Evaluation of Cytopathological Techniques for the Diagnosis of Canine Visceral Leishmaniosis with Lymph Node Samples.

The identification of the parasite in cytological smears of lymph node aspirates is a widely applied technique for the direct diagnosis of Leishmania spp. infection, especially in endemic areas. Although very specific, this method has limited sensitivity, and improving the technique would be highly desirable. This study aimed to evaluate the efficacy of conventional smear cytology (SC), liquid-based cytology (LBC), cell block (CB) stained with haematoxylin and eosin (HE) and immunocytochemistry (ICC), and formalin-fixed paraffin wax-embedded tissue immunohistochemistry (FFPE-IHC) compared with serology and polymerase chain reaction for the diagnosis of canine visceral leishmaniosis (CVL) in lymphoid tissue. The use of a preservative medium and centrifugation for cytological samples reduced the number of unsatisfactory artefacts/background. Moreover, LBC allowed excellent cellular preservation and the application of ancillary techniques, such as CB and ICC. SC was the most accurate morphological diagnostic method (45.0%). CB-ICC alone or associated with SC demonstrated significantly higher sensitivity (70.0% and 72.0%, respectively) when compared with SC alone (34.00%). CB-ICC was found to be more effective in the detection of infected animals with mild clinical signs, similar to FFPE-IHC. The specificity and positive predictive value were similar between all methods. Finally, the detection limit for CB-ICC and SC + CB-ICC was identical (18.46 amastigotes/mm2). Our study suggests that CB-ICC is a promising tool for improvement of the cytopathological diagnosis of CVL and may be applied in routine epidemiological screening.

Periphytic algae dynamics in lentic ecosystems in the Brazilian semiarid / Estrutura da comunidade de algas perifíticas do semiárido brasileiro

Abstract The hydrological periods drive the structure and organization of aquatic communities in semiarid regions. We hypothesize that a decrease of the precipitation during the dry period will favor the development of the periphytic algal community, leading to higher richness and density in this period. To test this hypothesis, we investigated the changes in the periphytic algal community structure in three shallow and eutrophic ecosystems of the Brazilian semiarid. The sampling was performed between 2007 and 2010 at two-mensal intervals. The sampling of periphytic algal was performed in aquatic macrophytes and rocks. The abiotic variables were analyzed simultaneously. Dominance in diatoms, cyanobacteria and chlorophytes, respectively, was observed in two periods. In the dry period, waters were alkaline and had high concentrations of nitrate and total phosphorus associated with the highest densities of Bacillariophyceae. In the rainy period the water was warmer, oxygenated and high concentrations of ammonia and soluble reactive phosphorus with diatoms remained dominant but with reduced density, while cyanobacteria and chlorophytes increased. Overall, periphytic algal community composition no responded to changes in the hydrological periods. However, the hydrological periods altered the dynamics of periphytic algal community, supported by the alternation of the most representative classes (diatoms and cyanobacteria) between the hydrologic periods. Our data suggest that the morphometric and chemical and physical characteristics of lentic aquatic ecosystems studied were more important in the dynamics of periphytic algal community than the hydrological periods and types of substrates.

Resumo Os períodos hidrológicos impulsionam a estrutura e organização das comunidades aquáticas em regiões semiáridas. Nós hipotetizamos que uma diminuição da precipitação durante o período seco irá favorecer o desenvolvimento da comunidade de algas perifíticas, levando a uma maior riqueza e densidade nesse período. Para testar esta hipótese, nós investigamos as mudanças na estrutura da comunidade de algas perifíticas em três ecossistemas rasos e eutróficos do semiárido brasileiro. As amostragens foram realizadas entre 2007 e 2010 em intervalos de bimensais. A amostragem das algas perifíticas foi realizada em macrófitas aquáticas e rochas. As variáveis abióticas foram analisadas simultaneamente. A dominância de diatomáceas, cianobactérias e clorófitas, respectivamente, foi observada em todos os períodos. No período seco, as águas foram alcalinas com altas concentrações de nitrato e fósforo total associado às maiores densidades de Bacillariophyceae. Durante o período chuvoso as águas apresentaram-se mais quentes, oxigenadas e com altas concentrações de amônia e fósforo reativo solúvel. As diatomáceas permaneceram dominantes, mas com densidade reduzida, enquanto as cianobactérias e clorofíceas aumentaram. No geral, a composição da comunidade de algas perifíticas não respondeu as mudanças nos períodos hidrológicos. No entanto, os períodos hidrológicos alteraram a dinâmica da comunidade de algas perifíticas, apoiados pela alternância das classes mais representativas (diatomáceas e cianobactérias) entre os períodos hidrológicos. Nossos dados sugerem que as características morfométricas, químicas e físicas dos ecossistemas aquáticos lênticos estudados foram mais importantes na dinâmica da comunidade de algas perifíticas do que os períodos hidrológicos e tipos de substratos.

