Effect of angiotensin II and losartan on the phagocytic activity of peritoneal macrophages from Balb/C mice

Angiotensin II (AII), a product of rennin-angiotensin system, exerts an important role on the function of immune system cells. In this study, the effect of AII on the phagocytic activity of mouse peritoneal macrophages was assessed. Mice peritoneal macrophages were cultured for 48 h and the influence of different concentrations of AII (10(-14) to 10(-7) M) and/or losartan, 10(-16) to 10(-6) M), an AT1 angiotensin receptor antagonist, on phagocytic activity and superoxide anion production was determined. Dimethylthiazoldiphenyltetrazolium bromide reduction and the nucleic acid content were used to assess the cvtotoxicity of losartan. A stimulatory effect on phagocytic activity (P < 0.05) was observed with 10(-13) M and 10(-12 M) AII concentrations. The addition of losartan (up to10(-14) M) to the cell cultures blocked (P < 0.001) the phagocytosis indicating the involvement of AT1 receptors. In contrast, superoxide anion production was not affected by AII or losartan. The existence of AT1 and AT2 receptors in peritoneal macrophages was demonstrated by immunofluorescence microscopy. These results support the hypothesis that AII receptors can modulate murine macrophage activity and phagocytosis, and suggest that AII may have a therapeutic role as an immunomodulatory agent in modifying the host resistance to infection.     

Survey of Sand Flies (Diptera: Psychodidae) in an Environmentally Protected Area in Brazil

Brazil is one of the most important endemic areas for leishmaniasis worldwide. Protected areas that are tourist attractions likely present an important risk of transmission of cutaneous leishmaniasis (CL). Furthermore, with the geographical expansion of visceral leishmaniasis (VL), several studies have recorded the occurrence of its vector, Lutzomyia longipalpis, and cases of human and canine VL in such tourist areas. The Parque Estadual do Sumidouro is an environmentally protected area located in the Brazilian Cerrado biome and in an important area endemic for leishmaniasis in the state of Minas Gerais. The purpose of this study was to monitor the sand fly fauna in areas of tourist activity in the park. Sampling was performed every month, from September 2011 to August 2013, using CDC light traps at six sites of differing environmental characteristics. Sampled specimens were identified following Galati (2003), and females were submitted to molecular techniques for the detection and identification of Leishmania DNA. A total of 4,675 sand fly specimens of 25 species belonging to nine genera were collected. The most abundant species were Micropygomyia quinquefer, Lutzomyia renei and Pintomyia pessoai, although only Pi. pessoai is implicated in the transmission of Leishmania braziliensis. The species accumulation curve reached saturation on the 16th sampling event. Species richness, diversity and evenness differed among the sampled areas. The seasonal curve was not determined by a single unique species, and no single species was the most abundant in all environments sampled. The main vector of Leishmania (Leishmania) infantum, Lutzomyia longipalpis, accounted for only 5.35% of the specimens collected. Proven or suspected vectors of Leishmania (Viannia) braziliensis were recorded, and one female of the cortellezzii complex tested positive for Le. braziliensis DNA. Even with a low infection rate (0.62%), these data indicate the circulation of the parasite and reinforce the need for entomological and epidemiological surveillance in the park and its surroundings.     

Purification and properties of a coagulant thrombin-like enzyme from the venom of Bothrops leucurus

A thrombin-like enzyme from Bothrops leucurus venom, named leucurobin (leuc), was purified by gel filtration, affinity and ion exchange chromatographies. Physicochemical studies indicated that the purified enzyme is a 35 kDa monomeric glycoprotein on SDS-PAGE under reducing conditions, which decreased to 29 kDa after deglycosylation with N-glycosidase F (PNGase F). The amino acid sequence of leuc was determined by automated sequencing of the intact native protein and peptides produced by digestion of the S-pyridyl-ethylated protein with trypsin. The protein sequence exhibits significant similarities with other serine proteases reported from snake venoms, and contains two potential sites of N-linked glycosylation. The proteinase split off fibrinopeptide A (FPA) rapidly from human fibrinogen; however, only negligible traces of fibrinopeptide B (FPB) were observed. In addition, the enzyme released the N-terminal peptide (Mr=4572) containing the first 42 residues from the Bbeta-chain. Leuc could neither activate factor XIII nor release kinins from heat-treated bovine plasma. Its specific clotting activity was equivalent to 198 NIH thrombin U/mg on human fibrinogen. Kinetic properties of leuc were determined using representative chromogenic substrates. The enzyme evoked the gyroxin syndrome when injected into the tail veins of mice at levels of 0.143 microg/g mouse. The inhibitory effects of PMSF and benzamidine on the amidolytic activity suggest that leuc is a serine proteinase, and inhibition by beta-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Antibothropic serum, SBTI and EDTA had little or no effect on its amidolytic activity. However, the clotting effect of the enzyme was strongly inhibited by antibothropic serum. A Dixon plot showed that the hydrolysis of Bz-L-Arg-pNA by leuc was competitively inhibited by benzamidine (Ki=1.61+/-0.25 mM).     

