Artificial Feeding of Ornithodoros fonsecai and O. brasiliensis (Acari: Argasidae) and Investigation of the Transstadial Perpetuation of Anaplasma marginale.

Anaplasma marginale is a Gram-negative, obligate intraerythrocytic bacterium that causes bovine anaplasmosis. While hard ticks of the genera Dermacentor and Rhipicephalus can be biological vectors, transmitting this pathogen via saliva during blood meals, blood-sucking insects, and fomites play a role as mechanical vectors. Little is known about the interaction between Anaplasma marginale and Argasidae ticks. Among soft ticks, Ornithodoros fonsecai (Labruna and Venzal) and Ornithodoros brasiliensis Aragão inhabit environments surrounding localities where many cases of bovine anaplasmosis have been reported. Ticks of the species O. fonsecai parasitize bats, while O. brasiliensis can parasitize different vertebrate species. Therefore, the present study aimed to feed third-instar nymphs artificially (N3) of O. fonsecai and O. brasiliensis using blood samples obtained from a calf naturally infected with A. marginale and rabbit blood added to A. marginale-containing bovine erythrocytes, to investigate the ability of these nymphs to acquire, infect and transstadially perpetuate this agent. For the artificial feeding system, adapted chambers and parafilm membranes were used. Nymphs of both tick species were submitted to different replications weighed before and after each feeding. Blood samples and molted ticks were submitted to DNA extraction, quantitative real-time PCR for the msp1ß gene to detect A. marginale DNA, while a semi-nested polymerase chain reaction for the msp1α gene was performed for genotyping. Using calf blood naturally infected with A. marginale, among the three artificial feeding replications performed with O. fonsecai and O. brasiliensis nymphs, the DNA of A. marginale was detected in both nymphs after 30-50 days of molting. For artificial feeding with rabbit blood added to bovine erythrocytes containing A. marginale, the DNA of this pathogen was also detected in both nymph species. As for the assay for the msp1α gene, strains were found Is9; 78 24-2; 25; 23; α; and ß. It was concluded that nymphs (N3) of O. fonsecai and O. brasiliensis could feed artificially through a parafilm membrane using blood from calves and rabbits infected by A. marginale. The DNA of A. marginale was detected in nymphs fed artificially of both tick species studied after molt. However, further studies are needed to confirm transstadial perpetuation in other instars and their host transmission capacity.

Antitumoral effects of Amblyomma sculptum Berlese saliva in neuroblastoma cell lines involve cytoskeletal deconstruction and cell cycle arrest / Efeito antitumoral da saliva do carrapato Amblyomma sculptum Berlese em células de neuroblastoma envolve desconstrução do citoesqueleto e parada do ciclo celular

Abstract The antitumor properties of ticks salivary gland extracts or recombinant proteins have been reported recently, but little is known about the antitumor properties of the secreted components of saliva. The goal of this study was to investigate the in vitro effect of the saliva of the hard tick Amblyomma sculptum on neuroblastoma cell lines. SK-N-SK, SH-SY5Y, Be(2)-M17, IMR-32, and CHLA-20 cells were susceptible to saliva, with 80% reduction in their viability compared to untreated controls, as demonstrated by the methylene blue assay. Further investigation using CHLA-20 revealed apoptosis, with approximately 30% of annexin-V positive cells, and G0/G1-phase accumulation (>60%) after treatment with saliva. Mitochondrial membrane potential (Δψm) was slightly, but significantly (p < 0.05), reduced and the actin cytoskeleton was disarranged, as indicated by fluorescent microscopy. The viability of human fibroblast (HFF-1 cells) used as a non-tumoral control decreased by approximately 40%. However, no alterations in cell cycle progression, morphology, and Δψm were observed in these cells. The present work provides new perspectives for the characterization of the molecules present in saliva and their antitumor properties.

