RESUMO
Neutralizing monoclonal antibodies against three major toxic components of Bothrops atrox venom were produced and tested. The mAbs against phospholipase A2, hemorrhagic metalloprotease, and thrombin-like enzymes were produced in large amounts and purified with caprylic acid followed by ammonium sulfate precipitation. Purified mAbs were analyzed by SDS-PAGE and their ability to neutralize the respective toxins was tested. Five Swiss mice were injected i.p. with 13.5 mg of pooled mAbs and challenged via s.c. route with venom. Survival rate was recorded for the next 48 h. All mice treated and challenged with venom survived, whereas only one mouse in the control group survived. Bleeding time in mice treated with mAbs was similar to that observed in control mice. Our results show that monoclonal antibodies neutralized the lethal toxicity of Bothrops venom and indicate that there is a reasonable possibility of developing antivenoms based on humanized mAbs to treat victims of venomous animals in the future.
Assuntos
Anticorpos Monoclonais/farmacologia , Antivenenos/farmacologia , Bothrops , Venenos de Crotalídeos/antagonistas & inibidores , Sulfato de Amônio/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Antivenenos/imunologia , Coagulação Sanguínea/efeitos dos fármacos , Caprilatos/metabolismo , Venenos de Crotalídeos/imunologia , Venenos de Crotalídeos/toxicidade , Eletroforese em Gel de Poliacrilamida , Camundongos , Testes de NeutralizaçãoRESUMO
Chagas disease is caused by Trypanosoma cruzi and affects 18 million people in Central and South America. Here we analyzed the exposure of phosphatidylserine by the different forms of the parasite life cycle. Only the infective trypomastigotes, but not the epimastigotes or intracellular amastigotes, expose this phospholipid. This triggers a transforming growth factor beta signaling pathway, based on phosphorylated Smad 2 nuclear translocation, leading to iNOS disappearance in infected macrophages. This macrophage deactivation favors the survival of this intracellular parasite. Thus, phosphatidylserine exposure may be used by T. cruzi to evade innate immunity and be a common feature of obligate intracellular parasites that have to deal with activated macrophages.
Assuntos
Macrófagos/parasitologia , Fosfatidilserinas/metabolismo , Trypanosoma cruzi/fisiologia , Transporte Ativo do Núcleo Celular , Animais , Macrófagos/metabolismo , Camundongos , Óxido Nítrico Sintase Tipo II/metabolismo , Transdução de Sinais , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America. The experimental murine model has been used to approach the disease, with susceptible and resistant mice strains that reproduce most of the main human immunological features. Since the hypergammaglobulinemia observed in susceptible mice and humans might have an influence on B1 cells, we investigated its role during the experimental infection with Paracoccidiodes brasiliensis. CBA/Nxid mice, deficient in B1 cells, and CBA/Nxid reconstituted with B1 cells isolated from the non-mutant CBA/J strain were infected with 106 yeast forms of P. brasiliensis. At the 8th and 22nd week post infection the DTH response of CBA/Nxid mice was significantly higher after 24 h of P. brasiliensis antigens inoculation and the specific humoral response was reduced, in comparison to CBA/J or recCBA/Nxid. Production of NAbs is a hallmark of the B1 subset. Higher Ig productions to auto antigens such as DNA, MBP and RBC were observed in CBA/J infected mice or recCBA/Nxid. Anti P. brasiliensis IgG2a was produced by CBA/Nxid mice early in infection, while CBA/J or recCBA/Nxid presented increased levels of this isotype only after the 8th week of infection. Furthermore, western blotting analysis showed that CBA/Nxid mice expanded less clones against P. brasiliensis antigens, with weakly detectable anti-gp43 antibodies while CBA/J mice produce IgM anti-gp43 at the 2nd week of infection and IgG anti-gp43 at the 2nd and 8th week. On the other hand, recognition of gp70, a fungal antigen that, as gp43, inhibits macrophage activation was not compromised in B1 deficient mice. These results suggest that B1 cells might have influence in the kinetic of production of protective isotypes of immunoglobulins and their repertoire that could contribute to an early drive towards a Th2 response, affecting the cellular response in susceptible mice during experimental paracoccidiodomycosis.
Assuntos
Anticorpos Antifúngicos/sangue , Subpopulações de Linfócitos B/imunologia , Suscetibilidade a Doenças , Isotipos de Imunoglobulinas/sangue , Paracoccidioides/imunologia , Paracoccidioidomicose/imunologia , Animais , Antígenos de Fungos/imunologia , Western Blotting , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Proteínas Fúngicas/imunologia , Glicoproteínas/imunologia , Hipersensibilidade Tardia , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Camundongos , Camundongos Endogâmicos CBARESUMO
Paracoccidiodomycosis (PCM) is a systemic mycosis that presents a wide spectrum of clinical manifestations caused by Paracoccidiodes brasiliensis. The experimental murine model has been used to approach the disease with susceptible and resistant mouse strains that reproduce most of the main human immunological features. We investigated whether the pattern of apoptosis of peritoneal cells from two polar strains of mice after infection with P. brasiliensis could be associated with the susceptibility or resistance to this pathogen. Apoptosis of A/J mouse cells (resistant), cultured in the presence or absence of LPS as stimuli, was observed as early as on the first day of infection. Cells from the infected susceptible strain BALB/c did not exhibit apoptosis in absence of LPS and persistently at a lesser degree than that observed in resistant mice. The apoptosis induced by the infection in resistant mice was not due to nitric oxide, since its blockage either in vitro or in vivo did not revert it. Analysis of additional strains of polar susceptibilities to PCM assured the dissociation of NO production and apoptosis. Interestingly, IL-6 and IL-10 were secreted in high amounts, by BALB/c cells and might be involved in shielding cells from apoptosis induced by P. brasiliensis. Furthermore, IFNgamma(-/-) mice did not show apoptosis of peritoneal cells while the Wt controls presented levels similar to those of A/J strain that secreted high amounts of IFNgamma and IL-1beta. The expression of Fas was increased in both strains and in Wt mice, whereas FasL was decreased in the susceptible strain and not significantly modulated in TNFRI and IFNgamma KO mice. These results suggest that apoptosis might be a mechanism of control of engagement of cells that could otherwise contribute to the susceptible phenotype observed in some strains of mice.
