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Find video protocols related to scientific articles indexed in Pubmed.
Associated factors and prevalence of erectile dysfunction in hemodialysis patients.
Int Braz J Urol
PUBLISHED: 03-20-2014
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The proposal of this study was to determine the prevalence and the associated factors of erectile dysfunction (ED) among hemodialysis (HD) patients.
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A multicentric evaluation of the recombinant Leishmania infantum antigen-based immunochromatographic assay for the serodiagnosis of canine visceral leishmaniasis.
Parasit Vectors
PUBLISHED: 03-16-2014
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Visceral leishmaniasis (VL) is a serious public health challenge in Brazil and dogs are considered to be the main urban reservoir of the causative agent. The culling of animals to control VL in some countries makes the accurate diagnosis of canine VL (CVL) essential. Recombinant antigens rLci1A and rLci2B were selected from a cDNA library of Leishmania infantum amastigotes due to their strong potential as candidates in diagnostic testing for CVL. The present multicentric study aimed to evaluate the sensitivity of a prototype test using these antigens (DPP rLci1A/rLci2B) against 154 sera obtained from symptomatic dogs within three endemic areas of VL in Brazil. The specificity was evaluated using 40 serum samples from negative dogs and dogs infected with other pathogens. Sensitivity and specificity rates of DPP rLci1A/rLci2B prototype were compared to rates from other diagnostic tests currently in use by the Brazilian Ministry of Health, including DPPLVC, EIELVC.
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Automated segmentation of optic nerve head structures with optical coherence tomography.
Invest. Ophthalmol. Vis. Sci.
PUBLISHED: 01-30-2014
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To quantify and characterize the difference between automated and manual segmentation of optic nerve head structures with spectral-domain optical coherence tomography (SD-OCT).
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Shotgun proteomics to unravel the complexity of the Leishmania infantum exoproteome and the relative abundance of its constituents.
Mol. Biochem. Parasitol.
PUBLISHED: 01-27-2014
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The exoproteome of some Leishmania species has revealed important insights into host-parasite interaction, paving the way for the proposal of novel disease-oriented interventions. The focus of the present investigation constituted the molecular profile of the L. infantum exoproteome revealed by a shotgun proteomic approach. Promastigotes under logarithmic phase of growth were obtained and harvested by centrifugation at different time points. Cell integrity was evaluated through the counting of viable parasites using propidium iodide labeling, followed by flow cytometry analysis. The 6h culture supernatant, operationally defined here as exoproteome, was then conditioned to in solution digestion and the resulting peptides submitted to mass spectrometry. A total of 102 proteins were identified and categorized according to their cellular function. Their relative abundance index (emPAI) allowed inference that the L. infantum exoproteome is a complex mixture dominated by molecules particularly involved in nucleotide metabolism and antioxidant activity. Bioinformatic analyses support that approximately 60% of the identified proteins are secreted, of which, 85% possibly reach the extracellular milieu by means of non-classic pathways. At last, sera from naturally infected animals, carriers of differing clinical forms of Canine Visceral Leishmaniasis (CVL), were used to test the immunogenicity associated to the L. infantum exoproteome. Western blotting experiments revealed that this sub-proteome was useful at discriminating symptomatic animals from those exhibiting other clinical forms of the disease. Collectively, the molecular characterization of the L. infantum exoproteome and the preliminary immunoproteomic assays opened up new research avenues related to treatment, prognosis and diagnosis of CVL.
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Immunological profile of resistance and susceptibility in naturally infected dogs by Leishmania infantum.
Vet. Parasitol.