Periphytic algae dynamics in lentic ecosystems in the Brazilian semiarid.

The hydrological periods drive the structure and organization of aquatic communities in semiarid regions. We hypothesize that a decrease of the precipitation during the dry period will favor the development of the periphytic algal community, leading to higher richness and density in this period. To test this hypothesis, we investigated the changes in the periphytic algal community structure in three shallow and eutrophic ecosystems of the Brazilian semiarid. The sampling was performed between 2007 and 2010 at two-mensal intervals. The sampling of periphytic algal was performed in aquatic macrophytes and rocks. The abiotic variables were analyzed simultaneously. Dominance in diatoms, cyanobacteria and chlorophytes, respectively, was observed in two periods. In the dry period, waters were alkaline and had high concentrations of nitrate and total phosphorus associated with the highest densities of Bacillariophyceae. In the rainy period the water was warmer, oxygenated and high concentrations of ammonia and soluble reactive phosphorus with diatoms remained dominant but with reduced density, while cyanobacteria and chlorophytes increased. Overall, periphytic algal community composition no responded to changes in the hydrological periods. However, the hydrological periods altered the dynamics of periphytic algal community, supported by the alternation of the most representative classes (diatoms and cyanobacteria) between the hydrologic periods. Our data suggest that the morphometric and chemical and physical characteristics of lentic aquatic ecosystems studied were more important in the dynamics of periphytic algal community than the hydrological periods and types of substrates.

Experimental Tityus serrulatus scorpion envenomation: age- and sex-related differences in symptoms and mortality in mice

Among the various methods for evaluating animal venom toxicity, the calculation of the median lethal dose (LD50) is the most widely used. Although different protocols can be used to calculate the LD50, the source of the venom and the method of extraction, as well as the strain, age, and sex of the animal model employed, should be taken into consideration. The objective of the present study was to evaluate the influence of sex and age on the toxicity of Tityus serrulatus scorpion venom in Swiss mice. Although the symptoms of envenomation were similar in male and female animals, female mice proved to be more resistant to the venom. In females, age had no impact on the susceptibility to scorpion envenomation. Male mice were more sensitive to T. serrulatus venom. Moreover, in males, age was an important parameter since sensitivity to the venom increased with age.

Salinity and freshwater discharge determine rotifer distribution at the Mossoró River Estuary (Semiarid Region of Brazil).

The present study aimed at evaluating differences in rotifer distribution in three estuarine zones in an inverse estuary located in the Semiarid Region of Brazil. Zones were chosen based on their proximity to the ocean and river border as a means of reflecting a horizontal salinity gradient. High freshwater discharge during the rainy season was the major determinant of rotifer composition. On the other hand, due to higher salinity values during the dry season, very low values of species richness and abundance were observed in all zones. Therefore, the study highlights the constraints of salinity and the positive influence of seasonality and river proximity on rotifer species in a semiarid estuarine environment.

Salinity and freshwater discharge determine rotifer distribution at the Mossoró River Estuary (Semiarid Region of Brazil) / Salinidade e descarga de água doce determinam a distribuição de rotíferos no estuário do Rio Mossoró (região semiárida do Brasil)

The present study aimed at evaluating differences in rotifer distribution in three estuarine zones in an inverse estuary located in the Semiarid Region of Brazil. Zones were chosen based on their proximity to the ocean and river border as a means of reflecting a horizontal salinity gradient. High freshwater discharge during the rainy season was the major determinant of rotifer composition. On the other hand, due to higher salinity values during the dry season, very low values of species richness and abundance were observed in all zones. Therefore, the study highlights the constraints of salinity and the positive influence of seasonality and river proximity on rotifer species in a semiarid estuarine environment.

