmoidal behavior at low substrate concentrations. The BrTX-I reached its maximal PLA2 activity at pH 8.0 and 37 °C. Maximum PLA2 activity required Ca2+ (mM) and substitution of Ca2+ by zn+2, Mn+2, Mg+2 or Cd+2 showed reduced enzymatic activity. Sequence homology studies of BrTX-I showed extremely conserved positions in the molecule. ln positions 1 and 2, there is a predominance of the amino acids sequence (DL), and in position 4 (Q). One of the highly conserved regions in the amino acid sequences of PLA2 is the Ca2+ -binding loop, segment ... YGCYCGXGG. Residues forming the Ca2+-binding loop and the catalytic network of BrTX-I PLA2 showed a high conservation grade, reflecting the non-decreased catalytic activity. The region related to the presynaptic neurotoxic activity (80-110), showed mainly the presence of hydrophobic residues. ln ex vivo studies, the whole venom and BrTX-I caused a fast blockade of the neuromuscular transmission in young chick biventer cervicis preparations m a similar way to other Bothrops species, without alters significantly the contractures induced by ACh and KCL at doses of 5 and 20 'mi'g/mL, respectively, indicating presynaptic neurotoxic activity. ln mice, BrTX-I induced local myotoxicity, determined by increase in CK serum leveis, and showed proinflammatory effects analyzed through edema-forming activity and citokines IL-1 , IL-6, and TNF'alpha' release. Once BrTX-I induces a strong pro-inflammatory effect, the enzymatic phospholipid hydrolysis may be relevant for envenomation pathophysiology
Doutorado
Bioquimica
Doutor em Biologia Funcional e Molecular

L'inflammation joue un rôle prépondérant dans bon nombre de pathologies. Elle débute par la libération d'Acide Arachidonique grâce à une enzyme appelée la Phospholipase A2. Cet acide est alors transformé sous l'action de multiples enzymes en médiateurs inflammatoires très puissants. A ce jour, les seuls médicaments capables d'inhiber la PLA2 sont les glucocorticoi͏̈des comme la cortisone, mais leurs effets indésirables reconnus ont incité la recherche à créer d'autres médicaments : les Anti-infammatoires non stéroi͏̈diens (AINS), inhibant des enzymes en aval de la PLA2 dans la réaction d'inflammation. Non dépourvus d'effets secondaires, les AINS n'ont pas cependant la propriété de bloquer les autres voies importantes de l'inflammation, enparticulier la formation du facteur d'activation des plaquettes (PAF). Basée sur des analogies avec le substrat, notre laboratoire a découvert depuis 1999 une série de molécules capables de bloquer spécifiquement la PLA2. [. . . ]
The inflammatory reaction is a benefic process because of its self-defense property. However it can induce serious complications qualified as inflammatory pathologies. SPLA2-IIA plays a pivotal role in the propagation and amplification of inflammation. In many pathological situations, circulating sPLA2-IIA level correlates with the severity and illness outcome. Human non pancreatic secretory PLA2 (hnps-PLA2) of group II is associated with pathologies as acute pancreatitis, rhumatisms, septic shock. . . But until now, its role is not completely clarified [. . . ]

The sphingolipid, ceramide-1-phosphate (C1P), directly binds and activates Group IVA cytosolic phospholipase A2 (cPLA2a) to generate eicosanoids. Due to the role of eicosanoids in wound healing, we choose to use our novel genetic mouse model expressing cPLA2a with an ablated C1P interaction site (KI) to examine the cPLA2a/C1P interaction in wound healing. Wound closure rate was not affected, but wound maturation was dramatically enhanced by loss of the C1P/cPLA2α interaction based on the following findings. Wounds in KI mice displayed: i) increased infiltration of dermal fibroblasts into the wound environment; ii) increased wound tensile strength; and iii) higher Type I/Type III collagen ratios. These findings were recapitulated in vitro as primary dermal fibroblasts (pDFs) from KI mice showed significantly increased collagen deposition and migration velocity compared to WT and KO pDFs. Additionally, the KI showed an altered eicosanoid profile of reduced pro-inflammatory prostaglandins (e.g., PGE2) and increased levels of specific HETE species (e.g., 5-HETE). Elevated 5-HETE levels promoted increased dermal fibroblast migration and collagen deposition. This “gain of function” role for the mutant cPLA2a was also linked to differential cellular localization of cPLA2α and 5-HETE biosynthetic factors. These studies demonstrate regulation of key in vivo biological mechanisms by a defined protein:lipid interaction and provide new insights into cPLA2a function.

Orientador: Marcos Hikari Toyama
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Mesmo após décadas do descobrimento dos anti-inflamatórios inibidores de fosfolipase continua a busca por novas moléculas que sejam capazes de apresentar esse efeito terapêutico sem a indução de efeitos colaterais. O objetivo deste estudo foi obter extrato polar de partes aéreas de Tithonia diversifolia, viabilizando um padrão de qualidade para esse extrato a partir da identificação de seus principais constituintes e após essa determinação, avaliar o seu efeito sobre a atividade de frações de fosfolipase A2 básicas secretórias (sPLA2) obtidas de Bothrops jararacussu (Bj) e Crotalus durissus ssp (Cd). Os extratos foram obtidos por infusão e decocção e após a identificação dos constituintes por cromatografia e LC/MS-MS, determinou-se que o extrato de melhor rendimento foi obtido por decocção e que seus principais constituintes são derivados do ácido cinâmico: cafeoil-glicosídico, tagitinina C e ácido quínico. A técnica de molecular imprint (MP) permitiu uma separação dos constituintes sendo uma metodologia prática. O extrato polar apresentou atividade anti-agregante plaquetária na concentração de 0,6 a 20µg/mL, frente ao estímulo de indução por trombina, o que representa que o chá não é tão inerte e que pode promover complicações em indivíduos com distúrbios de coagulação. A purificação de Bj e Cd foi realizada por técnicas cromatográficas, com rendimento de aproximadamente 30% (p<0,05) de sPLA2. Ensaio enzimático in vitro, que utilizou substrato cromogênico sintético de PLA2 o 4-nitro-3-octanoiloxi-benzóico (NOBA) identificou que o extrato polar foi capaz de reduzir em 60% (p<0,05) a atividade enzimática de sPLA2 de Bj, independente do tipo de tratamento aplicado. A redução da atividade de sPLA2 de Cd foi reduzida em 30% (p<0,05) quando a mesma foi incubada por 30 min em presença do extrato. As modificações dos efeitos patológicos causados por sPLA2s foram avaliadas por ensaios in vivo. A ação edematogênica de sPLA2 de Bj foi reduzida em 50% (p<0,05), quando esta estava em presença do extrato, independe de prévia incubação, já a mesma ação para sPLA2 de Cd foi reduzida drasticamente pelo extrato, no entanto, após 60 min da administração do agente indutor de inflamação, a capacidade miotóxica com consequente liberação de creatina-quinase (CK) foi modulada pelo extrato de forma distinta dependendo da fonte de sPLA2. Para sPLA2 de Bj o extrato promoveu uma atividade protetora de liberação de CK, pois reduziu a liberação da enzima quando foi aplicado intraperitoneal 30 min antes do agente agressor, no entanto, a sPLA2 de Cd apresentou capacidade reduzida de liberação de CK quando foi incubada com o extrato por 30min antes da aplicação do mesmo no músculo do cobaio. Diante da ação do extrato sobre a inflamação induzida por sPLA2 buscou-se, através de técnica da reação da polimerase em cadeia em tempo real (PCR em tempo real), determinar a influência do extrato na expressão de genes envolvidos na inflamação. Determinou-se que o gene Nf-Kb, embora responda a presença do extrato, é o que mais tardiamente é ativado (ou expresso), possivelmente por ser nuclear. Através desses ensaios foi possível determinar a ação desses extratos sobre a resposta inflamatória aguda induzida pela ação de sPLA2s de venenos de serpentes
Abstract: Even after decades of the discovery of the anti-inflammatory inhibitors of phospholipase, there is a continuous search for new molecules which are able to provide this therapeutic effect without inducing side effects. The aim of this study was to obtain polar extract of the aerial parts of Tithonia diversifolia, enabling a quality standard for this extract from the identification of its main constituents and after this determination, to evaluate its effect on the activity of fractions A2 phospholipase basic secretory (sPLA2) obtained from Bothrops jararacussu (Bj) and Crotalus durissus ssp (Cd). The extracts were obtained by decoction and infusion and after the identification of the chromatography and LC / MS-MS. It was determined that the best yield of extract was obtained by decoction and its main constituents are cinnamic acid derivatives: caffeoyl - glycosidic, tagitinina C and quinic acid. Molecular imprint technique (MP) enabled separation of the constituents being a practical methodology. The polar extract showed anti-platelet activity at a concentration of 0.6 to 20 ?g/mL, opposite the stimulus induced by thrombin, which indicates that tea is not as inert and may promote complications in patients with coagulation disorders. Purification of Bj and Cd was performed by chromatographic techniques, with a yield of approximately 30% (p<0,05) of sPLA2. In vitro enzyme assay, which used synthetic chromogenic substrate of sPLA2 4-nitro-3-octanoyloxy benzoic acid (NOBA), it was identified that the polar extract was able to reduce by 60% (p<0,05) the enzymatic activity of sPLA2 Bj, regardless of the type of treatment applied, the reduction of the activity of sPLA2 Cd was reduced by 30% (p<0,05) when it was incubated for 30 min in presence of the extract. The modifications of the pathological effects caused by sPLA2s were evaluated by in vivo tests. The edematous action of sPLA2 Bj was reduced by 50% (p<0,05) when it was in presence of the extract, independent of incubation, since the same action for sPLA2 Cd was drastically reduced by the extract, however, after 60 min of administration of the agent inducer of inflammation, the myotoxic capacity with consequent release of creatine - kinase (CK) was modulated by the statement differently depending on the source of sPLA2, sPLA2 Bj to extract promoted a protective activity of CK release, because it reduced the release of enzyme, when applied intraperitoneally, 30 min before the offending agent, however, sPLA2 Cd showed reduced ability to release when CK was incubated with the extract for 30 min before application of the same muscle. Before the action of the extract on the sPLA2 induced inflammation was sought through the technique of polymerase chain reaction in real time (real time PCR), to determine the influence of the extract on the expression of genes involved in inflammation. We determined that Nf - Kb gene was the answer that although the presence of the extract is that the later is activated (or expressed), possibly because it was nuclear. Through these studies it was possible to determine the effect of these extracts on the acute inflammatory response induced by the action of sPLA2s from snake venoms
Doutorado
Bioquimica
Doutora em Biologia Funcional e Molecular