PUBLISHED: 01-23-2014
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Visceral leishmaniasis has a great impact on public health, and dogs are considered the main domestic reservoir of Leishmania infantum, the causal parasite. In this study, 159 animals naturally infected by L. infantum from an endemic area of Brazil were evaluated through an analysis of cellular responses, using flow cytometry, and of the hematological parameters. The results confirmed that disease progression is associated with anemia and reductions in eosinophils, monocytes and lymphocytes. The investigation of the immune response, based on the immunophenotypic profile of peripheral blood, showed declines in the absolute numbers of T lymphocytes CD5(+) and their subsets (CD4(+) and CD8(+)) and a drop of B lymphocytes in asymptomatic seropositive (AD-II) and symptomatic seropositive (SD) dogs. Neutrophils, when stimulated with soluble antigen of L. infantum, showed higher synthesis of interferon (IFN)-?(+) in AD-II and SD groups, with decreased production of interleukin (IL)-4(+) in asymptomatic seronegative dogs positive for L. infantum infection based on polymerase chain reaction testing (AD-I group). In the AD-II and SD groups, subpopulations of stimulated lymphocytes (CD4(+) and CD8(+)) also exhibited greater synthesis of IFN-?(+) and IL-4(+) in culture. These results suggest that the animals of the AD-II and SD groups exhibited a mixed immune response (Type 1 and 2) and the AD-I group presenting an immune profile very similar to normal control animals.
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LBSapSal-vaccinated dogs exhibit increased circulating T-lymphocyte subsets (CD4? and CD8?) as well as a reduction of parasitism after challenge with Leishmania infantum plus salivary gland of Lutzomyia longipalpis.
Parasit Vectors
PUBLISHED: 01-18-2014
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The development of a protective vaccine against canine visceral leishmaniasis (CVL) is an alternative approach for interrupting the domestic cycle of Leishmania infantum. Given the importance of sand fly salivary proteins as potent immunogens obligatorily co-deposited during transmission of Leishmania parasites, their inclusion in an anti-Leishmania vaccine has been investigated in the last few decades. In this context, we previously immunized dogs with a vaccine composed of L. braziliensis antigens plus saponin as the adjuvant and sand fly salivary gland extract (LBSapSal vaccine). This vaccine elicited an increase in both anti-saliva and anti-Leishmania IgG isotypes, higher counts of specific circulating CD8? T cells, and high NO production.
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Immunotherapy and Immunochemotherapy in Visceral Leishmaniasis: Promising Treatments for this Neglected Disease.
Front Immunol
PUBLISHED: 01-01-2014
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self-healing or chronic cutaneous leishmaniasis or post-kala-azar dermal leishmaniasis; mucosal leishmaniasis; visceral leishmaniasis (VL), which is fatal if left untreated. The epidemiology and clinical features of VL vary greatly due to the interaction of multiple factors including parasite strains, vectors, host genetics, and the environment. Human immunodeficiency virus infection augments the severity of VL increasing the risk of developing active disease by 100-2320 times. An effective vaccine for humans is not yet available. Resistance to chemotherapy is a growing problem in many regions, and the costs associated with drug identification and development, make commercial production for leishmaniasis, unattractive. The toxicity of currently drugs, their long treatment course, and limited efficacy are significant concerns. For cutaneous disease, many studies have shown promising results with immunotherapy/immunochemotherapy, aimed to modulate and activate the immune response to obtain a therapeutic cure. Nowadays, the focus of many groups centers on treating canine VL by using vaccines and immunomodulators with or without chemotherapy. In human disease, the use of cytokines like interferon-? associated with pentavalent antimonials demonstrated promising results in patients that did not respond to conventional treatment. In mice, immunomodulation based on monoclonal antibodies to remove endogenous immunosuppressive cytokines (interleukin-10) or block their receptors, antigen-pulsed syngeneic dendritic cells, or biological products like Pam3Cys (TLR ligand) has already been shown as a prospective treatment of the disease. This review addresses VL treatment, particularly immunotherapy and/or immunochemotherapy as an alternative to conventional drug treatment in experimental models, canine VL, and human disease.
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Evaluation of change in canine diagnosis protocol adopted by the visceral leishmaniasis control program in Brazil and a new proposal for diagnosis.
