Transferência de imunidade passiva (TIP) e dinâmica de anticorpos específicos em bezerros naturalmente expostos para as viroses respiratórias / Passive immune transfer (PIT) and dynamics of specific antibodies in calves naturally exposed to the respiratory viruses

Esta pesquisa avaliou a TIP e a dinâmica de anticorpos (ACs) específicos em bezerros naturalmente expostos aos agentes causadores da doença respiratória bovina (DRB). Foram selecionados 19 bezerros Holandeses alimentados com colostro proveniente de doadoras vacinadas para DRB. Amostras de soro foram obtidas antes e após a ingestão do colostro (48h) para a soroneutralização (SN). Os valores médios (log2) detectados após colostragem foram de 11,5±1,6 (BVDV), 8,8±1,3 (BoHV-1), 5,5±1,6 (BRSV) e 8,4±1,5 (BPIV-3). Cinco bezerros foram criados do nascimento aos 240 dias de vida, observando-se decréscimo nos títulos de ACs para BVDV, BoHV-1 e BPIV-3 ao longo do tempo (P≤0,001). As taxas de infecções detectadas entre o D14 e o D240 foram de 40% (2/5), 20% (1/5), 80% (4/5), e 60% (3/5), respectivamente, para BVDV, BoHV-1, BRSV e BPIV-3. A maioria dos bezerros manifestou broncopneumonia após as infecções virais. Os bezerros apresentaram ACs para todas as viroses às 48 horas de vida, porém os títulos adquiridos para o BRSV foram baixos. A susceptibilidade para as infecções variou de acordo com os níveis e a duração dos títulos de ACs maternos.(AU)

This research evaluated the PIT and the dynamics of specific antibody (Ab) for calves naturally exposed to the viral agents involved in Bovine Respiratory Disease (BRD). Nineteen Holstein calves fed colostrum from vaccinated donors for DRB. Serum samples were obtained before and after colostrum intake (48h) for serum neutralization (SN). Mean values (log2) detected after colostrum feeding were 11.5±1.6 (BVDV), 8.8 ±1.3 (BoHV-1) 5.5±1.6 (BRSV) and 8.4±1.5 (BPIV-3). Five calves were raised from birth to 240 days of life and presented a decrease in Ab titers for BVDV, BoHV-1 and BPIV-3 over time (P≤ 0.001). Infection rates from D14 to D240 were of 40% (2/5), 20% (1/5), 80% (4/5) and 60% (3/5), respectively for BVDV, BoHV-1, BRSV and BPIV-3. Most of the calves presented bronchopneumonia after seroconversion to the virus. Calves presented Ab for all viruses at 48 hours of life, however BRSV Ab titer were low. Levels and persistence of maternal antibody titers determined the susceptibility to viral infections.(AU)

Sn-protoporphyrin inhibits both heme degradation and hemozoin formation in Rhodnius prolixus midgut.

Hematophagy is a feeding habit that involves the ingestion of huge amounts of heme. The hematophagous hemipteran Rhodnius prolixus evolved many genetic resources to protect cells against heme toxicity. The primary barrier against the deleterious effects of heme is the aggregation of heme into hemozoin in the midgut lumen. Hemozoin formation is followed by the enzymatic degradation of heme by means of a unique pathway whose end product is dicysteinyl-biliverdin IX-γ (Rhodnius prolixus biliverdin, RpBv). These mechanisms are complemented by a heme-binding protein (RHBP) in the hemolymph that attenuates the pro-oxidant effects of heme. In this work, we show that when insects are fed with blood enriched with a heme analog, Sn-protoporphyrin (SnPP-IX), both hemozoin synthesis and RpBv production are inhibited in a dose-dependent manner. These effects are accompanied by increased oxidative damage to the midgut epithelium and inhibition of oviposition, indicating that hemozoin formation and heme degradation are protective mechanisms that work together and contributed to the adaptation of this insect to successfully feed on vertebrate blood.

Heme requirement and intracellular trafficking in Trypanosoma cruzi epimastigotes.