L’inflammation est l’une des premières lignes de défense ayant pour but d’éradiquer un pathogène lors d’un processus infectieux. Cependant, l’inhalation de Bacillus anthracis engendre une pathologie souvent fatale associée à un état septique et toxémique : c’est le charbon d’inhalation ou anthrax pulmonaire. Cette observation suggère que les systèmes de défense pulmonaire, comme l’inflammation, sont inefficaces vis-à-vis d’une infection par le bacille du charbon. Il était donc nécessaire d’identifier les mécanismes impliqués dans la dérégulation de la réponse inflammatoire. En absence de toxines, nous avons montré que le bacille du charbon a la capacité à induire l’expression de la phospholipase A2 sécrétée de type IIA (sPLA-II2) et de l’interleukine-8 (IL-8). Nous avons également démontré que ce microorganisme modifie le comportement des cellules épithéliales et phagocytaires en sécrétant les toxines œdématogène (ET) et létale (LT). Nous avons, en particulier, étudié les mécanismes moléculaires et cellulaires de l’inflammation qui expliquent, en partie, le maintien de l’intégrité pulmonaire. Par l’utilisation conjuguée de modèles expérimentaux de charbon pulmonaire et de modèles in vitro, nous avons pu identifier les mécanismes par lesquels ET et LT dérégulent différents aspects de la défense anti-infectieuse pulmonaire. ET inhibe l’expression de la sPLA2-IIA, enzyme inflammatoire produite par les macrophages alvéolaires et possédant un fort pouvoir bactéricide vis-à-vis de B. Anthracis. Notre étude nous a également permis d’identifier la voie AMPc/PKA comme étant la voie de signalisation par laquelle ET réprime l’expression de la sPLA2-IIA. LT inhibe quant à elle l’expression de l’IL-8 probablement en régulant le degré de compaction de la chromatine. En inhibant la phosphorylation de l’histone H3, LT aurait ainsi la capacité à « verrouiller » l’accès de la région promotrice du gène codant pour l’IL-8 au facteur de transcription NF-B. Dans un contexte infectieux, nous suggérons qu’en fonction du cycle bactérien, B. Anthracis est capable d’induire la réponse inflammatoire caractérisée notamment par la sécrétion de la sPLA2-IIA et de l’IL-8. Puis, plus tardivement dans le cycle bactérien, cette bactérie peut inhiber l’expression de ces mêmes facteurs inflammatoires en sécrétant LT et ET lui permettant ainsi de proliférer au sein de l’organisme infecté.

Os venenos de serpentes contêm concentrações elevadas de fosfolipases A2 secretadas (sFLA2), que apresentam homologia com as FLA2s de mamíferos, cujos níveis estão aumentados em doenças inflamatórias. Neste estudo, investigou-se a ativação e a expressão de fatores envolvidos na formação de corpúsculos lipídicos (CLs) em células fagociticas e o papel desses fatores na resposta imune inata, induzida pela MT-III, uma sFLA2s de veneno. A MT-III induziu aumento dos níveis de triacilglicerol, colesterol e lisofosfolipideos e a ativação e expressão dos fatores PPAR-g, PPAR-d/b, SREBP2 e do CD36. Sob estimulo da MT-III, o receptor PPAR-b/d, as enzimas DGAT, ACAT e FAS foram relevantes para a formação de CLs e para a expressão da PLIN2. O CD36 participa da expressão da COX-2, sem modificar a liberação de PGE2. O TLR2 e a MyD88 foram essenciais para a formação de CLs e síntese da IL-1b e IL-10. Ainda, o TLR2 foi relevante para a liberação de PGE2, PGD2 e LTB4, enquanto MyD88 foi fundamental somente para a liberação de PGE2 e expressão da PLIN2, induzidas pela MT-III.
Snake venoms contain high concentrations of secreted phospholipase A2 (sPLA2) with homology to mammalian PLA2s, whose levels are elevated in inflammatory diseases. In this study, we investigated activation and expression of factors involved in lipid droplets formation (LDs) and participation that factors in the innate immune response induced by MT-III, sPLA2s from snake venom, in phagocytic cells. MT-III induced increase of triacylglycerol, cholesterol and lysophospholipids levels and activation and expression of factors PPAR-g, PPAR-d/b, SREBP2 and CD36. PPAR-b/d receptor, DGAT, ACAT and FAS enzymes were relevant to LDs formation and critical to PLIN2 expression induced by MT-III. CD36 participates in COX-2 expression without modifying PGE2 release stimulated by MT-III. TLR2 and MyD88 were essential to LDs formation and IL-1b and IL-10 synthesis stimulated by MT-III. Moreover, TLR2 was relevant to PGE2, PGD2 and LTB4 biosynthesis, while MyD88 is essential only for PGE2 release and PLIN2 expression induced by MT-III.