PLoS ONE
PUBLISHED: 01-01-2014
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The techniques used for diagnosis of canine visceral leishmaniasis (CVL) in Brazil ELISA and IFAT have been extensively questioned because of the accuracy of these tests. A recent change in the diagnosis protocol excluded IFAT and included the Dual-Path Platform (DPP). We evaluated the prevalence and incidence rates of Leishmania spp. before and after the change in the protocol. In addition, based on our results, we propose a new alternative that is less expensive for the screening and confirmation of CVL. Plasma samples were obtained from a serobank from dogs evaluated in a cross-sectional study (1,226 dogs) and in a cohort study of susceptible animals (n = 447), followed for 26 months. Serology testing was performed using ELISA, IFAT, and DPP. The incidence and prevalence of CVL were determined by using the protocol of the Visceral Leishmaniasis Control and Surveillance Program until 2012 (ELISA and IFAT using filter paper) and the protocol used after 2012 (DPP and ELISA using plasma). The prevalence was 6.2% and the incidence was 2.8 per 1,000 dog-months for the protocol used until 2012. For the new diagnosis protocol for CVL resulted in an incidence of 5.4 per 1,000 dog-months and a prevalence of 8.1%. Our results showed that the prevalence and incidence of infection were far greater than suggested by the previously used protocol and that the magnitude of infection in endemic areas has been underestimated. As tests are performed sequentially and euthanasia of dogs is carried out when the serological results are positive in both tests, the sequence does not affect the number of animals to be eliminated by the Control Program. Then we suggest to municipalities with a large demand of exams to use ELISA for screening and DPP for confirmation, since this allows easier performance and reduced cost.
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Oxygen-limited cellobiose fermentation and the characterization of the cellobiase of an industrial Dekkera/Brettanomyces bruxellensis strain.
Springerplus
PUBLISHED: 01-01-2014
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The discovery of a novel yeast with a natural capacity to produce ethanol from lignocellulosic substrates (second-generation ethanol) is of great significance for bioethanol technology. While there are some yeast strains capable of assimilating cellobiose in aerobic laboratory conditions, the predominant sugar in the treatment of lignocellulosic material, little is known about this ability in real industrial conditions. Fermentations designed to simulate industrial conditions were conducted in synthetic medium with glucose, sucrose, cellobiose and hydrolyzed pre-treated cane bagasse as a different carbon source, with the aim of further characterizing the fermentation capacity of a promising Dekkera bruxellensis yeast strain, isolated from the bioethanol process in Brazil. As a result, it was found (for the first time in oxygen-limiting conditions) that the strain Dekkera bruxellensis GDB 248 could produce ethanol from cellobiose. Moreover, it was corroborated that the cellobiase activity characterizes the enzyme candidate in semi-purified extracts (?-glucosidase). In addition, it was demonstrated that GDB 248 strain had the capacity to produce a higher acetic acid concentration than ethanol and glycerol, which confirms the absence of the Custer effect with this strain in oxygen-limiting conditions. Moreover, it is also being suggested that D. bruxellensis could benefit Saccharomyces cerevisiae and outcompete it in the industrial environment. In this way, it was confirmed that D. bruxellensis GDB 248 has the potential to produce ethanol from cellobiose, and is a promising strain for the fermentation of lignocellulosic substrates.
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Evaluation of a prototype flow cytometry test for serodiagnosis of canine visceral leishmaniasis.
Clin. Vaccine Immunol.
PUBLISHED: 10-09-2013
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Diagnosing canine visceral leishmaniasis (CVL) is a critical challenge since conventional immunoserological tests still present some deficiencies. The current study evaluated a prototype flow cytometry serology test, using antigens and fluorescent antibodies that had been stored for 1 year at 4°C, on a broad range of serum samples. Noninfected control dogs and Leishmania infantum-infected dogs were tested, and the prototype test showed excellent performance in differentiating these groups with high sensitivity, specificity, positive and negative predictive values, and accuracy (100% in all analyses). When the CVL group was evaluated according to the dogs clinical status, the prototype test showed outstanding accuracy in all groups with positive serology (asymptomatic II, oligosymptomatic, and symptomatic). However, in dogs which had positive results by PCR-restriction fragment length polymorphism (RFLP) but negative results by conventional serology (asymptomatic I), serological reactivity was not observed. Additionally, sera from 40 dogs immunized with different vaccines (Leishmune, Leish-Tec, or LBSap) did not present serological reactivity in the prototype test. Eighty-eight dogs infected with other pathogens (Trypanosoma cruzi, Leishmania braziliensis, Ehrlichia canis, and Babesia canis) were used to determine cross-reactivity and specificity, and the prototype test performed well, particularly in dogs infected with B. canis and E. canis (100% and 93.3% specificities, respectively). In conclusion, our data reinforce the potential of the prototype test for use as a commercial kit and highlight its outstanding performance even after storage for 1 year at 4°C. Moreover, the prototype test efficiently provided accurate CVL serodiagnosis with an absence of false-positive results in vaccinated dogs and minor cross-reactivity against other canine pathogens.
