Methodological approaches to planar and volumetric scintigraphic imaging of small volume targets with high spatial resolution and sensitivity

Single-photon emission computed tomography (SPECT) is a non-invasive imaging technique, which provides information reporting the functional states of tissues. SPECT imaging has been used as a diagnostic tool in several human disorders and can be used in animal models of diseases for physiopathological, genomic and drug discovery studies. However, most of the experimental models used in research involve rodents, which are at least one order of magnitude smaller in linear dimensions than man. Consequently, images of targets obtained with conventional gamma-cameras and collimators have poor spatial resolution and statistical quality. We review the methodological approaches developed in recent years in order to obtain images of small targets with good spatial resolution and sensitivity. Multipinhole, coded mask- and slit-based collimators are presented as alternative approaches to improve image quality. In combination with appropriate decoding algorithms, these collimators permit a significant reduction of the time needed to register the projections used to make 3-D representations of the volumetric distribution of target’s radiotracers. Simultaneously, they can be used to minimize artifacts and blurring arising when single pinhole collimators are used. Representation images are presented, which illustrate the use of these collimators. We also comment on the use of coded masks to attain tomographic resolution with a single projection, as discussed by some investigators since their introduction to obtain near-field images. We conclude this review by showing that the use of appropriate hardware and software tools adapted to conventional gamma-cameras can be of great help in obtaining relevant functional information in experiments using small animals.

Transport pathways in the malaria-infected erythrocyte: characterization and their use as potential targets for chemotherapy

The intraerythrocytic malarial parasite is involved in an extremely intensive anabolic activity while it resides in its metabolically quiescent host cell. The necessary fast uptake of nutrients and the discharge of waste product, are guaranteed by parasite-induced alterations of the constitutive transporters of the host cell and the production of new parallel pathways. The membrane of the host cell thus becomes permeable to phospholipids, purine bases and nucleosides, small non-electrolytes, anions and cations. When the new pathways are quantitatively unimportant, classical inhibitors of native transporters can be used to inhibit parasite growth. Several compounds were found to effectively inhibit the new pathways and consequently, parasite growth. The pathways have also been used to introduce cytotoxic agents. The parasitophorous membrane consists of channels which are highly permeable to small solutes and display no ion selectivity. Transport of some cations and anions across the parasite membrane is rapid and insensitive to classical inhibitors, and in some cases it is mediated by specific antiporters which respond to their respective inhibitors. Macromolecules have been shown to reach the parasitophorous space through a duct contiguous with the host cell membrane, and subsequently to be endocytosed at the parasite membrane. The simultaneous presence of the parasitophorous membrane channels and the duct, however, is incompatible with experimental evidences. No specific inhibitors were found as yet that would efficiently inhibit transport through the channels or the duct.

Interleukin-4 and interleukin-5 as targets for the inhibition of eosinophilic inflammation and allergic airways hyperreactivity

Clinical and experimental investigations suggest that allergen-specific CD4+ T-cells, IgE and the cytokines IL-4 and IL-5 play central roles in initiating and sustaining an asthmatic response by regulating the recruitment and/or activation of airways mast cells and eosinophils. IL-5 plays a unique role in eosinophil development and activation and has been strongly implicated in the aetiology of asthma. The present paper summarizes our recent investigations on the role of these cytokines using cytokine knockout mice and a mouse aeroallergen model. Investigations in IL-5-/- mice indicate that this cytokine is critical for regulating aeroallergen-induced eosinophilia, the onset of lung damage and airways hyperreactivity during allergic airways inflammation. While IL-4 and allergen-specific IgE play important roles in the regulation of allergic disease, recent investigations in IL4-/- mice suggest that allergic airways inflammation can occur via pathways which operate independently of these molecules. Activation of these IL-4 independent pathways are also intimately associated with CD4+ T-cells, IL-5 signal transduction and eosinophilic inflammation. Such IL-5 regulated pathways may also play a substantive role in the aetiology of asthma. Thus, evidence is now emerging that allergic airways disease is regulated by humoral and cell mediated processes. The central role of IL-5 in both components of allergic disease highlights the requirements for highly specific therapeutic agents which inhibit the production or action of this cytokine.