Neste estudo foram avaliados os efeitos da subunidade CB (FLA2 isolada do veneno da serpente C. d. terrificus) em células endoteliais quanto: 1) síntese de PGI2 e mecanismos e 2) mecanismos inibitórios sobre moléculas de adesão. Os resultados mostraram que a liberação de PGI2 induzida pela CB depende de COX-1, PGIS, cFLA2, ERK1/2, mas não de COX-2, NF-kB, p38, JNK, iFLA2, receptor IP, AC ou PKA. Em adição, a CB aumentou os níveis de PGIS, mas não de COX-1 ou COX-2. Ainda, a CB inibiu a expressão de ICAM-1 e VCAM-1, mas não de PECAM-1, induzidas pelo LPS. Em relação aos mecanismos da ação inibitória da CB, foi demonstrado que o PPAR-α e -β/δ, IP, AC, PKA e a PGI2 são relevantes para inibição de ICAM-1, mas não de VCAM-1, induzida pelo LPS. Além disso, a CB inibiu a expressão gênica de ICAM-1, VCAM-1, TNF-α e IL-6, induzida pelo LPS. Este estudo evidenciou importantes vias na ação inibitória da FLA2 crotálica, em um evento fundamental da resposta inflamatória e trouxe a melhor compreensão das atividades biológicas desencadeadas por esta FLA2 em células endoteliais.
In this study the effect of CB, a phospholipase A2 (PLA2) isolated from Crotalus durissus terrificus snake venom, in cultured endothelial cells was investigated, with focus on: i) biosynthesis of prostacyclin (PGI2) and related mechanisms, and ii) inhibitory mechanisms on expression of ICAM-1, VCAM-1 and PECAM-1 adhesion molecules. Results showed that COX-1, PGI2 synthase (PGIS), cPLA2, ERK1/2 and MEK1/2, but not COX-2, NF-kB, p38, JNK, iPLA2, IP receptor, cyclase adenylate nor PKA, are involved CB-induced PGI2 biosynthesis. In addition, CB up-regulated PGIS, but not COX-1 nor COX-2 protein levels. Moreover, CB was able to inhibit LPS-induced ICAM-1 and VCAM-1, but not PECAM-1 protein expression. PPAR-α and -β/δ, IP receptor, cyclase adenylate, PKA and PGI2, but not PPAR-γ, are essential for CB-induced inhibition of ICAM-1. Furthermore, CB inhibited LPS-induced gene expression of ICAM-1, VCAM-1, TNF-α and IL-6. Inhibition of these cytokines by CB may be related to down regulation of both VCAM-1 and ICAM-1 seen in endothelial cells incubated with this venom PLA2.

Ceramide-1-phosphate (C1P) is a bioactive lipid that has been implicated in many biological processes. Our laboratory has conclusively demonstrated its role in inflammation via activation of cPLA2α. The only known enzyme to date responsible for direct synthesis of C1P is ceramide kinase. Very little was known about this enzyme in terms of its enzyme kinetics and substrate specificity. As CERK is an enzyme that acts on membrane lipids, its kinetics cannot be studied using standard bulk dilutions methods. Thus we developed a surface dilution approach using Triton X 100 mixed micelles for studying the kinetics of CERK. We discovered that ceramide kinase has an affinity for naturally occurring long chain ceramides while ceramides containing shorter than 8 carbons are very poor substrates for the enzyme. Also of note is the discovery that there is no discrimination between the naturally occurring long chain ceramides leading to the conclusion that the preponderance of D-e-C16 C1P in cells are due to an availability effect. We also investigated the chain length specificity of interaction between C1P and cPLA2α. Our data indicate that cPLA2α is activated by C1P’s containing acyl chains longer than two carbons. The study showed C2 C1P as being unable to activate cPLA2α thus establishing a tool for the investigation of cPLA2α dependent and independent effects of C1P. In the course of the study we investigated the ethanol/dodecane delivery system as a means of safely delivering lipids to cells. Our data conclusively demonstrate that this delivery system successfully delivers lipids to the internal membranes where their biological action takes place and that at low lipid concentration (<1µM), is non toxic to cells. A significant technical hurdle in the study of C1P was the lack of accurate and reproducible method of quantitatively and qualitatively analyzing the lipid. Using a mass spectrometric approach we developed an accurate technique that now allows us to quantify the lipids in cells. Using this and radiolabeling studies we discovered evidence for production of C1P from S1P via an acyl transferase pathway. Further studies are currently being carried out to identify the enzyme/s responsible for this pathway.

A ação de fosfolipases A2 (FLA2s): miotoxinas (MTs) ?II e III, isoladas de Bothrops asper (VBa) e CB2, de Crotalus durissus terrificus (VCdt) e os venenos brutos, sobre a expressão de ciclooxigenases (COXs) e síntese de prostaglandina (PG) E2 e PGD2 foi avaliada. As MTs e VBa mas não CB2 e nem VCdt induziram a expressão de COX-2 por leucócitos. Em estudos in vitro ocorreram a liberação de PGs e expressão de COX-2, após a incubação de macrófagos (M?s) com FLA22s e, de neutrófilos (N?s) e M?s, com VBa. A CB2 induziu somente a liberação de PGs. A inibição de FLA2 citosólica (cFLA2), diminuiu os níveis de PG induzidos pelas MTs, mas não pela CB2, e não afetou a expressão de COX-2 induzida pelas MTs. O envolvimento do NF-?kB na expressão de COX-2 foi mostrado com inibidores. Em conclusão, MTs, CB2 e VBa estimulam a síntese de PGs in vivo e in vitro e MTs e VBa, mas não CB2, induzem a expressão de COX-2. O VCdt não afeta estes parâmetros. O efeito das MTs sobre a expressão de COX-2 e PGs é mediado pelo NF-kB e pela cFLA2, respectivamente. Os efeitos de CB2 na produção de PGs são independentes de cFLA2s e COX-2. O fato da MT-II ser destituída de atividade enzimática sugere que a atividade catalítica per se, não seja relevante para os efeitos observados.
Action of the phospholipase A2 (PLA22): myotoxins (MTs) -II and -III, from Bothrops asper (BaV) and CB2, from Crotalus durissus terrificus (CdtV) and these venoms on cyclooxygenases (COXs) and synthesis of prostaglandins (PGs) E2 and D2 were studied in vivo and in vitro. Intraperitoneal injection of sPLA22s and BaV but not CdtV released PGD2 and PGE2. MTs and BaV but neither CB2 nor CdtV induced expression of COX-2 by leukocytes. Release of PGs and expression of COX-2 occurred in vitro after incubation of macrophages (M?s) with PLA2 and neutrophils (N?) and M?s with BaV. CB2 induced only PGs release. Inhibition of cytosolic PLA2 (cPLA2), reduced PG levels caused by MTs, but not by CB2 while did not affect MTs-induced COX-2 expression. Involvement of NF-kB in COX-2 was showed using with inhibitors. In conclusion MTs, CB2 and BaV stimulate the synthesis of PGs in vivo and in vitro and MTs and BaV, but not CB2, induce COX-2 expression. VCdt does not affect these parameters. Effect of MTs on COX-2 is mediated by NF-kB, and on PGs by cPLA2. Effects of CB2 on PGs are independent of cPLA2 and OX-2. Since MT-II lacks catalytic activity the PLA2 activity per se is not relevant for activation of this cascade.

