Elucidating in vitro and in vivo phenotypic behaviour of L. infantum/L. major natural hybrids.

The clinical manifestation and course of Leishmania infections depend on factors such as species, virulence and host-immunity. Although trypanosomatids are considered to have clonal propagation, genetic hybridization has produced successful natural hybrid lineages. Hybrids displaying strong selective advantages may have an impact on pathogenesis and the eco-epidemiology of leishmaniasis. Thus, characterization of phenotypic properties of Leishmania hybrids could bring significant insight into the biology, infectivity, pathogenicity and transmission dynamics of these atypical strains. The present study focuses on phenotypic features and survival capacity of Leishmania infantum/Leishmania major hybrid isolates as compared with representative putative parental species, L. infantum and L. major. In vitro assays (growth kinetics, susceptibility to different conditions) and in vivo infection (parasite detection and histopathological alterations) showed that hybrids present higher growth capacity and decreased susceptibility to reactive oxygen species. Furthermore, evaluation of infected spleen tissue suggests that hybrids induce a stronger immune reaction than their putative parents, leading to the development of white pulp hyperplasia in B-lymphocyte compartments. Overall, these hybrids have shown high plasticity in terms of their general behaviour within the different phenotypic parameters, suggesting that they might have acquired genetic features conferring different mechanisms to evade host cells.

Plasma lipoproteins in visceral leishmaniasis and their effect on Leishmania-infected macrophages.

This work aimed at investigating the lipid profile of zoonotic visceral leishmaniasis (VL) patients' sera and the effect of lipoproteins on the in vitro production of tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, IL-10 and IL-12 by Leishmania infantum-infected and uninfected macrophages. Lipids were quantified in 26 VL patients' sera and 26 healthy controls from a VL endemic area. The patients' sera had higher triglyceride and very low density lipoprotein (VLDL) levels, and much lower apolipoprotein A1, total cholesterol, low density lipoprotein (LDL) and high density lipoprotein (HDL) levels than the control sera. Lipoprotein fractions were obtained by ultracentrifugation of sera. The addition of LDL and HDL to Leishmania-infected and uninfected macrophages, in physiological concentrations, enhanced the production of IL-6 and IL-10, but not of IL-12. LDL stimulated the production of TNF-alpha only in infected macrophages, whereas HDL stimulated the production of lower amounts of TNF-alpha in both infected and uninfected macrophages. VLDL stimulated only the production of IL-10. It is proposed herein that LDL may influence the development of VL by promoting the production of TNF-alpha by infected macrophages. A decrease in plasma LDL in some VL patients (to 20 mg/mL or less); however, would tend to reduce the production of TNF-alpha and therefore to limit the development of immune-mediated pathology, not withstanding the fact that it would perhaps increase the permissiveness of macrophages to Leishmania growth.

How do sexual expression, reproductive phenology and reproductive success relate to sexual systems in Fissidens Hedw. (Fissidentaceae)? A case study comparing two different sexual systems in mosses.

Reproductive performance is known to differ between co-sexual and non-co-sexual species. Thus, our aim was to determine whether: (i) the distance between sex structures is negatively associated with sex expression; (ii) male gametangia take longer to mature in rhizautoicous species than in gonioautoicous species; and (iii) the gonioautoicous sexual system has greater reproductive success (i.e. percentage of ramets with sporophyte) than the rhizautoicous sexual system. One population each of the mosses Fissidens scariosus and F. submarginatus, rhizautoicous and gonioautoicous, respectively, in a remnant of Atlantic Forest in Brazil were sampled monthly from September 2016 until August 2017. The number and phenophases of antheridia, archegonia and sporophytes were analysed for each species. Sexual expression and reproductive success were calculated, and reproductive phenology compared across environmental variables. As expected, sexual expression was significantly higher for the gonioautoicous species, which produced antheridia throughout the year and archegonia over many months, while gametangia production by the rhizautoicous species occurred only during the rainy season. Mean number of gametangia per perigonium and perichaetium were slightly higher for the rhizautoicous species (6.84 antheridia; 11.38 archegonia) than for the gonioautoicous species (4.39; 7.62). Gametangia and sporophyte production in the rhizautoicous species were markedly seasonal compared to that of the gonioautoicous species, although reproductive success did not differ. Therefore, we infer that the rhizautoicy (i.e. a functional dioicy) promotes lower expression of gametangia compared to gonioautoicy but is more efficient and so obtains the same reproductive success.

