Artificial Feeding of Amblyomma cajennense (Fabricius, 1787) (Acari: Ixodidae) through Silicone Membrane

An artificial feeding system was used where citrated bovine blood was offerred to male and female Amblyomma cajennense. Vestiges of blood, sweat, hair and exfoliated skin were used as phago-stimulants placed on the surface of the silicone membrane. The ticks were collected, as engorged nymphs, from naturally infested equines, with the ecdysis occurring in the laboratory. Four hundred ticks were used, 50% being female, at three to four weeks post-ecdysis. Vestiges of blood on the silicone membrane were the most efficient phago-stimulant and the association of vestiges of blood and sweat residue smears yielded better results compared to the other phago-stimulants used

IL-5 and IL-5 receptor in asthma

Eosinophils, along with mast cells are key cells involved in the innate immune response against parasitic infection whereas the adaptive immune response is largely dependent on lymphocytes. In chronic parasitic disease and in chronic allergic disease, IL-5 is predominantly a T cell derived cytokine which is particularly important for the terminal differentiation, activation and survival of committed eosinophil precursors. The human IL-5 gene is located on chromosome 5 in a gene cluster that contains the evolutionary related IL-4 family of cytokine genes. The human IL-5 receptor complex is a heterodimer consisting of a unique a subunit (predominantly expressed on eosinophils) and a beta subunit which is shared between the receptors for IL-3 & GM-CSF (more widely expressed). The a subunit is required for ligand-specific binding whereas association with the beta subunit results in increased binding affinity. The alternative splicing of the alphaIL-5R gene which contains 14 exons can yield several alphaIL-5R isoforms including a membrane-anchored isoform (alphaIL-5Rm) and a soluble isoform (alphaIL-5Rs). Cytokines such as IL-5 produce specific and non-specific cellular responses through specific cell membrane receptor mediated activation of intracellular signal transduction pathways which, to a large part, regulate gene expression. The major intracellular signal transduction mechanism is activation of non-receptor associated tyrosine kinases including JAK and MAP kinases which can then transduce signals via a novel family of transcriptional factors named signal transducers and activators of transcription (STATS). JAK2, STAT1 and STAT 5 appear to be particularly important in IL-5 mediated eosinophil responses. Asthma is characterized by episodic airways obstruction, increased bronchial responsiveness, and airway inflammation. Several studies have shown an association between the number of activated T cells and eosinophils in the airways and abnormalities in FEV1, airway reactivity and clinical severity in asthma. It has now been well documented that IL-5 is highly expressed in the bronchial mucosa of atopic and intrinsic asthmatics and that the increased IL-5 mRNA present in airway tissues is predominantly T cell derived. Immunocytochemical staining of bronchial biopsy sections has confirmed that IL-5 mRNA transcripts are translated into protein in asthmatic subjects. Furthermore, the number of activated CD 4 + T cells and IL-5 mRNA positive cells are increased in asthmatic airways following antigen challenge and studies that have examined IL-5 expression in asthmatic subjects before and after steroids have shown significantly decreased expression following oral corticosteroid treatment in steroid-sensitive asthma but not in steroid resistant and chronic severe steroid dependent asthma. The link between T cell derived IL-5 and eosinophil activation in asthmatic airways is further strengthened by the demonstration that there is an increased number of alphaIL-5R mRNA positive cells in the bronchial biopsies of atopic and non-atopic asthmatic subjects and that the eosinophil is the predominant site of this increased alphaIL-5R mRNA expression. We have also shown that the subset of activated eosinophils that expressed mRNA for membrane bound alpha IL5r inversely correlated with FEV1, whereas the subset of activated eosinophils that expressed mRNA for soluble alphaIL5r directly correlated with FEV1. Hence, not only does this data suggest that the presence of eosinophils expressing alphaIL-5R mRNA contribute towards the pathogenesis of bronchial asthma, but also that the eosinophil phenotype with respect to alphaIL-5R isoform expression is of central importance. Finally, there are several animal, and more recently in vitro lung explant, models of allergen induced eosinophilia, late airway responses(LARS), and bronchial hyperresponsiveness(BHR) - all of which support a link between IL-5 and airway eosinophila and bronchial hyperresponsiveness. The most direct demonstration of T cell involvement in LARS is the finding that these physiological responses can be transferred by CD4+ but not CD8+ T cells in rats. The importance of IL-5 in animal models of allergen induced bronchial hyperresponsiveness has been further demonstrated by a number of studies which have indicated that IL-5 administration is able to induce late phase responses and BHR and that anti-IL-5 antibody can block allergen induced late phase responses and BHR. In summary, activated T lymphocytes, IL5 production and eosinophil activation are particularly important in the asthmatic response. Human studies in asthma and studies in allergic animal models have clearly emphasised the unique role of IL-5 in linking T lymphocytes and adaptive immunity, the eosinophil effector cell, and the asthma phenotype. The central role of activated lymphocytes and eosinophils in asthma would argue for the likely therapeutic success of strategies to block T cell and eosinophil activation (eg steroids). Importantly, more targeted therapies may avoid the complications associated with steroids. Such therapies could target key T cell activation proteins and cytokines by various means including blocking antibodies (eg anti-CD4, anti-CD40, anti-IL-5 etc), antisense oligonucleotides to their specific mRNAs, and/or selective inhibition of the promoter sites for these genes. Another option would be to target key eosinophil activation mechanisms including the aIL5r. As always, the risk to benefit ratio of such strategies await the results of well conducted clinical trials.