As fosfolipases A2 (PLA2s) catalisam a hidrólise de ácidos graxos na posição sn-2 das membranas fosfolipídicas e liberam, como subprodutos, ácidos graxos livres. As PLA2s do grupo IIA são encontradas em peçonhas de serpentes da família Viperidae e desempenham diversas atividades apresentando potencial miotóxico, neurotóxico, hemolítico, edematogênico, citotóxico, hipotensivo, anticoagulante, inibição/ativação da agregação plaquetária, bactericida e pró-inflamatório. Esse trabalho teve como objetivo o isolamento e a caracterização funcional de uma PLA2 isolada da peçonha de Bothrops jararaca. Para a purificação dessa proteína, denominada BJ-PLA2-I, foram necessários três passos cromatográficos consecutivos: cromatografia de exclusão molecular em Sephacryl S-200, cromatografia de troca iônica em Source TM 15Q/50mL e cromatografia de troca iônica em MonoQ TM 5/50 GL. A BJ-PLA2-I apresentou elevado grau de pureza por SDS-PAGE e por cromatografia de fase reversa C18, em HPLC. Apresentou ainda, características ácidas, com pI em torno de 4,4 e teve a sua massa molecular determinada por dois métodos, obtendo-se valores bem próximos de 14,8 kDa (SDS-PAGE) e 14,2 kDa (MALDI-TOF). Esse fato é comum considerando que a espectrometria de massas é um método mais preciso e determina de maneira mais exata a massa molecular. O sequenciamento N-terminal da BJ-PLA2-I resultou em 60 resíduos de aminoácidos. O alinhamento múltiplo com outras fosfolipases A2 de serpentes do mesmo gênero mostrou similaridade entre elas, mostrando identidade de 100% com a BJ-PLA2, fosfolipase A2 Asp-49, também isolada da Bothrops jararaca. Esse dado levanta a hipótese de que a BJ-PLA2-I purificada neste trabalho e a BJ-PLA2 se tratam da mesma proteína, entretanto essa hipótese só poderá ser confirmada quando a sequência completa da BJ-PLA2-I for obtida. Outros dados encontrados neste trabalho reforçam essa hipótese, isso porque, avaliando a atividade fosfolipásica, o efeito sobre as plaquetas e o pI, tanto a BJ-PLA2-I quanto a BJ-PLA2 apresentaram características semelhantes. A BJ-PLA2-I, sendo uma Asp-49, mostrou alta atividade catalítica e efeito inibidor da agregação plaquetária induzida por ADP (20,5 ?g/mL inibiu 50 % da agregação plaquetária). Ela também foi capaz de induzir a migração leucocitária após a administração de diferentes concentrações (5, 10 e 20 ?g/mL) da BJ-PLA2-I. Esse dado também foi encontrado no ensaio em que a concentração de 10 ?g/mL foi fixada e variou-se o tempo de 2, 4 e 24 horas, observando-se principalmente a migração de neutrófilos. Além disso, verificou-se a liberação das citocinas IL-6 e IL-1?, de proteínas totais e de prostaglandina E2 na reação inflamatória induzida pela BJ-PLA2-I. No entanto, não foi observado a produção de TNF-?, IL-10 e leucotrieno B4. A BJ-PLA2-I caracterizou-se como uma PLA2 pró-inflamatória, produzindo inflamação local aguda. A BJ-PLA2-I foi avaliada quanto ao seu potencial antitumoral em três linhagens celulares distintas (PBMC, HL-60 e HepG2). Observou-se que a enzima em questão possui baixo potencial antitumoral para a linhagem HL-60, reduzindo o número de células tumorais em apenas cerca de 20% nas concentrações testadas. Verificou-se pequena alteração na viabilidade celular das células de PMBC, nas maiores concentrações testadas (160 e 80 ?g/mL) e, na linhagem HepG2 não foi encontrada nenhuma alteração. Concluindo, as informações adquiridas neste trabalho são de suma importância para a melhor compreensão dos mecanismos envolvidos nas atividades biológicas desempenhadas pelas PLA2s. Além disso, a BJ-PLA2-I pode servir como modelo molecular para a formulação de fármacos mais eficazes a serem utilizados no tratamento de várias doenças.
Phospholipases A2 (PLA2s) catalyze the hydrolysis of fatty acids in the sn-2 position of membrane phospholipids, releasing free fatty acids as by-products. PLA2s of group IIA are found in snake venoms of the Viperidae family and perform various activities, including myotoxic, neurotoxic, hemolytic, edematogenic, cytotoxic, hypotensive, anticoagulant, inhibition/activation of platelet aggregation, bactericidal and proinflammatory effects. This work aimed at the isolation and functional characterization of a PLA2 isolated from Bothrops jararaca venom. For the purification of this protein, called BJ-PLA2-I, three consecutive chromatographic steps were used (size exclusion chromatography on Sephacryl S-200, ion exchange chromatography on Source 15Q/50 mL, ion exchange chromatography on MonoQ 5/50 GL). Confirmation of the purity of BJ-PLA2-I was evaluated by SDS-PAGE and reverse phase HPLC using a C18 column. BJ-PLA2-I has acidic characteristics, with pI around 4.4, and its molecular mass was determined by two methods, obtaining values close to 14.8 kDa (SDS-PAGE) and 14.2 kDa (MALDI-TOF). The N-terminal sequencing of BJ-PLA2-I resulted in 60 amino acid residues. Multiple alignment with other phospholipases A2 of snakes of the same genus showed high similarity between them, showing 100% identity with BJ-PLA2, an Asp-49 phospholipase A2 previously isolated from Bothrops jararaca venom. This finding raises the possibility that the PLA2 purified in this work is the same protein previously described (BJ-PLA2), however, this assumption can only be confirmed when the complete sequence of BJ-PLA2-I is obtained. Other data obtained in this study support this hypothesis, considering that the phospholipase activity, the effect on platelets and pI of both BJ-PLA2-I and BJ-PLA2 showed to be similar. BJ-PLA2-I, being an Asp-49 PLA2, showed high catalytic activity and inhibitory effect on the platelet aggregation induced by ADP (20.5 ?g/mL inhibited 50% of the platelet aggregation). It was also able to induce leukocyte migration after the administration of different concentrations (5, 10 and 20 ?g/mL) of BJ-PLA2-I. This fact was also found when the concentration of 10 ?g/mL was fixed and response times were varied (2, 4 and 24 hours), observing especially neutrophil migration. Furthermore, there was a release of IL-6 and IL-1?, total proteins and prostaglandin E2 in the inflammatory reaction induced by BJ-PLA2-I, however, the production of TNF-?, IL-10 and leukotriene B4 was not observed. BJ-PLA2-I was characterized as a proinflammatory PLA2 producing acute local inflammation. BJ-PLA2-I was evaluated for its antitumor potential on three different cell lines (PBMC, HL-60 and HepG2). It was observed that this enzyme showed a low antitumor potential on HL-60 tumor cell line, reducing the number of tumor cells in only about 20% at the concentrations tested. There was little change in cell viability of PBMC cells in the higher concentrations tested (80 and 160 ?g/mL), but no change was found on HepG2 tumor cell line. In conclusion, the information obtained in this work are of utmost importance for better understanding the mechanisms involved in the biological activities induced by PLA2s. Furthermore, BJ-PLA2-I may serve as a molecular model for the formulation of more effective drugs to be used in the treatment of various diseases.

