VEGF Promotes Malaria-Associated Acute Lung Injury in Mice

The spectrum of the clinical presentation and severity of malaria infections is broad, ranging from uncomplicated febrile illness to severe forms of disease such as cerebral malaria (CM), acute lung injury (ALI), acute respiratory distress syndrome (ARDS), pregnancy-associated malaria (PAM) or severe anemia (SA). Rodent models that mimic human CM, PAM and SA syndromes have been established. Here, we show that DBA/2 mice infected with P. berghei ANKA constitute a new model for malaria-associated ALI. Up to 60% of the mice showed dyspnea, airway obstruction and hypoxemia and died between days 7 and 12 post-infection. The most common pathological findings were pleural effusion, pulmonary hemorrhage and edema, consistent with increased lung vessel permeability, while the blood-brain barrier was intact. Malaria-associated ALI correlated with high levels of circulating VEGF, produced de novo in the spleen, and its blockage led to protection of mice from this syndrome. In addition, either splenectomization or administration of the anti-inflammatory molecule carbon monoxide led to a significant reduction in the levels of sera VEGF and to protection from ALI. The similarities between the physiopathological lesions described here and the ones occurring in humans, as well as the demonstration that VEGF is a critical host factor in the onset of malaria-associated ALI in mice, not only offers important mechanistic insights into the processes underlying the pathology related with malaria but may also pave the way for interventional studies.

Detection of mechanical and disease stresses in citrus plants by fluorescence spectroscopy

We have investigated the detection of mechanical and disease stresses in citrus plants (Citrus limonia [L.] Osbeck) using laser-induced fluorescence spectroscopy. Due to its economic importance we have chosen to investigate the citrus canker disease, which is caused by the Xanthomonas axonopodis pv. citri bacteria. Mechanical stress was also studied because it plays an important role in the plant's infection by such bacteria. A laser-induced fluorescence spectroscopy system, composed of a spectrometer and a 532 nm 10 mW excitation laser was used to perform fluorescence spectroscopy. The ratio of two chlorophyll fluorescence bands allows us to detect and discriminate between mechanical and disease stresses. This ability to discriminate may have an important application in the field to detect citrus canker infected trees. (c) 2008 Optical Society of America.

Influence of Ecto-Nucleoside Triphosphate Diphosphohydrolase Activity on Trypanosoma cruzi Infectivity and Virulence

Background: The protozoan Trypanosoma cruzi is the causative agent of Chagas disease. There are no vaccines or effective treatment, especially in the chronic phase when most patients are diagnosed. There is a clear necessity to develop new drugs and strategies for the control and treatment of Chagas disease. Recent papers have suggested the ecto-nucleotidases (from CD39 family) from pathogenic agents as important virulence factors. In this study we evaluated the influence of Ecto-Nucleoside-Triphosphate-Diphosphohydrolase (Ecto-NTPDase) activity on infectivity and virulence of T. cruzi using both in vivo and in vitro models. Methodology/Principal Findings: We followed Ecto-NTPDase activities of Y strain infective forms (trypomastigotes) obtained during sequential sub-cultivation in mammalian cells. ATPase/ ADPase activity ratios of cell-derived trypomastigotes decreased 3- to 6-fold and infectivity was substantially reduced during sequential sub-cultivation. Surprisingly, at third to fourth passages most of the cell-derived trypomastigotes could not penetrate mammalian cells and had differentiated into amastigote-like parasites that exhibited 3- to 4-fold lower levels of Ecto-NTPDase activities. To evidence the participation of T. cruzi Ecto-NTPDase1 in the infective process, we evaluated the effect of known Ecto-ATPDase inhibitors (ARL 67156, Gadolinium and Suramin), or anti-NTPDase-1 polyclonal antiserum on ATPase and ADPase hydrolytic activities in recombinant T. cruzi NTPDase-1 and in live trypomastigotes. All tests showed a partial inhibition of Ecto-ATPDase activities and a marked inhibition of trypomastigotes infectivity. Mice infections with Ecto-NTPDase-inhibited trypomastigotes produced lower levels of parasitemia and higher host survival than with non-inhibited control parasites. Conclusions/Significance: Our results suggest that Ecto-ATPDases act as facilitators of infection and virulence in vitro and in vivo and emerge as target candidates in chemotherapy of Chagas disease.

