Les cellules progénitrices endothéliales formant des colonies (EFCFs) sont issues decellules CD34+ de la moelle osseuse humaine. Peu de données concernent l’expression dufacteur tissulaire (FT) lors de cette différenciation endothéliale. Outre son rôle dansl’initiation de la génération de thrombine, le FT est impliqué dans l’angiogenèse.Nous montrons que les cellules CD34+ expriment le FT mais non ses isoformes. LesECFCs expriment peu de FT à l’état basal. En revanche, leur stimulation par le TNF-α induitune augmentation de l’expression de FT, et la génération de microparticules pro-coagulantes.Nous avons analysé les modifications fonctionnelles induites par cette stimulation. Nosrésultats montrent que l’expression de FT par les ECFCs est responsable d’une activité procoagulante majeure, alors que les propriétés angiogéniques ne semblent pas affectées.L’expression du tissue factor pathway inhibitor (TFPI) a été évaluée, ainsi que la capacité desmicroparticules issues de ECFCs à générer des métalloprotéinases (MMP2-, MMP-9).Une évaluation de la stabilité chromosomique des cb-ECFCs durant leur expansion aété réalisée, mettant en évidence des anomalies de nombre, mais pas d’anomalies destructures. Les conséquences de ces résultats en termes de thérapie cellulaire appliquées auxpathologies cardio-vasculaires sont discutées. Enfin, nous évoquons la possibilité deconsidérer l’expression de FT comme un marqueur de différenciation cellulaire. / Endothelial colony-forming cells (ECFCs) can be obtained from human bone marrowCD34+ cells. In spite of the essential role of the tissue factor (TF) in coagulation triggeringand angiogenesis, its expression during endothelial differentiation is not established. We showthat CD34+ cells express TF, but not TF splicing forms. ECFCs express a small amount of TFat baseline level. In contrast, ECFCs express TF high levels of TF on response to TNF-α andcan generated highly pro-coagulant microparticles. We have examined the functionalproperties induced by TNF-α stimulation. TF expression confers to ECFCs a strong thrombingeneration capacity without influencing their non-coagulant properties. We have examinedthe co-expression of the tissue factor pathway inhibitor (TFPI) and the ability of ECFCs togenerate microparticles producing metalloproteins (MMP-2, MMP-9).We have performed an evaluation of cb-ECFCs chromosomal stability during theirexpansion. We found quantitative but no structural chromosomal abnormalities. Theconsequences of our observations in the use of cell therapy in cardiovascular diseases arediscussed. We conclude that TF expression may be considered as cell differentiation marker

Monocytes are no longer regarded as a homogenous cell population but can be divided, both phenotypically and functionally, into different subsets. In rheumatoid arthritis, the subpopulation of CD14bright/CD16+ monocytes is expanded and prone towards generation of Th17 cells. CD56+ monocytes represent a different subpopulation, which is also expanded in conditions associated with autoimmunity like inflammatory bowel diseases. The aim of the study was the quantification and functional characterization of the CD56+ monocyte subset in rheumatoid arthritis (RA). The work at hand shows that the frequency of CD56+ monocytes is also expanded in RA; moreover, this subpopulation seems to increase with age in healthy controls. This age association is completely lost in patients suffering from RA. Further functional investigations could demonstrate a dysregulated cytokine response to lipopolysaccharide (LPS) with an increased production of pro-inflammatory cytokines like TNFα as well as an increased spontaneous reactive oxygen intermediate (ROI) production. A longitudinal treatment study using Etanercept as an established TNFα-blocking agent revealed a decrease of the frequency of that cell population under therapy. This decrease was more pronounced in patients with a good treatment response as judged by the reduction of the disease activity score (DAS) 28. Summing up those results, the CD56+ monocyte subset might be involved in immunosenescence as well as in the pathogenesis of RA.

Die vorliegende Untersuchung hatte zur Zielsetzung, bei Kühen die Unterschiede zwischen hypokalzämischen und normokalzämischen Festliegern zu analysieren. Es sollte geklärt werden, welche klinischen und labordiagnostischen Veränderungen, außer der Kalzium- (Ca) Konzentration, dem normokalzämische Festliegen zugrunde liegen. Des Weiteren wurden die TNF-α-, Haptoglobin- (Hp-) und TEAC-Konzentrationen in Beziehung zum Festliegen, den Mineralstoffkonzentrationen sowie hinsichtlich diagnostischer Information geprüft. Außerdem wurden die Festlieger mit Nachgeburtsverhaltung (Ret. sec.) und die Kühe mit Exitus letalis labordiagnostisch genauer analysiert.