O presente estudo objetivou avaliar diferenças na distribuição de rotíferos em três zonas estuarinas em um estuário inverso localizado na região semiárida do Brasil. As zonas foram escolhidas com base em suas proximidades com o oceano e a margem do rio de forma a representar um gradiente de salinidade horizontal. A forte entrada de água doce durante o período chuvoso foi o maior determinante da composição de rotíferos. Do outro lado, devido aos maiores valores de salinidade durante o período seco, valores muito baixos de riqueza de espécies e abundância foram observados em todas as zonas. Dessa forma, o estudo demonstra as restrições da salinidade e a influência positiva da sazonalidade e da proximidade com o rio sobre as espécies de rotíferos em um ambiente estuarino do semiárido.

Expression of recombinant human antibody fragments capable of inhibiting the phospholipase and myotoxic activities of Bothrops jararacussu venom.

Phospholipases A(2) are components of Bothrops venoms responsible for disruption of cell membrane integrity via hydrolysis of its phospholipids. This study used a large nonimmune human scFv library named Griffin.1 (MRC, Cambridge, UK) for selection of recombinant antibodies against antigens present in Bothrops jararacussu venom and identification of specific antibodies able to inhibit phospholipase activity. Four clones were identified as capable of inhibiting this activity in vitro. These clones were able to reduce in vivo the myotoxic activity of BthTX-I and BthTX-II PLA(2), but had no effect on the in vitro anticoagulant activity of BthTX-II. This work shows the potential of using recombinant scFv libraries in the search for antibodies that neutralize relevant venom components.

Reduced erythrocyte complement receptor type 1 in systemic lupus erythematosus is related to a disease activity index and not to the presence or severity of renal disease.

The present study investigated the expression of the complement receptor type 1 (CR1) on the membrane of erythrocytes (CR1/E) of patients with systemic lupus erythematosus (SLE) by flow cytometry. We found a significant reduction in CR1/E numbers in SLE patients (n = 52), compared to controls (512 +/- 171 and 689 +/- 146, respectively, P = 0.0001). Reduction was more pronounced in active disease patients. The mean CR1/E number observed in patients with inactive disease was 546 +/- 163 CR1/E, while active SLE patients presented a mean of 385 +/- 133 CR1/E (P = 0.001). Patients with SLE with similar activity indexes tend to have similar CR1/E numbers, irrespective of disease severity. We also observed a trend to CR1/E reduction in severe nephritis patients. A small group of SLE patients with chronic renal failure and inactive disease showed CR1/E numbers nearly identical to controls (689 +/- 146 versus 686 +/- 123, respectively, P = 0.95). This was the only group of SLE patients with normal CR1/E numbers. These results confirm the CR1/E reduction in SLE patients as previously described, and also suggest that this reduction is related to disease activity and not to disease severity.

Ontogeny of complement regulatory proteins - concentrations of factor h, factor I, c4b-binding protein, properdin and vitronectin in healthy children of different ages and in adults.

Previous studies of human in vivo complement protein levels have only compared data for neonates with that from adult sera. Here, we establish the normal concentration ranges of the following complement regulatory proteins in healthy Brazilian children of different age groups (neonates: 1 month-1 year, 1-6 years and 6-13 years) and in adults: factor H (fH), factor I (fI), C4b-binding protein (C4 BP), properdin and vitronectin. We found that the concentrations of fH, fI, properdin and vitronectin in neonates are significantly lower than in adults. Remarkably, the concentration of C4 BP is below the method resolution (<50 micro g/ml) in 76% of the sera from neonates, while adults presented 199-532 microg/ml of C4 BP in their sera. The concentration of properdin in the sera from neonates and up to 1-year-old children was less than that observed in older children. Adults presented vitronectin levels significantly higher than all the other age groups in the study. No significant sex differences in the concentrations of all the studied regulatory proteins were detected. This study reveals the ontogeny of complement system in greater detail than previously available and may point to the reasons why neonates have higher susceptibility to develop life-threatening pyogenic infections. These reference values will be of use in clinical and laboratory investigations of disorders associated with low levels of these regulatory proteins.