Role of the gp85/Trans-Sialidases in Trypanosoma cruzi Tissue Tropism: Preferential Binding of a Conserved Peptide Motif to the Vasculature In Vivo

Background: Transmitted by blood-sucking insects, the unicellular parasite Trypanosoma cruzi is the causative agent of Chagas' disease, a malady manifested in a variety of symptoms from heart disease to digestive and urinary tract dysfunctions. The reasons for such organ preference have been a matter of great interest in the field, particularly because the parasite can invade nearly every cell line and it can be found in most tissues following an infection. Among the molecular factors that contribute to virulence is a large multigene family of proteins known as gp85/trans-sialidase, which participates in cell attachment and invasion. But whether these proteins also contribute to tissue homing had not yet been investigated. Here, a combination of endothelial cell immortalization and phage display techniques has been used to investigate the role of gp85/trans-sialidase in binding to the vasculature. Methods: Bacteriophage expressing an important peptide motif (denominated FLY) common to all gp85/trans-sialidase proteins was used as a surrogate to investigate the interaction of this motif with the endothelium compartment. For that purpose phage particles were incubated with endothelial cells obtained from different organs or injected into mice intravenously and the number of phage particles bound to cells or tissues was determined. Binding of phages to intermediate filament proteins has also been studied. Findings and Conclusions: Our data indicate that FLY interacts with the endothelium in an organ-dependent manner with significantly higher avidity for the heart vasculature. Phage display results also show that FLY interaction with intermediate filament proteins is not limited to cytokeratin 18 (CK18), which may explain the wide variety of cells infected by the parasite. This is the first time that members of the intermediate filaments in general, constituted by a large group of ubiquitously expressed proteins, have been implicated in T. cruzi cell invasion and tissue homing.

Novel insights into the genomic basis of citrus canker based on the genome sequences of two strains of Xanthomonas fuscans subsp aurantifolii

Background: Citrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002. Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C. Results: We have sequenced the genomes of strains B and C to draft status. We have compared their genomic content to X. citri subsp. citri and to other Xanthomonas genomes, with special emphasis on type III secreted effector repertoires. In addition to pthA, already known to be present in all three citrus canker strains, two additional effector genes, xopE3 and xopAI, are also present in all three strains and are both located on the same putative genomic island. These two effector genes, along with one other effector-like gene in the same region, are thus good candidates for being pathogenicity factors on citrus. Numerous gene content differences also exist between the three cankers strains, which can be correlated with their different virulence and host range. Particular attention was placed on the analysis of genes involved in biofilm formation and quorum sensing, type IV secretion, flagellum synthesis and motility, lipopolysacharide synthesis, and on the gene xacPNP, which codes for a natriuretic protein. Conclusion: We have uncovered numerous commonalities and differences in gene content between the genomes of the pathogenic agents causing citrus canker A, B, and C and other Xanthomonas genomes. Molecular genetics can now be employed to determine the role of these genes in plant-microbe interactions. The gained knowledge will be instrumental for improving citrus canker control.

Structure-Function Analysis of the HrpB2-HrcU Interaction in the Xanthomonas citri Type III Secretion System

Bacterial type III secretion systems deliver protein virulence factors to host cells. Here we characterize the interaction between HrpB2, a small protein secreted by the Xanthomonas citri subsp. citri type III secretion system, and the cytosolic domain of the inner membrane protein HrcU, a paralog of the flagellar protein FlhB. We show that a recombinant fragment corresponding to the C-terminal cytosolic domain of HrcU produced in E. coli suffers cleavage within a conserved Asn264-Pro265-Thr266-His267 (NPTH) sequence. A recombinant HrcU cytosolic domain with N264A, P265A, T266A mutations at the cleavage site (HrcU(AAAH)) was not cleaved and interacted with HrpB2. Furthermore, a polypeptide corresponding to the sequence following the NPTH cleavage site also interacted with HrpB2 indicating that the site for interaction is located after the NPTH site. Non-polar deletion mutants of the hrcU and hrpB2 genes resulted in a total loss of pathogenicity in susceptible citrus plants and disease symptoms could be recovered by expression of HrpB2 and HrcU from extrachromossomal plasmids. Complementation of the Delta hrcU mutant with HrcU(AAAH) produced canker lesions similar to those observed when complemented with wild-type HrcU. HrpB2 secretion however, was significantly reduced in the Delta hrcU mutant complemented with HrcU(AAAH), suggesting that an intact and cleavable NPTH site in HrcU is necessary for total functionally of T3SS in X. citri subsp. citri. Complementation of the Delta hrpB2 X. citri subsp. citri strain with a series of hrpB2 gene mutants revealed that the highly conserved HrpB2 C-terminus is essential for T3SS-dependent development of citrus canker symptoms in planta.