This dissertation focuses on rationalised DNA design as a tool for the discovery and development of new therapeutic entities, as well as understanding the biological function of DNA beyond the storage of genetic information. The study is comprised of two main areas of study: (i) the use of DNA as a coding unit to illustrate the relationship between code-diversity and dynamics of self-assembly; and (ii) the use of DNA as an active unit that interacts and regulates a target protein. In the study of DNA as a coding unit in code-diversity and dynamics of self-assembly, we developed the DNA-Based Diversity Modelling and Analysis (DDMA) method. Using Polymerase Chain Reaction (PCR) and Real Time Polymerase Chain Reaction (RT-PCR), we studied the diversity and evolution of synthetic oligonucleotide populations. The manipulation of critical conditions, with monitoring and interpretation of their effects, lead to understanding how PCR amplification unfolding could reshape a population. This new take on an old technology has great value for the study of: (a) code-diversity, convenient in a DNA-based selection method, so semi-quantitation can evaluate a selection development and the population\'s behaviour can indicate the quality; (b) self-assembly dynamics, for the simulation of a real evolution, emulating a society where selective pressures direct the population's adaptation; and (c) development of high-entropy DNA structures, in order to understand how similar unspecific DNA structures are formed in certain pathologies, such as in auto-immune diseases. To explore DNA as an active unit in Tumour Necrosis Factor α (TNF-α) interaction and activity modulation, we investigate DNA's influence on its spatial conformation by physical environment regulation. Active TNF-α is a trimer and the protein-protein interactions between its monomers are a promising target for drug development. It has been hypothesised that TNF-α forms a very intricate network after its activation between its subunits and receptors, but the mechanism is still not completely clear. During our research, we estimate the non-specific DNA binding to TNF-α in the low micro-molar range. Cell toxicity assays confirm this interaction, where DNA consistently enhances TNF-α's cytotoxic effect. Further binding and structural studies lead to the same conclusion that DNA binds and interferes with TNF-α structure. From this protein-DNA interaction study, a new set of tools to regulate TNF-α's biological activity can be developed and its own biology can be unveiled.

Cette étude a pour objectif d'apporter des éléments de réponse à la compréhension de la nature et de l'origine des effets biologiques des nanoparticules (NP) de boehmite en réalisant des tests de contacts in vitro sur des macrophages de culture (RAW 264.7), les macrophages étant la cible cellulaire privilégiée au niveau du tractus respiratoire. Différents domaines de la réponse cellulaire en relation avec les propriétés physico-chimiques des particules ont été étudiés. Dans une première partie l’activité biologique a été mesurée sur des NP de boehmite puis après broyage ou dispersion permettant d’observer en particulier l’influence de la taille des particules dans la réponse cellulaire.Les résultats soulignent que les particules industrielles de boehmite sont principalement caractérisées par une activité modérée au niveau de la réponse inflammatoire, mais sans effet cytotoxique ou sans stress oxydant significatif. Des différences en fonction de la taille des particules ont été observées sur les paramètres inflammatoire et cytotoxique.Toutefois, les mesures des biomolécules libérées dans les surnageants de culture cellulaire peuvent être biaisées par l’adsorption de ces biomolécules sur les NP en présence. Ainsi, ce mécanisme d'adsorption doit être pleinement compris pour éviter une interprétation erronée des données obtenues. Dans un second temps l’objectif a donc été d’élaborer une méthodologie afin d’évaluer les interactions organo-minérales en terme d’affinité et de quantité de biomolécules adsorbées sur les nanoparticules à l’équilibre thermodynamique et de déterminer une loi de correction. Dans ce contexte nous nous sommes intéressés en particulier au TNF-α. / This study aimed at a better understanding of the biological effects of boehmite nanoparticles by carrying out in vitro tests using a macrophage-like cell line (RAW 264.7), as macrophages represent the first target cells in the respiratory tract. Different cellular responses: inflammation (evaluated by the release of TNF-α), cell death (assessed by the release of LDH), and oxidative stress (production of ROS) were studied in relation to physico-chemical properties of the particles. In the first part of this work, the biological activity was measured using boehmite nanoparticles obtained either from industrial environment or by hydrothermal synthesis, and after grinding or dispersion that enabled us to observe the influence of particle size on the cellular response.The results point out that industrial boehmite particles are mainly characterized by a moderate pro-inflammatory activity, no cytotoxic effect and no significant oxidative stress. Differences depending on the size of the particles were observed on the inflammatory and cytotoxic parameters.However, the assessment of biomolecules released into cell culture supernatants may be biased by the adsorption of biomolecules on the nanoparticles. Thus, the adsorption mechanism must be fully understood to avoid misinterpretation of the results. Therefore, the second aims of this work were to develop a methodology to assess the organo-mineral interactions in terms of affinity and quantity of biomolecules adsorbed on the nanoparticles at the thermodynamic equilibrium and to determine a correction law. We focused more specifically our study on TNF-α.