Influence of the electric charge of the antigen and the immune complex (IC) lattice on the IC activation of human complement.

In order to understand the mechanism of complement (C) activation by immune complexes (ICs), the anti-complementary effect of ICs containing cationized antigens was compared in vitro to that using ICs formed by native antigens. ICs were prepared with affinity-purified rabbit polyclonal IgG antibovine serum albumin (BSA) antibody and either native BSA (isoelectric point 4.2) or BSA rendered cationic by treatment with ethylenediamine (isoelectric point 9.4). Native and cationized antigens were characterized by isoelectric focusing. ICs containing anti-BSA IgG or F(ab')2, formed either at equivalence or in excess of native or cationized antigen, were submitted to ultracentrifugation in a sucrose gradient for mesh size determination. The anti-complementary effect of ICs was evaluated by kinetic determination of haemolytic activity of human serum on haemolysin-sensitized sheep red blood cells. In conditions of antigen excess, the ICs formed by cationized BSA were significantly more efficient in activating human complement than those formed by native antigen. This higher activity was dependent on cationized antigen complexed with complete antibody molecules, as non-complexed cationized BSA or ICs prepared with F(ab')2 fragments were inactive under the same experimental conditions. Furthermore, this difference did not depend on the mesh size of the immune complexes. Our results suggest that the balance between antigen, antibody and C may be of importance in vivo for the onset and course of infections and other pathological processes involving IC formation. ICs containing cationized antigens should be proven of value in experimental models for studies on the regulation of C activation.

Expression of complement receptor type 1 (CR1) on erythrocytes of paracoccidiodomycosis patients.

Complement receptor type 1 (CR1) is a membrane glycoprotein that acts as a receptor for the C3b, iC3b and C4b fragments of complement. In primates, one function of erythrocytes is to promote safe clearance of immunocomplexes (IC) from the circulation through CR1. Theoretically, in diseases characterized by high levels of circulating IC, an erythrocyte CR1 (CR1/E) deficiency may favor IC deposition in tissues or facilitate inappropriate activation of leukocytes in the circulation. Depression of the cell immune response occurs in paracoccidioidomycosis (PCM), especially in the more severe cases, and is frequently associated with high serum IC levels. In the present study we quantified the number of CR1/E in patients with the acute and chronic forms of PCM before and after treatment and correlated it with serum IC levels and CD4+ and CD8+ T cell concentration in the peripheral blood of these patients. Patients with PCM, particularly those with active disease and who had received treatment for shorter periods of time, had low numbers of CR1/E. In addition, an increase in serum IC concentration and a reduction in the CD4+/CD8+ T cell ratio were observed. After treatment there was a significant increase in mean CR1/E number and a reduction in serum IC levels. In patients with the chronic form of the disease the CD4+/CD8+ T cell ratio tended to increase after treatment and was associated with increased CR1/E levels. These results suggest that the reduction in CR1/E observed in patients is a phenomenon acquired with the disease and that CR1 could play a role in the pathogenesis of PCM.

Intravenous immunoglobulin (IVIG) attenuates antibody binding in acute haemorrhagic immunopneumonitis in a rat model of complement-dependent lung injury.

Administration of rabbit anti-rat lung serum (PNTS) to rats produces a fulminant haemorrhagic pneumonitis sensitive to the availability of complement. The present experiments were undertaken to assess whether a high dose of IVIG can affect the development of this kind of cytotoxic reaction. The experimental design included groups of Wistar rats pretreated intravenously with physiologic saline, IVIG or a preparation of human F(ab')2 fragments. One hour later the animals were challenged with either saline or PNTS. At 30 min after challenge, blood was collected and the lungs were removed. Pulmonary damage was evaluated by light microscopy; C3 deposits and the binding of immunoglobulins to the alveolar septa were assayed by immunofluorescence. The serum complement activity of the classical and alternative pathways was estimated by a kinetic technique. Pretreatment with IVIG decreased binding of rabbit anti-lung antibodies to alveolar septa and prevented the deposition of C3. These results indicate that pretreatment with IVIG inhibits the binding of the pathogenic antibody to lung tissue. Human IgG binding was not detected in any animal. The protection against lung injury afforded by pretreatment with IVIG, in contrast to the pneumotoxic effect of PNTS observed in control animals, was evident despite the administration of F(ab')2 to the rats. Since pretreatment with F(ab')2 failed to prevent the acute lung lesion, our results indicate that the attenuation afforded by IVIG in this model of complement-dependent tissue injury seems to be related to the integrity of the IgG molecule.