Purification and properties of a coagulant thrombin-like enzyme from the venom of Bothrops leucurus

A thrombin-like enzyme from Bothrops leucurus venom, named leucurobin (leuc), was purified by gel filtration, affinity and ion exchange chromatographies. Physicochemical studies indicated that the purified enzyme is a 35 kDa monomeric glycoprotein on SDS-PAGE under reducing conditions, which decreased to 29 kDa after deglycosylation with N-glycosidase F (PNGase F). The amino acid sequence of leuc was determined by automated sequencing of the intact native protein and peptides produced by digestion of the S-pyridyl-ethylated protein with trypsin. The protein sequence exhibits significant similarities with other serine proteases reported from snake venoms, and contains two potential sites of N-linked glycosylation. The proteinase split off fibrinopeptide A (FPA) rapidly from human fibrinogen; however, only negligible traces of fibrinopeptide B (FPB) were observed. In addition, the enzyme released the N-terminal peptide (Mr = 4572) containing the first 42 residues from the Bβ-chain. Leuc could neither activate factor XIII nor release kinins from heat-treated bovine plasma. Its specific clotting activity was equivalent to 198 NIH thrombin U/mg on human fibrinogen. Kinetic properties of leuc were determined using representative chromogenic substrates. The enzyme evoked the gyroxin syndrome when injected into the tail veins of mice at levels of 0.143 μg/g mouse. The inhibitory effects of PMSF and benzamidine on the amidolytic activity suggest that leuc is a serine proteinase, and inhibition by β-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Antibothropic serum, SBTI and EDTA had little or no effect on its amidolytic activity. However, the clotting effect of the enzyme was strongly inhibited by antibothropic serum. A Dixon plot showed that the hydrolysis of Bz-l-Arg-pNA by leuc was competitively inhibited by benzamidine (K_i = 1.61 ± 0.25 mM).     

5' flanking region of var genes nucleate histone modification patterns linked to phenotypic inheritance of virulence traits in malaria parasites

In the human malaria parasite Plasmodium falciparum antigenic variation facilitates long-term chronic infection of the host. This is achieved by sequential expression of a single member of the 60-member var family. Here we show that the 5' flanking region nucleates epigenetic events strongly linked to the maintenance of mono-allelic var gene expression pattern during parasite proliferation. Tri- and dimethylation of histone H3 lysine 4 peak in the 5' upstream region of transcribed var and during the poised state (non-transcribed phase of var genes during the 48 h asexual life cycle), 'bookmarking' this member for re-activation at the onset of the next cycle. Histone H3 lysine 9 trimethylation acts as an antagonist to lysine 4 methylation to establish stably silent var gene states along the 5' flanking and coding region. Furthermore, we show that competition exists between H3K9 methylation and H3K9 acetylation in the 5' flanking region and that these marks contribute epigenetically to repressing or activating var gene expression. Our work points to a pivotal role of the histone methyl mark writing and reading machinery in the phenotypic inheritance of virulence traits in the malaria parasite.     

Reduced‐risk insecticides in Neotropical stingless bee species: impact on survival and activity

As honeybees are the main pollinator subject to an intense research regarding effects of pesticides, other ecologically important native bee pollinators have received little attention in ecotoxicology and risk assessment of pesticides in general, and insecticides in particular, some of which are perceived as reduced‐risk compounds. Here, the impact of three reduced‐risk insecticides – azadirachtin, spinosad and chlorantraniliprole – was assessed in two species of stingless bees, Partamona helleri and Scaptotrigona xanthotrica, which are important native pollinators in Neotropical America. The neonicotinoid imidacloprid was used as a positive control. Spinosad exhibited high oral and contact toxicities in adult workers of both species at the recommended label rates, with median survival times (LT₅₀s) ranging from 1 to 4 h, whereas these estimates were below 15 min for imidacloprid. Azadirachtin and chlorantraniliprole exhibited low toxicity at the recommended label rates, with negligible mortality that did not allow LT₅₀ estimation. Sublethal behavioural assessments of these two insecticides indicated that neither one of them affected the overall group activity of workers of the two species. However, both azadirachtin and chlorantraniliprole impaired individual flight take‐off of P. helleri and S. xanthotrica worker bees, which may compromise foraging activity, potentially leading to reduced colony survival. These findings challenge the common perception of non‐target safety of reduced‐risk insecticides and bioinsecticides, particularly regarding native pollinator species.     