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Dogs infected with the blood trypomastigote form of Trypanosoma cruzi display an increase expression of cytokines and chemokines plus an intense cardiac parasitism during acute infection.
Mol. Immunol.
PUBLISHED: 10-07-2013
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The recent increase in immigration of people from areas endemic for Chagas disease (Trypanosoma cruzi) to the United States and Europe has raised concerns about the transmission via blood transfusion and organ transplants in these countries. Infection by these pathways occurs through blood trypomastigotes (BT), and these forms of T. cruzi are completely distinct of metacyclic trypomastigotes (MT), released by triatomine vector, in relation to parasite-host interaction. Thus, research comparing infection with these different infective forms is important for explaining the potential impacts on the disease course. Here, we investigated tissue parasitism and relative mRNA expression of cytokines, chemokines, and chemokine receptors in the heart during acute infection by MT or BT forms in dogs. BT-infected dogs presented a higher cardiac parasitism, increased relative mRNA expression of pro-inflammatory and immunomodulatory cytokines and of the chemokines CCL3/MIP-1?, CCL5/RANTES, and the chemokine receptor CCR5 during the acute phase of infection, as compared to MT-infected dogs. These results suggest that infection with BT forms may lead to an increased immune response, as revealed by the cytokines ratio, but this kind of immune response was not able to control the cardiac parasitism. Infection with the MT form presented an increase in the relative mRNA expression of IL-12p40 as compared to that of IL-10 or TGF-?1. Correlation analysis showed increased relative mRNA expression of IFN-? as well as IL-10, which may be an immunomodulatory response, as well as an increase in the correlation of CCL5/RANTES and its CCR5 receptor. Our findings revealed a difference between inoculum sources of T. cruzi, as vectorial or transfusional routes of T. cruzi infection may trigger distinct parasite-host interactions during the acute phase, which may influence immunopathological aspects of Chagas disease.
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Development of a fluorescent based immunosensor for the serodiagnosis of canine leishmaniasis combining immunomagnetic separation and flow cytometry.
PLoS Negl Trop Dis
PUBLISHED: 08-01-2013
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An accurate diagnosis is essential for the control of infectious diseases. In the search for effective and efficient tests, biosensors have increasingly been exploited for the development of new and highly sensitive diagnostic methods. Here, we describe a new fluorescent based immunosensor comprising magnetic polymer microspheres coated with recombinant antigens to improve the detection of specific antibodies generated during an infectious disease. As a challenging model, we used canine leishmaniasis due to the unsatisfactory sensitivity associated with the detection of infection in asymptomatic animals where the levels of pathogen-specific antibodies are scarce.
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Analysis using canine peripheral blood for establishing in vitro conditions for monocyte differentiation into macrophages for Leishmania chagasi infection and T-cell subset purification.
Vet. Parasitol.