Complement haemolytic activity (classical and alternative pathways), C3, C4 and factor B titres in healthy children.

Values of complement lytic activity of classical and alternative pathways, assessed by measuring the time required to lyse 50% of target red blood cells, and the concentration of complement components C3, C4 and factor B were estimated in the sera of 103 healthy children aged 3 to 14 y. Age-dependent variations were seen in the C3 and factor B concentrations, but not in C4, with the highest values found among 5-6-y-old children. Variations in classical and alternative lytic activity were not detected in this group of children, although the values are significantly different from our previously published data on adults, using the same kinetic assay (1). We also evaluated the relationship between the lytic activity of the classical (CPT) and alternative pathways (APT) and the levels of complement components. There were significant correlations between: APT and factor B, APT and C3, C3 and C4, C3 and factor B, and C4 and factor B concentrations. The normal ranges measured here can be used in the initial screening of Brazilian children presenting diseases involving the complement system. This study also contributes to a better understanding of the complement system ontogeny.

Interactions of F1 fractions from different strains of Paracoccidioides brasiliensis with human complement and with human neutrophils.

The aim of our study was to investigate differences that might exist in the activation of the human complement system by F1 fractions from four different isolates of P. brasiliensis. Isolates HC and 18 (virulent), 265 (low virulence), and 9 (intermediate virulence, attenuated) were used; before the experiments, the virulence of isolates HC and 18 was recovered by in vivo passage in guinea pigs. The four isolates of the fungus were processed for purification of F1 fractions and the activation of the human complement system was studied by a kinetic method of hemolytic activity measurement. The incubation of F1 fractions in normal human serum resulted in different degrees of inhibition of the classical and alternative pathways. The F1 fraction from the low virulence isolate was more efficient than the F1 fraction from the virulent isolates (HC and 18). Previous absorption of sera with F1 fractions completely abolished classical pathway activation. Using zymosan, instead of F1, in the absorption process caused the same phenomenon, suggesting that natural or nonspecific antibodies are responsible for the classical pathway activation. The alternative pathway activation did not depend on these antibodies, but was enhanced by their presence. On the other hand, F1 fractions from virulent isolates were more active in the stimulation of neutrophil chemiluminescence compared with the F1 fraction from the low virulence isolate. Whole P. brasiliensis yeast cells (WYC) from two distinct strains, 18 and 265, showed the same patterns of response of those observed with the F1 fractions in the functions tested. These differences in the behavior of the F1 fractions as well as WYC in relation to human complement activation and consequently to neutrophil stimulation may correlate with the virulence of individual isolates and may contribute to the understanding of the inflammatory response generation and maintenance processes in paracoccidioidomycosis.

CR1 stump peptide and terminal complement complexes are found in the glomeruli of lupus nephritis patients.

The number of CR1 on podocytes is reduced in nephropathies with severe glomerular damage, especially in the diffuse proliferative glomerulonephritis (DPGN) of systemic lupus erythematosus (SLE). Reduction of CR1 number on erythrocytes is due to proteolysis of CR1 by macrophage proteases activated by the reaction of their complement receptors, which leaves a 'CR1 stump peptide' on the erythrocyte. In the present study, we demonstrated the presence of the terminal complement complex (TCC) and the CR1 stump in histological sections of biopsies from patients with SLE by the indirect immunoperoxidase technique. Less severe glomerular lesions presented TCC deposits mainly in the mesangium (mesangial pattern). In lupus nephritis, with more severe glomerular damage, TCC deposits were detected both in the mesangium and in the capillary loops with podocyte involvement (mixed pattern). Patients with highly active DPGN presented a marked reduction of intact podocyte CR1 receptors in association with increased reactivity to the anti-CR1 stump antibody and with glomerular TCC deposits of mixed histological pattern. These results suggest that the decrease in the number of podocyte CR1 receptors in severe glomerular lesions of SLE may be due to a local proteolytic activity associated with activation and deposition of TCC.