Thesis (MSc)--University of Stellenbosch, 2006.
ENGLISH ABSTRACT: Both, the cytokine Tumor Necrosis Factor-α (TNF-α) and the enzyme cytosolic phospholipase A2 (cPLA2) are crucial driving forces in mediating the cellular inflammatory response and are involved in ischaemic injury. During an ischaemic insult, TNF-α is endogenously generated. Apart from the recognized effects of TNF- α, such as the induction of apoptosis, proliferation and differentiation, if present in low dosages, it also mediates cytoprotective effects. Upon activation, cPLA2 contributes to the ischaemic challenge with the generation of mediators of cellular injury and apoptosis. Upon stimulation, this calcium dependent enzyme translocates to the phospholipid compartment of the cell membrane and induces the hydrolysis of sn-2 ester bonds in phospholipids. It governs the release of free fatty acids and lysophospholipids and generates role players of inflammation. We suggest a role for cPLA2 in the TNF-α mediated cytoprotection, with a distinct phosphorylation and translocation pattern. Aims The involvement of cPLA2 in TNF-α mediated cytoprotection in the C2C12 murine skeletal muscle cell line in tolerance to ischaemia was examined. To investigate the nature of the cPLA2 phosphorylation pattern, the mitogen activated protein kinases (MAPKs) p38 and extracellular regulated kinase (ERK) as contributors to cPLA2 phosphorylation and activation, were examined at appropriate time points. To dissect out the cPLA2 interplay and dependencies with these MAPKs within the pathway context, the selective cPLA2 inhibitor arachidonyl trifluoromethyl ketone (AACOCF3) was employed and its effect on cell viability was examined. Fluorescence microscopy was used to substantiate cPLA2 activation, by assessing its cellular distribution, translocation and cell organelle target preference, using co-localization and z-stack techniques. In addition, the induction of the apoptotic pathway through analysis of caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage was examined. The role of caspase-3 in cPLA2 turnover was addressed employing the caspase inhibitor, Z-DEVD-FMK. Methods Cells were grown in Dulbecco’s Modified Eagles Medium (DMEM) with 10% fetal bovine serum (FBS), and incubated under 5% CO2 conditions, until 50%-70% confluent. Using DMEM supplemented with 1% horse serum, cell differentiation into myotubes was induced. Differentiated cells were preconditioned for 30 min classically, with 0.5 ng/ml TNF-α or the cPLA2 selective inhibitor AACOCF3 (10 μM) respectively. Followed by a 60 min washout period the cells were subjected to 8 hrs simulated ischaemia. Cellular viability; and cPLA2 phosphorylation- and translocation events were assessed using Western blots and advanced immunocytochemistry and imaging techniques. Results Preconditioning with TNF-α, ischaemic preconditioning; and the use of the cPLA2 inhibitor AACOCF3, attenuated the decrease is cell viability brough about by ischaemia. Western blot analysis indicates the induction of the apoptotic pathway with caspase-3 and PARP cleavage. A significantly reduced translocation of pcPLA2 to the nuclear region in the TNF-α preconditioned group compared to the ischaemic group, as reflected by reduced mean nuclear fluorescence intensity, was observed. A z-stack analysis confirmed that the nuclear and endonuclear region was the target organelle for cPLA2. 3-dimensional co-localazation analysis of pcPLA2 with the nuclear marker nucleoporin p62 mirrored these results. Discussion and conclusion Our results provide evidence that there is a role for cPLA2 in TNF-α mediated cytoprotection. Although we do not observe a differential activation pattern in terms of cPLA2 phosphorylation at various time points within the ischaemic event, and no differential inactivation of cPLA2 via caspase-mediated cPLA2 cleavage, we describe a differential cPLA2 translocation pattern, similar to that in IPC. Through inhibition of cPLA2 translocation, a functional cPLA2 inhibition might be achieved. This would imply inhibition of the inflammatory pathway and a subsequent reduction in the generation of inflammatory mediators. In addition we describe an effect of TNF-α preconditioning on the efficacy of the caspase inhibitor Z-DEVD-FMK. Our results shed light on the survival mechanisms employed by the ischaemically challenged cell in a setting of TNF-α mediated cytoprotection. This might lead to novel approaches in the context of inflammation treatment, through agents that control differential cPLA2 trafficking within the cell.
AFRIKAANSE OPSOMMING: Beide, die sitokien “Tumor Necrosis Factor-α (TNF-α)” en die ensiem, sitosoliese fosfolipase A2 (cPLA2) is uiters belangrike bemiddelaars van die sellulêre inflammatoriese respons en is verder ook betrokke by isgemiese selskade. TNF-α word endogeen gegenereer tydens ‘n isgemiese intervensie. Afgesien van ‘n verskeidenheid effekte, soos die inisiëring van apoptose, sel-proliferasie en - differensiasie, bemiddel dit ook selbeskermende meganismes indien dit in lae konsentrasies in die sel teenwoordig is. Na aktivering dra cPLA2 by tot die isgemiese intervensie deur die vorming van bemiddelaars van selskade en apoptose. Hierdie kalsium-afhanklike ensiem translokeer na die fosfolipied membraankomponent na stimulering en induseer die hidrolise van die sn-2 esterbinding in die fosfolipied. Die vrystelling van vry vetsure en lisofosfolipiede word sodoende bewerkstellig wat verder gemetaboliseer kan word tot inflammatoriese bemiddelaars. Ons stel voor dat cPLA2 ‘n rol in TNF-α bemiddelde selbeskerming speel en dat dit gepaardgaan met kenmerkende fosforilerings- en translokeringspatrone. Doelwitte Die rol van cPLA2 tydens TNF-α bemiddelde selbeskerming is in ‘n C2C12 skeletspiersellyn na blootstelling aan isgemie ondersoek. Die rol van die MAPKs, p38 en ERK, is ondersoek om vas te stel of hulle betrokke is by die aktivering van cPLA2. Die selektiewe cPLA2 inhibitor, AACOCF3, is gebruik om te bepaal of die fosforilering van MAPKs ook cPLA2-afhanklik is. Die sellulêre cPLA2 verspreiding, translokering en teiken selorganelle is ook ondersoek met behulp van fluoresensie mikroskopie deur gebruik te maak van ko-lokalisering en z-plaat tegnieke. Verder, is die indusering van die apoptotiese paaie ondersoek deur tegnieke wat kaspase- en PARP kliewing meet. Die kaspase inhibitor, Z-DEVD-FMK, is gebruik om vas te stel of kaspase-3 ‘n rol speel in cPLA2 kliewing in ons selmodel. Metodes Selle is gekweek in Dulbecco’s gemodifiseerde Eagles Medium (DMEM) waarby 10% fetale kalf serum (FBS) gevoeg is, en wat geïnkubeer is in 5% CO2 totdat dit 50%-70% konfluent was. Die selle is verder gedifferensieer in miobuise deur gebruik te maak van DMEM waarby 1% perdeserum gevoeg is. Gedifferensieerde selle is vir 30 min klassiek geprekondisioneer asook respektiewelik met 0.5 ng/ml TNF-α en die cPLA2 selektiewe inhibitor, AACOCF3 (10 μM). Na ‘n 60 minute uitwas periode is die selle blootgestel aan 8 h gesimuleerde isgemie. Sellulêre lewensvatbaarheid, cPLA2 fosforilering- and translokering is ondersoek deur onderskeidelik gebruik te maak van die “Western” klad metode en gesofistikeerde immunositochemiese beeld tegnieke. Resultate Prekondisionering met TNF-α, isgemiese prekondisionering asook inhibisie van as cPLA2 met die inhibitor, AACOCF3, het ‘n beduidende toename in sellewensvatbaarheid tot gevolg gehad. Daar is ook dmv die “Western” klad tegniek bewys dat apoptose geïduseer word deur middel van kaspase-3- en PARP kliewing. Daar is insiggewend minder translokasie van cPLA2 na die nukluêre fraksie in die isgemiese groep in vergelyking met die TNF-α geprekondisioneerde groep waargeneem (die gemiddelde nukluêre fluoreserende intensiteit is bepaal om voorafgaande feit te staaf). Die cPLA2 teiken organel is geverifieer as die nukleus en die endonukluêre gebied deur middel van z-plaat analises. Drie-dimensionele kolokaliserings analises van pcPLA2 met die nukluêre merker, nucleoporin p62 het hierdie resultate bevestig. Bespreking en Gevolgtrekking Ons resultate verskaf bewyse vir ‘n rol vir cPLA2 in TNF-α bemiddelde selbeskerming. Alhoewel daar nie ‘n differensiële aktiveringspatroon in terme van cPLA2 fosforilering tydens verskeie tydspunte in die isgemiese intervensie waargeneem is nie, en ook geen kaspase-3 bemiddelde kliewing van cPLA2 nie, word ‘n differensiële translokeringspatroon soorgelyk aan die isgemiese prekondisioneringsgroep, waargeneem. Funsksionele cPLA2 inhibisie kan dus moontlik bewerkstellig word deur inhibisie van cPLA2 translokasie. Die inflammatoriese respons kan dus moontlik so inhibieer word en die vorming van minder inflammatoriese bemiddelaars tot gevolg hê. Verder het TNF-α prekondisionering ook ‘n effek op die effektiwiteit van die kaspase-inhibitor, ZDEVD- FMK. Ons resultate werp ook lig op die meganismes wat deur selle onder isgemiese toestande uitgeoefen word tydens TNF-α bemiddelde selbeskerming. Hierdie resultate mag lei tot nuwe benaderings in die konteks van behandeling teen inflammasie deur gebruik te maak van middels wat cPLA2 translokering in die sel beheer.

