CD8(+) T cell adjuvant effects of Salmonella FliCd flagellin in live vaccine vectors or as purified protein

Salmonella flagellin, the flagellum structural subunit, has received particular interest as a vaccine adjuvant conferring enhanced immunogenity to soluble proteins or peptides, both for activation of antibody and cellular immune responses. In the present study, we evaluated the Salmonella enterica FliCd flagellin as a T cell vaccine adjuvant using as model the 9-mer (SYVPSAEQI) synthetic H2(d)-restricted CD8(+) T cell-specific epitope (CS(280-288)) derived from the Plasmodium yoelii circumsporozoite (G) protein. The FliCd adjuvant effects were determined under two different conditions: (i) as recombinant flagella, expressed by orally delivered live S. Dublin vaccine strains expressing the target CS(280-288) peptide fused at the central hypervariable domain, and (ii) as purified protein in acellular vaccines in which flagellin was administered to mice either as a recombinant protein fused or admixed with the target CS(280-288) peptide. The results showed that CS(280-288)-specific cytotoxic CD8(+) T cells were primed when BALB/c mice were orally inoculated with the expressing the CS280-288 epitope S. Dublin vaccine strain. In contrast, mice immunized with purified FliCd admixed with the CS280-288 peptide and, to a lesser extent, fused with the target peptide developed specific cytotoxic CD8(+) T cell responses without the need of a heterologous booster immunization. The CD8(+) T cell adjuvant effects of flagellin, either fused or not with the target peptide, correlated with the in vivo activation of CD11c(+) dendritic cells. Taken together, the present results demonstrate that Salmonella flagellins are flexible adjuvant and induce adaptative immune responses when administered by different routes or vaccine formulations. (C) 2009 Elsevier Ltd. All rights reserved.

Naturally Acquired Antibodies to Plasmodium vivax Duffy Binding Protein (DBP) in Rural Brazilian Amazon

Duffy binding protein (DBP), a leading malaria vaccine candidate, plays a critical role ill Plasmodium vivax erythrocyte invasion. Sixty-eight of 366 (18.6%) subjects had IgG anti-DBP antibodies by enzyme-linked immunosorbent assay (ELISA) in a community-based cross-sectional survey ill the Brazilian Amazon Basin. Despite Continuous exposure to low-level malaria transmission, the overall seroprevalence decreased to 9.0% when the Population was reexamined 12 months later. Antibodies from 16 of 50 (360%) Subjects who were ELISA-positive at the baseline were able to inhibit erythrocyte binding to at least one of two DBP variants tested. Most (13 of 16) of these subjects still had inhibitory antibodies when reevaluated 12 months later. Cumulative exposure to malaria was the strongest predictor of DBP seropositivity identified by Multiple logistic regression models in this population. The poor antibody recognition of DBP elicited by natural exposure to P. vivax in Amazonian populations represents a challenge to be addressed by vaccine development strategies.

Seroprevalence of anti-Leishmania spp. antibodies in rural dogs from the city of Monte Negro, State of Rondonia, Brazil

The present study assessed the prevalence of anti-Leishmania spp antibodies in dogs from the city of Monte Negro, State of Rondonia, Brazil ELISA (NE >= 3) and IFAT (>= 1 40) were used to evaluate 161 serum samples collected from rural dogs from Monte Negro Forty-five (27 9%) dogs were positive by ELISA tests and five (3 1%) were positive by IFAT The present study showed for the first time the frequency of exposure to Leishmania spp in dogs in the State of Rondonia, Amazon Region

In vitro sensitivity of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis Brazilian isolates to meglumine antimoniate and amphotericin B

Resistance of Leishmania parasites to specific chemotherapy has become a well-documented problem in the Indian subcontinent in recent years but only a few studies have focused on the susceptibility of American Leishmania isolates. Our susceptibility assays to meglumine antimoniate were performed against intracellular amastigotes after standardizing an in vitro model of macrophage infection appropriate for Leishmania (Viannia) braziliensis isolates. For the determination of promastigote susceptibility to amphotericin B, we developed a simplified MTT-test. The sensitivity in vitro to meglumine antimoniate and amphotericin B of 13 isolates obtained from Brazilian patients was determined. L. (V.) braziliensis isolates were more susceptible to meglumine antimoniate than Leishmania (Leishmania) amazonensis. EC(50), EC(90) and activity indexes (calculated over the sensitivity of reference strains), suggested that all isolates tested were susceptible in vitro to meglumine antimoniate, and did not show association with the clinical outcomes. Isolates were also uniformly susceptible in vitro to amphotericin B.

