Anti-complement activity in salivary glands and midgut of Chagas disease vector, Panstrongylus megistus (Hemiptera, Triatominae)

The triatomine insect Panstrongylus megistus, one of the most important Chagas disease vectors in Brazil, presents salivary molecules pharmacologically active to counteract homeostatic responses from the host, including inhibitors of the human complement system, a major effector of immune responses. The aim of the present study was to investigate the effect of P. megistus salivary gland extract (SGE) on the complement system from different host species and characterize the inhibitory effect of SGE and intestinal contents on human complement. Glands and midguts from fourth instar nymphs were used. Hemolytic assays were performed with sheep erythrocytes as complement activators by using human, rats and chickens sera in the presence or absence of SGE. An ELISA assay was carried out detect deposition of the C3b component on IgG- or agarose-sensitized microplates, in the presence or absence of SGE or midgut contents. P. megistus SGE was able to significantly inhibit the complement of the three studied species (human, rat and chiken). Both, SGE and midgut contents inhibited C3b deposition in either the classical or the alternative pathways. As conclusions, SGE and midgut from P. megistus possess anti-complement activity. The inhibitors are effective against different host species and act on the initial steps of the complement system cascade. These inhibitors may have a role in blood feeding and Trypanosoma cruzi transmission by the vector

Citotoxicity evaluation of three dental adhesives on vero cells in vitro

To evaluate, in vitro, the potential cytotoxicity of three different dental adhesives systems (Adper Single Bond 2 -SB, Silorane System Adhesive Bond -SSAB and Single Bond Universal -SBU) on cultivated Vero cells after different contact times. The cells were cultured in a concentration of 2 x 105 cells/mL for 24h and grown to sub-confluent monolayers. VERO cells were exposed to 25µl of conditioned extracts obtained from 24h, 48h and 72h immersion of adhesive samples in culture medium (DMEM), immediately after polymerization. Fresh DMEM was used as negative control. Cell metabolism was evaluated by the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2, 5diphenyl-tetrazolium bromide). The data were analyzed statistically by ANOVA, considering a significance of 5%. The values of cell viability ranged from 94.2% at 72h (SBU) to 109.6% at 48h (SB). The mean percentage of viability after exposure to the extracts of SB, SSAB and SBU were 103.2%, 100.63% and 97.43%, respectively. There was no statistically significant difference (p= 0.342) between the experimental and negative control groups. At all exposure times, all adhesives tested in this study presented no cytotoxicity to Vero cells in vitro

The Role of Salivary and Intestinal Complement System Inhibitors in the Midgut Protection of Triatomines and Mosquitoes

Saliva of haematophagous arthropods contain biomolecules involved directly or indirectly with the haematophagy process, and among them are encountered some complement system inhibitors. The most obvious function for these inhibitors would be the protection of the midgut against injury by the complement. To investigate this hypothesis, Triatoma brasiliensis nymphs were forced to ingest human serum in conditions in which the protection of midgut by the inhibitors is bypassed. In these conditions, the anterior midgut epithelium was injured by the complement, causing cell death. Once some insects such as Aedes aegypti have no salivary inhibitors, we hypothesized the existence of intestinal inhibitors. The inhibitory activity was investigated in the intestine of A. aegypti as well as in the saliva and intestine of other three triatomine species (T. brasiliensis, T. infestans and Rhodnius prolixus) using an immunological method able to determine the level of deposition of some complement factors (C1q, C3b, or C4b) on the surface of complement activator molecules linked to microplates. This methodology permitted to identify which points along the activation phase of the complement cascade were inhibited. As expected, soluble contents of A. aegypti's intestine was capable to inhibit C3b deposition by the classical and alternative pathways. Saliva or soluble intestinal contents, obtained from triatomines were unable to inhibit C1q deposition by the classical pathway. C4b deposition by the classical pathway was inhibited by the intestinal contents from the three triatomines. On the other hand, only T. brasiliensis saliva inhibited C4b deposition. Both, saliva and intestinal contents from all triatomines were able to inhibit C3b deposition in the classical and alternative pathways. None of the material extracted from the intestinal cell membranes from the triatomines inhibited C3b deposition in the classical pathway. The existence of complement inhibitors may have important biological consequences which are discussed in detail

O papel de inibidores salivares e intestinais do complemento humano na proteção do intestino médio de triatomíneos