Resumo As propriedades antitumorais de extratos de glândulas salivares de carrapatos ou proteínas recombinantes foram relatadas recentemente, mas pouco se sabe sobre as propriedades antitumorais dos componentes secretados da saliva. O objetivo deste estudo foi investigar o efeito in vitro da saliva bruta do carrapato duro Amblyomma sculptum sobre as linhagens celulares de neuroblastoma. Células SK-N-SK, SH-SY5Y, Be(2)-M17, IMR-32 e CHLA-20 foram suscetíveis à saliva, com redução de 80% na sua viabilidade em comparação com controles não tratados, como demonstrado pelo ensaio de Azul de Metileno. Investigações posteriores utilizando CHLA-20 revelaram apoptose, com aproximadamente 30% de células positivas para anexina-V, e G0/G1 (> 60%) após tratamento com saliva. O potencial de membrana mitocondrial (Δψm) foi reduzido significativamente (p <0,05), e o citoesqueleto de actina foi desestruturado, como indicado pela microscopia de fluorescência. A viabilidade do fibroblasto humano (células HFF-1), usado como controle não tumoral, diminuiu em aproximadamente 40%. No entanto, não foram observadas alterações na progressão do ciclo celular, morfologia e Δψm nestas células. O presente trabalho fornece novas perspectivas para a caracterização das moléculas presentes na saliva e suas propriedades antitumorais.

Antitumoral effects of Amblyomma sculptum Berlese saliva in neuroblastoma cell lines involve cytoskeletal deconstruction and cell cycle arrest.

The antitumor properties of ticks salivary gland extracts or recombinant proteins have been reported recently, but little is known about the antitumor properties of the secreted components of saliva. The goal of this study was to investigate the in vitro effect of the saliva of the hard tick Amblyomma sculptum on neuroblastoma cell lines. SK-N-SK, SH-SY5Y, Be(2)-M17, IMR-32, and CHLA-20 cells were susceptible to saliva, with 80% reduction in their viability compared to untreated controls, as demonstrated by the methylene blue assay. Further investigation using CHLA-20 revealed apoptosis, with approximately 30% of annexin-V positive cells, and G0/G1-phase accumulation (>60%) after treatment with saliva. Mitochondrial membrane potential (Δψm) was slightly, but significantly (p < 0.05), reduced and the actin cytoskeleton was disarranged, as indicated by fluorescent microscopy. The viability of human fibroblast (HFF-1 cells) used as a non-tumoral control decreased by approximately 40%. However, no alterations in cell cycle progression, morphology, and Δψm were observed in these cells. The present work provides new perspectives for the characterization of the molecules present in saliva and their antitumor properties.

Antiproliferative and antiangiogenic effect of Amblyomma sculptum (Acari: Ixodidae) crude saliva in endothelial cells in vitro.

Neovascularization, a process that includes vasculogenesis and angiogenesis, may be a physiological or pathologic event, but in any cases the phenomenon is related to the formation of vascular net and sprouting of endothelial cells from preexisting blood vessel. The tumor environment, which counts on the tumor cell proliferation, is plenty of proangiogenic factors, such as angiogenin, TGF (α and ß), FGF, VEGF, all of them playing a crucial role in angiogenesis, an important hallmark of cancer frequently related to a poor prognosis. Therefore, therapies focusing the inhibition of cancer neovasculogenesis have become an interesting strategy for the development of antitumor therapies. In this work, we investigate the effect of tick saliva on the human endothelial cells, in order to understand its inhibitory effects on angiogenesis. To this end, the HUVEC cells were used as model of angiogenesis in vitro and the anti-proliferative, anti-migratory, cytotoxicity was evaluated. Our data depicts that saliva impairs cell development by causing structural changes while precludes cell proliferation and migration, that are crucial events related to angiogenesis. Aiming the identification of the bioactive components related to antiangiogenic activity, saliva was analyzed through the Mass Spectrometry and among all molecules identified, disintegrins and cathepsin L seems to be primarily responsible for the antiangiogenic effects of saliva.

Gut transcriptome analysis on females of Ornithodoros mimon (Acari: Argasidae) and phylogenetic inference of ticks.

Ornithodoros mimon is an argasid tick that parasitizes bats, birds and opossums and is also harmful to humans. Knowledge of the transcripts present in the tick gut helps in understanding the role of vital molecules in the digestion process and parasite-host relationship, while also providing information about the evolution of arthropod hematophagy. Thus, the present study aimed to know and ascertain the main molecules expressed in the gut of argasid after their blood meal, through analysis on the gut transcriptome of engorged females of O. mimon using 454-based RNA sequencing. The gut transcriptome analysis reveals several transcripts associated with hemoglobin digestion, such as serine, cysteine, aspartic proteases and metalloenzymes. The phylogenetic analysis on the peptidases confirmed that most of them are clustered with other tick genes. We recorded the presence a cathepsin O peptidase-coding transcript in ticks. The topology of the phylogenetic inferences, based on transcripts of inferred families of homologues, was similar to that of previous reports based on mitochondrial genome and nuclear rRNA sequences. We deposited 2,213 sequence of O. mimon to the public databases. Our findings may help towards better understanding of important argasid metabolic processes, such as digestion, nutrition and immunity.