O objetivo do projeto foi desenvolver linhagens celulares para um anticorpo terapêutico anti-hTNFα e comprovar sua funcionalidade. Os genes anti-hTNFα foram clonados em células CHO para seleção da população estável mista, demonstrando expressão de anticorpo com reconhecimento de hTNFα em estrutura tridimensional. A população de transfectantes de maior produtividade específica foi escolhida para geração de linhagem monoclonal utilizando a tecnologia robótica ClonePix FL. Não houve diferença estatística entre o anti-hTNFα purificado e o produto de referência na cinética de ligação ao TNFα e reconhecimento diferencial por FcγRs em ensaios por SPR O ensaio de atividade funcional mostrou que o anti-TNFα desenvolvido pôde neutralizar a citotoxicidade induzida em células L929 e inibir a expressão de ELAM-1 em HUVEC. Os resultados finais permitiram identificar os três melhores clones, estáveis por 60 gerações. A comparabilidade entre o anti-TNFα desenvolvido e a referência permite admiti-lo como não inferior, um dos requisitos para o desenvolvimento de biossimilar. / The aim of the project was to develop a therapeutic anti-hTNFα antibody and evaluate its functionality. The anti-hTNFα synthezised genes were cloned into CHO cells and stable pools were selected, producing antibodies able to hTNFα three-dimensional structure recognition. The stable pools displaying higher antibody yields were the source for the generation of monoclonal lineage by ClonePix FL robotic technology. The clones selection proceeded using different criteria as cell density, specific productivity, fed-batch performance, kinetics measured by surface plasmonic resonance, hTNFα binding through ELISA, western-blotting and SPR, FcγRs binding by SPR. Besides, a small number of clones was tested in functional assays by the impairment of cytotoxicity of hTNFα over L929 cells and the inhibition of ELAM-1 expression by HUVEC. The long term stability testing allowed to finally select 3 top clones, not inferior to adalimumab reference by the above criteria.

Os papilomavírus são pequenos vírus de DNA dupla-fita, não envelopados, mucoepiteliotrópicos, capazes de infectar inúmeros vertebrados superiores de maneira espécie-específica. A infecção por estes vírus está associada a uma série de desordens proliferativas que levam desde de a formação de verrugas comuns até a do carcinoma invasivo. Aproximadamente 200 tipos de papilomavírus humano (HPVs) foram identificados, sendo que cerca de 40 deles infectam o trato genital. Dentre estes, os chamados HPVs de alto-risco estão associados etiologicamente ao carcinoma de colo de útero, enquanto que os de baixo-risco estão relacionados às lesões epiteliais benignas. A infecção por HPVs de alto-risco é muito comum, no entanto, a maioria destas é transitória e somente uma pequena proporção de mulheres desenvolvem o carcinoma. Entretanto, algumas mulheres são incapazes de eliminar esta infecção, levando a persistência viral e o conseqüente desenvolvimento da neoplasia. Para que a infecção pelo HPV persista é necessário um mecanismo de escape ao sistema imune do hospedeiro. O mecanismo de escape à resposta imune inata parece ser característico da infecção pelo HPV, pois o ciclo infeccioso deste vírus não promove inflamação. A infecção por HPV promove a liberação de citocinas, tal como o fator de necrose tumoral (TNF). Esta citocina possui um potente efeito citostático em queratinócitos normais e imortalizados com HPV, enquanto que em queratinóctos imortalizados com HPV18 este efeito não é observado. Do mesmo modo, observamos que a expressão do oncogene E6 de HPV16 ou 18 é suficiente para promover resistência ao efeito antiproliferativo do TNF em culturas em monocamada e organotípica. A expressão aumentada e contínua destes ocogenes é sabidamente o principal evento favorável ao desenvolvimento do câncer de colo de útero. Estas proteínas são essenciais na indução da transformação celular, visto que interferem na regulação do ciclo celular e apoptose. O produto dos genes E6 e E7 se liga ao produto dos genes supressores de tumor p53 e pRb, respectivamente, levando a sua degradação pela via de proteólise dependente de ubiquitina. As bases moleculares desta resistência ao TNF ainda são pouco conhecidas. Neste estudo, comparamos o efeito desta citocina em queratinócitos normais e que expressam E6 ou E7. Observamos através de cDNA Microarray a expressão de um grupo de genes, entre eles TCN1, DEK, HMGB2, INHBA, MCM2, MCM5 e MMP9, com expressão diferencial entre as células sensíveis e as resistentes ao TNF. / Papillomaviruses are small, non-enveloped, epitheliotropic, double-stranded DNA viruses