Characterization of spliced leader genes of Trypanosoma (Megatrypanum) theileri: phylogeographical analysis of Brazilian isolates from cattle supports spatial clustering of genotypes and parity with ribosomal markers

Trypanosoma (Megatrypanum) theileri from cattle and trypanosomes of other artiodactyls form a clade of closely related species in analyses using ribosomal sequences. Analysis of polymorphic sequences of a larger number of trypanosomes from broader geographical origins is required to evaluate the Clustering of isolates as suggested by previous studies. Here, we determined the sequences of the spliced leader (SL) genes of 21 isolates from cattle and 2 from water buffalo from distant regions of Brazil. Analysis of SL gene repeats revealed that the 5S rRNA gene is inserted within the intergenic region. Phylogeographical patterns inferred using SL sequences showed at least 5 major genotypes of T. theileri distributed in 2 strongly divergent lineages. Lineage TthI comprises genotypes IA and IB from buffalo and cattle, respectively, from the Southeast and Central regions, whereas genotype IC is restricted to cattle from the Southern region. Lineage Tth II includes cattle genotypes IIA, which is restricted to the North and Northeast, and IIB, found in the Centre, West, North and Northeast. PCR-RFLP of SL genes revealed valuable markers for genotyping T. theileri. The results of this study emphasize the genetic complexity and corroborate the geographical structuring of T. theileri genotypes found in cattle.

Comparison of two methods for collecting free-living ticks in the Amazonian forest

In the present study, we compared 2 methods for collecting ixodid ticks on the verges of animal trails in a primary Amazon forest area in northern Brazil. (i) Dragging: This method was based on passing a 1-m(2) white flannel over the vegetation and checking the flannel for the presence of caught ticks every 5-10 m. (ii) Visual search: This method consisted of looking for guesting ticks on the tips of leaves of the vegetation bordering animal trails in the forest. A total of 103 adult ticks belonging to 4 Amblyomma species were collected by the visual search method on 5 collecting dates, while only 44 adult ticks belonging to 3 Amblyomma species were collected by dragging on 5 other collecting dates. These values were statistically different (Mann-Whitney Test, P = 0.0472). On the other hand, dragging was more efficient for subadult ticks, since no larva or nymph was collected by visual search, whereas 18 nymphs and 7 larvae were collected by dragging. The visual search method proved to be suitable for collecting adult ticks in the Amazon forest: however, field studies should include a second method, such as dragging in order to maximize the collection of subadult ticks. Indeed, these 2 methods can be performed by a single investigator at the same time, while he/she walks on an animal trail in the forest. (C) 2010 Elsevier GmbH. All rights reserved.

Phylogenetic Validation of the Genera Angomonas and Strigomonas of Trypanosomatids Harboring Bacterial Endosymbionts with the Description of New Species of Trypanosomatids and of Proteobacterial Symbionts

We comparatively examined the nutritional, molecular and optical and electron microscopical characteristics of reference species and new isolates of trypanosomatids harboring bacterial endosymbionts. Sequencing of the V7V8 region of the small subunit of the ribosomal RNA (SSU rRNA) gene distinguished six major genotypes among the 13 isolates examined. The entire sequences of the SSU rRNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) genes were obtained for phylogenetic analyses. In the resulting phylogenetic trees, the symbiont-harboring species clustered as a major clade comprising two subclades that corresponded to the proposed genera Angomonas and Strigomonas. The genus Angomonas comprised 10 flagellates including former Crithidia deanei and C. desouzai plus a new species. The genus Strigomonas included former Crithidia oncopelti and Blastocrithidia cuiicis plus a new species. Sequences from the internal transcribed spacer of ribosomal DNA (ITS rDNA) and size polymorphism of kinetoplast DNA (kDNA) minicircles revealed considerable genetic heterogeneity within the genera Angomonas and Strigomonas. Phylogenetic analyses based on 16S rDNA and ITS rDNA sequences demonstrated that all of the endosymbionts belonged to the Betaproteobacteria and revealed three new species. The congruence of the phylogenetic trees of trypanosomatids and their symbionts support a co-divergent host-symbiont evolutionary history. (C) 2011 Elsevier GmbH. All rights reserved.