Participation of interleukin-5, interleukin-8 and leukotriene B4 in eosinophil accumulation in two different experimental models

There are several experimental models describing in vivo eosinophil (EO) migration, including ip injection of a large volume of saline (SAL) or Sephadex beads (SEP). The aim of this study was to investigate the mechanisms involved in the EO migration in these two models. Two consecutive injections of SAL given 48 hr apart, induced a selective recruitment of EO into peritoneal cavity of rats, which peaked 48 hr after the last injection. SEP, when injected ip, promoted EO accumulation in rats. The phenomenom was dose-related and peaked 48 hr after SEP injection. To investigate the mediators involved in this process we showed that BW A4C, MK 886 and dexamethasone (DXA) inhibited the EO migration induced by SAL and SEP. To investigate the source of the EO chemotactic factor we showed that mast cells, macrophages (MO), but not lymphocytes, incubated in vitro in presence of SAL released a factor which induced EO migration. With SEP, only mast cells release a factor that induced EO migration, which was inhibited by BW A4C, MK 886 and DXA. Furthermore, the chemotactic activity of SAL-stimulated mast cells was inhibited by antisera against IL-5 and IL-8 (interleukin). SAL-stimulated MO were only inhibited by anti-IL-8 antibodies as well SEP-stimulated mast cells. These results suggest that the EO migration induced by SAL may be dependent on resident mast cells and MO and mediated by LTB4, IL-5 and IL-8. SEP-induced EO migration was dependent on mast cells and may be mediated by LTB4 and IL-8. Furthermore, IL-5 and IL-8 induced EO migration, which was also dependent on resident cells and mediated by LTB4 . In conclusion, EO migration induced by SAL is dependent on mast cells and MO, whereas that induced by SEP is dependent on mast cells alone. Stimulated mast cells release LTB4, IL-5 and IL-8 while MO release LTB4 and IL-8. The IL-5 and IL-8 release by the SAL or SEP-stimulated resident cells may act in an autocrine fashion, thus potentiating LTB4 release.

Role of eosinophilic airway inflammation in models of asthma

Eosinophils play a central role in the establishment and outcome of bronchial inflammation in asthma. Animal models of allergy are useful to answer questions related to mechanisms of allergic inflammation. We have used models of sensitized and boosted guinea pigs to investigate the nature of bronchial inflammation in allergic conditions. These animals develop marked bronchial infiltration composed mainly of CD4+ T-lymphocytes and eosinophils. Further provocation with antigen leads to degranulation of eosinophils and ulceration of the bronchial mucosa. Eosinophils are the first cells to increase in numbers in the mucosa after antigen challenge and depend on the expression of alpha 4 integrin to adhere to the vascular endothelium and transmigrate to the mucosa. Blockage of alpha4 integrin expression with specific antibody prevents not only the transmigration of eosinophils but also the development of bronchial hyperresponsiveness (BHR) to agonists in sensitized and challenged animals, clearly suggesting a role for this cell type in this altered functional state. Moreover, introduction of antibody against Major Basic Protein into the airways also prevents the development of BHR in similar model. BHR can also be suppressed by the use of FK506, an immunosuppressor that reduces in almost 100% the infiltration of eosinophils into the bronchi of allergic animals. These data support the concept that eosinophil is the most important pro-inflammatory factor in bronchial inflammation associated with allergy.