La réaction inflammatoire est un processus généralement bénéfique pour l'organisme dans la mesure où elle assure sa défense. Cependant, elle induit aussi des complications sévères qualifiées de pathologies inflammatoires. Des anti-inflammatoires stéroïdîens ou non ont été synthétisés, mais leurs effets secondaires en limitent l'utilisation thérapeutique, Les PLA₂ humaines sécrétées non pancréatiques (snp-PLA2) jouent un rôle majeur dans la propagation et l'amplification de l'inflammation. Ces enzymes hydrolysent la liaison ester en position sn-2 des phospholipides et entraînent la libération de médiateurs lipidiques de l'Inflammation. Partant du PMS 1062 qui possède une forte lipophilie (log P = 7), des études de structure-activité ont conduit à la synthèse de molécules moins lipophiles. Les nouveaux PMS induisent une inhibition spécifique de la PLA2~il avec des concentrations inhibitrices de l'ordre de 31 nM à 2,5 μM. L'étude pharmaco-toxicologique réalisée sur des lignées cellulaires RAW 264. 7 et HepG2 nous a permis de préciser le mécanisme d'action anti-PLA₂ des nouveaux PMS. Nous montrons que ces composés sont non seulement dépourvus de toxicité in vitro mais protègent aussi contre le stress oxydant, la perturbation du fonctionnement mitochondrial et Tapoptose. Le PMS 1398 de la série des pipérazines est un puissant inhibiteur des HPLA₂ des groupes II, V et X. Moins toxique que le PMS 1062, le PMS 1398 maintient un bon fonctionnement mitochondrial par son effet antioxydant. Par ailleurs, avec un log P de 4,9 il offre aussi une meilleure biodisponibilité. Son activité pharmacoiogique nécessite cependant d'être vérifiée in vivo
The inflammatory reaction is generally a beneficial process. However, it also induces severe complications described as inflammatory diseases. To fight them, steroid and non-steroid anti-inflammatory drugs have been synthesized, but their side effects limit their therapeutic use, The human non-pancreatic secreted PLA2 (nps-PLA2), which are involved in inflammatory diseases, have a major role in their spread and magnification. These ubiquitous enzymes hydrolyze the ester link on the sn-2 position of phospholipids and induce the release of lipid mediators of inflammation. Based on the leader, PMS 1062, which has a high lipophilicity {log P = 7), structure-activity studies were conducted and led to less lipophilic molecules with a better bioavailability. Some new molecules induce a specific inhibition of PLA2-II with inhibitory concentrations from 31 nM to 2. 5|μM. The pharmaco-toxicological study conducted on macrophages and HepG2 cells, allowed us to clarify the mechanism of anti-PLA2 action of new compounds and their effects on inflammatory mediators. We have found that these potent inhibitors of HPLA₂are not only devoid of in vitro but also protect against oxidative stress, disruption of mitochondrial function and apoptosis. One of them, PMS 1398, in the piperazine series, is a potent inhibitor of HPLA2 groups II, V and X. It also appears as a better inhibitor of pro-inflammatory cytokines. Less toxic than PMS 1062, PMS 1398 maintains good mitochondrial function by its antioxidant effect With a log P of 4. 9 it also offers improved bioavailability. Its pharmacological activity requires, however, to be verified in vivo

Heightened and sustained neutrophilic airway inflammation is central to the pathogenesis of CF lung disease. Studies evaluating corticosteroids and ibuprofen have demonstrated potential benefits associated with antiinflammatory therapy. The challenge lies in finding drugs that combine effective antiinflammatory activity in the CF lung with an acceptable risk for adverse effects. Drugs targeting single cytokines or receptors might prove less effective due to the redundancies of inflammatory processes involved. Drugs affecting multiple molecules or key inflammatory pathway intermediates could be more effective, but their use will need to be weighed against the risk of impairing innate immunity. Indirect approaches to manage inflammation, such as neutralizing cytotoxic substances in the lung, could be used in combination with other approaches. Development of an orally active, small-molecule antiproteinase drug would be highly desirable in this regard. Rescue/correction of the Cftr defect is a longterm goal with the potential to address multiple facets of CF pathology, including inflammation. Combining one or more of these strategies with the existing treatments of pulmonary toilet and antibiotics may lead to improved clinical outcome.

As fosfolipases A2 (PLA2s) são enzimas que induzem vários efeitos farmacológicos e geralmente, correspondem a maior porcentagem do conteúdo protéico dos venenos de serpentes. Desta forma, o isolamento e a caracterização bioquímica, funcional e estrutural de PLA2s poderão gerar informações importantes para o melhor entendimento dos efeitos farmacológicos e de efeitos tóxicos ocasionados por estas proteínas. Através de dois métodos cromatográficos (troca-iônica em CM-Sepharose e hidrofóbica em Phenyl-Sepharose) foi isolada uma isoforma de fosfolipase A2 ácida presente na peçonha da serpente Bothrops moojeni, denominada de BmooPLA2. Quando submetida à eletroforese em gel de poliacrilamida com agente desnaturante, BmooPLA2 apresentou massa molar relativa de aproximadamente 14.000. A proteína isolada, BmooPLA2, possui uma única cadeia polipeptídica, pI~5,2, é rica em aminoácidos hidrofóbicos, ácidos e possui 14 resíduos de cisteína. Esta isoforma pH-termoestável apresentou alta atividade fosfolipásica. A enzima induziu edema moderado in vivo, na concentração de 25 g. Além disso, a BmooPLA2 foi capaz de inibir a agregação plaquetária de modo dose dependente e induzir efeito hipotensor nas concentrações de 15 e 30 g . Foi realizada também a construção da biblioteca de cDNA da glândula de peçonha da serpente Bothrops moojeni, onde o cDNA que codifica a proteína BmooPLA2 foi clonado e a proteína recombinante expressa em E. coli. Todos os ESTs (Expressed Sequence Tags) foram classificados de acordo com a homologia de sua estrutura primária com sequências conhecidas. As sequencias codificando para toxinas representaram cerca de 30% do total de sequências identificadas. De acordo com o transcriptoma, as toxinas mais expressas pela serpente Bothrops moojeni são as metaloproteases (SVMP), as quais correspondem a aproximadamente 77% das toxinas encontradas. A proteína recombinante apresentou a mesma sequência de aminoácidos, atividade fosfolipásica e efeito inibitório sobre plaquetas, observados para a proteína nativa BmooPLA2, sugerindo que a recBmooPLA2 foi expressa, purificada e reenovelada em sua forma ativa. Como nenhum estudo abordou a participação das PLA2s ácidas de Bothrops nos processos inflamatórios e os mecanismos envolvidos na liberação desses mediadores, particularmente os prostanóides, as toxinas nativa e recombinante foram avaliadas quanto ao seu efeito sobre a resposta inflamatória (ensaios sobre leucócitos in vitro), avaliando a expressão da enzima COX-2 e liberação do prostanóide PGE2, além de outros mediadores como TXB4 e LTB2, após a incubação com macrófagos isolados, in vitro. Com estes resultados foi possível uma melhor compreensão da composição da peçonha desta serpente, bem como um melhor entendimento da participação das PLA2s ácidas envenenamento ofídico, abrindo novas perspectivas para sua aplicação biotecnológica.
The phospholipase A2 (PLA2s) are enzymes that induce various pharmacological effects and usually correspond to a higher percentage of the protein content of snake venoms. Thus, isolation, biochemical, functional and structural characterization of PLA2s may generate important information for a better understanding of the pharmacological effects and toxicity induced by these proteins. Through two chromatographic steps (ion exchange on CMSepharose and hydrophobic in Phenyl-Sepharose) an acidic phospholipase A2 isoform was isolated from the venom of the snake Bothrops moojeni and named BmooPLA2. Its biochemical and partial functional characterization were also performed. When submitted to electrophoresis on polyacrylamide gel with denaturing agent (SDS-PAGE), BmooPLA2 presented relative molar mass of approximately 14,000. The isolated protein, BmooPLA2, has a single polypeptidic chain, pI ~ 5.2, is rich in hydrophobic amino acids and has 14 cysteine residues. This pH-thermostable isoform showed high phospholipasic activity. The enzyme induced moderate edema in vivo, at the concentration of 25 g. In addition, BmooPLA2 was able to inhibit platelet aggregation in a dose dependent manner and showed hypotensive effect at different concentrations (15 and 30 g). It was also carried out the construction of the cDNA library from the venom gland of the snake Bothrops moojeni, where the cDNA encoding the protein BmooPLA2 was cloned and a recombinant protein expressed in E. coli. All ESTs (Expressed Sequence Tags) were classified according to their primary structure homology with known sequences. The sequences coding for toxins accounted for approximately 30% of all identified sequences. According to the transcriptome, the majority of the toxins expressed by the snake Bothrops moojeni are metalloproteases (SVMP), which correspond to approximately 77% of the toxins found. The recombinant protein presented the same amino acid sequence, phospholipase activity and inhibitory effect on platelets, observed for the native BmooPLA2, suggesting that recBmooPLA2 was expressed, purified and refolded in its active form. Since no study has addressed the involvement of acidic PLA2s from Bothrops genus upon inflammatory processes and the mechanisms involved in the release of such mediators, particularly prostanoids, native and recombinant toxins were evaluated for their effects on the inflammatory response (essays on leukocytes in vitro), evaluating the expression of COX-2 and prostanoid release of PGE2, as well as other mediators as TXB4 and LTB2, after incubation with isolated macrophages, in vitro. With these results it was possible to better understand the composition of the venom of this snake, and a better understanding of the role of acidic PLA2s on the snake envenomation, opening new perspectives for its biotechnological application.