Molluscicidal and ovicidal activities of plant extracts of the Piperaceae on Biomphalaria glabrata (Say, 1818)

Schistosomiasis is a tropical disease caused by Schistosoma and occurs in 54 countries, mainly in South America, the Caribbean region, Africa and the eastern Mediterranean. Currently, 5 to 6 million Brazilian people are infected and 30,000 are under infection risk. Typical of poor regions, this disease is associated with the lack of basic sanitation and very frequently to the use of contaminated water in agriculture, housework and leisure. One of the most efficient methods of controlling the disease is application of molluscicides to eliminate or to reduce the population of the intermediate host snail Biomphalaria glabrata. Studies on molluscicidal activity of plant extracts have been stimulated by issues such as environmental preservation, high cost and recurrent resistance of snails to synthetic molluscicides. The aim of this study was to determine the molluscicide action of extracts from Piperaceae species on adult and embryonic stages of B. glabrata. Fifteen extracts from 13 Piperaceae species were obtained from stems, leaves and roots. Toxicity of extracts was evaluated against snails at two different concentrations (500 and 100 ppm) and those causing 100% mortality at 100 ppm concentration were selected to obtain the LC(90) (lethal concentration of 90% mortality). Piper aduncum, P. crassinervium, P. cuyabanum, P. diospyrifolium and P. hostmannianum gave 100% mortality of adult snails at concentrations ranging from 10 to 60 ppm. These extracts were also assayed on embryonic stages of B. glabrata and those from P. cuyabanum and P. hostmannianum showed 100% ovicidal action at 20 ppm.

New genes of Xanthomonas citri subsp citri involved in pathogenesis and adaptation revealed by a transposon-based mutant library

Background: Citrus canker is a disease caused by the phytopathogens Xanthomonas citri subsp. citri, Xanthomonas fuscans subsp. aurantifolli and Xanthomonas alfalfae subsp. citrumelonis. The first of the three species, which causes citrus bacterial canker type A, is the most widely spread and severe, attacking all citrus species. In Brazil, this species is the most important, being found in practically all areas where citrus canker has been detected. Like most phytobacterioses, there is no efficient way to control citrus canker. Considering the importance of the disease worldwide, investigation is needed to accurately detect which genes are related to the pathogen-host adaptation process and which are associated with pathogenesis. Results: Through transposon insertion mutagenesis, 10,000 mutants of Xanthomonas citri subsp. citri strain 306 (Xcc) were obtained, and 3,300 were inoculated in Rangpur lime (Citrus limonia) leaves. Their ability to cause citrus canker was analyzed every 3 days until 21 days after inoculation; a set of 44 mutants showed altered virulence, with 8 presenting a complete loss of causing citrus canker symptoms. Sequencing of the insertion site in all 44 mutants revealed that 35 different ORFs were hit, since some ORFs were hit in more than one mutant, with mutants for the same ORF presenting the same phenotype. An analysis of these ORFs showed that some encoded genes were previously known as related to pathogenicity in phytobacteria and, more interestingly, revealed new genes never implicated with Xanthomonas pathogenicity before, including hypothetical ORFs. Among the 8 mutants with no canker symptoms are the hrpB4 and hrpX genes, two genes that belong to type III secretion system (TTSS), two hypothetical ORFS and, surprisingly, the htrA gene, a gene reported as involved with the virulence process in animal-pathogenic bacteria but not described as involved in phytobacteria virulence. Nucleic acid hybridization using labeled cDNA probes showed that some of the mutated genes are differentially expressed when the bacterium is grown in citrus leaves. Finally, comparative genomic analysis revealed that 5 mutated ORFs are in new putative pathogenicity islands. Conclusion: The identification of these new genes related with Xcc infection and virulence is a great step towards the understanding of plant-pathogen interactions and could allow the development of strategies to control citrus canker.

Elemental composition changes in citrus affected by the CVC disease

The citrus variegated chlorosis (CVC) disease results in serious economical losses for the Brazilian citriculture. The influence of CVC disease on the elemental composition of citrus plants was investigated. Leaves of sweet orange varieties Hamlin, Pera Rio and Valencia were collected from healthy and CVC-affected trees for chemical characterization by instrumental neutron activation analysis (INAA). Significant differences between healthy and CVC-affected leaves were identified for Ca, Ce, Co, Eu, Fe, K, La, Na, Nd, Rb, Sc and Sm. Rare earth elements presented consistently higher mass fractions in the healthy leaves.

