Pathology of the spleen in hepatosplenic schistosomiasis. Morphometric evaluation and extracellular matrix changes

Histological, ultrastructural, morphometric and immunohistochemical data obtained from the study of spleens removed by splenectomy from 34 patients with advanced hepatosplenic schistosomiasis revealed that the main alterations were congestive dilatation of the venous sinuses and diffuse thickening of the splenic cords. Splenic cord thickening was due to an increase of its matrix components, especially type IV collagen and laminin, with the conspicuous absence of interstitial collagens, either of type I or type III. Deposition of interstitial collagens (types I and III) occurred in scattered, small focal areas of the red pulp, but in the outside of the walls of the venous sinuses, in lymph follicles, marginal zone, in the vicinity of fibrous trabeculae and in sidero-sclerotic nodules. However, fibrosis was not a prominent change in schistosomal splenomegaly and thus the designation "fibro-congestive splenomegaly" seems inadequate. Lymph follicles exhibited variable degrees of atrophy, hyperplasia and fibrous replacement, sometimes all of them seen in different follicles of the same spleen and even in the same examined section. Changes in white pulp did not seem to greatly contribute to increasing spleen size and weight, when compared to the much more significant red pulp enlargement.

Quantitave and qualitative interferences of pentoxifillyne on hepatic Schistosoma mansoni granulomas: effects on extracellular matrix and eosinophil population

Mast cells and eosinophils actively participate in tissue repair and are prominent components of Schistosoma mansoni granulomas. Since pentoxifillyne (PTX) is an immunomodulatory and antifibrotic substance, we aimed to characterize, by morphological techniques, the effect of this drug on fibrosis developed inside murine hepatic schistosomal granulomatous reaction, beyond the quantification of eosinophil and mast cell populations. The drug (1 mg/100 g animal weight) was administrated from 35 to 90 days post-infection, when the animals were killed. The intragranulomatous interstitial collagen network was analyzed by confocal laser scanning microscopy, the number of eosinophils and mast cells was quantified and the results were validated by t-student test. Treatment did not interfere on the granuloma evolution but caused a significant decrease in the total and involutive number of hepatic granulomas (p = 0.01 and 0.001, respectivelly), and in the intragranulomatous accumulation of eosinophils (p = 0.0001). Otherwise, the number of mast cells was not significantly altered (p = 0.9); however, it was positively correlated with the number of granulomatous structures (r = 0.955). In conclusion, PTX does not affect development and collagen deposition in S. mansoni murine granuloma, but decreases the intragranulomatous eosinophil accumulation possibly due to its immunomodulatory capability, interfering in cellular recruitment and/or differentiation.

Fibrogenesis and collagen resorption in the heart and skeletal muscle of Calomys callosus experimentally infected with Trypanosoma cruzi: immunohistochemical identification of extracellular matrix components

Intense inflammatory lesions and early development of interstitial fibrosis of the myocardium and skeletal muscle with spontaneous regression, have been described in Calomys callosus infected with Trypanosoma cruzi. The genetic types of collagen present in this model were investigated through immunohistochemistry using specific antibodies, combined with histopathology and Picro-Sirius staining of collagen. Thirty-five calomys were infected with the Colombian strain of T. cruzi and sacrificed at 24, 30, 40, 60 and 90 days post-infection. Inflammatory lesions and fibrogenesis were prominent at the early phase of infection and significantly decreased during late infection. Immunoisotyping of the matrix components was performed by indirect immunofluorescence on 5 µm thick cryostat sections using specific antibodies against laminin, fibronectin and isotypes I, III and IV of collagen. In the early phase, positive deposits of all the matrix components were present, with predominance of fibronectin, laminin and collagens types I and III in the myocardium and of types III and IV in the skeletal muscles. From the 40th day, type IV collagen predominates in the heart. At the late phase of infection (60th to 90th day), a clear fragmentation and decrease of all the matrix components were detected. Findings of the present study indicate that a modulation of the inflammatory process occurs in the model of C. callosus, leading to spontaneous regression of fibrosis independent of the genetic types of collagen involved in this process.