that infect mucosal and cutaneous epithelia in a wide variety of higher vertebrates in a species-specific manner. Papillomavirus infections are associated to a series of proliferative disorders that range from common warts to invasive carcinomas. Almost 200 types of human papillomaviruses (HPVs) have been identified and approximately 40 of them infect the genital tract. Only the so-called high-risk HPV types mediate human carcinogenesis, whereas the low-risk HPVs have been linked to benign epithelial lesions. High-risk genital HPV infection is very common, and the majority of individuals clear their infection with time. However, a proportion of women cannot effectively clear the virus, and the persistence of a high-risk HPV is the major risk factor for the development of anogenital malignancies. To persist, HPV must escape the host immune system. Effective evasion of innate immune recognition seems to be the hallmark of HPV infections, since the infectious cycle is one in which viral replication and virion release is not associated with inflammation. Furthermore, HPV infections promote cytokine release, as tumor necrosis factor-alpha (TNF). This cytokine has a potent cytostatic effect on normal and HPV16 immortalized keratinocytes, while it does not affect HPV18 immortalized keratinocytes proliferation. In addition, we have observed that expression of HPV 16 or 18 E7 oncogene is sufficient to overcome TNF antiproliferative effect in monolayer and organotypic cell cultures. The increased and sustained expression of HPV oncogenes, E6 and E7, is the main contributor to the development of cervical cancer. Both E6 and E7 proteins are essential to induce and maintain cellular transformation, due to their interference with cell-cycle and apoptosis regulation. The most manifest function of the E6 protein is to promote the degradation of p53, while E7 is known to bind to and promote the proteasomal degradation of the retinoblastoma tumor suppressor gene product, pRb, and its family members. The molecular basis of TNF resistance is not well understood. In this study we compared the effect of TNF between normal and HPV16 E6 or E7 expressing keratinocytes. We observed by cDNA Microarray the differential expression of a common set of genes in TNF-sensitive cell lines, including TCN1, DEK, HMGB2, INHBA, MCM2, MCM5 and MMP9, that differs from those modulated in TNF-resistant cells.

Fragestellung: Wir gingen der Frage nach, inwieweit genetische Variationen im Gen für den Tumor Nekrose Faktor-alpha einen Einfluß auf die Krankheitsprogression oder die Entstehung bzw. Ausprägung HIV-assoziierter Erkrankungen haben. Methoden: Die Promotorregion sowie die kodierenden Sequenzen des TNF-alpha-Gens wurden mittels SSCP-Analyse auf genetische Variationen untersucht. Anschließend erfolgte die Charakterisierung der häufigsten bekannten Promotorpolymorphismen mittels Restriktionsfragment-Längenpolymorphismus-Analyse (RFLP). Die Bestätigung der Polymorphismen der RFLP-Analyse erfolgte an ausgewählten Proben durch DNA-Fluoreszenzsequenzierung. In sämtlichen Fällen handelte es sich um singuläre Basentransitionen von Guanin zu Adenin. Ergebnisse: Unter Einbeziehung verschiedener Progressionsparameter wie der CD4-Zellzahl, des Zeitraumes vom ARC-Stadium zum AIDS-Stadium und AIDS-assoziierter Krankheiten wie dem Wasting Syndrom und der HIV-Enzephalopathie, erfolgte anschließend die statistische Analyse in Korrelation mit den ermittelten Genotypen. Es zeigte sich bei keiner der statistischen Analysen eine signifikante Assoziation mit einem bestimmten TNF-alpha-Genotyp. Schlußfolgerung: Es ist kritisch anzumerken, daß für einige Subanalysen die Größe der untersuchten Patientengruppe zu gering war, um eine statistische Aussagekraft für seltene Allele zu erreichen. Anhand der hier vorgelegten Ergebnisse hat der TNF-alpha-Genotyp weder einen Einfluß auf die Progression der HIV-Erkrankung noch auf die Ausbildung HIV-assoziierter Erkrankungen wie dem Wasting Syndrom oder der HIV-Enzephalopathie. / Objective: We determined whether variation of the tumor necrosis factor-alpha gene had an impact on HIV disease progression or the prevalence of hiv-associated diseases. Methods: The promotor region of the TNF-alpha gene were examined with SSCP analysis for polymorphisms in the promotor region. The most common promotor polymorphisms were characterized with restriction fragment length polymorphism analysis (RFLP). To confirm RFLP results DNA fluorescence sequenzing analyses were performed with selected samples. In all cases with diagnosis of promotor polymorphisms single base transitions from guanine to adenine were confirmed. Results: Statistical analyses correlated the genotypes with different markers for disease progression e.g. CD4-count, the period from ARC to AIDS and the occurance of HIV associated diseases (wasting syndrome, hiv encephalopathy). In none of the statistical analyses significant association with a certain TNF-alpha genotyp could be demonstrated. Conclusion: For some subanalysis the sample sizes were too small in order to be able to make safe statistical statements concerning rare allels. Regarding our results, none of the examined tumor necrosis factor-alpha promotor polymorphisms had an impact on HIV disease progression or the prevalence of hiv-associated diseases.