Epimastigotes multiplies in the insect midgut by taking up nutrients present in the blood meal including heme bound to hemoglobin of red blood cell. During blood meal digestion by vector proteases in the posterior midgut, hemoglobin is clipped off into amino acids, peptides, and free heme. In this paper, we compared the heme and hemoglobin uptake kinetics and followed their intracellular trafficking. Addition of heme to culture medium increased epimastigote proliferation in a dose-dependent manner, while medium supplemented with hemoglobin enhanced growth after 3-day lag phase. Medium supplemented with globin-derived peptides stimulated cell proliferation in a dose-independent way. Using Palladium mesoporphyrin IX (Pd-mP) as a fluorescent heme-analog, we observed that heme internalization proceeded much faster than that observed by hemoglobin-rhodamine. Binding experiments showed that parasites accumulated the Pd-mP into the posterior region of the cell whereas hemoglobin-rhodamine stained the anterior region. Finally, using different specific inhibitors of ABC transporters we conclude that a P-glycoprotein homologue transporter is probably involved in heme transport through the plasma membrane.

Tracing heme in a living cell: hemoglobin degradation and heme traffic in digest cells of the cattle tick Boophilus microplus.

Heme is present in all cells, acting as a cofactor in essential metabolic pathways such as respiration and photosynthesis. Moreover, both heme and its degradation products, CO, iron and biliverdin, have been ascribed important signaling roles. However, limited knowledge is available on the intracellular pathways involved in the flux of heme between different cell compartments. The cattle tick Boophilus microplus ingests 100 times its own mass in blood. The digest cells of the midgut endocytose blood components and huge amounts of heme are released during hemoglobin digestion. Most of this heme is detoxified by accumulation into a specialized organelle, the hemosome. We followed the fate of hemoglobin and albumin in primary cultures of digest cells by incubation with hemoglobin and albumin labeled with rhodamine. Uptake of hemoglobin by digest cells was inhibited by unlabeled globin, suggesting the presence of receptor-mediated endocytosis. After endocytosis, hemoglobin was observed inside large digestive vesicles. Albumin was exclusively associated with a population of small acidic vesicles, and an excess of unlabeled albumin did not inhibit its uptake. The intracellular pathway of the heme moiety of hemoglobin was specifically monitored using Palladium-mesoporphyrin IX (Pd-mP) as a fluorescent heme analog. When pulse and chase experiments were performed using digest cells incubated with Pd-mP bound to globin (Pd-mP-globin), strong yellow fluorescence was found in large digestive vesicles 4 h after the pulse. By 8 h, the emission of Pd-mP was red-shifted and more evident in the cytoplasm, and at 12 h most of the fluorescence was concentrated inside the hemosomes and had turned green. After 48 h, the Pd-mP signal was exclusively found in hemosomes. In methanol, Pd-mP showed maximal emission at 550 nm, exhibiting a red-shift to 665 nm when bound to proteins in vitro. The red emission in the cytosol and at the boundary of hemosomes suggests the presence of heme-binding proteins, probably involved in transport of heme to the hemosome. The existence of an intracellular heme shuttle from the digestive vesicle to the hemosome acting as a detoxification mechanism should be regarded as a major adaptation of ticks to a blood-feeding way of life. To our knowledge, this is the first direct observation of intracellular transport of heme in a living eukaryotic cell. A similar approach, using Pd-mP fluorescence, could be applied to study heme intracellular metabolism in other cell types.

Binding and storage of heme by vitellin from the cattle tick, Boophilus microplus.