Gut transcriptome analysis on females of Ornithodoros mimon (Acari: Argasidae) and phylogenetic inference of ticks / Transcriptoma do intestino de fêmeas de Ornithodoros mimon (Acari: Argasidae) e inferência filogenética de carrapatos

Abstract Ornithodoros mimon is an argasid tick that parasitizes bats, birds and opossums and is also harmful to humans. Knowledge of the transcripts present in the tick gut helps in understanding the role of vital molecules in the digestion process and parasite-host relationship, while also providing information about the evolution of arthropod hematophagy. Thus, the present study aimed to know and ascertain the main molecules expressed in the gut of argasid after their blood meal, through analysis on the gut transcriptome of engorged females of O. mimon using 454-based RNA sequencing. The gut transcriptome analysis reveals several transcripts associated with hemoglobin digestion, such as serine, cysteine, aspartic proteases and metalloenzymes. The phylogenetic analysis on the peptidases confirmed that most of them are clustered with other tick genes. We recorded the presence a cathepsin O peptidase-coding transcript in ticks. The topology of the phylogenetic inferences, based on transcripts of inferred families of homologues, was similar to that of previous reports based on mitochondrial genome and nuclear rRNA sequences. We deposited 2,213 sequence of O. mimon to the public databases. Our findings may help towards better understanding of important argasid metabolic processes, such as digestion, nutrition and immunity.

Resumo Ornithodoros mimon é um carrapato argasídeo parasita de morcegos, aves e marsupiais, além de ser bastante agressivo aos humanos. O conhecimento dos transcritos presentes no intestino dos carrapatos auxilia no entendimento do papel de moléculas vitais no processo de digestão e na relação parasito-hospedeiro, além de fornecer também informações sobre a evolução dos artrópodes hematófagos. Desta maneira, o presente estudo teve como objetivo conhecer e identificar as principais moléculas expressas no intestino de uma espécie de carrapato argasídeo após o repasto sanguíneo, através de uma análise transcritômica descritiva do intestino de fêmeas ingurgitadas de O. mimon, utilizando um sequenciamento de RNA de nova geração da plataforma 454. Além de inferir a relação filogenética de carrapatos através de um conjunto de dados transcritômicos. O transcriptoma do intestino revelou diversos transcritos associados com a digestão da hemoglobina, como proteinases das classes serino, cisteína, aspártica e metalo. Registramos a presença de um transcrito de uma cisteína peptidase do tipo catepsina O em carrapatos. A inferência filogenética baseada em conjunto de dados transcritos homólogos tem uma resolução topológica similar a de outros conjuntos de dados moleculares. Foram depositados no banco de dados gênico público 2213 transcritos de O. mimon. Os achados obtidos no presente estudo podem contribuir para compreensão dos importantes processos, como digestão, nutrição e imunidade dos carrapatos da família Argasidae, além de fornecer informações sobre a filogenia da ordem Ixodida.

Laboratory life cycle of Ornithodoros brasiliensis (Acari: Argasidae): An endemic tick from southern Brazil.

ORNITHODOROS BRASILIENSIS: Aragão is an endemic tick restricted to the highlands of the state of Rio Grande do Sul, Brazil. This species is very aggressive toward humans, causing fever, great pain and intense inflammatory response at the bite site. It is also very aggressive toward dogs, and tick toxicosis syndrome has been reported in this host. In order to elucidate the biology of this tick, the present study describes its life cycle under laboratory conditions, using guinea pigs as hosts for two generations. In the nonparasitic phase, the ticks were maintained in sand, in an incubator under controlled conditions. The larvae molted to the nymphal stage without feeding, and five nymphal stages (N1, N2, N3, N4 and N5) were observed in both generations. In both generations emergence of adults started from N3 when sex ratio was 0.85:1 (23 males and 27 females) in F1 and 0.63:1 (34 males and 54 females) in F2. For both generations, N4 generated more females, while N5 only produced females. The pre-ecdysis period of the nymphs ranged from 31.1 to 38.6 days. Two gonotrophic cycles were observed, and the first one presented a higher average number of eggs deposited (N=139) than the second (N=73.8). The mean duration of the life cycle (egg to egg) of O. brasiliensis was 215.4 days for the first generation and 195 days for the second.