Trypanosoma cruzi-elicited CD8+ T cell-mediated myocarditis: chemokine receptors and adhesion molecules as potential therapeutic targets to control chronic inflammation?

In Chagas disease, during the acute phase, the establishment of inflammatory processes is crucial for Trypanosoma cruzi control in target tissues and for the establishment of host/parasite equilibrium. However, in about 30% of the patients, inflammation becomes progressive, resulting in chronic disease, mainly characterized by myocarditis. Although several hypothesis have been raised to explain the pathogenesis of chagasic myocardiopathy, including the persistence of the parasite and/or participation of autoimmune processes, the molecular mechanisms underlying the establishment of the inflammatory process leading to parasitism control but also contributing to the maintenance of T. cruzi-elicited chronic myocarditis remain unsolved. Trying to shed light on these questions, we have for several years been working with murine models for Chagas disease that reproduce the acute self-resolving meningoencephalitis, the encephalitis resulting of reactivation described in immunodeficient individuals, and several aspects of the acute and chronic myocarditis. In the present review, our results are summarized and discussed under the light of the current literature. Furthermore, rational therapeutic intervention strategies based on integrin-mediated adhesion and chemokine receptor-driven recruitment of leukocytes are proposed to control T. cruzi-elicited unbalanced inflammation.

The use of biodiversity as source of new chemical entities against defined molecular targets for treatment of malaria, tuberculosis, and T-cell mediated diseases: a review

The modern approach to the development of new chemical entities against complex diseases, especially the neglected endemic diseases such as tuberculosis and malaria, is based on the use of defined molecular targets. Among the advantages, this approach allows (i) the search and identification of lead compounds with defined molecular mechanisms against a defined target (e.g. enzymes from defined pathways), (ii) the analysis of a great number of compounds with a favorable cost/benefit ratio, (iii) the development even in the initial stages of compounds with selective toxicity (the fundamental principle of chemotherapy), (iv) the evaluation of plant extracts as well as of pure substances. The current use of such technology, unfortunately, is concentrated in developed countries, especially in the big pharma. This fact contributes in a significant way to hamper the development of innovative new compounds to treat neglected diseases. The large biodiversity within the territory of Brazil puts the country in a strategic position to develop the rational and sustained exploration of new metabolites of therapeutic value. The extension of the country covers a wide range of climates, soil types, and altitudes, providing a unique set of selective pressures for the adaptation of plant life in these scenarios. Chemical diversity is also driven by these forces, in an attempt to best fit the plant communities to the particular abiotic stresses, fauna, and microbes that co-exist with them. Certain areas of vegetation (Amazonian Forest, Atlantic Forest, Araucaria Forest, Cerrado-Brazilian Savanna, and Caatinga) are rich in species and types of environments to be used to search for natural compounds active against tuberculosis, malaria, and chronic-degenerative diseases. The present review describes some strategies to search for natural compounds, whose choice can be based on ethnobotanical and chemotaxonomical studies, and screen for their ability to bind to immobilized drug targets and to inhibit their activities. Molecular cloning, gene knockout, protein expression and purification, N-terminal sequencing, and mass spectrometry are the methods of choice to provide homogeneous drug targets for immobilization by optimized chemical reactions. Plant extract preparations, fractionation of promising plant extracts, propagation protocols and definition of in planta studies to maximize product yield of plant species producing active compounds have to be performed to provide a continuing supply of bioactive materials. Chemical characterization of natural compounds, determination of mode of action by kinetics and other spectroscopic methods (MS, X-ray, NMR), as well as in vitro and in vivo biological assays, chemical derivatization, and structure-activity relationships have to be carried out to provide a thorough knowledge on which to base the search for natural compounds or their derivatives with biological activity.