Orientador: Marcos Hikari Toyama
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Os Anti-inflamatórios não esteroidais (AINES) constituem uma classe de medicamentos com prescrição mundial, pois são fármacos que auxiliam na terapia de vários processos patológicos visto que oferecem ações farmacológicas: anti-inflamatória, analgésica e antipirética, como efeitos primários. A venda dos AINES no Brasil é de forma ''livre", porém, nos EUA é realizada somente sob prescrição médica. Apesar do grande número de informações acumuladas durante anos de pesquisa a respeito destas drogas sobre as atividades das Ciclooxigenases 1 e 2 (COX 1 e COX 2), respectivamente, do seu papel como ferramenta molecular para o estudo das vias biossintéticas das prostaglandinas e dos avanços em relação a sua síntese e síntese de novos AINES, não se tem conhecimento da atuação dessas drogas sobre a estrutura e função das Fosfolipases A2 secretórias (PLA2s), responsáveis pela produção do acido araquidônico precursor de vários tipos de derivados lipídicos biologicamente ativos e responsável pelo processo inflamatório. O objetivo deste trabalho é responder questões relacionadas à ação dos AINES sobre a ação de PLA2 de serpentes, responsáveis por eventos farmacológicos principalmente relacionados com edema. Esta atividade inflamatória pode ser desencadeada tanto por PLA2 enzimaticamente ativas, como não ativas, ambas induzindo o efeito de inflamação aguda por mecanismos ainda controversos. O trabalho utilizou PLA2 farmacologicamente ativa (destacadamente no processo inflamatório), isoladas do veneno de serpentes Bothrops leucurus, em ensaios de cinética enzimática, atividade farmacológicas relacionados ao edema, agregação plaquetária, mionecrose e neurotoxicidade conduzidos em presença das toxinas puras, das toxinas em presença dos AINES, em ensaios com prévio tratamento dos AINES e posterior aplicação das PLA2. Para uma avaliação mais precisa das alterações induzidas por estes AINES (princípios ativos puros ou drogas comerciais) foram empregadas técnicas de varredura em espectrômetro de UV-Vis (220-700mn) para averiguar possíveis modificações do espectro de absorbância da PLA2 e do AINES após a incubação o que poderia indicar modificações estruturais na PLA2, induzidas por estas drogas. Além de avaliar a ação específica dos AINES sobre as PLA2, secundariamente, o trabalho possibilitou conhecer alguns aspectos físico-químicos e estruturais de PLA2 isoladas do veneno de Bothrops leucuros. Os resultados determinaram que, embora não ocorram interações estruturais entre os AINES e as PLA2, foram evidenciadas alterações de atividade farmacológica
Abstract: Anti-inflammatory drugs (NSAIDs) are a class of prescription drugs worldwide, as are drugs that help in the therapy of various pathological processes as they offer pharmacological actions: anti-inflammatory, analgesic and antipyretic, such as primary effects. The sale of NSAIDs in Brazil are ''free", but the U.S. is preferred only by prescription. Despite the large amount of information accumulated through years of research on these drugs on the activities of the cyclooxygenases 1 and 2 (COX 1 and COX 2), respectively, of their role as a molecular tool for studying the biosynthetic pathways of prostaglandins and advances in respect of its synthesis and synthesis of new NSAIDs, there are no known performance of these drugs on the structure and function of secretory phospholipase A2 (PLA2s), responsible for the production of arachidonic acid precursor of various biologically active lipid derivatives and responsible for the inflammatory process. This paper aims to answer questions related to the action of NSAIDs on the action of PLA2 of snakes responsible for pharmacological events mainly related to edema. This inflammatory activity can be triggered by PLA2 enzymatically active and non active, inducing both the effect of acute inflammation by still controversial. The paper uses PLA2 pharmacologically active (highlighted in the inflammatory process), isolated from Bothrops leucurus snake venom and Bothrops jararacussu in kinetics assays, pharmacological activity related to edema, platelet aggregation, and neurotoxicity myonecrosis conducted in the presence of pure toxins, the toxins in the presence of NSAIDs, previous treatment trials with NSAIDs and the subsequent application of PLA2. For a more accurate assessment of changes induced by these NSAIDs (pure active ingredients or drugs trade) is employed techniques of scanning UV-Vis spectrophotometer (220-700nm) to ascertain possible modification of the absorbance spectrum of PLA2 and NSAIDs after incubation which could indicate structural changes in PLA2, induced by these drugs. In addition to assessing the specific action of NSAIDs on PLA2 secondarily work enables to know some physical-chemical aspects and structures of PLA2 isolated from the venom of Bothrops leucurus. The results determine that although there occur structural interactions between NSAIDs and PLA2 activity were observed pharmacological changes
Doutorado
Bioquimica
Doutor em Biologia Funcional e Molecular

Os envenenamentos causados pelas serpentes do gênero Bothrops são os mais importantes do ponto de vista médico e econômico na América Central. Dentre estas, a serpente Bothrops asper é responsável por 90% dos envenenamentos ofídicos registrados no Panamá anualmente. Apesar da relevância médica e econômica, só a peçonha de populações de B. asper da Costa Rica e Guatemala tem sido estudadas em detalhes. Neste trabalho apresentamos a caracterização da peçonha da B. asper do Panamá e o isolamento, a caracterização funcional e estrutural de quatro fosfolipases A2 básicas denominadas MTX-I, MXT-II, MTX-III e MXT-IV e de uma fosfolipase A2acídica denominada Basp-I-PLA2. As fosfolipases A2 foram isoladas da peçonha em duas etapas usando cromatografia de troca iônica em CM-Sepharose (0,05 M NH4HCO3 pH 8,1), e cromatografia hidrofóbica em Fenil-Sepharose (0,05 M Tris-HCl pH 7,4) seguida de gradiente de concentração de 4 a 0 M NaCl no mesmo tampão a temperatura ambiente (25°C). A isoforma acídica demonstrou maior atividade catalítica que as isoformas básicas, quando atuou sobre fosfatidilcolina e fosfatidilglicerol. A focalização isoelétrica evidencia pIs de 8,1 a 8,3 para as MTXs e 4,6 para isoforma Basp-I-PLA2. A determinação da massa molecular por espectrometria de massa mostrou que MTX-1 14,156.5; MTX-2 14,249.5 e MTX-3 14,253.0 e Basp-I-PLA2 14,246.0 Da. As PLA2s (MTX-I, II, III e IV) induziram atividade miotóxica, reação inflamatória com migração de leucócitos ao músculo e ativação de macrófagos para exercer fagocitose e produção de superóxido. MTX-II exerceu um forte efeito citotóxico contra células tumorais JURKAT, C. albicans e E. coli. A fosfolipase A2 acídica, testada no plasma enriquecido com plaquetas, mostrou potente efeito inibitório na agregação plaquetária induzida pela ADP e colágeno. A análise de seqüência N- terminal demonstrou que as proteínas MTX-I, MTX-III e Basp-I-PLA2 pertencem à subclasse de fosfolipases A2 Asp49 cataliticamente ativas, enquanto que, MTX-II e MTX-IV pertencem à subclasse de fosfolipases A2 Lys49-homólogas cataliticamente inativas. Além disso, a sequência N-terminal das fosfolipases A2 básicas isoladas, demonstrou claramente que as miotoxinas isoladas neste trabalho são similares às miotoxinas previamente isoladas da peçonha da B. asper da Costa Rica. A Basp-I-PLA2 é uma nova fosfolipase A2 acídica isolada da peçonha de B. asper do Panamá e sua seqüência N-terminal revelou uma alta homologia com outras PLA2s acídicas Asp49 isoladas de peçonhas de serpentes.
Envenoming by Bothrops snakes is the most serious kind of envenoming from the medical and economical point of view in Central America. Bothrops asper snake is responsible for 90% of the snakebites registered in Panama every year. Despite its medical and economical relevance, only the venom of Costa Rica and Guatemala populations of this species has been studied to some detail, and there is very little information on intraspecies variability in venom composition and toxicity. In this study the crude venom of B. asper from Panama was characterized and its pharmacological and biochemistry activities were investigated with standard laboratory assays. Furthermore, we describe the isolation, functional and structural characterization of four basic phospholipases A2, named MTX-I, MTX-II, MTX-III, MTX-IV and a new acid phospholipase A2 named Basp-I-PLA2. The proteins were isolated from the crude venom by a combination of two chromatographic steps, using ion-exchange chromatography on CM-Sepharose (0.05 M NH4HCO3 pH 8.1 buffer), and hydrophobic chromatography on Phenyl-Sepharose (0.05 M Tris-HCl pH 7.4), followed by concentration gradient from 4 to 0 M NaCl at 25°C in the same buffer. Analyses of phospholipids hydrolyzed by these enzymes have shown that all phospholipases belong to type A2. The acidic isoform demostraded more catalytic activity than the basic PLA2s. This enzyme was more active on substrates as phosphotidylcholine and phosphatidylglycerol. The isoelectric focusing evidenced pIs beetwen 8.1 to 8.3 for the MTXs and 4.6 for the isoform Basp-I-PLA2. Its mol. Wt was estimated by Mass spectrometry to be MTX-1 14,156.5; MTX-2 14,249.5 and MTX-3 14,253.0 and Basp-I-PLA2 14,246.0 Da. The PLA2s (MTX-I, II, III and IV) induced myotoxic activity, inflammatory reaction mainly leukocyte migration to the muscle and activation of macrophages to exert phagocytic activity and production of superoxide. MTX-II, the most abundant one showed to be cytotoxic against JURKAT tumor cell line, C. albicans and E. coli. The acidic phospholipases A2 when tested in platelet rich plasma, showed a potent inhibitory effect on aggregation induced by ADP and collagen. The analysis of the sequence N-terminal demonstrated that the MTX-I, MTX-III and BASP-I-PLA2 belong to the subclass of Asp49 phospholipases A2 catalytically active, whereas, MTX-II and MTX-IV belong to proteins of the subclass of the enzymatically inactive Lys49 PLA2 s-like. In addition, a sequence of the region N-terminal of the PLA2s basic isolated, demonstrated clearly, that the isolated myotoxins in this work are similar of the previously isolated myotoxins of the snake venom Bothrops asper from Costa Rica. The Basp-I-PLA2 is a new acidic PLA2 and his sequence N-terminal revealed a high homology with other Asp49 acidic PLA2 s from snake venoms.