PUBLISHED: 07-11-2013
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Canine visceral leishmaniasis (CVL) is a parasitic disease endemic in many countries, and dogs present as the major natural reservoir of the parasite, Leishmania chagasi (syn. L. infantum). Biomarkers in the canine immune system is an important technique in the course of developing vaccines and treatment strategies against CVL. New methodologies for studying the immune response of dogs during Leishmania infection and after receiving vaccines and treatments against CVL would be useful. In this context, we used peripheral blood mononuclear cells (PBMCs) from healthy dogs to evaluate procedures related to (i) establishment of in vitro conditions of monocytes differentiated into macrophages infected with L. chagasi and (ii) purification procedures of T-cell subsets (CD4(+) and CD8(+)) using microbeads. Our data demonstrated that after 5 days of differentiation, macrophages were able to induce significant phagocytic and microbicidal activity after L. chagasi infection and also showed increased frequency of parasitism and a higher parasite load. Although N-acetyl-?-d-glucosaminidase (NAG) levels presented similar levels of macrophage culture and L. chagasi infection, a progressive decrease in myeloperoxidase (MPO) levels was a hallmark over 5 days of culture. High purity levels (>90%) of CD4 and CD8 T cells were obtained on a magnetic separation column. We concluded that monocytes differentiated into macrophages at 5 days and displayed an intermediate frequency of parasitism and parasite load 72 h after L. chagasi infection. Furthermore, the purification system using canine T-lymphocyte subsets obtained after 5 days of monocyte differentiation proved efficient for CD4 or CD8 T-cell purification (?90%). The in vitro analysis using L. chagasi-infected macrophages and purified T cells presented a prospective methodology that could be incorporated in CVL vaccine and treatment studies that aim to analyze the microbicidal potential induced by specific CD4(+) and/or CD8(+) T cells.
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Cellular immunophenotypic profile in the splenic compartment during canine visceral leishmaniasis.
Vet. Immunol. Immunopathol.
PUBLISHED: 06-23-2013
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To determine the role of the spleen in the pathogenesis of canine visceral leishmaniasis (CVL), we analyzed cellular immunophenotypic profiles of 52 dogs naturally infected with Leishmania infantum, clinically classified as follows: asymptomatic dogs-I (AD-I), seronegative/PCR+; asymptomatic dogs-II (AD-II), seropositive/PCR+; oligosymptomatic dogs (OD) and symptomatic dogs (SD). Seven non-infected dogs (CD) were included as a control group. AD-II presented higher levels of CD8+ T splenocytes and lower TCD4+/TCD8+ ratio in comparison with CD. OD and SD showed lower percentages of CD21+ as compared with AD-II. All seropositive dogs presented lower levels of CD45RA+ than CD. Regardless of the stimuli used, the proliferation index from splenocytes in vitro was inversely correlated with clinical status. After LSA stimulation, there was a higher percentage of specific CD8+ T in AD-II than CD and non-stimulated culture. In contrast, splenocytes from SD under in vitro LSA stimulation induced decreased MHC-II+ expression in comparison with all groups, and non-stimulated culture. In conclusion, the role of CD8+ T splenocytes seems to be important for an effective immunological response, a hallmark of asymptomatic CVL, whereas the pronounced loss of MHC-II expression upon LSA stimulation is a biomarker of symptomatic CVL.
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Sensitive and specific serodiagnosis of Leishmania infantum infection in dogs by using peptides selected from hypothetical proteins identified by an immunoproteomic approach.
Clin. Vaccine Immunol.
PUBLISHED: 04-03-2013
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In Brazil, the percentage of infected dogs living in areas where canine visceral leishmaniasis (CVL) is endemic ranges from 10 to 62%; however, the prevalence of infection in dogs is probably higher than figures reported from serological studies. In addition, problems with the occurrence of false-positive or false-negative results in the serodiagnosis of CVL have been reported. The present work analyzed the potential of synthetic peptides mapped from hypothetical proteins for improvement of the serodiagnosis of Leishmania infantum infection in dogs. From 26 identified leishmanial proteins, eight were selected, considering that no homologies between these proteins and others from trypanosomatide sequence databases were encountered. The sequences of these proteins were mapped to identify linear B-cell epitopes, and 17 peptides were synthesized and tested in enzyme-linked immunosorbent assays (ELISAs) for the serodiagnosis of L. infantum infection in dogs. Of these, three exhibited sensitivity and specificity values higher than 75% and 90%, respectively, to differentiate L. infantum-infected animals from Trypanosoma cruzi-infected animals and healthy animals. Soluble Leishmania antigen (SLA) showed poor sensitivity (4%) and specificity (36%) to differentiate L. infantum-infected dogs from healthy and T. cruzi-infected dogs. Lastly, the three selected peptides were combined in different mixtures and higher sensitivity and specificity values were obtained, even when sera from T. cruzi-infected dogs were used. The studys findings suggest that these three peptides can constitute a potential tool for more sensitive and specific serodiagnosis of L. infantum infection in dogs.