Inflammation and structural changes of splenic lymphoid tissue in visceral leishmaniasis: a study on naturally infected dogs.

The aim of this study was to identify splenic immuno-inflammatory patterns associated with natural infection by Leishmania chagasi. Spleen samples were obtained from 72 stray dogs from an endemic area of visceral leishmaniasis. The animals were grouped into four categories as follows: (i) potentially resistant to visceral leishmaniasis, with a positive leishmanin skin test result, and negative splenic culture for Leishmania parasites (ii) potentially susceptible to visceral leishmaniasis, with a negative leishmanin skin test and positive splenic culture for Leishmania (iii) infected with undefined susceptibility status, with a positive leishmanin skin test and positive splenic culture for Leishmania, and (iv) noninfected, with a negative leishmanin skin test, negative splenic culture for Leishmania, and negative serology for anti-Leishmania antibodies. Histopathological analyses showed that there was a higher frequency of perisplenitis (18/25, P < 0.0001), granuloma (7/25, P = 0.0102), structural disorganization (14/25, P < 0.0001), and atrophy of the lymphoid follicles (20/25, P = 0.0036) and of the marginal zone (15/25, P = 0.0025) in the potentially susceptible group than in the other groups. The data presented here show changes in the white pulp of the spleen that are associated with naturally acquired visceral leishmaniasis.

Associations among immunological, parasitological and clinical parameters in canine visceral leishmaniasis: Emaciation, spleen parasitism, specific antibodies and leishmanin skin test reaction.

Associations among parameters commonly used as markers of infection by Leishmania sp., or of susceptibility to visceral leishmaniasis, were investigated in 325 stray dogs from an area where this disease is endemic. Evidence of infection (presence of Leishmania in splenic cultures, positive leishmanin skin test (LST) or detection of anti-Leishmania antibody activity in the serum) was found in 57% of the animals. Both evidence of weight loss (chi(2)-test, P=0.0005) and presence of specific antibody activity in the serum (chi(2)-test, P<0.0001) were directly associated with positive splenic culture. The frequencies of animals with positive splenic culture were directly correlated with the intensities of antibody activity in the serum as measured by ELISA (relative risk of 3.4 for animals with moderate antibody levels and relative risk of 8.43 for animals with high-antibody levels). A negative association was observed between positive leishmanin skin test results and emaciation (chi(2), P=0.0089). Furthermore, animals with positive splenic cultures and negative leishmanin skin test results had higher levels of total serum IgG (Kruskal-Wallis test, P=0.001) and IgG2 (Kruskal-Wallis test, P=0.05) than animals with negative splenic cultures, and were more emaciated than animals with negative LST results and positive splenic cultures. The data presented herein suggest that associating these common parameters may improve their performance in predicting susceptibility to canine visceral leishmaniasis.

IgA nephropathy in Brazil: apropos of 600 cases.