Structure and Mode of Action of Microplusin, a Copper II-chelating Antimicrobial Peptide from the Cattle Tick Rhipicephalus (Boophilus) microplus

Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. Here we describe the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single alpha-helical globular domain, microplusin consists of five alpha-helices: alpha 1 (residues Gly-9 to Arg-21), alpha 2 (residues Glu-27 to Asn-40), alpha 3 (residues Arg-44 to Thr-54), alpha 4 (residues Leu-57 to Tyr-64), and alpha 5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied of mode of action of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also demonstrated that microplusin affects M. luteus respiration, a copper-dependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II.

Comparative phylogeography of Trypanosoma cruzi TCIIc: New hosts, association with terrestrial ecotopes, and spatial clustering

We characterized 28 new isolates of Trypanosoma cruzi IIc (TCIIc) of mammals and triatomines from Northern to Southern Brazil, confirming the widespread distribution of this lineage. Phylogenetic analyses using cytochrome b and SSU rDNA sequences clearly separated TCIIc from TCIIa according to terrestrial and arboreal ecotopes of their preferential mammalian hosts and vectors. TCIIc was more closely related to TCIId/e, followed by TCIIa, and separated by large distances from TCIIb and TCI. Despite being indistinguishable by traditional genotyping and generally being assigned to Z3, we provide evidence that TCIIa from South America and TCIIa from North America correspond to independent lineages that circulate in distinct hosts and ecological niches. Armadillos, terrestrial didelphids and rodents, and domestic dogs were found infected by TCIIc in Brazil. We believe that, in Brazil, this is the first description of TCIIc from rodents and domestic dogs. Terrestrial triatomines of genera Panstrongylus and Triatoma were confirmed as vectors of TCIIc. Together, habitat, mammalian host and vector association corroborated the link between TCIIc and terrestrial transmission cycles/ecological niches. Analysis of ITS1 rDNA sequences disclosed clusters of TCIIc isolates in accordance with their geographic origin, independent of their host species. (C) 2009 Elsevier B.V. All rights reserved.

Recurrent Parasitemias and Population Dynamics of Plasmodium vivax Polymorphisms in Rural Amazonia

Clinical trials documented alarming post-treatment Plasmodium vivax recurrence rates caused by recrudescence of surviving asexual blood stages, relapse from hypnozoites, or new infections. Here we describe high rates of P vivax recurrence (26-40% 180 days after treatment) in two cohorts of rural Amazonians exposed to low levels of malaria transmission after a vivax malaria episode treated with chloroquine-primaquine. Microsatellite analysis of 28 paired acute infection and recurrence parasites showed only two pairs of identical haplotypes (consistent with recrudescences or reactivation of homologous hypnozoites) and four pairs of related haplotypes (sharing alleles at 11-13 of 14 microsatellites analyzed). Local isolates of P vivax were extraordinarily diverse and rarely shared the same haplotype, indicating that frequent recurrences did not favor the persistence or reappearance of clonal lineages of parasites in the Population. This fast haplotype replacement rate may represent the typical population dynamics Of neutral polymorphisms in parasites from low-endemicity areas.

(1)H, (15)N and (13)C assignments of the Rhipicephalus (Boophilus) microplus anti-microbial peptide microplusin

Microplusin, a Rhipicephalus (Boophilus) microplus anti-microbial peptide (AMP) is the first member of a new family of cysteine-rich AMPs with histidine-rich regions at the N- and C-termini, which is being fully characterized by biophysical and biochemical methods. Here we report the NMR resonance assignments for (1)H, (15)N, and (13)C nuclei in the backbone and side chains of the microplusin as basis for further studies of structure, backbone dynamics and interactions mapping.