Ultrastructural alterations in adult Schistosoma mansoni caused by artemether

Progress has been made over the last decade with the development and clinical use of artemether as an agent against major human schistosome parasites. The tegument has been identified as a key target of artemether, implying detailed studies on ultrastructural damage induced by this compound. We performed a temporal examination, employing a transmission electron microscope to assess the pattern and extent of ultrastructural alterations in adult Schistosoma mansoni harboured in mice treated with a single dose of 400 mg/kg artemether. Eight hours post-treatment, damage to the tegument and subtegumental structures was seen. Tegumental alterations reached a peak 3 days after treatment and were characterized by swelling, fusion of distal cytoplasma, focal lysis of the tegumental matrix and vacuolisation. Tubercles and sensory organelles frequently degenerated or collapsed. Typical features of subtegumental alterations, including muscle fibres, syncytium and parenchyma tissues, were focal or extensive lysis, vacuolisation and degeneration of mitochondria. Severe alterations were also observed in gut epithelial cells and vitelline cells of female worms. Our findings of artemether-induced ultrastructural alterations in adult S. mansoni confirm previous results obtained with juvenile S. mansoni and S. japonicum of different ages.

Intestinal fibrovascular nodules caused by Schistosoma mansoni infection in Calomys callosus Rengger, 1830 (Rodentia: Cricetidae): a model of concomitant fibrosis and angiogenesis

Human schistosomiasis develops extensive and dense fibrosis in portal space, together with congested new blood vessels. This study demonstrates that Calomys callosus infected with Schistosoma mansoni also develops fibrovascular lesions, which are found in intestinal subserosa. Animals were percutaneously infected with 70 cercariae and necropsied at 42, 45, 55, 80, 90 and 160 days after infection. Intestinal sections were stained for brightfield, polarization microscopy, confocal laser scanning, transmission and scanning electron microscopies. Immunohistological analysis was also performed and some nodules were aseptically collected for cell culture. Numerous intestinal nodules, appearing from 55 up to 160 days after infection, were localized at the interface between external muscular layer and intestinal serosa, consisting of fibrovascular tissue forming a shell about central granuloma(s). Intranodular new vessels were derived from the vasculature of the external vascular layer and were positive for laminin, chondroitin-sulfate, smooth muscle alpha-actin and FVIII-RA. Fibroblastic cells and extracellular matrix components (collagens I, III and VI, fibronectin and tenascin) comprised the stroma. Intermixed with the fibroblasts and vessels there were variable number of eosinophils, macrophages and haemorrhagic foci. In conclusion, the nodules constitute an excellent and accessible model to study fibrogenesis and angiogenesis, dependent on S. mansoni eggs. The fibrogenic activity is fibroblastic and not myofibroblastic-dependent. The angiogenesis is so prominent that causes haemorrhagic ascites.

Extra-cellular matrix changes in Schistosoma mansoni-infected Biomphalaria glabrata

Reactivity of snails against parasites exhibits a primitive focal reaction, with encapsulation, phagocytosis and destruction of parasite larvae by macrophage-like cells - the hemocytes. This reaction mimics granulomatous inflammation seen in higher animals. However, different from the latter, little is known about the participation of extra-cellular matrix in such snail defense reactions. Normal and Schistosoma mansoni-infected Biomphalaria glabrata of different strains were submitted to cytological, histological, ultrastructural and biochemical methods in order to investigate the behavior of extra-cellular tissues at the site of anti-parasite reactions. In spite of the presence of two cell-types in peripheral hemolymph, only one cell-type was present at the sites of tissue reactions. Although pre-existent collagen and elastic fibers and microfibrils sometimes appeared slightly compressed around focal reactions, no evidences of duplication, synthesis or deposition of connective-tissue extra-cellular components were observed within or around the zones of reactive cell accumulations. Thus, tissue reactions against S. mansoni in the snail B. glabrata appeared exclusively dependent on one specific population of hemocytes.