Identification of Novel Membrane Structures in Plasmodium falciparum Infected Erythrocytes

Little is known about the molecular mechanisms underlying the release of merozoites from malaria infected erythrocytes. In this study membranous structures present in the culture medium at the time of merozoite release have been characterized. Biochemical and ultrastructural evidence indicate that membranous structures consist of the infected erythrocyte membrane, the parasitophorous vacuolar membrane and a residual body containing electron dense material. These are subcellular compartments expected in a structure that arises as a consequence of merozoite release from the infected cell. Ultrastructural studies show that a novel structure extends from the former parasite compartment to the surface membrane. Since these membrane modifications are detected only after merozoites have been released from the infected erythrocyte, it is proposed that they might play a role in the release of merozoites from the host cell

Molecular and Antigenic Characterization of the Leishmania (Viannia) panamensis Kinetoplastid Membrane Protein-11

The kinetoplastid membrane protein 11 (KMP-11) has been recently described in Leishmania (Leishmania) donovani as a major component of the promastigote membrane. Two oligonucleotide primers were synthesized to PCR-amplify the entire coding region of New World Leishmania species. The Leishmania (Viannia) panamensis amplification product was cloned, sequenced and the putative amino acid sequence determined. A remarkably high degree of sequence homology was observed with the corresponding molecule of L. (L) donovani and L. (L) infantum (97% and 96%, respectively). Southern blot analysis showed that the KMP-11 locus is conformed by three copies of the gene. The L. (V) panamensis ORF was subsequently cloned in a high expression vector and the recombinant protein was induced and purified from Escherichia coli cultures. Immunoblot analysis showed that 80%, 77% and 100% sera from cutaneous, mucocutaneous and visceral leishmaniasis patients, respectively, recognized the recombinant KMP-11 protein. In a similar assay, 86% of asymptomatic Leishmania-infected individuals showed IgG antibodies against the rKMP-11. We propose that KMP-11 could be used as a serologic marker for infection and disease caused by Leishmania in America.

Internalization of components of the host cell plasma membrane during infection by Trypanosoma cruzi

Epimastigote and trypomastigote forms of Trypanosoma cruzi attach to the macrophage surface and are internalized with the formation of a membrane bounded vacuole, known as the parasitophorous vacuole (PV). In order to determine if components of the host cell membrane are internalized during formation of the PV we labeled the macrophage surface with fluorescent probes for proteins, lipids and sialic acid residues and then allowed the labeled cells to interact with the parasites. The interaction process was interrupted after 1 hr at 37ºC and the distribution of the probes analyzed by confocal laser scanning microscopy. During attachment of the parasites to the macrophage surface an intense labeling of the attachment regions was observed. Subsequently labeling of the membrane lining the parasitophorous vacuole containing epimastigote and trypomastigote forms was seen. Labeling was not uniform, with regions of intense and light or no labeling. The results obtained show that host cell membrane lipids, proteins and sialoglycoconjugates contribute to the formation of the membrane lining the PV containing epimastigote and trypomastigote T. cruzi forms. Lysosomes of the host cell may participate in the process of PV membrane formation.

Experimental models of Schistosoma mansoni infection

Experimental models of Schistosoma mansoni infections in mammals have contributed greatly to our understanding of the pathology and pathogenesis of infection. We consider here hepatic and extrahepatic disease in models of acute and chronic infection. Experimental schistosome infections have also contributed more broadly to our understanding of granulomatous inflammation and our understanding of Th1 versus Th2 related inflammation and particularly to Th2-mediated fibrosis of the liver.