IgA nephropathy (IgAN) is th e commonest primary glomerular disease worldwide. Studies on its prevalence in Brazil are however scarce. Databases and clinical records from 10 reference centres were retrospectively reviewed. Clinical and laboratory features at the moment of the biopsy were retrieved (age, gender, presence of hematuria, serum creatinine [mg/dL], proteinuria [g/24 h]). Renal biopsy findings were classified according to Haas single grade classification scheme and the Oxford Classification of IgAN. 600 cases of IgAN were identified, of which 568 (94.7 %) were on native kidneys. Male to female ratio was 1.24:1. Patients averaged 32.76 ± 15.12 years old (range 4-89, median 32). Proteinuria and hematuria were observed, respectively in 56.63 and 72.29 % of patients. The association of both these findings occurred in 37.95 % of the cases. Serum creatinine averaged 1.65 ± 0.67 mg/dL (median 1.5 mg/dL) at diagnosis. Segmental sclerosis and mesangial hypercellularity were the main glomerular findings (47.6 and 46.2 %) The commonest combination by Oxford Classification of IgAN, was M0 E0 S0 T0 (22.4 %). Chronic tubulo-interstitial lesions with an extension wider than 25 % of the renal cortex could be identified in 32.2 % of the cases. Tubular atrophy and interstitial fibrosis were more strongly associated with higher 24-h proteinuria and serum creatinine levels. Segmental sclerosis (S1) showed a stronger tendency of association with the presence of tubulo-interstitial lesions (T1 and T2) than other glomerular variables. To the best of our knowledge this is the largest series of IgAN in Brazil. It depicts the main biopsy findings and their possible clinical correlates. Our set of data is comparable to previous reports.

Control of lymphocyte adhesion to brain and aortic endothelium: ICAM-1, VCAM-1 and negative charge.

Lymphocyte adhesion to CNS endothelium is low by comparison with non-CNS endothelium. It has been proposed that this could be due to the high surface charge of brain endothelium, or a low constitutive expression of adhesion molecules. In this study we compared the influence of these factors on lymphocyte adhesion to BEC and aortic endothelium (AEC) in culture. Brain endothelium expresses very low levels of VCAM-1, and lower levels of ICAM-1 than aortic endothelium. The negative charge differed between the endothelia, but this had a minimal effect on lymphocyte adhesion. The anionic sites were, however, more stable on brain endothelium, remaining unchanged after endothelial cell activation with cytokines, while redistribution was observed on cytokine-activated aortic endothelium.

SV40 large T immortalised cell lines of the rat blood-brain and blood-retinal barriers retain their phenotypic and immunological characteristics.

In the central nervous system the blood-brain and blood-retinal barriers (BBB and BRB respectively) are instrumental in maintaining homeostasis of the neural parenchyma and controlling leucocyte traffic. These cellular barriers are formed primarily by the vascular endothelium of the brain and retina although in the latter the pigmented epithelial cells also form part of the barrier. From primary cultures of rat brain endothelium, retinal endothelium and retinal pigment epithelium (RPE) we have generated temperature sensitive SV40 large T immortalised cell lines. Clones of brain (GP8.3) and retinal (JG2.1) endothelia and RPE (LD7.4) have been derived from parent lines that express the large T antigen at the permissive temperature. The endothelial cell (EC) lines expressed P-glycoprotein, GLUT-1, the transferrin receptor, von Willebrand factor and the RECA-1 antigen and exhibited high affinity uptake of acetylated LDL and stained positive with the lectin Griffonia simplicifolia. The RPE cell line was positive for cytokeratins and for the rat RPE antigen RET-PE2. All the cell lines expressed major histocompatibility complex (MHC) class 1 and intercellular adhesion molecule (ICAM)-1 constitutively and could be induced to express MHC class II and vascular cell adhesion molecule (VCAM)-1 following cytokine activation. The EC also expressed platelet endothelial cell adhesion molecule (PECAM)-1. Monolayers of these cells could support the migration of antigen-specific T cell lines. The generation of immortalised cell lines derived from the rat BBB and BRB should prove to be useful tools for the study of these specialised cellular barriers.

Cohort study on canine emigration and Leishmania infection in an endemic area for American visceral leishmaniasis. Implications for the disease control.