Detection of Brazilian spotted fever infection by polymerase chain reaction in a patient from the state of São Paulo

Brazilian spotted fever (BSF) cases have been increasing in the state of São Paulo but no genomic information about local rickettsia isolated from humans has been well documented. We recovered spotted-fever group rickettsiae from a sample of patient blood cultured in Vero cells using the shell vial technique. Rickettsial DNA fragments (gltA, ompA, and, ompB genes) were detected, and analysis of the ompB gene base sequences showed identity with the Rickettsia rickettsii ompB sequence available in the GenBank.

Identity of care in a Psychosocial Care Center for Children and Adolescents who uses drugs

Objective To associate the territory of identity with the production of care within a PCC focusing on children and adolescents with drug abuse and their institutional identity. Method We used the “ process tracing methodology” in four research categories: focus groups, characterization of professionals, observing the everyday and interviewing two members of emblematic cases of the service. Results territory of identity of the institution, which operates the production of care is crossed by the difficulty of dealing with the complexity brought by the users and the performance of the PCC network. This paper is also permeated by different conceptions of care and small problematization of these issues in collective spaces of service. Conclusion The discussion in focus groups and other devices can be powerful resources to reframe the meaning of care and identity of collective service.

INTERNAL STRUCTURE OF A VERMICULAR IRONSTONE AS DETERMINED BY X-RAY COMPUTED TOMOGRAPHY SCANNING

Ironstones or petroplinthites are common materials in soils under humid tropical climate, generally defined as the result of Fe oxide accumulation in areas where the water table oscillates, and may exhibit considerable morphological variability. The aim of this study was to examine the internal structure and porosity of an ironstone fragment from a Petroferric Acrudox in Minas Gerais, Brazil, by computed tomography (CT) and conventional techniques. The sample analyzed had total porosity of 59.5 %, with large macropores in the form of tubular channels and irregular vughs, the latter with variable degrees of infilling by material released from the ironstone walls or the soil matrix. The CT scan also showed that the ironstone has wide variation in the density of the solid phase, most likely due to higher concentrations or thick intergrowths of hematite and magnetite/maghemite, especially in its outer rims. The implications of these results for water retention and soil formation in ironstone environments are briefly discussed.

Survival in soil and detection of co-transformed Trichoderma harzianum by nested PCR

The objective of this work was to evaluate the survival of two Trichoderma harzianum co-transformants, TE 10 and TE 41, carrying genes for green fluorescent protein (egfp) and for resistance to benomyl, during four weeks in a contained soil microcosm. Selective culture media were used to detect viable fungal material, whose identity was confirmed by the observation of the fluorescent phenotype by direct epifluorence microscopy. PCR using two nested primer pairs specific to the egfp gene was also used to detect the transformed fungi. Although it was not possible to reliably detect the egfp gene directly from soil extracts, an enrichment step involving selective culture of soil samples in liquid medium prior to DNA extraction enabled the consistent detection of the T. harzianum co-transformants by nested PCR for the duration of the incubation period.

Genetic distance estimated by RAPD markers and its relationship with hybrid performance in maize

The objective of this work was to evaluate the genetic diversity of 16 maize inbred lines, and to determine the correlation between genetic distance and hybrid performance, using random amplified polymorphic DNA (RAPD) molecular markers. Twenty-two different random primers were used, which resulted in the amplification of 265 fragments, 237 (84.44%) of them being polymorphic. A genetic similarity matrix was created from the RAPD data, using Jaccard coefficient, and a dendrogram was constructed. Hybrid analyses were carried out using random block design and Griffing method VI for diallel crossings. The genetic associations showed five distinct heterotic groups. Correlations between genetic divergences detected by RAPD, as well as the means observed in the diallel crossings were positive and significant for plant height, ear height, prolificacy, and grain weight. The correlation of genetic divergences, detected by RAPD, and the specific combining ability between heterotic group associations, showed significance in all characteristics under study, except prolificacy. A direct relationship between genetic divergence and productivity was found in 79.2% of the 120 hybrids confirming the hypothesis that genetic divergence is directly related to the performance of hybrids and is efficient in predicting it.