Structural findings and molecular modeling approach of a TFPI-like inhibitor.

Specific blood coagulation inhibitors from hematophagous organisms, with different structures and novel mechanism of action, have been described and they represent promising agents for the treatment of a variety of human diseases related to coagulation and cancer. In our lab, the salivary glands transcriptome of the adult Amblyomma cajennense tick was previously characterized by expressed sequence tags (EST). A transcript that codes for a tissue factor pathway inhibitor (TFPI)-like protein with unique structure was found, and the recombinant form of this protein was named Amblyomin-X. This protein was able to inhibit the factor Xa amidolytic activity and the activation of factor X by the extrinsic tenase complex (FVIIa/TF). Herein, it was performed functional and structural evaluation of Amblyomin-X. The CD assay and molecular dynamics simulations revealed that Amblyomin-X is structurally stable and the naturally unfolded regions as well as the presence of three disulfide bridges in its Kunitz-type domain seem to sustain its inhibitory activity. Regarding the electrostatic potential mapping on the Kunitz-type region, the pattern of charged residues was not quite the same in comparison to human TFPI-1 and TFPI-2, pointing out there might be distinct functional and structural features, which are going to be experimentally exploited.

A Kunitz-type FXa inhibitor affects tumor progression, hypercoagulable state and triggers apoptosis.

Cancer is linked to hypercoagulability, and many studies have shown that anticoagulant drugs affect tumor progression. In this study was demonstrated that the Amblyomin-X (which is a recombinant protein that exerts similarity to the Kunitz-type inhibitors and shows pro-apoptotic effects in different tumor cell lines) and heparin (a classic anticoagulant) have similar effects on cancer progression and on normalization of the hypercoagulable state. However, Amblyomin-X showed a distinct mechanism in triggering its effects in vitro, because it exerted a cytotoxic effect in cancer cells by inducing apoptosis and promoting cell cycle arrest.

A novel proteasome inhibitor acting in mitochondrial dysfunction, ER stress and ROS production.

In cancer-treatment, potentially therapeutic drugs trigger their effects through apoptotic mechanisms. Generally, cell response is manifested by Bcl-2 family protein regulation, the impairment of mitochondrial functions, and ROS production. Notwithstanding, several drugs operate through proteasome inhibition, which, by inducing the accumulation and aggregation of misfolded or unfolded proteins, can lead to endoplasmic reticulum (ER) stress. Accordingly, it was shown that Amblyomin-X, a Kunitz-type inhibitor identified in the transcriptome of the Amblyomma cajennense tick by ESTs sequence analysis of a cDNA library, obtained in recombinant protein form, induces apoptosis in murine renal adenocarcinoma (RENCA) cells by: inducing imbalance between pro- and anti-apoptotic Bcl-2 family proteins, dysfunction/mitochondrial damage, production of reactive oxygen species (ROS), caspase cascade activation, and proteasome inhibition, all ER-stress inductive. Moreover, there was no manifest action on normal mouse-fibroblast cells (NHI3T3), suggesting an Amblyomin-X tumor-cell selectivity. Taken together, these evidences indicate that Amblyomin-X could be a promising candidate for cancer therapy.

Pro-apoptotic effects of Amblyomin-X in murine renal cell carcinoma "in vitro".

Renal cell carcinoma (RCC) is one of the most lethal urologic cancers and is highly resistant to both radiotherapy and chemotherapy. The recombinant protein Amblyomin-X, characterized as a Kunitz-type protease inhibitor, was obtained from a cDNA library from the salivary glands of the Amblyomma cajennense tick. This paper reports the biological effect of Amblyomin-X on inducing cell death by apoptotic process in vitro. For this purpose, the changes in morphological aspects of cells, the phosphatidylserine exposition and DNA degradation were evaluated after treatment with Amblyomin-X. We found that Amblyomin-X was able to induce apoptosis in Renca cells in a dose-dependent manner. So, the results presented here open perspectives for new researches and developing for Amblyomin-X in the treatment of RCC.

