Bothropic antivenom based on monoclonal antibodies, is it possible?

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.

Mannose-binding lectin complement pathway plays a key role in complement activation by Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis (Pb) is a dimorphic fungal pathogen that causes paracoccidioidomycosis, the most severe deep mycosis from South America. Although cell mediated immunity is considered the most efficient protective mechanism against Pb infection, mechanisms of innate immunity are poorly defined. Herein, we investigated the interaction of the complement system with high and low virulence isolates of Pb. We demonstrated that Pb18, a high virulence Pb isolate, when incubated with normal human serum (NHS) induces consumption of hemolytic complement and, when immobilized, promotes binding of C4b, C3b and C5b-C9. Both, low virulence (Pb265) and high virulence (Pb18) isolates consumed C4, C3 and mannose-binding lectin (MBL) of MBL-sufficient, but not of MBL-deficient serum as revealed by deposition of residual C4, C3 and MBL on immune complexes and mannan. However, higher complement components consumption was observed with Pb265, as compared with Pb18. The suggested relationship between low virulence and significant complement activation properties of Pb isolates, was confirmed by the demonstration that virulence attenuation of Pb 18 results in acquisition of the ability to activate complement. Conversely, reactivation of attenuated Pb18, results in loss of the ability to activate complement. Our results demonstrate for the first time that Pb yeasts activate the complement system by the lectin pathway, and there is an inverse correlation between complement activating ability and Pb virulence. These differences could exert an influence on innate immunity and severity of the disease developed by infected hosts.

Mannose-binding lectin complement pathway plays a key role in complementactivation by Paracoccidioides brasiliensis

Paracoccidioides brasiliensis (Pb) is a dimorphic fungal pathogen that causes paracoccidioidomycosis, the most severe deep mycosis from South America. Although cell mediated immunity is considered the mostefficient protective mechanism against Pb infection, mechanisms of innate immunity are poorly defined. Herein, we investigated the interaction of the complement system with high and low virulence isolates of Pb. We demonstrated that Pb18, a high virulence Pb isolate, when incubated with normal human serum (NHS) induces consumption of hemolytic complement and, when immobilized, promotes binding of C4b, C3b and C5b-C9. Both, low virulence (Pb265) and high virulence (Pb18) isolates consumed C4, C3 and mannose-binding lectin (MBL) of MBL-sufficient, but not of MBL-deficient serum as revealed bydeposition of residual C4, C3 and MBL on immune complexes and mannan. However, higher complementcomponents consumption was observed with Pb265, as compared with Pb18. The suggested relationshipbetween low virulence and significant complement activation properties of Pb isolates, was confirmed by the demonstration that virulence attenuation of Pb 18 results in acquisition of the ability to activate complement. Conversely, reactivation of attenuated Pb18, results in loss of the ability to activate complement. Our results demonstrate for the first time that Pb yeasts activate the complement system by the lectin pathway, and there is an inverse correlation between complement activating ability and Pbvirulence. These differences could exert an influence on innate immunity and severity of the disease developed by infected hosts.

Emerging multidrug resistant Mycobacterium tuberculosis strains of the Beijing genotype circulating in Russia express a pattern of biological properties associated with enhanced virulence.

The epidemiologically important Mycobacterium tuberculosis Beijing genotype strains, highly endemic in East Asia, have become an emerging infection in certain geographic areas, including Russia, because of its increasing prevalence and association with multidrug resistance (MDR). The aim was to verify whether MDR Beijing strains circulating in the emerging regions present some biological particularities that could contribute to their success in causing disease in comparison with the sporadic strains from locations with low prevalence of the Beijing genotype. We evaluated virulence-associated characteristics of the MDR Beijing strains isolated in Russia and compared them with those of the drug-resistant and susceptible Beijing strains from Brazil and reference H37Rv strain. We found that Russian MDR strains demonstrated an increased bacterial fitness and growth in THP-1 macrophage-like cells, as well as a higher capacity to induce non-protective cytokine synthesis and necrotic macrophage death. By contrast, the biological properties of the strains isolated in Brazil largely resembled those of the H37Rv strain, with the exception of the drug-resistant isolates that presented significantly reduced fitness. The data demonstrate that the emerging MDR strains of the Beijing genotype circulating in Russia do express a pattern of properties associated with the enhanced virulence favouring its clonal dissemination in this region.