American visceral leishmaniasis is a main public health matter in Brazil. Since dogs have been incriminated as the main urban reservoir of AVL agent Leishmania chagasi, a cohort study aimed at understanding the dynamics of the canine infection was carried out in Jequié--an endemic community in the Northeast of Brazil. The inhabited urban and periurban areas of Jequié were divided into 140 clusters of 0.25 km2. All 1681 dogs domiciled in 34 randomly selected clusters were screened for Leishmania antibodies in an enzyme-linked immunosorbent assay. After the seropositive dogs were painlessly eliminated, a cohort of 1286 seronegative dogs was followed up for 18 months, yielding a total of 1739.7 dog-years. The overall incidence of Leishmania infection, as assessed by the detection of Leishmania antibodies in blood samples collected every six months, was 6.55 cases/100 dog-years (95% confidence interval; CI 6.04-7.26). Two subsets of clusters, with 0.70 and 1.35 relative risks of infection, were identified. The annual emigration rate was 2.26 cases/100 dog-years (95% CI 1.86-2.66). The implications of these findings for the control of American visceral leishmaniasis are discussed.

In vivo protective effect of the lectin from Canavalia brasiliensis on BALB/c mice infected by Leishmania amazonensis.

In vivo administration of Canavalia brasiliensis lectin (at the time of infection, or maintained throughout the infection) reduced the lesions of highly susceptible BALB/c mice infected by Leishmania amazonensis. At the doses used C. brasiliensis lectin (ConBr) does not interfere with penetration or fate of Leishmania in the macrophages in vitro. Since Interferon-gamma (IFN-gamma) is the major macrophage activating factor, and considered a critical element in the successful immune response against leishmaniasis, we explored its participation in this phenomenon. ConBr either in vivo or in vitro induced IFN-gamma production in normal or in leishmania-infected BALB/c mice. However we were unable to change the course of disease by in vivo IFN-gamma administration (although IFN-gamma preparations were effective in inducing a leishmanicidal effect in vitro on L. amazonensis-infected peritoneal macrophages). Additionally, IFN-gamma neutralization with anti-IFN-gamma monoclonal antibody did not alter the protection conferred by ConBr administration. These data show that lectin administration in vivo is protective in the otherwise unchecked L. amazonensis infection of BALB/c mice, and suggest that such effect is not mediated by IFN-gamma.

Connective tissue changes in rheumatic heart disease.

The connective matrix of 17 surgically excised mitral complexes from patients with clinical diagnosis of rheumatic carditis was evaluated by semi-quantitative histopathological, immunohistochemical and ultrastructural parameters. Different and concomitant patterns of loose and dense fibrosis were observed with variable constitution and organization of collagen I, III, IV, procollagen III, laminin, fibronectin and elastin. Loose fibrosis exhibited codistribution of all matrix components, Initial phase of fibrosis was characterized by deposition of all matrix components organized in a network pattern. In dense fibrosis a parallel disposition of type I collagen bundles predominated. In the denser (hyalin) fibrosis, the collagen exhibited abnormalities in fiber diameters and in fiber conformation (hyperfibers) and there was reduction or disappearance of other matrix components. The presence of these different kinds of connective matrix and the ultrastructural alterations in collagen fibers are associated to different stages of fibrosis organization and probably reflect changes in collagen susceptibility to degradation. These morphologic patterns may be related to the evolution (stability or reversibility) of rheumatic sequelae.

Administration of a nondepleting anti-CD25 monoclonal antibody reduces disease severity in mice infected with Trypanosoma cruzi.

The role of CD25+ regulatory T cells during the course of Trypanosoma cruzi infection has been previously analyzed, and the bulk of results have shown a limited role for this T cell subpopulation. In this study, we have used an IgM, nondepleting monoclonal antibody (mAb) aiming at blocking interleukin (IL)-2 activity on CD25+ T cells. The administration of this antibody 10 days before infection increased the resistance of outbred Swiss mice to the Colombian strain of T. cruzi. Anti-CD25-treated mice had lower parasitemia and augmented numbers of effector memory T cells. In addition, these animals showed higher numbers of splenic T cells secreting IFN-γ and TNF-α, both cytokines described to be involved in the resistance to T. cruzi infection. The same treatment also increased the numbers of splenic T cells that produced homeostatic and regulatory cytokines, such as IL-2 and IL-10, and CD4+CD25+ T cells. The administration of nondepleting anti-CD25 mAb at the beginning of the chronic phase, when parasites were cleared from the blood, halted the inflammatory process in the heart, without any signs of infection reactivation. These results indicate that nondepleting anti-CD25 monoclonal antibodies may be useful to treat chronic Chagas' disease.