Chronisch entzündliche Darmerkrankungen stellen eine Gruppe von chronischen, häufig in Schüben verlaufenden Erkrankungen mit rezidivierenden Entzündungen des Gastrointestinaltraktes dar. Es konnte gezeigt werden, dass eine gestörte Barrierefunktion einen wichtigen Schritt für die Pathogenese darstellt und dass das Zytokin Tumornekrosefaktor alpha (TNF) eine entscheidende Rolle dabei spielt. Die Rolle der kleinen GTPase RhoA bei der TNF-induzierten Barrieredysfunktion ist aufgrund der Komplexität der Signalwege nicht vollständig verstanden. Daher sollte der Einfluss von RhoA und der RhoA Effektorkinase PKN auf diese Prozesse in vitro mit Hilfe eines induzierbaren Expressionssystems untersucht werden, welches die kontrollierte Expression einer konstitutiv aktiven (KA) RhoA- und PKN-Mutante sowie einer dominant negativen (DN) PKN-Mutante ermöglichte. Die Induktion der KA RhoA Expression führte zu einer Störung der epithelialen Barriere. Eine simultane Interferon-gamma und TNF-Behandlung resultierte ebenfalls in einer gestörten Barrierefunktion, welche in KA RhoA Zellen weniger stark ausgeprägt war. Die TNF-Behandlung führte zu einer Aktivierung von PKN, weshalb dieses Protein ein Kandidat für die Vermittlung dieser Effekte darstellte. Inhibition von PKN mit Inhibitoren oder der Expression der DN Mutante führten zu einer Aggravierung der TNF-induzierten Barrieredysfunktion, welche durch eine Verringerung des transepithelialen elektrischen Widerstandes und eine erhöhte Ionenpermeabilität charakterisiert war. Diese Veränderungen wurden von einer Erhöhung des Myosin Leichtketten und NF-kappaB p65-Phosphorylierungsniveaus sowie von morphologischen Veränderungen begleitet. Im Gegensatz dazu konnten diese Veränderungen durch die Expression der KA PKN Variante abgeschwächt bzw. verhindert werden. Diese Ergebnisse liefern Hinweise auf eine potenzielle Rolle der RhoA Effektorkinase PKN bei der Modulation der TNF-induzierten Barrieredysfunktion in intestinalen Epithelzellen. / Inflammatory bowel diseases are relapsing systemic inflammatory diseases of the gastrointestinal tract associated with high morbidity and costs. A plethora of studies demonstrated that impaired intestinal barrier function is a key step in the pathogenesis of inflammatory bowel diseases and that the cytokine tumor necrosis factor alphpa (TNF) is of pivotal importance for this effect. Although the small GTPase RhoA has been implicated in the control of tight junction function, its role in TNF induced barrier dysfunction is not entirely understood due to the complexity of its downstream signaling pathways. Therefore, the contribution of RhoA and its effector kinase PKN on TNF induced barrier dysfunction was investigated in vitro. An inducible expression system that allowed the doxycyline controlled expression of a constitutively active (CA) RhoA and PKN mutant as well as a dominant negative (DN) PKN mutant was generated. Induction of CA RhoA expression led to an impaired epithelial barrier. Simultaneous Interferon-gamma and TNF treatment also resulted in barrier perturbation, but this defect was attenuated when CA RhoA was expressed. As treatment with TNF resulted in activation of the RhoA effector kinase PKN, this protein constitutes a candidate molecule for the mediation of these effects. Inhibition of PKN by inhibitory compounds as well as expression of a dominant negative PKN mutant aggravated TNF-induced barrier dysfunction, characterized by a decline in transepithelial electrical resistance and increased ion permeability. These alterations were accompanied by an increase in myosin light-chain and NF-kappaB p65 subunit phosphorylation level as well as morphological changes of the tight junctions. Conversely, expression of a CA PKN mutant attenuated or prevented these changes. These results provide support for a potential role of the RhoA effector kinase PKN in modulating the barrier disrupting effects of TNF in the intestinal epithelium.