Polymerase chain reaction with two molecular targets in mucosal leishmaniasis' diagnosis: a validation study

We validated the polymerase chain reaction (PCR) with a composite reference standard in 61 patients clinically suspected of having mucosal leishmaniasis, 36 of which were cases and 25 were non-cases according to this reference standard. Patient classification and test application were carried out independently by two blind observers. One pair of primers was used to amplify a fragment of 120 bp in the conserved region of kDNA and another pair was used to amplify the internal transcript spacers (ITS) rDNA. PCR showed 68.6% (95% CI 59.2-72.6) sensitivity and 92% (95% CI 78.9-97.7) specificity; positive likelihood ratio: 8.6 (95% CI 2.8-31.3) and negative likelihood ratio: 0.3 (95% CI 0.3-0.5), when kDNA molecular target was amplified. The test performed better on sensitivity using this target compared to the ITS rDNA molecular target which showed 40% (95% CI 31.5-42.3) sensitivity and 96% (95% CI 84.1-99.3) specificity; positive likelihood ratio: 10 (95% CI 2.0-58.8) and negative likelihood ratio: 0.6 (95% CI 0.6-0.8). The inter-observer agreement was excellent for both tests. Based upon results obtained and due to low performance of conventional methods for diagnosing mucosal leishmaniasis, we consider PCR with kDNA as molecular target is a useful diagnostic test and the ITS rDNA molecular target is useful when the aim is to identify species.

Chromatin regulation in schistosomes and histone modifying enzymes as drug targets

Only one drug is currently available for the treatment and control of schistosomiasis and the increasing risk of selecting strains of schistosome that are resistant to praziquantel means that the development of new drugs is urgent. With this objective we have chosen to target the enzymes modifying histones and in particular the histone acetyltransferases and histone deacetylases (HDAC). Inhibitors of HDACs (HDACi) are under intense study as potential anti-cancer drugs and act via the induction of cell cycle arrest and/or apoptosis. Schistosomes like other parasites can be considered as similar to tumours in that they maintain an intense metabolic activity and rate of cell division that is outside the control of the host. We have shown that HDACi can induce apoptosis and death of schistosomes maintained in culture and have set up a consortium (Schistosome Epigenetics: Targets, Regulation, New Drugs) funded by the European Commission with the aim of developing inhibitors specific for schistosome histone modifying enzymes as novel lead compounds for drug development.

Meliponini neotropicais: o gênero Partamona Schwarz, 1939 (Hymenoptera, Apidae, Apinae) - bionomia e biogeografia