Searching for Moniliophthora perniciosa pathogenicity genes

The basidiomycete Moniliophthora perniciosa is the causal agent of witches` broom disease of Theobroma cacao (cacao). Pathogenesis mechanisms of this hemibiotrophic fungus are largely unknown. An approach to identify putative pathogenicity genes is searching for sequences induced in mycelia grown under in vitro conditions. Using this approach, genes from M. perniciosa induced under limiting nitrogen and light were identified from a cDNA library enriched by suppression subtractive hybridization as potential putative pathogenicity genes. From the 159 identified unique sequences, 59 were annotated and classified by gene ontology. Two sequences were categorized as ""Defence genes, Virulence, and Cell response"" presumably coding for allergenic proteins, whose homologues from other fungi are inducers of animal or plant defences. Differential gene expression was evaluated by quantitative amplification of reversed transcripts (RT-qPCR) of the putative identified genes coding for the two allergenic proteins (Aspf13 and 88KD), and for the enzymes Arylsulfatase (AS); Aryl-Alcohol Oxidase; Aldo-Keto Reductase (AK); Cytochrome P450 (P450); Phenylalanine Ammonia-Lyase; and Peroxidase from mycelia grown under contrasting N concentrations. All genes were validated for differential expression, except for the putative Peroxidase. The same eight genes were analysed for expression in susceptible plants inoculated with M. perniciosa, and six were induced during the early asymptomatic stage of the disease. In infected host tissues, transcripts of 88KD and AS were found more abundant at the biotrophic phase, while those from Aspf13, AK, PAL, and P450 accumulated at the necrotrophic phase, enabling to suggest that mycelia transition from biotrophic to necrotrophic might occur earlier than currently considered. These sequences appeared to be virulence life-style genes, which encode factors or enzymes that enable invasion, colonization or intracellular survival, or manipulate host factors to benefit the pathogen`s own survival in the hostile environment. (C) 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Development of novel microsatellites from Moniliophthora perniciosa, causal agent of the witches` broom disease of Theobroma cacao

Moniliophthora perniciosa is the causal agent of the witches` broom disease of cacao. Based on available genomic sequences, we identified 30 new microsatellite loci, which were analysed using 50 isolates from four populations sampled over a wide geographical area in Brazil, including three populations from the Amazon, the fungal putative centre of diversity, plus one from Bahia. Nine loci were polymorphic, with an average of 2.9 alleles per locus. The level of polymorphism observed was low, but these markers may allow the evaluation of pathogen diversity and the establishment of molecular standards for isolate fingerprinting to support cacao breeding.

The accuracy of the Clock Drawing Test compared to that of standard screening tests for Alzheimer`s disease: results from a study of Brazilian elderly with heterogeneous educational backgrounds

Background: Although the Clock Drawing Test (CDT) is the second most used test in the world for the screening of dementia, there is still debate over its sensitivity specificity, application and interpretation in dementia diagnosis. This study has three main aims: to evaluate the sensitivity and specificity of the CDT in a sample composed of older adults with Alzheimer`s disease (AD) and normal controls; to compare CDT accuracy to the that of the Mini-mental State Examination (MMSE) and the Cambridge Cognitive Examination (CAMCOG), and to test whether the association of the MMSE with the CDT leads to higher or comparable accuracy as that reported for the CAMCOG. Methods: Cross-sectional assessment was carried out for 121 AD and 99 elderly controls with heterogeneous educational levels from a geriatric outpatient clinic who completed the Cambridge Examination for Mental Disorder of the Elderly (CAMDEX). The CDT was evaluated according to the Shulman, Mendez and Sunderland scales. Results: The CDT showed high sensitivity and specificity. There were significant correlations between the CDT and the MMSE (0.700-0.730; p < 0.001) and between the CDT and the CAMCOG (0.753-0.779; p < 0.001). The combination of the CDT with the MMSE improved sensitivity and specificity (SE = 89.2-90%; SP = 71.7-79.8%). Subgroup analysis indicated that for elderly people with lower education, sensitivity and specificity were both adequate and high. Conclusions: The CDT is a robust screening test when compared with the MMSE or the CAMCOG, independent of the scale used for its interpretation. The combination with the MMSE improves its performance significantly, becoming equivalent to the CAMCOG.