A terapia endodôntica visa o selamento biológico do complexo sistema apical, contribuindo para isso, as substâncias usadas no tratamento e a resposta imune do paciente. A LILT tem mostrado atividade antiinflamatória, favorecendo o processo reparacional. Sendo assim, este trabalho objetivou analisar o efeito da LILT na atividade secretória de macrófagos, previamente ativados por IFN-? e LPS de E.coli, e estimulados por substâncias liberadas de três tipos de cimentos endodônticos, um a base de óxido de zinco e eugenol, outro a base de hidróxido de cálcio e um terceiro resinoso. A citotoxicidade dessas substâncias foi avaliada usando a técnica de análise do MTT. Macrófagos ativados foram estimulados por essas substâncias ou não (controle) e então, irradiados ou não (controle) e a secreção de proteínas próinflamatórias (interleucina-1 b, fator de necrose tumoral-a e metaloproteinase da matriz-1) foram analisadas pelo teste ELISA. As irradiações foram realizadas com um laser GaAlAs (780 nm, 70 mW, ponta da fibra de 4 mm2, 1.67 seg, 3 J/cm2). Foram usadas duas aplicações de irradiação com intervalo de 6 h. Os dados obtidos foram tratados por Análise de Variância, quando de distribuição normal, ou teste de Friedman, quando de distribuição não normal, com nível de significância de 5 % (p = 0,05). A viabilidade dos controles e células tratados pelos cimentos endodônticos foi similar. Produção de IL-1 b e TNF-a foram observadas. Houve alta produção de MMP-1. Entretanto, sem diferenças estatísticas entre os grupos experimentais. Os grupos irradiados apresentaram resultados similares aos não irradiados. Substâncias liberadas pelos cimentos endodônticos testados não se mostraram citotóxicas nas condições deste experimento. Essas substâncias, bem como a LILT, no parâmetro utilizado, não causam alteração na atividade de secreção de MMP-1, IL-1 b e TNF-a por macrófagos ativados. / The endodontic therapy seeks the dental root canal biological sealing, depending on substances used in this process and patient’s defense immune factors. LILT has shown an anti-inflammatory activity, improving the periapical repair process. This in vitro study aimed to analyze the effect of LILT at the secretory activity of macrophages previously activated by interferon-gamma and lypopolisaccharide from E.coli, and stimulated by substances leached from three endodontic sealers (zinc oxide-eugenol based, resinous and calcium hydroxide-based). Cytotoxicity of these substances was assessed by the MTT test. Activated macrophages were stimulated by the substances or not (control) and then, irradiated or not (control) and the secretion of pro-inflammatory proteins (interleukin-1 b, tumor necrosis factor-a and matrix metalloproteinase-1) was analyzed by ELISA test. The LILT was performed using a GaAlAs laser (780 nm, 70 mW, focal spot of 4.0 mm2, 1.67 sec, 3 J/cm2). Two irradiations with 6 h-intervals were done. The data was compared by either ANOVA test or Friedman’s test. The cell viabilities of controls and cells treated by the sealers were similar. Production of IL -1 b and TNF-a were observed. There was a high production of MMP-1. However, statistical differences were not observed amongst the groups. The irradiated groups presented results similar to those of non irradiated groups. Substances leached from the endodontic sealers are non cytotoxic at these experiments conditions . These substances, as well as the LILT, at the parameter used, were not able to change the secretion of MMP-1, IL-1 b e TNF-a by activated macrophages.