We have previously shown (, Curr. Biol. 9, 703-706) that the cattle tick Boophilus microplus does not synthesize heme, relying solely on the recovery of the heme from the diet to make all its hemeproteins. Here we present evidence that Vitellin (VN(1)), the main tick yolk protein, is a reservoir of heme for embryo development. VN was isolated from eggs at different days throughout embryogenesis. Immediately after oviposition, Boophilus VN contains approximately one mol of heme/mol of protein. During embryo development about one third of egg VN is degraded. The remaining VN molecules bind part of the heme released. These results suggest that VN functions as a heme reservoir, binding any free heme that exceeds the amount needed for development. In vitro measurement of the binding of heme to VN showed that each VN molecule binds up to 31 heme molecules. The association of heme with VN strongly inhibits heme-induced lipid peroxidation, suggesting that binding of heme is an important antioxidant mechanism to protect embryo cells from oxidative damage. This mechanism allows this hematophagous arthropod to safely store heme obtained from a blood meal inside their eggs for future use. Taken together our data suggest that, besides its known roles, VN also plays additional functions as a heme deposit and an antioxidant protective molecule.

On the biosynthesis of Rhodnius prolixus heme-binding protein.

The biosynthesis of Rhodnius prolixus heme-binding protein (RHBP), which is present in the hemolymph and oocytes of Rhodnius prolixus, was investigated. Fat bodies of female insects incubated in vitro with 14C-leucine were able to synthesize and secrete 14C-RHBP to the culture medium. Titrtion of synthesized RHBP with hemin showed that the protein secreted by the fat bodies is bound to heme, despite the presence of apo-RHBP in the hemolymph. The sequence of the RHBP cDNA encodes a pre-protein of 128 amino acids with no significant homology to any known protein. Northern-blot assays revealed that RHBP expression was limited to fat bodies. The levels of both RHBP mRNA and secreted protein increased in response to blood meal. In addition, the time-course of RHBP secretion in vitro paralleled mRNA accumulation observed in vivo. The inhibition of the de novo heme biosynthesis by treatment of fat bodies with succinyl acetone (SA), an irreversible inhibitor of delta-aminolevulinic acid-dehydratase, led to a significant decrease of heme-RHBP secretion. Nevertheless, the levels of RHBP mRNA were not modified by SA treatment, suggesting that the heme availability is involved in a post-transcriptional control of the RHBP synthesis.

Production of reactive oxygen species by hemocytes from the cattle tick Boophilus microplus.

We have investigated the phagocytic activity and the production of reactive oxygen species (ROS) by hemocytes from the cattle tick Boophilus microplus. Two main types of hemocytes were detected in tick hemolymph: plasmatocytes and granulocytes. The plasmocytes were the most abundant cells, being responsible for the in vivo phagocytosis of yeast. ROS production was evaluated by luminol-amplified luminescence and phenol red oxidation. The luminescence increased when hemocytes were incubated with bacteria, zymosan, or phorbol 12-miristate 13-acetate (PMA). The luminescence was inhibited by superoxide dismutase and catalase, which are antioxidant enzymes that remove superoxide and hydrogen peroxide, respectively. The phenol red oxidation assay also showed an increase in the level of hydrogen peroxide produced by hemocytes stimulated with bacteria and PMA. Taken all together, our data indicate that tick hemocytes are able to produce ROS during the phagocytic process similarly to vertebrate phagocytes.

The Brazilian investment in science and technology

An analysis of Brazilian federal expenditures in science and technology is presented is this study. The 1990-1999 data were compiled from records provided by two federal agencies (MCT and CNPq) responsible for managing most of the national budget related to these activities. The results indicate that the federal investments in Brazilian science and technology stagnated during the last decade (US$ 2.32 billion in 1990, US$ 2.39 billion in 1996, and US$ 2.36 billion in 1999). In contrast, a great increase in private investments in research was acknowledged both by industry and by the government during the same period, from US$ 2.12 to US$ 4.64 billion. However, this investment did not result in an increase in invention patents granted to residents (492 in 1990 and only 232 in 1997) or in a reduction of patent costs. Despite this unfavorable scenario, the number of graduate programs in the country has increased two-fold in the last decade and the contribution of Brazilians to the database of the Institute for Scientific Information has increased 4.7-fold from 1990 (2,725 scientific publications) to 2000 (12,686 scientific publications). Unstable federal resources for science, together with the poor returns of private resources in terms of developing new technologies, may jeopardize the future of Brazilian technological development

The Brazilian investment in science and technology.