Exportado OPUSMade available in DSpace on 2019-08-12T23:07:49Z (GMT). No. of bitstreams: 1 tese_veruska_parasitologia_2009.pdf: 1638146 bytes, checksum: fa73e8f8ab07bd724b0d4b6537685bf8 (MD5) Previous issue date: 11A saliva de artrópodes hematófagos contém biomoléculas envolvidas diretamente ou indiretamente no processo de hematofagia, entre as quais estão moléculas inibidoras do sistema complemento. A função mais óbvia para estes inibidores seria a proteção do intestino médio contra danos causados pelo complemento. Para investigar esta hipótese, ninfas de Triatoma brasiliensis foram forçadas a ingerir soro humano em condições nas quais os inibidores salivares eram incapazes de proteger o intestino. Nestas condições, o epitélio do intestino médio anterior foi marcado e danificado pelo complemento, causando morte celular. Uma vez que a saliva de Aedes aegypti não contém inibidores do complemento, foi formulada a hipótese da possível existência destes inibidores no intestino médio. Assim, a atividade inibitória foi investigada no intestino de A. aegypti, na saliva e no intestino de três espécies de triatomíneos (T. brasiliensis, T. infestans e Rhodnius prolixus) por meio de um método imunológico capaz de determinar o nível de deposição de alguns fatores do complemento (C1q, C3b, ou C4b) na superfície de moléculas ativadoras em microplacas. Esta metodologia permitiu identificar em quais pontos da cascata do complemento as moléculas presentes na saliva ou no conteúdo intestinal de triatomíneos podem inibir o complemento. Foi verificado que tanto a saliva quanto o conteúdo intestinal solúvel das espécies de triatomíneos estudadas não inibiram a deposição de C1q pela via clássica. O conteúdo intestinal solúvel das três espécies de triatomíneos foi capaz de inibir a deposição de C4b pela via clássica. Somente a saliva de T. brasiliensis foi capaz de inibir a deposição de C4b. Ambos, saliva e conteúdo intestinal das três espécies de triatomíneos puderam inibir a deposição de C3b nas vias clássica e alternativa. Nenhum dos materiais extraídos das membranas de células intestinais dos triatomíneos inibiu a deposição de C3b na via clássica. Como esperado, o conteúdo intestinal solúvel de A. aegypti foi capaz de inibir a deposição de C3b pelas vias clássica e alternativa. A existência de inibidores de complemento pode apresentar consequências biológicas importantes tanto para o sucesso na hematofagia dos insetos quanto para o seu papel na interação com seus parasitos

Scheme of the activation phase of the complement system showing the points potentially targeted by the inhibitors.

<p>The classic pathway is initiated by the binding of C1q, C1r and C1s (the C1 complex) to antibodies linked to the activation surface. The proteolytic activity of C1r is automatically activated by interaction with C1q. C1r then cleaves and activates C1s, which is another serine protease. C1s acts specifically on C2 and C4 activating them. Once cleaved, the fragment C4b is capable to bind covalently to the activation surface creating a binding site to C2a. The active serine protease C2a, in the C4b-C2a complex, acts on C3 producing C3b molecules which are also capable to bind covalently to the activation surface nearby its site of activation. The complex C4b-C2a-C3b acts as a C3 convertase as well as a C5 convertase generating the MAC. For activation of the alternative pathway, a small fraction of C3 present in the extracelular fluids slowly undergoes spontaneous reaction with H₂O molecules generating C3-H₂O. These molecules can interact with the protein B generating the B-C3-H₂O complex which is a substrate for D, a plasmatic serine protease. The Bb-C3-H₂O complex acts on C3 cleaving it to C3b and C3a. Most of the C3b molecules generated will combine with H₂O or other self molecules becoming inactive. On the other hand, if a C3b molecule is generated near an adequate surface such as a bacterium, it will covalently bind to it creating a binding site for factor B. The C3b-B complex is activated by the protease D generating C3b-Bb, an efficient protease capable to activate other C3 molecules. The complex C3b-Bb-C3b is an efficient C3 convertase as well as a C5 convertase generating the MAC. To simplify, the lectin pathway as well as the normal regulatory proteins were omitted.</p

Influence of pH in the operation of the human complement system.

<p>Operation of the classical (A) and alternative (B) pathways at different pHs when compared with the control at pH 7.4 (100%).</p

Percentage of inhibition of the complement system by saliva from <i>Triatoma brasiliensis, T. infestans</i> and <i>Rhodnius prolixus</i>.

<p>(n) = number of independent experiments performed for each treatment.</p><p>Asterisk (*) indicates statistical difference from control (P<0.05).</p><p>AP: Alternative Pathway, CP: Classical Pathway.</p

Apyrase activity from the crop soluble contents of <i>Rhodnius prolixus</i> after artificial and forced feeding.

<p>The activity was expressed as ng of inorganic phosphate (Pi) released in a minute by 1/12 of anterior midgut contents±standard error. The T test indicated a significant difference between groups (P<0.05).</p

Protection of anterior midgut of <i>T. brasiliensis</i> against the complement system.

<p>A- MAC deposition onto the anterior midgut wall after normal blood ingestion, B- natural fluorescence observed on the midgut wall, C- Apparatus used for the forced feeding procedure, D- MAC deposition onto the anterior midgut wall after forced feeding of 50 µL of normal human sera, E- Increased deposition of MAC onto the anterior midgut wall after forced feeding of 50 µL of 2 fold concentrated normal human sera, F- MAC deposition onto the anterior midgut wall after forced feeding of 50 µL of inactivated 2 fold concentrated normal human sera, G- Cell death in the anterior midgut epithelium after forced feeding of 2 fold concentrated normal human serum containing propidium iodide. H- Absence of cell death after forced feeding of inactivated 2 fold concentrated normal human sera containing propidium iodide.</p