This work, dedicated to the study of nesting habits of the species of the Neotropical genus Partamona Schwarz, is a sequence to the taxonomic revision recently published elsewhere. A total of 214 nests and nest aggregations of 18 species [Partamona epiphytophila Pedro & Camargo, 2003; P. testacea (Klug, 1807); P. mourei Camargo, 1980; P. vicina Camargo, 1980; P. auripennis Pedro & Camargo, 2003; P. combinata Pedro & Camargo, 2003; P. chapadicola Pedro & Camargo, 2003; P. nhambiquara Pedro & Camargo, 2003; P. ferreirai Pedro & Camargo, 2003; P. pearsoni (Schwarz, 1938); P. gregaria Pedro & Camargo, 2003; P. batesi Pedro & Camargo, 2003; P. ailyae Camargo, 1980; P. cupira (Smith, 1863); P. mulata Moure in Camargo, 1980; P. seridoensis Pedro & Camargo, 2003; P. criptica Pedro & Camargo, 2003; P. helleri (Friese, 1900)] were studied , including data about habitat, substrate, structural characteristics, construction materials and behavior. The descriptions of the nests are illustrated with 48 drawings. Partial data of the nests of P. bilineata (Say, 1837), P. xanthogastra Pedro & Camargo, 1997, P. orizabaensis (Strand, 1919), P. peckolti (Friese, 1901), P. aequatoriana Camargo, 1980, P. musarum (Cockerell, 1917) and P. rustica Pedro & Camargo, 2003 are also presented. Nests of P. grandipennis (Schwarz, 1951), P. yungarum Pedro & Camargo, 2003, P. subtilis Pedro & Camargo, 2003, P. vitae Pedro & Camargo, 2003, P. nigrior (Cockerell, 1925), P. sooretamae Pedro & Camargo, 2003 and P. littoralis Pedro & Camargo, 2003 are unknown. The species of Partamona build notable nest entrance structures, with special surfaces for incoming / exiting bees; some of them are extremely well-elaborated and ornamented, serving as flight orientation targets. All species endemic to western Ecuador to Mexico with known nesting habits (P. orizabaensis, P. peckolti, P. xanthogastra, P. bilineata, P. aequatoriana and P. musarum) build their nests in several substrates, non-associated with termitaria, such as cavities and crevices in walls, among roots of epiphytes and in bases of palm leaves, in abandoned bird nests, under bridges, and in other protected places, except P. peckolti that occasionally occupies termite nests. In South America, on the eastern side of the Andes, only P. epiphytophila and P. helleri nest among roots of epiphytes and other substrates, non-associated with termitaria. All other species studied (P. batesi, P. gregaria, P. pearsoni, P. ferreirai, P. chapadicola, P. nhambiquara, P. vicina, P. mourei, P. auripennis, P. combinata, P. cupira, P. mulata, P. ailyae, P. seridoensis, P. criptica and P. rustica) nest inside active termite nests, whether epigeous or arboreous. The only species that builds obligate subterranean nests, associated or not with termite or ant nests (Atta spp.) is P. testacea. Nests of Partamona have one vestibular chamber (autapomorphic for the genus) closely adjacent to the entrance, filled with a labyrinth of anastomosing pillars and connectives, made of earth and resins. One principal chamber exists for food and brood, but in some species one or more additional chambers are filled with food storage pots. In nests of P. vicina, there is one atrium or "false nest", between the vestibule and the brood chamber, which contains involucral sheaths, cells and empty pots. All structures of the nest are supported by permanent pillars made of earth and resins (another autapomorphy of the genus). The characters concerning nesting habits were coded and combined with morphological and biogeographic data, in order to hypothesize the evolutive scenario of the genus using cladistic methodology. The phylogenetic hypothesis presented is the following: (((((P. bilineata (P. grandipennis, P. xanthogastra)) (P. orizabaensis, P. peckolti)) (P. aequatoriana, P. musarum)) P. epiphytophila, P. yungarum, P. subtilis, P. vitae) (((((P. testacea (P. mourei, P. vicina)) (P. nigrior (P. auripennis, P. combinata))) (P. ferreirai (P. pearsoni (P. gregaria (P. batesi (P. chapadicola, P. nhambiquara)))))) ((((P. ailyae, P. sooretamae) P. cupira, P. mulata) P. seridoensis) P. criptica, P. rustica, P. littoralis)) P. helleri))). One area cladogram is presented. Dates of some vicariance / cladogenesis events are suggested. For bilineata / epiphytophila group, which inhabits the Southwestern Amazonia and the Chocó-Mexican biogeographical components, the origin of ancestral species is attributed to the Middle Miocene, when the transgressions of the Maracaibo and Paranense seas isolated the tropical northwestern South America from the eastern continental land mass. The next cladogenic event in the history of the bilineata / epiphytophila group is attributed to the Plio-Pleistocene, when the Ecuadorian Andes reached more than 3000 m, and the ancestral species was fragmented in two populations, one occupying the western Andes (ancestral species of the bilineata subgroup) and other the southwestern Amazon (ancestral species of the epiphytophila subgroup). Other aspects of the history of Partamona are also discussed.