An analysis of Brazilian federal expenditures in science and technology is presented is this study. The 1990-1999 data were compiled from records provided by two federal agencies (MCT and CNPq) responsible for managing most of the national budget related to these activities. The results indicate that the federal investments in Brazilian science and technology stagnated during the last decade (US$ 2.32 billion in 1990, US$ 2.39 billion in 1996, and US$ 2.36 billion in 1999). In contrast, a great increase in private investments in research was acknowledged both by industry and by the government during the same period, from US$ 2.12 to US$ 4.64 billion. However, this investment did not result in an increase in invention patents granted to residents (492 in 1990 and only 232 in 1997) or in a reduction of patent costs. Despite this unfavorable scenario, the number of graduate programs in the country has increased two-fold in the last decade and the contribution of Brazilians to the database of the Institute for Scientific Information has increased 4.7-fold from 1990 (2,725 scientific publications) to 2000 (12,686 scientific publications). Unstable federal resources for science, together with the poor returns of private resources in terms of developing new technologies, may jeopardize the future of Brazilian technological development.

Hydrogen peroxide detoxification in the midgut of the blood-sucking insect, Rhodnius prolixus.

Here we investigated H2O2 production and detoxification in the hematophagous hemiptera, Rhodnius prolixus. Superoxide dismutase (SOD) catalyzes the dismutation of superoxide radical (O2-). This reaction produces hydrogen peroxide, which is scavenged by antioxidant enzymes such as catalase (CAT). SOD and CAT activities were found in all tissues studied, being highest in the midgut. CAT was dose-dependently inhibited in vivo by injections of 3-amino-1,2,4-triazole (AT). Insects treated with AT showed a twofold increase in H2O2 levels. Injection of DL-buthionine-[S, R]-sulfoximine (BSO), an inhibitor of glutathione synthesis, also resulted in a fourfold increase in H2O2, together with stimulation of CAT activity. Simultaneous administration of both AT and BSO had a synergistic effect on midgut H2O2 content. Taken all together, our results suggest that CAT and glutathione-dependent mechanisms cooperate to control H2O2 concentration in the midgut cell and prevent hydroxyl radical generation by Fenton reaction in this tissue.

Crystallization and preliminary X-ray study of haem-binding protein from the bloodsucking insect Rhodnius prolixus.

Rhodnius haem-binding protein (RHBP) from the bloodsucking insect Rhodnius prolixus, a 15 kDa protein, has been crystallized using polyethylene glycol as a precipitant. X-ray diffraction data have been collected at a synchrotron source. The crystals belong to the space group P4(1(3))2(1)2, with unit-cell parameters a = b = 64.98, c = 210.68 A, and diffract beyond 2.6 A resolution.

Heme biosynthesis and oogenesis in the blood-sucking bug, Rhodnius prolixus.

We have previously shown that the pathway of porphyrin synthesis operates in the blood feeding triatomine bug Rhodnius prolixus but not in the cattle tick Boophilus microplus. In the present paper we studied the correlation between heme synthesis and egg development in Rhodnius. There is a sharp increase heme biosynthetic capability in the fat body (160%) and in the ovaries (360%) in response to a blood meal, as evaluated from the activity of the enzyme delta-aminolevulinate dehydratase (EC 4.2.1.24). The in vivo inhibition of ALA-D by succinyl acetone results in a dose dependent decrease of oviposition. Oviposition is recovered when porphobilinogen, the product of the impaired reaction, is added to the succinyl acetone enriched blood. Taken together, these results show that heme biosynthesis is a fundamental event to vitellogenic females. The demand for heme in this metabolic juncture cannot be supplied by the heme eventually absorbed during blood digestion and associated with Rhodnius heme binding protein (RHBP), which is then incorporated into growing oocytes. Inhibition of heme biosynthesis results in lower levels of RHBP in the hemolymph, suggesting that the synthesis of this protein is controlled by heme availability.