Distribution and analysis of surface charge on brain endothelium in vitro and in situ.

Vascular endothelial cells are associated with a number of anionic molecules. These anions are important in endothelial function, particularly in regulating permeability, haemostasis and cellular traffic. To explore the nature and distribution of anions on the brain endothelial cell (BEC) surface, we have examined rat brain endothelium in culture, and in situ. The anionic sites were probed with cationic colloidal gold and cationised ferritin, and visualised by light microscopy. Additionally we compared the distribution of the anionic sites on BEC with that present on other endothelial cell types in culture. The predominant anion detected on BEC was heparan sulphate (HS). This was distributed throughout the cell membrane, but was most densely associated with intercellular junctions. This pattern was distinct from the anionic locations observed in endothelia from aorta and epididymal fat microvessels. The distribution of anions was dependent on the age of cultured cells, with only minimal levels of HS seen at the periphery of younger cells. The nature and distribution of negative charge was different in situ. Here, sialic acid was the major surface anion, with only a small contribution from HS. The significance of these findings are discussed in relation to endothelial function in normal tissue and in pathological conditions.

Placental and fetal candidiasis. Presentation of a case of an abortus.

The authors present a case of intrauterine fetal infection by candida, in an abortion of four months, associated with an I.U.D. In the placenta and adnexa we observed an acute inflammation consisting of extraplacental membranitis, omphalitis, chorio-amnionitis and choriovasculitis with a marked villitis and intervillitis. In the fetus, involvement of the skin, lungs and pharynx was observed. This case represents, probably the 15th reported instance of congenital fetal candidiasis, and the first case of a candida hematogenic placental infection acquired from the fetal blood. The fetus undoubtedly acquired its infection by an ascending route, through the contamined amniotic fluid.

Experimental infection with Basidiobolus haptosporus.

The authors tried to reproduce experimentally the Basidiobolus haptosporus infection. Culture forms of the fungus were inoculated in 26 adult hamsters, in two newborn hamsters and in two marmosets. Oral, intratesticular, intrahepatic, intraperitonial and subcutaneous inoculations were made. Bethametasone was given prior to inoculation in a group of animals. The lesions produced were only of the foreign body type and there was no development of the fungus in the animal tissues. The AA concluded that an experimental model for the B. haptosporus infection has not yet been found.

Skin reactions to thimerosal and Leishmania in dogs from a leishmaniasis endemic area: it is better to keep them apart.

Positive Montenegro's skin test is a delayed type hypersensitivity reaction widely used as indicative of previous infection with Leishmania in both humans and dogs. Montenegro's antigen consists of a crude Leishmania antigen solution, usually containing thimerosal as preserving agent. In this work it is shown that a large proportion of dogs (11 out of 56) examined in an endemic area of leishmaniasis presented induration at the site of injection of a diluent containing thimerosal alone. This clearly demonstrates that thimerosal leads to a high number of false positive skin reactions in dogs and that its use in Montenegro's skin test antigenic preparations should be avoided.

Dissociation between vasodilation and Leishmania infection-enhancing effects of sand fly saliva and maxadilan.

In this study, the ability of maxadilan and Lutzomyia longipalpis salivary gland lysate to enhance the infection of CBA mice by Leishmania major and of BALB/c mice by L. braziliensis was tested. No difference was observed between sizes of lesion in CBA mice infected with L. major and treated or not with salivary gland lysate or maxadilan, although they were injected in concentrations that induced cutaneous vasodilation. Although parasites were more frequently observed in foot pads and spleens of animals treated with maxadilan than in the animals treated with salivary gland lysate or saline, the differences were small and not statistically significant. The lesions in BALB/c mice infected with L. braziliensis and treated with maxadilan were slightly larger than in animals that received Leishmania alone. Such differences disappeared 14 weeks after infection, and were statistically significant only in one of two experiments.