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Dogs immunized with LBSap vaccine displayed high levels of IL-12 and IL-10 cytokines and CCL4, CCL5 and CXCL8 chemokines in the dermis.
Mol. Immunol.
PUBLISHED: 03-27-2013
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The complex interplay between cytokines and chemokines regulates innate and adaptive immune responses against pathogens; specifically, cytokine and chemokine expression drives activation of immune effector cells and their recruitment to tissue infection sites. Herein, we inoculated dogs with Leishmania braziliensis antigens plus saponin (the LBSap vaccine), as well as with the vaccine components, and then used real-time PCR to evaluate the kinetics of dermal expression of mRNAs of cytokines (IL-12, IFN-?, TNF-?, IL-4, IL-13, TGF-? and IL-10) and chemokines (CCL2, CCL4, CCL5, CCL21 and CXCL8) 1, 12, 24 and 48 h after inoculation. We also evaluated the correlation between cytokine and chemokine expression and dermal cellularity. The LBSap vaccine induced high levels of IL-12 and IL-10 expression at 12 and 24 h, respectively. Furthermore, we observed positive correlations between IL-12 and IL-13 expression, IFN-? and IL-13 expression, and IL-13 and TGF-? expression, suggesting that a mixed cytokine microenvironment developed after immunization with the vaccine. Inoculation with the saponin adjuvant alone induced a chemokine and cytokine expression profile similar to that observed in the LBSap group. CCL4 and CXCL8 chemokine expression was up regulated by the LBSap vaccine. CCL5 expression was initially highest in the LBSap group, but at 48 h, expression was highest in the LB group. Information about the kinetics of the immune response to this vaccine gained using this dog model will help to elucidate the mechanisms of and factors involved in a protective response against Leishmania infection and will aid in establishing rational approaches for the development of vaccines against canine visceral leishmaniasis.
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Molecular diagnosis of canine visceral leishmaniasis: a comparative study of three methods using skin and spleen from dogs with natural Leishmania infantum infection.
Vet. Parasitol.
PUBLISHED: 03-23-2013
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Polymerase chain reaction (PCR) and its variations represent highly sensitive and specific methods for Leishmania DNA detection and subsequent canine visceral leishmaniasis (CVL) diagnosis. The aim of this work was to compare three different molecular diagnosis techniques (conventional PCR [cPCR], seminested PCR [snPCR], and quantitative PCR [qPCR]) in samples of skin and spleen from 60 seropositive dogs by immunofluorescence antibody test and enzyme-linked immunosorbent assay. Parasitological analysis was conducted by culture of bone marrow aspirate and optical microscopic assessment of ear skin and spleen samples stained with Giemsa, the standard tests for CVL diagnosis. The primers L150/L152 and LINR4/LIN17/LIN19 were used to amplify the conserved region of the Leishmania kDNA minicircle in the cPCR, and snPCR and qPCR were performed using the DNA polymerase gene (DNA pol ?) primers from Leishmania infantum. The parasitological analysis revealed parasites in 61.7% of the samples. Sensitivities were 89.2%, 86.5%, and 97.3% in the skin and 81.1%, 94.6%, and 100.0% in spleen samples used for cPCR, snPCR, and qPCR, respectively. We demonstrated that the qPCR method was the best technique to detect L. infantum in both skin and spleen samples. However, we recommend the use of skin due to the high sensitivity and sampling being less invasive.
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Canine visceral leishmaniasis: incidence and risk factors for infection in a cohort study in Brazil.
Vet. Parasitol.