INFLUENCE OF THE GLYPHOSATE FORMULATIONS ON WETTABILITY AND EVAPORATION TIME OF DROPLETS ON DIFFERENT TARGETS

ABSTRACTEfficiency of weed control can be increased if the herbicide formulation provides higher target coverage and evaporation time that enable an adequate distribution of herbicide on the target plant, allowing the absorption to continue even after the droplets evaporation. The aim of this research was to assess the influence of glyphosate formulations on the wetted area and evaporation time of droplets on different targets. Tests were conducted with droplets sizing from 500 μm containing three formulations of glyphosate (isopropylamine salt, ammonium salt and potassium salt) deposited on three surfaces, two leaves (Bidens pilosa and Cenchrus echinatus) and glass slides. Sequential images analyses were used to quantify the evaporation time and the wetted area. An experimental system was utilized that was composed of a droplet generator, a stereo microscope with a camera to capture images, as well as an environmental chamber controlled for temperature and relative humidity. The kind of glyphosate formulations and target surfaces are crucial in the wetted area and evaporation time. The isopropylamine salt decreased the wetted area and evaporation time when compared with ammonium salt and potassium salt for all the surfaces deposited on. Bidens pilosa allows an increased wetted area for all the glyphosate formulations when compared to Cenchrus echinatus and glass slides.

Inhibitory effects in the detection of 1 cpd jo targets superimposed to angular frequency stimuli or sinewave gratings

Independence among channels processing different aspects of spatial information, including orthogonal stimuli, has been generally assumed in the literature. We tested independence between the processing of jo targets and the processing of either vertical sinusoidal gratings or angular frequency stimuli with suprathreshold summation. We found the detection of a jo target at 1 cpd to be affected in an inhibitory fashion by either background angular frequencies in the range of 3-96 cycles or sinewave gratings in the range of 0.8-3.0 cpd. These results demonstrate interactions both among orthogonal stimuli and among channels processing vertical sinewave gratings and jo target stimuli. Our discussion focuses on the hypothesis of frequency decomposition in polar coordinates

Extracellular matrix molecules as targets for brown spider venom toxins

Loxoscelism, the term used to describe lesions and clinical manifestations induced by brown spider's venom (Loxosceles genus), has attracted much attention over the last years. Brown spider bites have been reported to cause a local and acute inflammatory reaction that may evolve to dermonecrosis (a hallmark of envenomation) and hemorrhage at the bite site, besides systemic manifestations such as thrombocytopenia, disseminated intravascular coagulation, hemolysis, and renal failure. The molecular mechanisms by which Loxosceles venoms induce injury are currently under investigation. In this review, we focused on the latest reports describing the biological and physiopathological aspects of loxoscelism, with reference mainly to the proteases recently described as metalloproteases and serine proteases, as well as on the proteolytic effects triggered by L. intermedia venom upon extracellular matrix constituents such as fibronectin, fibrinogen, entactin and heparan sulfate proteoglycan, besides the disruptive activity of the venom on Engelbreth-Holm-Swarm basement membranes. Degradation of these extracellular matrix molecules and the observed disruption of basement membranes could be related to deleterious activities of the venom such as loss of vessel and glomerular integrity and spreading of the venom toxins to underlying tissues.