Ochratoxin A in Brazilian roasted and instant coffees.

Thirty-four samples of roast and ground coffee, 14 samples of instant coffee and two samples of decaffeinated instant coffee were collected in markets and supermarkets in the city of Campinas, Brazil, and analysed for ochratoxin A using immunoaffinity columns for clean-up and HPLC with fluorescence detection for quantification. The limit of detection was 0.2 ng/g ochratoxin A. Twenty-three samples of ground and roast coffee were found to be contaminated with the toxin at levels ranging between 0.3 and 6.5 ng/g. The average concentration in all 34 samples was 0.9 ng/g. All samples of instant coffee contained ochratoxin A at levels ranging from 0.5 to 5.1 ng/g, with an average figure of 2.2 ng/g. Roast and ground coffee is the type of coffee most used by Brazilians for the preparation of the beverage. Considering that an average Brazilian adult takes five cups of coffee per day, which corresponds to 30 g of roast and ground coffee, the probable daily intake of ochratoxin A by a 70 kg adult would be 0.4 ng/kg bw, which is far below the current Provisional Tolerable Daily Intake of 14 ng/kg bw for ochratoxin A as set by the Codex Alimentarius. To study the transfer of ochratoxin A into coffee brew, the beverage was prepared by two methods: (a) the drip method and (b) the Brazilian country style method. No significant difference was observed between the two methods in terms of extraction of the toxin using five contaminated samples containing between 0.8 and 6.5 ng/g ochratoxin A. The drip method extracted 86 +/- 15% and the Brazilian country style 74 +/- 20% of the ochratoxin A initially present in the roast and ground coffee.

HeLp, a heme lipoprotein from the hemolymph of the cattle tick, Boophilus microplus.

The main protein of the hemolymph of the cattle tick Boophilus microplus has been isolated and shown to be a heme lipoprotein (HeLp). HeLp has an apparent molecular mass of 354,000 and contains two apoproteins (103 and 92 kDa) found in equal amounts. HeLp presents a pI of 5.8 and a density of 1.28 g/ml and contains 33% lipids, containing both neutral lipids and phospholipids, and 3% of sugars. A remarkable feature of HeLp is the abundance of cholesterol ester (35% of total lipids), a lipid not previously reported in invertebrate lipoproteins. Western blot analysis showed HeLp in hemolymph from adult females and males, but not in eggs. Although HeLp contains 2 heme molecules, it is capable of binding 6 additional molecules of heme. Boophilus feeds large amount of blood, and we recently showed that this tick is unable to perform de novo synthesis of heme (Braz, G. R. C., Coelho, H. S. L., Masuda, H., and Oliveira, P. L. (1999) Curr. Biol. 9, 703-706). Injection of tick females with (55)Fe-labeled heme-HeLp indicated that this protein transports heme from hemolymph to tissues. HeLp is suggested to be an essential adaptation to the loss of the heme synthesis pathway.

A heme-binding aspartic proteinase from the eggs of the hard tick Boophilus microplus.

An aspartic proteinase that binds heme with a 1:1 stoichiometry was isolated and cloned from the eggs of the cattle tick Boophilus microplus. This proteinase, herein named THAP (tick heme-binding aspartic proteinase) showed pepstatin-sensitive hydrolytic activity against several peptide and protein substrates. Although hemoglobin was a good substrate for THAP, low proteolytic activity was observed against globin devoid of the heme prosthetic group. Hydrolysis of globin by THAP increased as increasing amounts of heme were added to globin, with maximum activation at a heme-to-globin 1:1 ratio. Further additions of heme to the reaction medium inhibited proteolysis, back to a level similar to that observed against globin alone. The addition of heme did not change THAP activity toward a synthetic peptide or against ribonuclease, a non-hemeprotein substrate. The major storage protein of tick eggs, vitellin (VT), the probable physiological substrate of THAP, is a hemeprotein. Hydrolysis of VT by THAP was also inhibited by the addition of heme to the incubation media. Taken together, our results suggest that THAP uses heme bound to VT as a docking site to increase specificity and regulate VT degradation according to heme availability.