PUBLISHED: 03-05-2013
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Zoonotic visceral leishmaniasis in Brazil is caused by Leishmania infantum parasites and is transmitted by sand flies of the Phlebotominae family. Dogs are the main urban reservoirs and represent the major source of contagion for the vectors. Studies have shown that most infected dogs are polymerase chain reaction-positive months before seroconversion. Herein, we describe a cohort study designed to identify the incidence of and risk factors for L. infantum infection as detected by polymerase chain reaction-restriction fragment length polymorphism. To determine the risk factors for infection, we conducted a baseline canine survey (n=1443) from which dogs were selected for the cohort study (n=282) involving three evaluations over the course of a 26-month follow-up period. Serology, molecular tests, and a structured questionnaire were used. The risk factors for infection were identified by means of the Cox regression model. The overall infection incidence was 5.8 per 100 dog-months (95% confidence interval 5.1-6.5). Increased risk of infection was associated with the presence of previous cases of canine visceral leishmaniasis in the domiciles (hazard ratio [HR] 1.4; 95% confidence interval [CI] 1.1-1.8) and unplastered house walls (HR 3.6; 95% CI 1.6-8.1). These risk factors suggest that insecticide spraying in cracks and crevices in unplastered walls can reduce biting rates within and around homes. Furthermore, our results demonstrate that the Visceral Leishmaniasis Control and Surveillance Program should adopt environmental management measures in homes with previous cases of canine visceral leishmaniasis, because these homes are more likely to maintain the transmission cycle.
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Cytokine and nitric oxide patterns in dogs immunized with LBSap vaccine, before and after experimental challenge with Leishmania chagasi plus saliva of Lutzomyia longipalpis.
Vet. Parasitol.
PUBLISHED: 02-20-2013
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In the studies presented here, dogs were vaccinated against Leishmania (Leishmania) chagasi challenge infection using a preparation of Leishmania braziliensis promastigote proteins and saponin as adjuvant (LBSap). Vaccination with LBSap induced a prominent type 1 immune response that was characterized by increased levels of interleukin (IL-) 12 and interferon gamma (IFN-?) production by peripheral blood mononuclear cells (PBMC) upon stimulation with soluble vaccine antigen. Importantly, results showed that this type of responsiveness was sustained after challenge infection; at day 90 and 885 after L. chagasi challenge infection, PBMCs from LBSap vaccinated dogs produced more IL-12, IFN-? and concomitant nitric oxide (NO) when stimulated with Leishmania antigens as compared to PBMCs from respective control groups (saponin, LB- treated, or non-treated control dogs). Moreover, transforming growth factor (TGF)-? decreased in the supernatant of SLcA-stimulated PBMCs in the LBSap group at 90 days. Bone marrow parasitological analysis revealed decreased frequency of parasitism in the presence of vaccine antigen. It is concluded that vaccination of dogs with LBSap vaccine induced a long-lasting type 1 immune response against L. chagasi challenge infection.
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Development of a label-free immunosensor based on surface plasmon resonance technique for the detection of anti-Leishmania infantum antibodies in canine serum.
Biosens Bioelectron
PUBLISHED: 01-24-2013
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In this work, a surface plasmon resonance (SPR) immunosensor was developed using an 11-mercaptoundecanoic acid (11-MUA) modified gold SPR sensor chip for the detection of anti-Leishmania infantum antibodies. The soluble antigens of L. infantum were securely immobilized on an SPR gold disk by an 11-MUA self-assembled monolayer. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and scanning electrochemical microscopy (SECM) techniques were employed in the characterization of the antigen immobilization. After the immunosensor construction, canine serum positive for visceral leishmaniasis was added to its surface and showed significant variation in the SPR angle, indicating excellent sensitivity of the technique for antigen-antibody interaction detection. Moreover, the addition of negative serum was accompanied by a smaller response, demonstrating that the immunosensor shows good specificity against anti-L. infantum antibodies. Therefore, this work demonstrates the successful development of an SPR sensor for anti-L. infantum antibodies detection in short time, showing a great perspective as a sensing system of visceral leishmaniasis in endemic regions.
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Effect of the preservative and temperature conditions on the stability of Leishmania infantum promastigotes antigens applied in a flow cytometry diagnostic method for canine visceral leishmaniasis.
Diagn. Microbiol. Infect. Dis.
