Ecto-Nucleotidase Activities of Promastigotes from Leishmania (Viannia) braziliensis Relates to Parasite Infectivity and Disease Clinical Outcome

Background

Leishmania (Viannia) braziliensis has been associated with a broad range of clinical manifestations ranging from a simple cutaneous ulcer to destructive mucosal lesions. Factors leading to this diversity of clinical presentations are not clear, but parasite factors have lately been recognized as important in determining disease progression. Given the fact that the activity of ecto-nucleotidases correlates with parasitism and the development of infection, we evaluated the activity of these enzymes in promastigotes from 23 L. braziliensis isolates as a possible parasite-related factor that could influence the clinical outcome of the disease.

Methodology/Principal Findings

Our results show that the isolates differ in their ability to hydrolyze adenine nucleotides. Furthermore, we observed a positive correlation between the time for peak of lesion development in C57BL/6J mice and enzymatic activity and clinical manifestation of the isolate. In addition, we found that L. (V.) braziliensis isolates obtained from mucosal lesions hydrolyze higher amounts of adenine nucleotides than isolates obtained from skin lesions. One isolate with high (PPS6m) and another with low (SSF) ecto-nucleotidase activity were chosen for further studies. Mice inoculated with PPS6m show delayed lesion development and present larger parasite loads than animals inoculated with the SSF isolate. In addition, PPS6m modulates the host immune response by inhibiting dendritic cell activation and NO production by activated J774 macrophages. Finally, we observed that the amastigote forms from PPS6m and SSF isolates present low enzymatic activity that does not interfere with NO production and parasite survival in macrophages.

Conclusions/Significance

Our data suggest that ecto-nucleotidases present on the promastigote forms of the parasite may interfere with the establishment of the immune response with consequent impaired ability to control parasite dissemination and this may be an important factor in determining the clinical outcome of leishmaniasis.     

Molecular and morphological characterization of the predatory mite <Emphasis Type="Italic">Amblyseius largoensis</Emphasis> (Acari: Phytoseiidae): surprising similarity between an Asian and American populations

The accurate characterization of biological control agents is a key step in control programs. Recently, Amblyseius largoensis from Thailand were introduced in Brazil to evaluate their efficiency for the control of the red palm mite, Raoiella indica. The aim of this study was to confirm their identification and to characterize the population from Thailand, comparing it to populations of the Americas and Indian Ocean islands. In addition, a population of A. largoensis from New Caledonia, Oceania, of which DNA sequences were available, was included in phylogenetic analyses. Morphometric data obtained for the population of A. largoensis from Thailand were compared to those of populations from Reunion Island and the Americas through univariate and multivariate analyses. Two DNA fragments were amplified and sequenced: the nuclear ribosomal region ITSS and the mitochondrial 12S rRNA. Haplotypes (12S rRNA) and genotypes (ITSS) were identified and phylogenetic analyses using both fragments were conducted separately and combined using maximum likelihood and the Bayesian information criterion. The integrative approach reveals morphometric and molecular variabilities among populations of A. largoensis and shows that the population identified as A. largoensis collected in Thailand, as well as that from New Caledonia, are conspecific to the populations of the Americas and Indian Ocean islands. Populations from the Americas and Asia are more related to each other than with that from the Indian Ocean islands. Hypotheses to explain this clustering are proposed. Data on the molecular intraspecific variability of this predatory mite from remote areas will be helpful for the development of molecular diagnosis.     

Diagrammatic scale for severity evaluation of the Eremanthus erythropappus–Puccinia velata pathosystem

Candeia (Eremanthus erythropappus (DC.) MacLeish), a native forest species from South America, has garnered commercial interest due to its production of essential oil that contains alpha-bisabolol. This compound is widely used in the pharmaceutical and cosmetics industry, with approximately 80% of Brazilian production being exported. Since candeia rust (Puccinia velata) has only been reported in Brazil, little is known about its epidemiology and control. There is no methodology to quantify rust severity in candeia, justifying the elaboration and validation of a diagrammatic scale containing eight levels of disease severity based on leaf area coverage (0.25%, 0.5%, 1%, 2%, 4%, 8%, 16% and 32%). In a natural sampling of disease in the field, 95% of the leaves showed severity below 16%, with the remaining 5% showing severities between 16% and 32% of leaf area. Validation of the proposed diagrammatic scale was performed by assessing the results from 10 inexperienced evaluators, performing evaluations of three leaves with different severity levels. The evaluations were performed at 7-day intervals; in the first instance, severity values were assigned without the diagrammatic scale, and for the second attempt, the scale proposed in this study was used. The accuracy and precision of the severity estimates produced by each evaluator compared to the real severity was analysed by linear regression and by Lin's statistics. The reproducibility of the estimates was evaluated by analysing the coefficient of determination of linear regressions by pairs of evaluators. The scale provided adequate levels of accuracy, precision, repeatability and reproducibility, indicating the proposed scale was a suitable method for quantifying the severity of candeia rust.     