Synthesis, characterization and evaluation of antileishmanial activity of copper(II) with fluorinated alpha-hydroxycarboxylate ligands

In this study, Cu(II) complexes with fluorinated ligands were produced aiming at the development of new, less toxic antileishmanial metallodrugs. Complexes of the general formula CuL(2) (L = lactate, trifluorolactate, 2-hydroxyisobutyrate, trifluoro-2-hydroxyisobutyrate) were synthesized in methanolic medium, purified by crystallization and characterized by elemental analysis and electronic and infrared spectroscopies. In vitro experiments with Leishmania amazonensis promastigotes showed that the trifluorolactate derivative more active than its non-fluorinated counterpart. Our results indicate that fluorinated chelators may be interesting to increase metal toxicity and/or open new paths for metallodrug chemotherapy against leishmaniasis.

Transcriptional memory and switching in the Plasmodium falciparum rif gene family

The human malaria parasite Plasmodium falciparum expresses erythrocyte-surface directed variant antigens which are important virulence factors Many are transcribed from multigene families and presumably their mode of expression is strictly controlled to guarantee immune evasion in the human host. In order to elucidate the dynamics of rif transcription and to investigate if rif switching is comparable to var switching we monitored rif variant gene expression in parasites with different cytoadhesive properties as well as after a number of reinvasions. We found identical transcripts in parasite lines with different adhesive phenotypes suggesting that rif genes do not have a critical role in determining the cytoadhesion specificity of infected erythrocytes. We show for the first time that rif genes may show a conserved mode of transcription, maintaining the previously dominant rif transcript in subsequent reinvasions, but also observed rapid switching at rates up to 45% per generation, much higher than for the var gene family. (C) 2009 Elsevier B.V. All rights reserved.

Genes of cathepsin L-like proteases in Trypanosoma rangeli isolates: Markers for diagnosis, genotyping and phylogenetic relationships

We have sequenced genes encoding cathepsin L-like (CatL-like) cysteine proteases from isolates of Trypanosoma rangeli from humans, wild mammals and Rhodnius species of Central and South America. Phylogenetic trees of sequences encoding mature CatL-like enzymes of T rangeli and homologous genes from other trypanosomes, Leishmania spp. and bodonids positioned sequences of T rangeli (rangelipain) closest to T cruzi (cruzipain). Phylogenetic tree of kinetoplastids based on sequences of CatL-like was totally congruent with those derived from SSU rRNA and gGAPDH genes. Analysis of sequences from the CatL-like catalytic domains of 17 isolates representative of the overall phylogenetic diversity and geographical range of T rangeli supported all the lineages (A-D) previously defined using ribosomal and spliced leader genes. Comparison of the proteolytic activities of T rangeli isolates revealed heterogeneous banding profiles of cysteine proteases in gelatin gels, with differences even among isolates of the same lineage. CatL-like sequences proved to be excellent targets for diagnosis and genotyping of T rangeli by PCR. Data from CatL-like encoding genes agreed with results from previous studies of kDNA markers, and ribosomal and spliced leader genes, thereby corroborating clonal evolution, independent transmission cycles and the divergence of T rangeli lineages associated with sympatric species of Rhodnius. (c) 2009 Elsevier B.V. All rights reserved.

Inhibitory activity of limonene against Leishmania parasites in vitro and in vivo

Limonene is a monoterpene that has antitumoral, antibiotic and antiprotozoal activity. In this study we demonstrate the activity of limonene against Leishmania species in vitro and in vivo. Limonene killed Leishmania amazonensis promastigotes and amastigotes with 50% inhibitory concentrations of 252.0 +/- 49.0 and 147.0 +/- 46.0 mu M, respectively. Limonene was also effective against Leishmania major, Leishmania braziliensis and Leishmania chagasi promastigotes. The treatment of L. amazonensis-infected macrophages with 300 mu M limonene resulted in 78% reduction in infection rates. L. amazonensis-infected mice treated topically or intrarectally with limonene had significant reduction of lesion sizes. A significant decrease in the parasite load was shown in the lesions treated topically with limonene by histopathological examination. The intrarectal treatment was highly effective in decreasing the parasite burden, healing established lesions and suppressing the dissemination of ulcers. Limonene presents low toxicity in humans and has been shown to be effective as an agent for enhancing the percutaneous permeation of drugs. Our results suggest that limonene should be tested in different experimental models of infection by Leishmania. (C) 2009 Elsevier Masson SAS. All rights reserved.