Toll receptors type-2 and CR3 expression of canine monocytes and its correlation with immunohistochemistry and xenodiagnosis in visceral leishmaniasis

The aim of the present study was to investigate TLR2 expression in peripheral blood monocytes from dogs naturally infected with Leishmania (Leishmania) infantum to determine whether it correlates with CD11b/CD18 (CR3) expression, and to evaluate the potential of dogs as sources of infection using phlebotomine xenodiagnosis. Forty eight dogs were serologically diagnosed with L. infantum infection by indirect immunofluorescence antibody test (IFAT) and enzyme linked immunosorbent assay (ELISA). Parasitological exams from bone-marrow aspirates were positive by PCR analysis. All dogs were clinical defined as symptomatic. Ear skin tissue samples were obtained for immunohistochemistry (IHQ) analysis. The potential of these dogs as a source of infection using phlebotomine xenodiagnosis (XENO) was evaluated. Flow cytometry was carried out on peripheral blood mononuclear cells using superficial receptors including CD14, CD11b, TLR2 and MHCII. IHQ ear skin tissue parasite load and XENO where done where we found a strict correlation (r = 0.5373). Dogs with higher expression of MFI of CD11b inside CD14 monocytes were represented by dogs without parasite ear tissue load that were unable to infect phlebotomines (IHQ⁻/XENO⁻). Dogs with lower expression of MFI of CD11b inside CD14 monocytes were represented by dogs with parasite ear tissue load and able to infect phlebotomines (IHQ⁺/XENO⁺) (p = 0,0032). Comparable results were obtained for MFI of MHCII (p = 0.0054). In addition, considering the population frequency of CD11b⁺TLR2⁺ and CD11b⁺MHCII⁺, higher values were obtained from dogs with IHQ⁻/XENO⁻ than dogs with IHQ⁺/XENO⁺ (p = 0.01; p = 0.0048, respectively). These data, together with the TLR2 and NO assays results (CD11b⁺TLR2⁺ and NO with higher values for dogs with IHQ⁻/XENO⁻ than dogs with IHQ⁺/XENO⁺), led to the conclusion that IHQ⁻/XENO⁻ dogs are more resistant or could modulate the cellular immune response essential for Leishmania tissue clearance.     

Conventional Pap smear and liquid-based cytology as screening tools in low-resource settings in Latin America: experience of the Latin American screening study

OBJECTIVE: To evaluate the performance of the conventional Pap test and liquid-based cytology (LBC) in an ongoing multicenter trial testing optional screening tools (cytology, screening colposcopy, visual inspection with acetic acid, visual inspection with Lugol's Iodine, cervicography and Hybrid Capture II [HCII] (Digene Brazil, S?o Paulo, Brazil) conventional and self-sampling), for cervical cancer in Brazil and Argentina. STUDY DESIGN: A cohort of 12,107 women attending four clinics (Campinas, S?o Paulo, Porto Alegre, Buenos Aires) were randomized into the 8 diagnostic arms. Women testing positive with any of the tests were referred for colposcopy, and cervical biopsies were used as the gold standard to assess performance characteristics of the diagnostic tests. Conventional Pap smears were sampled by all clinics (n = 10,240), and LBC (Autocyte PREP, [TriPath Imaging, Burlington, North Carolina, U.S.A.], n=320, and DNA-Citoliq [Digene Brazil], n =1,346) was performed by 1 of the clinics. RESULTS: Conventional Pap smears showed no squamous intraepithelial lesions (normal) in 8,946 (87.4%) and LBC in 1,373 (82.4%). Using high grade squamous intraepithelial lesions (HSIL) as the cutoff, Pap smears predicted high grade (cervical intraepithelial neoplasia [CIN] 3) with OR 63.0 (95% CI, 36.90-107.70), standard error (SE) 59%, SP 97.8%, positive predictive value (PPV) 68.1% and negative predictive value (NPV) 96.7%. The same figures for Autocyte PREP were: OR 9.0 (95% CI, 2.43-33.24), sensitivity (SE) 33.3%, specificity (SP) 100%, PPV 100% and negative PV (NPV) 88.8%. DNA-Citoliq detected CIN 3 as follows: OR 11.8 (95% CI 2.60-53.26), SE 40.0%, SP 94.6%, PPV 40.0% and NPV 94.6%. Lowering the cutoff to low grade squamous intraepithelial lesions increased SE and NPV but compromised SP and PPV. The detection rates for high grade lesions after an atypical squamous cells of undetermined significance diagnosis were similar with the 3 techniques. In our settings, the 3 methods of cervical cytology were slightly different in performance. The conventional Pap smear had the highest SE, while Autocyte PREP had 100% SP and PPV in detecting CIN3 with the HSIL cutoff. All 3 tests had lower SE but higher SP as compared to HCII.     

Nuclear Localization of SP, CGRP, and NK 1 R in a Subpopulation of Dorsal Root Ganglia Subpopulation Cells in Rats

Signals generated by renal pelvic afferent nerves in response to stimulation are transmitted from peripheral processes of dorsal root ganglia neurons to their central terminals in the dorsal horn of the spinal cord to cause the release of neuropeptides, including SP and CGRP. All of the cellular activities of SP are considered to be mediated through interaction with NK₁R located on the cell surface. We have investigated the colocalization and subcellular distribution of NK₁R, SP, and CGRP in different subpopulations of neurons that innervate renal tissue. Our findings therefore provide the first evidence for the presence of NK₁R, SP, and CGRP in the nuclei of DGR neural cells. The physiological significance of this localization remains unknown. One possibility is that pelvic sensory neurons may regulate their responses to different stimuli by modulating the ratio of CGRP and SP release and/or nuclear NK₁R expression.