Assuntos
Apoptose , Paracoccidioides/imunologia , Paracoccidioidomicose/imunologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Proteína Ligante Fas/biossíntese , Imunidade Inata , Interferon gama/biossíntese , Interleucina-10/biossíntese , Interleucina-1beta/biossíntese , Interleucina-6/biossíntese , Macrófagos Peritoneais/fisiologia , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Camundongos Knockout , Óxido Nítrico/biossíntese , Paracoccidioidomicose/fisiopatologia , Receptor fas/biossínteseRESUMO
Toxoplasma gondii is an obligate intracellular parasite, able to disseminate into deep tissues and cross biological barriers, reaching immunoprivileged sites such as the brain and retina. In order to investigate whether the parasite uses leukocyte trafficking to disseminate throughout the host, the adhesive potential to extracellular matrix components, the expression of adhesion molecules and the in vivo migration of murine macrophages infected with RH strain of T. gondii were investigated. Cellular adhesion to fibronectin, laminin and collagen IV decreased after 24 h of T. gondii infection. However, the decrease in adhesion of infected macrophages observed at early infection was reversed after 48 h. Moreover, decreased adhesion was dependent on active penetration, since heat-killed parasites were unable to reproduce it. Expression of integrins alphaL, alpha4 and alpha5 chains was downmodulated early postinfection, but a progressive regain of expression was observed after 12 h of infection. Expression of beta2, alphav and alpha4 integrins by peritoneal macrophages at late infection was also gradually reestablished. The assessment of in vivo migration of infected macrophages labeled with the fluorescent dye 5-chloromethylfluorescein diacetate showed a 48-h delay in migration to cervical lymph nodes when compared to LPS pre-stimulated macrophages. Furthermore, cells that migrate to distal lymph nodes were loaded with live parasites. Taken together, these results provide insights about T. gondii escape from the host immune response, placing the macrophage as a "Trojan horse", contributing to parasite dissemination and access to immunoprivileged sites.
Assuntos
Adesão Celular , Movimento Celular/fisiologia , Macrófagos/parasitologia , Toxoplasma/patogenicidade , Animais , Antígenos CD18/biossíntese , Antígenos CD18/genética , Colágeno Tipo IV/metabolismo , Fibronectinas/metabolismo , Regulação da Expressão Gênica/imunologia , Cadeias alfa de Integrinas/biossíntese , Cadeias alfa de Integrinas/genética , Laminina/metabolismo , Linfonodos/citologia , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Macrophages are host cells for the pathogenic parasite Leishmania major. Neutrophils die and are ingested by macrophages in the tissues. We investigated the role of macrophage interactions with inflammatory neutrophils in control of L. major infection. Coculture of dead exudate neutrophils exacerbated parasite growth in infected macrophages from susceptible BALB, but killed intracellular L. major in resistant B6 mice. Coinjection of dead neutrophils amplified L. major replication in vivo in BALB, but prevented parasite growth in B6 mice. Neutrophil depletion reduced parasite load in infected BALB, but exacerbated infection in B6 mice. Exacerbated growth of L. major required PGE(2) and TGF-beta production by macrophages, while parasite killing depended on neutrophil elastase and TNF-alpha production. These results indicate that macrophage interactions with dead neutrophils play a previously unrecognized role in host responses to L. major infection.
Assuntos
Comunicação Celular/imunologia , Leishmania major/crescimento & desenvolvimento , Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Macrófagos Peritoneais/imunologia , Neutrófilos/imunologia , Animais , Morte Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Citocinas/biossíntese , Suscetibilidade a Doenças , Feminino , Genótipo , Imunidade Inata , Leishmaniose Cutânea/genética , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Elastase de Leucócito/fisiologia , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Neutrófilos/enzimologia , Neutrófilos/patologia , Neutrófilos/transplanteRESUMO
Treatment of cultures of Tritrichomonas foetus with 4 mM hydroxyurea (HU), a known DNA synthesis inhibitor, induced pseudocyst formation and caused a mitotic burst. An hour after drug release there was a characteristic, synchronous burst of cell division. T. foetus culture was arrested in the G2/M phase. The synchrony index varied from 66% to 69%. The synchrony was maintained for several cell cycles, even in thawed cultures which had been frozen for storage in liquid nitrogen. The synchronized cells were analyzed by light and scanning electron microscopy, as well by flow cytometry.