Thereza L. Kipnis

Development of snake antivenom antibodies in chickens and their purification from yolk

Adult white leghorn hens hyperimmunised with Brazilian snake venoms of the genus Bothrops and/or Crotalus produced antibodies capable of recognising, combining with and neutralising the toxic and lethal components of the venoms. The antibodies were first detected by an enzyme-linked immunosorbent assay two weeks after starting the immunisation schedule, reached the highest titres by the third week and remained high for at least 24 weeks. These antibodies are transferred to the egg yolk from which they were isolated as enriched IgY preparations by a combination of methods using positive and negative precipitation with sodium sulphate and/or caprylic acid. The yolk-derived IgY preparations contained antibodies which blocked the phospholipase A2-dependent haemolytic activity of both venoms and the haemorrhagic activity of Bothrops venom, and neutralised the toxic lethal activities of the venoms with good efficacy. The median effective dose (ED50) of the IgY anti-Bothrops venom was 592.5 μl/2LD50 and, 1.0 ml neutralised 0.0675 mg of venom. The ED50 of the IgY anti-Crotalus venom was 457.5 μl/3LD50 and 1.0 ml neutralised 0.075 mg of venom.

The activation of the complement system by Paracoccidioides brasiliensis in vitro: its opsonic effect and possible significance for an in vivo model of infection.

Abstract The yeast phase of Paracoccidioides brasiliensis is poorly phagocytosed by “stimulated” mouse peritoneal macrophages in culture. However, either heterologous or homologous antibodies greatly enhanced the phagocytosis of P. brasiliensis by macrophages. The same effect was observed when the fungus was pretreated with fresh normal sera. The following treatments abolished the opsonic activity of fresh normal serum: heating at 56°C for 30 min, addition of 10 mM EDTA. CoF treatment, and properdin or factor-B depletion. Conversely, EGTA had no effect. These data indicate that the alternative pathway of complement activation is involved in the expression of this phenomenon. When yeast cells of P. brasiliensis were incubated with fresh normal serum, the third component of complement changed its electrophoretic mobility of β1C to β1A. In an attempt to correlate these data with the in vivo conditions, fungi were inoculated into the subcutaneous tissues of normal mice. The resultant lesions reached their maximal size 48 hr after inoculation. Histological studies of these lesions showed a massive influx of polymorphonucleocytes. The histological picture was not modified conclusively when the mice were pretreated with CoF or when the fungi were inoculated in C5-deficient mice. The hypothesis that the fungus might secrete a chemotactic factor was tested in an in vitro system. It was verified that the activation of the complement system by P. brasiliensis elicited the formation of chemotactic substances for mouse polymorphonucleocytes. However, when the supernatant of cultures of the fungus in medium 199 was used as a chemotactic source, no effect could be detected. Thus, neither complement activation nor a fungus-secreted factor seems to explain the initial events of the lesion formation induced by P. brasiliensis.

Local inflammation, lethality and cytokine release in mice injected with Bothrops atrox venom

We have provided evidence that: (a) lethality of mice to crude Bothrops venom varies according the isogenic strain (A/J > C57Bl/6 > A/Sn > BALB/c > C3H/HePas > DBA/2 > C3H/He); (b)BALB/c mice (LD50=100.0 microg) were injected i.p. with 50 microg of venom produced IL-6, IL-10, INF-gamma, TNF-alpha and NO in the serum. In vitro the cells from the mice injected and challenged with the venom only released IL-10 while peritoneal macrophages released IL-10, INF-gamma and less amounts of IL-6; (c) establishment of local inflammation and necrosis induced by the venom, coincides with the peaks of TNF-alpha, IFN-gamma and NO and the damage was neutralized when the venom was incubated with a monoclonal antibody against a 60 kDa haemorrhagic factor. These results suggest that susceptibility to Bothrops atrox venom is genetically dependent but MHC independent; that IL-6, IL-10, TNF-alpha, IFN-gamma and NO can be involved in the mediation of tissue damage; and that the major venom component inducers of the lesions are haemorrhagins.

Activation of phosphatidylinositol 3-kinase and c-Jun-N-terminal kinase cascades enhances NF-κB-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)‐κB activation. The main transduction pathway responsible for NF‐κB activation has been established and involves the MyD88, interleukin‐1 receptor‐associated kinase, tumor necrosis factor receptor‐associated factor‐6, NF‐κB‐inducing kinase, and inhibitor of κB kinase complex. The role of other kinase cascades triggered by mycobacteria in the NF‐κB 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‐κB‐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‐κB‐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‐κB‐reporter activity. The effect of inhibitors was mediated by the down‐regulation of NF‐κB transcription activity and without effecting its nuclear translocation. These data suggest an indirect mechanism of the NF‐κB 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‐κB‐driven gene expression contributing to the proinflammatory macrophage response.

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.

Development of process to produce polyvalent IgY antibodies anti-African snake venom.

Polyvalent anti-Bitis and anti-Naja antivenom IgY antibodies were prepared using B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca, and N. mossambica venoms to immunize chickens. Blood and eggs were collected before and during the 10-month immunization period; the sera and yolk extracts were then prepared and assayed for the presence of antivenom antibodies by ELISA and Western blot methods. ELISA Antivenom antibody titers, referred to as U-ELISA/ml of serum or egg yolk extracts, absent in pre-immunization sera or yolk, increased sharply during the 4 weeks after immunization, reaching a plateau thereafter. Yolk extracts with high antivenom titers, as detected by ELISA were used to isolate and purify IgY. Purified IgY preparations recognized venom protein bands from 10 to 20 kDa to 60 and 70 kDa, as shown by Western blot. Recovery of antivenom antibodies from the whole yolk was over 80%. Final preparations exhibited high antivenom activity (>100,000 U-ELISA/ml) as well as efficacy in neutralizing venom lethality (1,440 microg of IgY neutralize 62.2 LD(50) of venom), and were free of toxic products, pyrogen or bacterial and fungal contaminations.