Prevalence of anti-cardiolipin antibody in habitual aborters.

An enzyme-linked immunosorbent assay was developed for the detection of anticardiolipin antibody (ACA) in habitual aborters. Results above the 98th percentile of a distribution of 100 blood donors were considered to be positive. Specific binding index for ACA was higher in 20 patients with at least 3 consecutive spontaneous abortions (group A) than in 20 women with at least one live birth without pregnancy wastage (group B). ACA was detected in 4 patients of group A and in none of the women of group B. Most of the pregnancy wastages occurred after the first trimester in ACA-positive patients and during the first trimester in ACA-negative patients.

The role of the complement system in the neutrophil functions stimulated in vitro by an alkali-insoluble cell wall fraction of Paracoccidioides brasiliensis.

We investigated the capacity of an alkali-insoluble cell wall polysaccharide fraction (F1) of Paracoccidioides brasiliensis to induce rat polymorphonuclear neutrophil (PMN) migratory and chemiluminescence (CL) responses. Normal rat serum pre-incubated with F1 induced a chemotactic neutrophil response which was fully abolished by heat-inactivation. The participation of the alternative complement pathway was more effective than that of the classical pathway since depletion of factor B by heating at 50 degrees C reduced PMN migration, whereas blockade of the classical pathway with EGTA left the migratory response practically unchanged. Opsonized serum F1 induced a significant release of oxygen radicals from PMN as measured by CL. The complement system was also found to be involved in this activity since serum inactivation at 56 degrees C altered the CL response. In addition to complement-derived fragments, other serum opsonins, probably cross-reacting antibodies, were required for optimal interaction between PMN and opsonized particles. These results contribute to the understanding of the role of fungal components and of the complement system in the inflammatory response observed in paracoccidioidomycosis.

An anti-peptide antibody that recognizes a neo-antigen in the CR1 stump remaining on erythrocytes after proteolysis.

Previous studies of erythrocyte CR1 levels in systemic lupus erythematosus (SLE) and other diseases with in vivo complement activation have led to the conclusion that CR1 levels fall because of loss of CR1 from erythrocytes by proteolysis--predominantly in the liver. In order to measure the existence of proteolysed CR1 remnants on erythrocytes an antibody was raised to a peptide corresponding to the CR1 sequence between the proximal standard consensus repeat (SCR) and the transmembrane segment. This antipeptide antibody recognizes a neo-antigen found on trypsinized erythrocytes which has been demonstrated to represent the 'CR1-stump'. The anti-'CR1-stump' antiserum detects proteolysed CR1 on the ex vivo erythrocytes of a patient with cold haemolytic antibody disease (CHAD). However, higher affinity antibodies will be needed to make anti-CR1-stump a satisfactory diagnostic reagent.

Effect of the in vitro ageing on the restriction of sheep erythrocyte lysis by horse serum.

Fresh sheep erythrocytes are resistant to lysis by horse serum but gradually became susceptible to this source of complement after storage (ageing) in the blood at 4 degrees C. This occurs faster when the fresh cells are stored in isotonic buffer. The supernatant of the buffer/red cell suspension stored at 4 degrees C for 10-15 days restrict lysis by horse serum when incubated back at 37 degrees C or 43 degrees C with the 'aged' sheep cells or fresh guinea pig red cells. In assays using fresh guinea pig erythrocytes this effect is described by reincubation of the cells in buffer, and is specific to horse serum when compared to human serum.

Serum haemolytic classical and alternative pathways of complement in infancy: age-related changes.

The haemolytic activity of complement was evaluated in the serum of healthy children from birth to 2 years of age using the kinetic method for the determination of the time needed to lyse 50% of target red cells (t 1/2). No sex-linked differences were observed in any of the age groups studied and the lowest lytic activity levels for both complement pathways were detected in neonates. The two pathways, however, showed different maturation patterns, i.e., lytic activity levels similar to those of adults were reached between the 1st and 3rd month of life (classical pathway) and around the 13th month (alternative pathway). In the age group of 7 to 24 months, the lytic activity of the classical pathway was higher than in adults. The present data permitted us to establish normal ranges of t 1/2 values for the classical and alternative pathways in serum of healthy neonates and children aged 1 to 24 months.