Studies of membrane fluidity and heart contractile force in Trypanosoma cruzi infected mice

In Chagas disease serious cardiac dysfunction can appear. We specifically studied the cardiac function by evaluating: ventricle contractile force and norepinephrine response, affinity and density of beta-adrenergic receptors, dynamic properties of myocardial membranes, and electrocardiography. Albino swiss mice (n = 250) were infected with 55 trypomastigotes, Tulahuen strain and studied at 35, 75, and 180 days post-infection, that correspond to the acute, indeterminate, and chronic phase respectively. Cardiac beta-adrenergic receptors' affinity, myocardial contractility, and norepinephrine response progressively decreased from the acute to the chronic phase of the disease (p < 0.01). The density (expressed as fmol/mg.prot) of the receptors was similar to non-infected mice (71.96 ± 0.36) in both the acute (78.24 ± 1.67) and indeterminate phases (77.28 ± 0.91), but lower in the chronic disease (53.32 ± 0.71). Electrocardiographic abnormalities began in the acute phase and were found in 65% of the infected-mice during the indeterminate and chronic phases. Membrane contents of triglycerides, cholesterol, and anisotropy were similar in all groups. A quadratic correlation between the affinity to beta-adrenergic receptors and cardiac contractile force was obtained. In conclusion the changes in cardiac beta-adrenergic receptors suggests a correlation between the modified beta-adrenergic receptors affinity and the cardiac contractile force.

The role of chemokines in Schistosoma mansoni infection: insights from human disease and murine models

Chemokines are a superfamily of low-molecular-weight cytokines that were initially described for their chemoattractant activity. It is now clear chemokines have several other activities that modulate immune processes. More than 50 chemokines ligands and at least 19 receptors have been described to date. Depending on the number of N-terminal cysteine residues, chemokines are grouped in the subfamilies CXC, CC, C or CX3C. A growing body of evidence suggests a role for chemokines in the pathogenesis of several inflammatory diseases. Our studies involving mice and humans infected with Schistosoma mansoni suggest an important role of the chemokine CCL3 and its receptors (CCR1 and CCR5) in the pathogenesis of severe schistosomiasis. We suggest that the differential activation of CCR1 or CCR5 during the course of schistosomiasis may dictate the outcome of the disease.

Transferrin uptake may occur through detergent-resistant membrane domains at the cytopharynx of Trypanosoma cruzi epimastigote forms

Uptake of transferrin by epimastigote forms of the protozoan Trypanosoma cruzi occurs mainly through a cytostome/ cytopharynx, via uncoated endocytic vesicles that bud off from the bottom of the cytopharynx. We have here examined whether detergent-resistant membrane (DRM) domains might be involved in this process. Purified whole cell membrane fractions were assayed for cholesterol levels and used in dot blot analyses. Detergent-resistant membrane markers (cholera B toxin and anti-flotillin-1 antibody) presented positive reaction by dot blots in cholesterol-rich/ protein-poor membrane sub-fractions. The positive dot blot fraction was submitted to lipid composition analysis, showing composition similar to that of raft fractions described for other eukaryotic cells. Immunofluorescence assays allowed the localization of punctual positive signal for flotillin-1, matching the precise cytostome/ cytopharynx location. These data were confirmed by immunofluorescence assays with the co-localization of flotillin-1 and the transferrin uptake site. Our data suggest that DRM domains occur and are integrated at the cytostome/ cytopharynx of T. cruzi epimastigotes, being the main route for transferrin uptake.

The utility of rhesus monkey (Macaca mulatta) and other non-human primate models for preclinical testing of Leishmania candidate vaccines

Leishmaniasis causes significant morbidity and mortality, constituting an important global health problem for which there are few effective drugs. Given the urgent need to identify a safe and effective Leishmania vaccine to help prevent the two million new cases of human leishmaniasis worldwide each year, all reasonable efforts to achieve this goal should be made. This includes the use of animal models that are as close to leishmanial infection in humans as is practical and feasible. Old world monkey species (macaques, baboons, mandrills etc.) have the closest evolutionary relatedness to humans among the approachable animal models. The Asian rhesus macaques (Macaca mulatta) are quite susceptible to leishmanial infection, develop a human-like disease, exhibit antibodies to Leishmania and parasite-specific T-cell mediated immune responses both in vivo and in vitro, and can be protected effectively by vaccination. Results from macaque vaccine studies could also prove useful in guiding the design of human vaccine trials. This review summarizes our current knowledge on this topic and proposes potential approaches that may result in the more effective use of the macaque model to maximize its potential to help the development of an effective vaccine for human leishmaniasis.