Active entry of ; bloodstream forms of Trypanosoma cruzi into macrophages.

The uptake of bloodstream forms of Trypanosoma cruzi, Y and CL stocks, by mouse peritoneal macrophages and their intracellular differentiation and multiplication has been compared in vitro. After 48 h the number of macrophages showing intracellular amastigote forms was higher when the Y stock was used. The number of parasitized cells increased with the time of contact between parasites and macrophages. Prior treatment of the parasites with anti-T. cruzi antibodies and/or complement increased the number of infected macrophages, but did not interfere with their subsequent differentiation within the macrophages. The number of parasitized cells was greater when macrophages were obtained from mice previously treated with lipopolysaccharide, peptone or thioglycollate. Uptake was not appreciably affected when macrophages were pre-treated with trypsin or anti-macrophage serum, or when the parasites and macrophages were incubated in the presence of cytochalasin B. In the same experimental conditions, epimastigotes of T. cruzi when not able to differentiate into amastigotes. Their uptake was potentiated by previous treatment with specific antibodies and/or complement and was blocked by cytochalasin B. These results confirm that epimastigotes derived from T. cruzi cultures are phagocytosed and suggest that bloodstream forms penetrate actively into macrophages.

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‐κB activation were important for the early cytoskeletal rearrangements observed, although suppression of these pathways is known to inhibit chemokine secretion by activated macrophages. Β2‐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.

Biochemical and biological properties of phospholipases A2 from Bothrops atrox snake venom

Phospholipases A(2) (PLA(2)s), of molecular mass 13-15kDa, are commonly isolated from snake venom. Two myotoxins with PLA(2) activity, BaPLA(2)I and BaPLA(2)III, with estimated molecular masses of 15kDa were isolated from the venom of Bothrops atrox using Sephacryl S-100-HR and reverse-phase chromatography. BaPLA(2)I was basic, with a pI of 9.1, while BaPLA(2)III was neutral with a pI of 6.9. On a molecular basis, BaPLA(2)III exhibited higher catalytic activity on synthetic substrates than BaPLA(2)I. Comparison of the N-terminal residues of BaPLA(2)I with other PLA(2) proteins from snake venoms showed that it has the highest homology (94%) with B. asper myotoxin II and homology with a PLA(2) Lys(49) from B. atrox (89%). In contrast, BaPLA(2)III demonstrated 75, 72, and 71% homology with PLA(2) from Vipera ammodytes meridionalis, B. jararacussu, and B. jararaca, respectively. BaPLA(2)I and BaPLA(2)III were capable, in vitro, of inducing mast cell degranulation and, in vivo, of causing creatine kinase release, edema, and myonecrosis typical of PLA(2)s from snake venoms, characterized by rapid disruption of the plasma membrane as indicated by clumping of myofilaments and necrosis of affected skeletal muscle cells. BaPLA(2)I- and BaPLA(2)III-specific monoclonal and polyclonal antibodies, although incapable of neutralizing PLA(2) edematogenic activity, blocked myonecrosis efficiently in an in vivo neutralization assay. The results presented herein suggest that the biological active site responsible for edema induction by these two PLA(2) enzymes is distinct from the myonecrosis active site and is not dependent upon the catalytic activity of the PLA(2) enzyme.

Pro-inflammatory activities in elapid snake venoms.

1 Snake venoms from the genera Micrurus (M. ibiboboca and M. spixii) and Naja (N. naja, N. melanoleuca and N. nigricollis) were analysed, using biological and immunochemical methods, to detect pro‐inflammatory activities, cobra venom factor (COF), proteolytic enzymes, thrombin‐like substances, haemorrhagic and oedema‐producing substances. 2 The venoms of the five snake species activate the complement system (C) in normal human serum (NHS) in a dose‐related fashion, at concentrations ranging from 5 μg to 200 μg ml−1 serum. Electrophoretic conversion of C3 was observed with all venoms in NHS containing normal concentrations of Ca2+ and Mg2+, but only by venoms from N. naja and N. melanoleuca when Ca2+ was chelated by adding Mg2+‐EGTA. 3 Purified human C3 was electrophoretically converted, in the absence of other C components, by the venoms from N. naja, N. nigricollis and M. ibiboboca. However, only the venoms from N. naja and N. melanoleuca contained a 144 kDa protein revealed in Western blot with sera against COF or human C3. 4 All venoms, at minimum concentrations of 30 ng ml−1, were capable of lysing sheep red blood cells, also in a dose‐related fashion, when incubated with these cells in presence of egg yolk as a source of lecithin. Although the venoms from M. spixii and N. nigricollis showed detectable thrombin‐like activity, these and the other venoms were free of proteolytic activity when fibrin, gelatin and casein, were used as substrates. 5 When tested on mice skin, all five venoms were capable of inducing an increase in vascular permeability and oedema, but were devoid of haemorrhagic producing substances (haemorrhagins). 6 These data provide evidence indicating that Elapidae venoms contain various pro‐inflammatory factors which may be important in the spreading of neurotoxins throughout the tissues of the prey or human victim.