Obtaining interspecific hybrids, and molecular analysis by microsatellite markers in grapevine

The objective of this work was to assess the potential of interspecific hybridization of Vitis labruscana and Muscadinia rotundifolia by using artificial cross-pollinations. Microsatellite markers were used to confirm interspecific hybridizations and the identity of the parental genotypes. In crosses in which M. rotundifolia was used as the female parent, no true hybrids were obtained. In the reciprocal crosses, 114 seedlings were identified as true V. labruscana x M. rotundifolia hybrids. Self pollination occurred in direct and in reciprocal crosses. The crossings between 'Bordo' x 'Carlos', 'Magnolia', 'Regale' and' Roanoke', and between' Isabel' x 'Bountiful', 'Carlos', 'Magnolia', 'Regale' and 'Roanoke' were confirmed. The 15 markers evaluated showed that two M. rotundifolia parental genotypes had the same fingerprint profile, indicating a like lyplanting error. The success of hybridization depends mainly on the species and on the cultivar used as the female parent. Microsatellite markers are efficient to confirm the paternity of interspecific F1 hybrids and to determine the correct identity of M. rotundifolia cultivars.

Molecular characterization of soybean cultivars by microsatellite markers with universal tail sequence

The objective of this work was to standardize a semiautomated method for genotyping soybean, based on universal tail sequence primers (UTSP), and to compare it with the conventional genotyping method that uses electrophoresis in polyacrylamide gels. Thirty soybean cultivars were genotypically characterized by both methods, using 13 microsatellite loci. For the UTSP method, the number of alleles (NA) was 50 (2-7 per marker) and the polymorphic information content (PIC) ranged from 0.40 to 0.74. For the conventional method, the NA was 38 (2-5 per marker) and the PIC varied from 0.39 to 0.67. The genetic dissimilarity matrices obtained by the two methods were highly correlated with each other (0.8026), and the formed groups were coherent with the phenotypic data used for varietal registration. The 13 markers allowed the distinction of all analyzed cultivars. The low cost of the UTSP method, associated with its high accuracy, makes it ideal for the characterization of soybean cultivars and for the determination of genetic purity.

THE LYCHEE TREE PROPAGATION BY LAYERING

The aim of the study was to assess the influence of season and different substrates on rooting of air layers of lychee (Litchi chinensisSonn.) for the production of seedlings to ensure the formation of uniform and productive orchards. Air layers were done in plants of the Bengal cultivar using leafy and healthy woody branches, with about 0.010 to 0.015 m in diameter, in which were performed complete girdling with 0.020 m wide at a distance of 0.30 to 0.40 m below the apex. Then the branches were wrapped in moistened substrate. The layering was made at six times of theyear (January, March, May, July, September and November) and two substrates were used (coconut fiber and sphagnum) in a 6 x 2 factorial design in a randomized block with ten replicates. After 90 days, layers were separated from the matrix plant and evaluated for rooting and callus formation, root number, considering only the primary roots, length, area and volume of the roots, beyond the dry weight of roots and calluses. The months of January, March, September and November showed the best results for all analyzed variables related to rooting. With respect to the substrates, the only difference was in January and March to the root number and dry weight of roots, where the sphagnum showed the best results. The month of July was more conducive to the formation of calluses. The period between September and March was more suitable to the propagation of lychee, when there were rooting percentages above 90%, in addition to the formation of large amount of roots.

Ultra-trace monitoring of copper in environmental and biological samples by inductively coupled plasma atomic emission spectrometry after separation and preconcentration by using octadecyl silica membrane disks modified by a new schiff's base

Ultra-trace amounts of Cu(II) were separated and preconcentrated by solid phase extraction on octadecyl-bonded silica membrane disks modified with a new Schiff,s base (Bis- (2-Hydroxyacetophenone) -2,2-dimethyl-1,3-propanediimine) (SBTD) followed by elution and inductively coupled plasma atomic emission spectrometric detection. The method was applied as a separation and detection method for copper(II) in environmental and biological samples. Extraction efficiency and the influence of sample matrix, flow rate, pH, and type and minimum amount of stripping acid were investigated. The concentration factor and detection limit of the proposed method are 500 and 12.5 pg mL-1, respectively.