Kallikrein-Kinin System Mediated Inflammation in Alzheimer`s Disease In Vivo

The Kallikrein-Kinin System (KKS) has been associated to inflammatory and immunogenic responses in the peripheral and central nervous system by the activation of two receptors, namely B1 receptor and B2 receptor. The B1 receptor is absent or under-expressed in physiological conditions, being up-regulated during tissue injury or in the presence of cytokines. The B2 receptor is constitutive and mediates most of the biological effects of kinins. Some authors suggest a link between the KKS and the neuroinflammation in Alzheimer`s disease (AD). We have recently described an increase in bradykinin (BK) in the cerebrospinal fluid and in densities of B1 and B2 receptors in brain areas related to memory, after chronic infusion of amyloid-beta (A beta) peptide in rats, which was accompanied by memory disruption and neuronal loss. Mice lacking B1 or B2 receptors presented reduced cognitive deficits related to the learning process, after acute intracerebroventricular (i.c.v). administration of A. Nevertheless, our group showed an early disruption of cognitive function by i.c.v. chronic infusion of A beta after a learned task, in the knock-out B2 mice. This suggests a neuroprotective role for B2 receptors. In knock-out B1 mice the memory disruption was absent, implying the participation of this receptor in neurodegenerative processes. The acute or chronic infusion of A beta can lead to different responses of the brain tissue. In this way, the proper involvement of KKS on neuroinflammation in AD probably depends on the amount of A beta injected. Though, BK applied to neurons can exert inflammatory effects, whereas in glial cells, BK can have a potential protective role for neurons, by inhibiting proinflammatory cytokines. This review discusses this duality concerning the KKS and neuroinflammation in AD in vivo.

Stress and coping in older people with Alzheimer`s disease

To investigate stress intensity and coping style in older people with mild Alzheimer`s disease. The potential risk assessment of a stress event and the devising of coping strategies are dependent on cognitive function. Although older individuals with Alzheimer`s disease present significant cognitive impairment, little is known about how these individuals experience stress events and select coping strategies in stress situations. Survey. A convenient sample of 30 cognitively healthy older people and 30 individuals with mild Alzheimer`s disease were given an assessment battery of stress indicators (Symptom Stress List, Cornell Scale for Depression in Dementia, State-Trait Anxiety Inventory), coping style (Jalowiec Coping Scale) and cognitive performance (mini-mental state exam) were applied in both groups. Statistical analysis of the data employed the Mann-Whitney test to compare medians of stress indicators and coping style, Fischer`s exact test to compare proportions when expected frequencies were lower than five, and Spearman`s correlation coefficient to verify correlation between coping style and cognitive performance. Both groups suffered from the same stress intensity (p = 0.254). Regarding coping styles, although differences were not statistically significant (p = 0.124), emotion-oriented coping was predominant in the patients with Alzheimer`s disease. However, those individuals displaying better cognitive performance in the Alzheimer`s disease group had selected coping strategies focused on problem solving (p = 0.0074). Despite a tendency for older people with Alzheimer`s disease to select escape strategies and emotional control, rather than attempting to resolve or lesser the consequences arising from a problem, coping ultimately depends on cognitive performance of the individual. The findings of this study provide information and data to assist planning of appropriate support care for individuals with Alzheimer`s disease who experience stress situations, based on their cognitive performance.

Relationship Between Cortisol Levels and Memory Performance may be Modulated by the Presence or Absence of Cognitive Impairment: Evidence from Healthy Elderly, Mild Cognitive Impairment and Alzheimer`s Disease Subjects

An inverted U-shape function between cortisol levels and memory performance has been reported in studies on both young animals and humans. Yet little is known about this relationship in normal aging or in older subjects with cognitive impairment. This issue is particularly significant since increased levels of cortisol have been reported in Alzheimer`s disease (AD). The present study examined the association between cortisol levels and visual memory performance in healthy subjects as well as in individuals presenting mild cognitive impairment (MCI) or AD. Salivary cortisol was measured in 40 healthy elderly subjects, 31 individuals with amnestic MCI, and 40 subjects with mild probable AD. Memory performance was evaluated using the Brief Cognitive Screening Battery. Higher cortisol levels were associated with better memory performance in healthy elderly (p = 0.005), while higher cortisol levels were correlated with poorer memory performance in MCI subjects (p = 0.011). No correlation between cortisol and memory was found in the AD group (p > 0.05). These results suggest that the relationship between cortisol levels and memory performance in the aging process could vary according to the presence or absence of cognitive impairment.