Haemozoin formation in the midgut of the blood-sucking insect Rhodnius prolixus.

Malaria parasites digest haemoglobin and detoxify the free haem by its sequestration into an insoluble dark-brown pigment known as haemozoin (Hz). Until recently, this pigment could be found only in Plasmodium parasites. However, we have shown that Hz is also present in the midgut of the blood-sucking insect Rhodnius prolixus [Oliveira et al. (1999) Nature 400, 517-518]. Here we show that Hz synthesis in the midgut of this insect is promoted by a particulate fraction from intestine lumen. Haem aggregation activity is heat-labile and is inhibited in vitro by chloroquine (CLQ). Inhibition of Hz formation in vivo by feeding insects with CLQ leads to increased levels of haem in the haemolymph of the insect, which resulted in increased lipid peroxidation. Taken together, these results indicate that a factor capable of promoting Hz crystallisation is present in R. prolixus midgut and that this activity represents an important physiological defence of this insect against haem toxicity.

A missing metabolic pathway in the cattle tick Boophilus microplus.

Heme proteins are involved in a wide variety of biological reactions, including respiration, oxygen transport and oxygen metabolism [1]. The heme prosthetic group is synthesized in almost all living organisms except for a few pathogenic bacteria and trypanosomatids that use blood as food [2] [3]. There is a general belief that all nucleated animal cells synthesize heme [1] [4]. However, blood-feeding arthropods ingest enormous amounts of vertebrate blood in a single meal and the heme pathway has not been studied in these animals. We have examined heme synthesis in two hematophagous arthropods - the blood-sucking bug Rhodnius prolixus and the cattle tick Boophilus microplus. We show that R. prolixus makes heme and has a fully operative heme biosynthetic pathway, while B. microplus does not. To our knowledge, this is the first report of an animal that does not synthesize its own heme and relies solely on the recovery of heme present in the diet. Because of the inability of Boophilus to synthesize heme and its ability to deal efficiently with large amounts of free heme, we propose this organism as a good model for studying heme transport and reutilization in animal cells.

Urate synthesis in the blood-sucking insect rhodnius prolixus. Stimulation by hemin is mediated by protein kinase C.

Hemin is a catalyst of the formation of reactive oxygen species. We proposed that hematophagous insects are exposed to intense oxidative stress because of hemoglobin hydrolysis in their midgut (Petretsky, M. D., Ribeiro, J. M. C., Atella, G. C., Masuda, H., and Oliveira, P. L. (1995) J. Biol. Chem. 270, 10893-10896). We have shown that hemin stimulates urate synthesis in the blood-sucking insect Rhodnius prolixus (Graça-Souza, A. V., Petretsky, J. H., Demasi, M., Bechara, E. J. H., and Oliveira, P. L. (1997) Free Radical Biol. Med. 22, 209-214). Once released by fat body cells, urate accumulates in the hemolymph, where this radical scavenger constitutes an important defense against blood-feeding derived oxidative stress. Incubation of Rhodnius fat bodies with okadaic acid raises the level of urate synthesis, suggesting that urate production can be controlled by protein phosphorylation/dephosphorylation. Urate synthesis is stimulated by dibutyryl cAMP and inhibited by N(2((p-bromocinnamil)amino)ethyl)-5-isoquinolinesulfonamide (H-89), an inhibitor of protein kinase A, as well as activated by the protein kinase C activator phorbol 12-myristate 13-acetate. In the presence of hemin, however, inhibition of urate synthesis by H-89 does not occur, suggesting that the hemin stimulatory effect is not mediated by protein kinase A. Calphostin C completely inhibits the hemin-induced urate production, suggesting that the triggering of urate antioxidant response depends on protein kinase C activation. This conclusion is reinforced by the observation that in fat bodies exposed to hemin, both protein kinase C activity and phosphorylation of specific endogenous polypeptides are significantly increased.