Emerging multidrug resistant Mycobacterium tuberculosis strains of the Beijing genotype circulating in Russia express a pattern of biological properties associated with enhanced virulence

The epidemiologically important Mycobacterium tuberculosis Beijing genotype strains, highly endemic in East Asia, have become an emerging infection in certain geographic areas, including Russia, because of its increasing prevalence and association with multidrug resistance (MDR). The aim was to verify whether MDR Beijing strains circulating in the emerging regions present some biological particularities that could contribute to their success in causing disease in comparison with the sporadic strains from locations with low prevalence of the Beijing genotype. We evaluated virulence-associated characteristics of the MDR Beijing strains isolated in Russia and compared them with those of the drug-resistant and susceptible Beijing strains from Brazil and reference H37Rv strain. We found that Russian MDR strains demonstrated an increased bacterial fitness and growth in THP-1 macrophage-like cells, as well as a higher capacity to induce non-protective cytokine synthesis and necrotic macrophage death. By contrast, the biological properties of the strains isolated in Brazil largely resembled those of the H37Rv strain, with the exception of the drug-resistant isolates that presented significantly reduced fitness. The data demonstrate that the emerging MDR strains of the Beijing genotype circulating in Russia do express a pattern of properties associated with the enhanced virulence favouring its clonal dissemination in this region.

The functional state of the complement system in leprosy.

Ninety-one patients with different clinical forms of leprosy, 36 lepromatous (LL), 33 tuberculoid (TL), and 22 dimorphic (DL), and 31 healthy volunteer donors were included in this study. Total complement system (CS) activity was assessed by hemolytic methods, whereas individual components were quantified by the enzyme-linked immunosorbent assay. Under conditions allowing initiation of cascade by the classic pathway (CP) but not alternative pathway (AP) activation, significant CS consumption was detected only in sera from patients with LL. In this group of patients, C4 but not factor B (fB) or C3 was significantly reduced, whereas mannose-binding lectin (MBL) serum levels were significantly higher. These results indicate that the CP is involved in CS activation in patients infected with Mycobacterium leprae manifesting LL clinical form of leprosy. An association is likely between circulating immune complexes and MBL high serum levels for initiation of CS activation in patients with LL form of leprosy.

Adjuvant and immunogenic activities of the 73kDa N-terminal alpha-domain of BrkA autotransporter and Cpn60/60kDa chaperonin of Bordetella pertussis.

A soluble fraction obtained from Bordetella pertussis was evaluated as adjuvant for the pertussis component of the Diphtheria-Pertussis-Tetanus (DPT) vaccine. High levels of antibodies were induced, and a 78% protection rate of mice challenged with live B. pertussis was observed. Two proteins were identified as the 73 kDa N-terminal alpha-domain of BrkA autotransporter protein and the Cpn60/60 kDa chaperonin. Both stimulated antibodies against pertussis and induced a 42% protection rate against the challenge. IgG1 and IgG2a were stimulated suggesting that the immune response could be modulated to produce Th1 or Th2.

Adjuvant and immunogenic activities of the 73 kDa N-terminal á-domain of BrkA autotransporter and Cpn60/60 kDa chaperonin of Bordetella pertussis

A soluble fraction obtained from Bordetella pertussis was evaluated as adjuvant for the pertussis component of the Diphtheria-Pertussis-Tetanus (DPT) vaccine. High levels of antibodies were induced, and a 78% protection rate of mice challenged with live B. pertussis was observed. Two proteins were identified as the 73 kDa N-terminal á-domain of BrkA autotransporter protein and the Cpn60/60 kDa chaperonin. Both stimulated antibodies against pertussis and induced a 42% protection rate against the challenge. IgG1 and IgG2a were stimulated suggesting that the immune response could be modulated to produce Th1 or Th2.