The action of Amblyomma cajennense tick saliva in compounds of the hemostatic system and cytotoxicity in tumor cell lines.

Ticks are blood-feeding arthropods that secrete anticoagulant molecules to maintain the fluidity of the blood during its feeding. Tick saliva has many compounds with biological activities that interact directly with host systems, such as blood clotting, platelet aggregation, cell death, among others. Some reports show that there are proteins with anticancer properties in tick saliva. This paper reports some of the biological roles of the Amblyomma cajennense tick saliva, including Factor Xa and thrombin inhibition, action on platelet aggregation, and also preliminary cytotoxic effects on tumor cell lines. The crude saliva was tested in the coagulation, fibrinolysis and platelet aggregation systems. The protein profile of the crude saliva was examined through anion exchange chromatography performed in a FPLC system. The chromatography separated seven protein fractions (Pools I to VII), which biological activities were evaluated. Moreover, the cytotoxic effects of the crude saliva were evaluated on SK-MEL-28 (melanoma cells) and MIA PaCa-2 (pancreas adenocarcinoma cells) using the MTT assay, flow cytometry and fluorescence microscopy. The crude saliva was able to induce cell death on both cancer cells lines, and, interestingly, the cytotoxic effects were not observed on human fibroblasts, which were used as control. The present work opens perspectives for the characterization and development of new molecules involved in the hemostatic system and in cancer control.

A new tick Kunitz type inhibitor, Amblyomin-X, induces tumor cell death by modulating genes related to the cell cycle and targeting the ubiquitin-proteasome system.

The aim of this study was to evaluate the anti-tumor activity of Amblyomin-X, a serine protease Kunitz-type inhibitor. Amblyomin-X induced tumor mass regression and decreased number of metastatic events in a B16F10 murine melanoma model. Alterations on expression of several genes related to cell cycle were observed when two tumor cell lines were treated with Amblyomin-X. PSMB2, which encodes a proteasome subunit, was differentially expressed, in agreement to inhibition of proteasomal activity in both cell lines. In conclusion, our results indicate that Amblyomin-X selectively acts on tumor cells by inducing apoptotic cell death, possibly by targeting the ubiquitin-proteasome system.

Immobilized kidney 28-kDa endostatin-related (KES28kDa) fragment promotes endothelial cell survival.

BACKGROUND/OBJECTIVE: Renal ischemia-hypoxia is a leading cause of acute kidney injury (AKI). Ischemia causes extracellular matrix breakdown of the tubular basement membrane. Endostatin (ES) is the C-terminal fragment of collagen XVIII generated by proteolytic cleavage. Recent studies have demonstrated that ES expression is upregulated in ischemic kidneys. The present study aimed to characterize ES from ischemic kidneys. METHODS: Ischemic renal failure was induced via 45 min of occlusion of the left renal artery and vein. After the ischemic period, blood was collected. Kidneys were harvested and used for immunohistochemical testing and protein extraction. Three-step purification was used. Soluble and immobilized purified ES were tested in cell viability and adhesion assays. results: The soluble KES28kDa inhibited endothelial cell proliferation: 25 versus 12.5 microg (p < 0.05); 12.5 versus 3.15 microg (p < 0.05). Immobilization of KES28kDa supports endothelial cell survival over the control (p = 0.021). Human umbilical vein endothelial cells plated on immobilized KES28kDa showed an increase in membrane ruffles and stress fibers. CONCLUSION: These data demonstrate the local synthesis of a 28-kDa ES-related fragment following AKI and suggest its role in endothelium survival.

Oviposition and eclosion periods of Ixodes didelphidis Fonseca and Aragäo, 1951 (Acari: Ixodidae) under laboratory conditions

Oviposition and eclosion periods for Ixodes didelphidis were observed under two temperatures (25o.C and 27o.C) and 90-95 percent humidity. Although there was a significant increase in the eclosion period (p<0.05) and a tendency to increase the oviposition period at 25o.C, there was neither significant differences in the interval (days), until maximum peak of eclosion nor in the number of emerging larvae during the peak nor the total number of emerged larvae. These temperature values are not critical for embryological development of the species. Because at 27o.C and under high humidity the oviposition and eclosion periods are shorter, and the percentage of emerged larvae is higher, we consider this to be the ideal temperature for laboratory studies