As fosfolipases A2 secretadas, particularmente do grupo II são abundantes nos venenos de serpentes, incluindo o gênero Bothrops e estão envolvidas em diversos processos fisiológicos e fisiopatológicos, como inflamação e dor, incluindo artrite. Contudo, não está totalmente caracterizado o papel da FLA2 para a gênese e manutenção dos quadros de inflamação articular. Nosso objetivo foi padronizar um novo modelo de artrite, utilizando sFLA2 do grupo IIA (miotoxina II) isolada do veneno da serpente Bothrops asper e avaliar a mediação química envolvida no processo nociceptivo deste quadro. Os resultados indicaram aumento de permeabilidade vascular, infiltrado celular e hiperalgesia. A hiperalgesia é um processo multimediado com a participação de prostanóides, sendo sua produção decorrente da ativação de FLA2 endógenas. Estes dados sugerem que esta FLA2 pode se tornar uma ferramenta científica importante para o entendimento dos mecanismos fisiopatológicos envolvidos nos processos de inflamação articular.
Secretory phospholipases A2 are abundant in different animal tissues and particularly group II are found in venom snakes and are proteins involved in many physiological and pathophysiological processes, like inflammation and pain, components of arthritis. However, the involvement of PLA2 in the genesis and maintenance of articular inflammation is not well characterized. Our aim is to characterize the articular inflammatory response induced by Lys 49-PLA2 (IIA group) isolated from B. asper snake venom. It was analyzed the nociceptive process involved, developing a new experimental model of articular inflammation. Our results indicated that sPLA2 induces increase in the vascular permeability, cell migration and hyperalgesia. Hyperalgesia is a multimediated process and prostanoids are involved in the nociceptive process, being its production dependent of the endogenous PLA2 activation. These data indicate that this PLA2 could be an important scientific tool for the understanding of the pathophysiological mechanisms involved in articular inflammation processes.

Les phospholipases A2 sécrétées (sPLA2) font partie d’une grande famille d’enzymes impliquées dans la synthèse d’écosanoïdes, de chimiokines et dans l’expression de molécules d’adhérence. Ce groupe comprend dix isoformes différentes (sPLA2-IB, -IIA, -IIC, -IID, -IIE, -IIF, -III, -V, -X et XII) dont la majorité sont surexprimées en présence de molécules pro-inflammatoires telles que l’interleukine-1β (IL-1 β) et le lipopolysaccharide bactérien (LPS). La sPLA2-IIA fut longtemps considérée comme la principale sPLA2 associée à l’inflammation. Toutefois, un nombre grandissant d’études suggère l’implication d’autres isoformes dans la réponse inflammatoire. Étant donné la similarité structurelle des différentes isoformes de sPLA2, la majorité des inhibiteurs présentement disponibles sont non spécifiques et bloquent simultanément plus d’une sPLA2. De ce fait, encore peu de choses sont connues quant au rôle précis de chacune des sPLA2 dans la réponse inflammatoire. Ayant accès à des souris génétiquement modifiées n’exprimant pas la sPLA2-V (sPLA2-V-/-), nous avons donc investigué le rôle spécifique de la sPLA2-V dans le recrutement leucocytaire induit par le LPS, ainsi que sa capacité à moduler l’expression de certaines molécules d’adhérence. Pour ce faire, nous avons utilisé le modèle inflammatoire de la poche d’air sous-cutanée. L’administration de LPS dans la poche d’air de souris contrôles (WT) entraîne un recrutement leucocytaire important. Cet appel de cellules inflammatoires est cependant significativement diminué chez les souris sPLA2-V-/-. De plus, l’expression des molécules d’adhérence VCAM-1 et ICAM-1 est également diminuée chez les souris sPLA2-V-/- comparativement aux souris WT. Nos résultats démontrent donc le rôle important de la sPLA2-V dans le recrutement leucocytaire et l’expression de molécules d’adhérence induits par le LPS, confirmant ainsi l’implication de cette enzyme dans le processus inflammatoire.
Secretory phospholipases A2 (sPLA2s) are well known for their contribution in the biosynthesis of inflammatory eicosanoids. These enzymes also participate in the inflammatory process by regulating chemokine production and protein expression of adhesion molecules. The majority of sPLA2 isoforms are up-regulated by proinflammatory stimuli such as bacterial lipopolysaccharide (LPS), which predominantly increases the expression of group V sPLA2 (sPLA2-V). Furthermore, it has recently been shown that sPLA2-V is a critical messenger in the regulation of cell migration during allergic airway responsiveness. Herein, we investigated the effect of sPLA2-V on LPS-mediated leukocyte recruitment and its capacity to modulate adhesion molecule expression. We conducted our study in the murine air pouch model, using sPLA2-V null mice (sPLA2-V-/-) and control wild-type (WT) littermates. We observed that LPS (1 μg/mL)-mediated leukocyte migration in sPLA2-V-/- was attenuated by 52 and 86% after 6 and 12 hours of treatment, respectively, as compared to WT mice. In WT mice, treatment with the cell-permeable sPLA2 inhibitor (12-epi-scalaradial; SLD) reduced LPS-mediated leukocyte recruitment by 67%, but had no additional inhibitory effect in sPLA2-V-/- mice. Protein analyses from the air pouch skin were carried out upon LPS-challenge, and the expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 were both significantly reduced in sPLA2-V-/- mice as compared to control WT mice. Together, our data demonstrate the role of sPLA2-V in LPS-induced ICAM-1 and VCAM-1 protein overexpression and leukocyte recruitment, supporting the contribution of sPLA2-V in the development of inflammatory innate immune responses.