Pulmonary Embolectomy

Embolectomy was carried out in eight patients with pulmonary emboli. Angiographic diagnosis was obtained in six, and in two cases pulmonary angiography could not be done because of the very critical condition of the patients. In the latter two, diagnosis was made based only on clinical findings. Two patients died in the operating room (25 percent). Six patients were discharged in good condition.It is emphasized that pulmonary embolectomy should be done in cases of pulmonary emboli when a clinical status of shock is present (systolic blood pressure less than 80 mm of mercury and the patient in low cardiac output syndrome) and when there is no response to medical treatment regardless of the degree of obstruction in the pulmonary arterial tree.     

Presence of fimH,mrkD, and irp2 Virulence Genes in KPC-2-Producing Klebsiella pneumoniae Isolates in Recife-PE,Brazil

Klebsiella pneumoniae strains can produce different virulence factors, such as fimbrial adhesins and siderophores, which are important in the colonization and development of the infection. The aims of this study were to determine the occurrence of fimH, mrkD, and irp2 virulence genes in 22 KPC-2-producing K. pneumoniae isolates as well as 22 not producing-KPC isolates, from patients from different hospitals in Recife-PE, Brazil, and also to analyze the clonal relationship of the isolates by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). The genes were detected by PCR and DNA sequencing. The bla _KPC-2 gene was identified in 22 KPC-positive isolates. On analyzing the antimicrobial susceptibility profile of the isolates, it was detected that polymyxin and amikacin were the antimicrobials of best activity against K. pneumoniae. On the other hand, five isolates exhibited resistance to polymyxin. In the KPC-positive group, was observed a high rate of resistance to cephalosporins, followed by carbapenems. Molecular typing by ERIC-PCR detected 38 genetic profiles, demonstrating a multiclonal spread of the isolates analyzed. It was observed that the virulence genes irp2, mrkD, and fimH were seen to have together a higher frequency in the KPC-positive group. The accumulation of virulence genes of KPC-positive K. pneumoniae isolates, observed in this study, along with the multi-resistance impose significant therapeutic limitations on the treatment of infections caused by K. pneumoniae.     

Pregnancy associated glycoprotein-1, -6, -7, and -17 are major products of bovine binucleate trophoblast giant cells at midpregnancy

Pregnancy associated glycoproteins (PAGs) are extensively glycosylated secretory proteins of ruminant trophoblast cells. In cattle placenta several PAG cDNAs are expressed, but the variety of correspondent proteins and their degree of glycosylation are not well characterized. Thus, we purified PAGs by using a protocol which included a lectin (Vicia villosa agglutinin) affinity chromatography. Due to their specific glycosylation pattern, PAGs derived from binucleate trophoblast giant cells were highly enriched by this protocol. PAGs were purified from cotyledons of 2 day 100 placentas and from a single placenta at day 155 and 180. In all samples three major bands (75; 66; 56 kDa) were detected by one-dimensional SDS-PAGE. Mass-spectrometric analysis identified the 75 kDa band as a mixture of PAG-7 and PAG-6, the 66 kDa band as PAG-1 and the 56 kDa band as PAG-17. N-terminal sequencing of the day 100 sample confirmed the mass spectrometric identifications. Enzymatic release of N-glycans with peptide-N-glycanase-F from PAGs reduced the molecular weight to approximately 37 kDa which corresponds to the theoretical molecular mass of PAGs. Limited peptide-N-glycanase-F treatment revealed that all four N-glycosylation sites are quantitatively occupied in PAG-1. Compared to PAG-1 the number of potential N-glycosylation sites is lower in PAG-17 (three sites) and higher in PAG-6 and -7 (five and six sites, respectively). This suggests that the number of attached N-glycans is the main determinant of molecular mass of bovine PAGs. The degree of glycosylation may be a major factor regulating the plasma half life of PAGs.     

The use of isobaric tag peptide labeling (iTRAQ) and mass spectrometry to examine rare,primitive hematopoietic cells from patients with chronic myeloid leukemia

Chronic Myeloid Leukemia (CML) is a hematopoietic stem cell disease, associated with a t(9, 22) chromosomal translocation leading to formation of the BCR/ABL chimeric protein, which has an intrinsic tyrosine kinase activity. Recently, the BCR/ABL tyrosine kinase inhibitor imatinib mesylate (imatinib) has been successfully used clinically, although, disease relapse can still occur. The precise detail of the mechanism by which CML cells respond to imatinib is still unclear. We therefore systematically examined the effects of imatinib on the primitive CML cell proteome, having first established that the drug inhibits proliferation and induces increased apoptosis and differentiation. To define imatinib-induced effects on the CML proteome, we employed isobaric tag peptide labeling (iTRAQ) coupled to two-dimensional liquid chromatography/tandem mass spectrometry. Given the limited clinical material available, the isobaric tag approach identified a large population of proteins and provided relative quantification on four samples at once. Novel consequences of the action of imatinib were identified using this mass spectrometric approach. DEAD-box protein 3, heat shock protein 105 kDa, and peroxiredoxin-3 were identified as potential protein markers for response to imatinib. Electronic Supplementary Material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. Stephen D. Griffiths and John Burthem contributed equally to this publication. This work is supported by The Leukaemia Research Fund (UK).