Stress-induced neuroinflammation: mechanisms and new pharmacological targets

Stress is triggered by numerous unexpected environmental, social or pathological stimuli occurring during the life of animals, including humans, which determine changes in all of their systems. Although acute stress is essential for survival, chronic, long-lasting stress can be detrimental. In this review, we present data supporting the hypothesis that stress-related events are characterized by modifications of oxidative/nitrosative pathways in the brain in response to the activation of inflammatory mediators. Recent findings indicate a key role for nitric oxide (NO) and an excess of pro-oxidants in various brain areas as responsible for both neuronal functional impairment and structural damage. Similarly, cyclooxygenase-2 (COX-2), another known source of oxidants, may account for stress-induced brain damage. Interestingly, some of the COX-2-derived mediators, such as the prostaglandin 15d-PGJ2 and its peroxisome proliferator-activated nuclear receptor PPARγ, are activated in the brain in response to stress, constituting a possible endogenous anti-inflammatory mechanism of defense against excessive inflammation. The stress-induced activation of both biochemical pathways depends on the activation of the N-methyl-D-aspartate (NMDA) glutamate receptor and on the activation of the transcription factor nuclear factor kappa B (NFκB). In the case of inducible NO synthase (iNOS), release of the cytokine TNF-α also accounts for its expression. Different pharmacological strategies directed towards different sites in iNOS or COX-2 pathways have been shown to be neuroprotective in stress-induced brain damage: NMDA receptor blockers, inhibitors of TNF-α activation and release, inhibitors of NFκB, specific inhibitors of iNOS and COX-2 activities and PPARγ agonists. This article reviews recent contributions to this area addressing possible new pharmacological targets for the treatment of stress-induced neuropsychiatric disorders.

Parameters of electrostatic spraying and its influence on the application efficiency

When the electrostatic spraying is used correctly, it provides advantages over conventional systems, however many factors can affect the system efficiency. Therefore, the objective of this study was to evaluate the charge/mass ratio (Q/M) at different spraying distances (0, 1, 2, 3, 4 and 5 m), and the liquid deposition efficiency on the target. Evaluating the Q/M ratio the Faraday cage method was used and to evaluate the liquid deposition efficiency the artificial targets were positioned longitudinally and transversely to the spray jet. It was found that the spraying distance affects the Q/M ratio, consequently, the liquid deposition efficiency. For the closest distance to the target the Q/M ratio was 4.11 mC kg-1, and at distances of 1, 2, 3, 4 and 5 m, the ratio decreased to 1.38, 0.64, 0.31, 0.17 and 0.005 mC kg-1, respectively. For the liquid deposition, the electrostatic system was affected by the target orientation and spraying distance. The target transversely to the jet of liquid did not improve the liquid deposition, but longitudinally increased the deposition up to 3 meters of distance.

Immunogenicity of WHO-17D and Brazilian 17DD yellow fever vaccines: a randomized trial

OBJECTIVE: To compare the immunogenicity of three yellow fever vaccines from WHO-17D and Brazilian 17DD substrains (different seed-lots). METHODS: An equivalence trial was carried out involving 1,087 adults in Rio de Janeiro. Vaccines produced by Bio-Manguinhos, Fiocruz (Rio de Janeiro, Brazil) were administered following standardized procedures adapted to allow blocked randomized allocation of participants to coded vaccine types (double-blind). Neutralizing yellow fever antibody titters were compared in pre- and post-immunization serum samples. Equivalence was defined as a difference of no more than five percentage points in seroconversion rates, and ratio between Geometric Mean Titters (GMT) higher than 0.67. RESULTS: Seroconversion rates were 98% or higher among subjects previously seronegative, and 90% or more of the total cohort of vaccinees, including those previously seropositive. Differences in seroconversion ranged from -0.05% to -3.02%. The intensity of the immune response was also very similar across vaccines: 14.5 to 18.6 IU/mL. GMT ratios ranged from 0.78 to 0.93. Taking the placebo group into account, the vaccines explained 93% of seroconversion. Viremia was detected in 2.7% of vaccinated subjects from Day 3 to Day 7. CONCLUSIONS: The equivalent immunogenicity of yellow fever vaccines from the 17D and 17DD substrains was demonstrated for the first time in placebo-controlled double-blind randomized trial. The study completed the clinical validation process of a new vaccine seed-lot, provided evidence for use of alternative attenuated virus substrains in vaccine production for a major manufacturer, and for the utilization of the 17DD vaccine in other countries.