Back to the future in Chagas disease: from animal models to patient cohort studies, progress in immunopathogenesis research

Despite the wealth of information generated by trans-disciplinary research in Chagas disease, knowledge about its multifaceted pathogenesis is still fragmented. Here we review the body of experimental studies in animal models supporting the concept that persistent infection by Trypanosoma cruzi is crucial for the development of chronic myocarditis. Complementing this review, we will make an effort to reconcile seemingly contradictory results concerning the immune profiles of chronic patients from Argentina and Brazil. Finally, we will review the results of molecular studies suggesting that parasite-induced inflammation and tissue damage is, at least in part, mediated by the activities of trans-sialidase, mucin-linked lipid anchors (TLR2 ligand) and cruzipain (a kinin-releasing cysteine protease). One hundred years after the discovery of Chagas disease, it is reassuring that basic and clinical research tends to converge, raising new perspectives for the treatment of chronic Chagas disease.

In vitro and in vivo experimental models for drug screening and development for Chagas disease

Chagas disease, a neglected illness, affects nearly 12-14 million people in endemic areas of Latin America. Although the occurrence of acute cases sharply has declined due to Southern Cone Initiative efforts to control vector transmission, there still remain serious challenges, including the maintenance of sustainable public policies for Chagas disease control and the urgent need for better drugs to treat chagasic patients. Since the introduction of benznidazole and nifurtimox approximately 40 years ago, many natural and synthetic compounds have been assayed against Trypanosoma cruzi, yet only a few compounds have advanced to clinical trials. This reflects, at least in part, the lack of consensus regarding appropriate in vitro and in vivo screening protocols as well as the lack of biomarkers for treating parasitaemia. The development of more effective drugs requires (i) the identification and validation of parasite targets, (ii) compounds to be screened against the targets or the whole parasite and (iii) a panel of minimum standardised procedures to advance leading compounds to clinical trials. This third aim was the topic of the workshop entitled Experimental Models in Drug Screening and Development for Chagas Disease, held in Rio de Janeiro, Brazil, on the 25th and 26th of November 2008 by the Fiocruz Program for Research and Technological Development on Chagas Disease and Drugs for Neglected Diseases Initiative. During the meeting, the minimum steps, requirements and decision gates for the determination of the efficacy of novel drugs for T. cruzi control were evaluated by interdisciplinary experts and an in vitro and in vivo flowchart was designed to serve as a general and standardised protocol for screening potential drugs for the treatment of Chagas disease.

Kinetoplastid membrane protein-11 is present in promastigotes and amastigotes of Leishmania amazonensis and its surface expression increases during metacyclogenesis

Kinetoplastid membrane protein-11 (KMP-11), a protein present in all kinetoplastid protozoa, is considered a potential candidate for a leishmaniasis vaccine. A suitable leishmaniasis vaccine candidate molecule must be expressed in amastigotes, the infective stage for mammals. However, the expression of KMP-11 in Leishmania amastigotes has been a subject of controversy. We evaluated the expression of this molecule in logarithmic and stationary growth phase promastigotes, as well as in amastigotes, of Leishmania amazonensis by immunoblotting, flow cytometry and immunocytochemistry, using a monoclonal antibody against KMP-11. We found that KMP-11 is present in promastigotes and amastigotes. In both stages, the protein was found in association with membrane structures (at the cell surface, flagellar pocket and intracellular vesicles). More importantly, its surface expression is higher in amastigotes than in promastigotes and increases during metacyclogenesis. The increased expression of KMP-11 in metacyclic promastigotes, and especially in amastigotes, indicates a role for this molecule in the parasite relationship with the mammalian host. The presence of this molecule in amastigotes is consistent with the previously demonstrated immunoprotective capacity of vaccine prototypes based on the KMP-11-coding gene and the presence of humoral and cellular immune responses to KMP-11 in Leishmania-infected humans and animals.