Mycobacteria directly induce cytoskeletal rearrangements for macrophage spreading and polarization through TLR2-dependent PI3K signaling.

Macrophage migration and adhesion are important for the control of mycobacterial infection and are critically dependent on the reorganization of the cytoskeleton. Mycobacteria elicit rapid morphological changes, such as cell spreading, a process relevant to in vivo changes of macrophage shape during extravasation and migration. In this study, we investigated the BCG mycobacteria-induced signaling events leading to macrophage cytoskeletal rearrangements employing specific pharmacological inhibitors to suppress distinct kinase pathways known to be elicited by infection. Viable or lysed mycobacteria, as well as purified cell wall lipoprotein p19, TLR2 agonist, induced RAW264.7 cells to extend actin-rich pseudopods, which impart radial spreading within 3 h, leading later to persistent cell polarization. BCG induced rapid activation of phosphatidylinositol 3-kinase, PI3K, activation that was recruited to the activated TLR2 receptor. TLR2- neutralizing antibody inhibited macrophage spreading and PI3K activation induced by p19. Additionally, BCG induced spreading and polarization of bone marrow-derived macrophages from TLR2- expressing mice in contrast to their TLR2-knockout counterparts. Neither MEK1/ERK, p38 MAPK, nor NF-kappaB activation were important for the early cytoskeletal rearrangements observed, although suppression of these pathways is known to inhibit chemokine secretion by activated macrophages. Beta2-integrins blockade with a corresponding antibody inhibited macrophage spreading and polarization but had no effect on pseudopodia protrusions demonstrating the downstream position of integrin-mediated adhesion in PI3K- dependent signaling pathway leading to the motility phenotype. The obtained data demonstrate that the direct effect of mycobacteria on macrophage shape might be mediated through TLR2-dependent PI3K activation.

Activation of phosphatidylinositol 3-kinase and c-Jun-N-terminal kinase cascades enhances NF-kappaB-dependent gene transcription in BCG-stimulated macrophages through promotion of p65/p300 binding.

The proinflammatory response of infected macrophages is an important early host defense mechanism against mycobacterial infection. Mycobacteria have been demonstrated to induce proinflammatory gene transcription through the Toll-like receptors, (TLR)2 and TLR 4, which initiate signaling cascades leading to nuclear factor (NF)-kappaB activation. The main transduction pathway responsible for NF-kappaB activation has been established and involves the MyD88, interleukin-1 receptor-associated kinase, tumor necrosis factor receptor-associated factor-6, NF-kappaB-inducing kinase, and inhibitor of kappaB kinase complex. The role of other kinase cascades triggered by mycobacteria in the NF-kappaB activation is less clear. We herein examine the role of the mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI-3K) cascades in the expression of the bacillus Calmette-Guerin (BCG) mycobacteria-induced NF-kappaB-dependent genes, macrophage-inflammatory protein-2 (MIP-2) and inducible nitric oxide (NO) synthase. Specific pharmacological inhibition of the PI-3K, c-jun-N-terminal kinase (JNK), and to a smaller extent, p38 MAPK but not extracellular-regulated kinase (ERK), suppressed NF-kappaB-dependent reporter gene transcription and MIP-2 and NO secretion in BCG-induced RAW264.7 macrophages. A similar effect was obtained following molecular inhibition of JNK via JNK-interacting protein-1 overexpression. In addition, a kinase-dead mutant of MEK kinase-1, the up-stream regulator of JNK, also proved to be a potent inhibitor of NF-kappaB-reporter activity. The effect of inhibitors was mediated by the down-regulation of NF-kappaB transcription activity and without effecting its nuclear translocation. These data suggest an indirect mechanism of the NF-kappaB regulation by these kinases, probably through p65 phosphorylation and improved binding to the p300 transcription coactivator. The data obtained demonstrate that PI-3K, JNK, and p38 MAPK activation by mycobacteria enhance NF-kappaB-driven gene expression contributing to the proinflammatory macrophage response.