PUBLISHED: 01-16-2013
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The control of canine visceral leishmaniasis (CVL) is imperative, but euthanasia of seropositive dogs has been highly criticized. Commonly used, immunodiagnostic tests, including Dual-Path Platform®, enzyme-linked immunosorbent assay, and immunofluorescent antibody test, have failed at detecting asymptomatic dogs in endemic areas. In this context, new serological methods are needed. Flow cytometry serology has demonstrated potential as a test with excellent performance for CVL. In this study, we proposed to establish the best conditions for preserving Leishmania infantum promastigote antigens employed in this serology test. During 12 months of follow-up, promastigotes were maintained in different preservatives (phosphate-buffered saline with 3% fetal bovine serum, phenol 0.35%, thimerosal 0.01%, and formaldehyde 0.5%) and stored at 3 distinct temperatures (25 °C, 4 °C, and -20 °C). During the study period, the morphological characteristics of the promastigotes were assessed by flow cytometry according to the forward and side scatter parameters and also under optical microscopic analysis. Reactivity performance was evaluated as the percentage of positive fluorescent parasites in the sera of naturally infected and noninfected dogs. Microbiological analysis was performed at 2 time points, the first and sixth months, to rule out contamination of stored promastigotes. Taken together, our results indicated that the best conditions to preserve fixed L. infantum antigens were storage in formaldehyde at 4 °C. Promastigotes presented the best morphological profile, with appropriate antigenic stability even at 4 °C, in an inexpensive preservative for a long period of conservation.
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Clinical Forms of Canine Visceral Leishmaniasis in Naturally Leishmania infantum-Infected Dogs and Related Myelogram and Hemogram Changes.
PLoS ONE
PUBLISHED: 01-01-2013
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Hematological analysis has limited applications for disease diagnosis in Leishmania infantum-infected dogs, but it can be very important in evaluating the clinical forms of the disease and in understanding the evolution of canine visceral leishmaniasis (CVL) pathogenesis. Recently, we demonstrated that alterations in leucopoiesis and erythropoiesis are related to clinical status and bone marrow parasite density in dogs naturally infected by L. infantum. To further characterize these alterations, we evaluated the association between the hematological parameters in bone marrow and peripheral blood alterations in groups of L. infantum-infected dogs: asymptomatic I (AD-I: serum negative/PCR+), asymptomatic II (AD-II: serum positive), oligosymptomatic (OD), and symptomatic (SD). Results were compared with those from noninfected dogs (NID). The SD group was found to present a decrease in erythropoietic lineage with concomitant reductions in erythrocytes, hemoglobin, and hematocrit parameters, resulting in anemia. The SD group also had increased neutrophils and precursors and decreased band eosinophils and eosinophils, leading to peripheral blood leucopenia. In the AD-II group, lymphocytosis occurred in both the peripheral blood and the bone marrow compartments. The SD group exhibited lymphocytosis in the bone marrow, with lymphopenia in the peripheral blood. In contrast, the AD-I group, showed no significant changes suggestive of CVL, presenting normal counts in bone marrow and peripheral blood. Our results showed for the first time that important changes in hematopoiesis and hematological parameters occur during ongoing CVL in naturally infected dogs, mainly in symptomatic disease. Taken together, our results based on myelogram and hemogram parameters enable better understanding of the pathogenesis of the anemia, lymphocytosis, and lymphopenia, as well as the leucopenia (eosinopenia and monocytopenia), that contribute to CVL prognosis.
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Risk factors for seroconversion by Leishmania infantum in a cohort of dogs from an endemic area of Brazil.
PLoS ONE
PUBLISHED: 01-01-2013
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Visceral leishmaniasis (VL) has recently emerged in various urban and peri-urban areas of Brazil and other countries. Understanding the urbanization of VL requires identification of risk factors associated with human and canine infection. To determine the predictors of risk for canine VL, a survey was conducted of 1,443 dogs, from which a cohort was selected (n = 455) and evaluated for approximately 26 months. Serology was conducted with two enzyme-linked immunosorbent assays (ELISA): one conducted in the Laboratory of Zoonosis of the Belo Horizonte Health Department (LZOON) and the other in the Laboratory of Immunopathology of the Federal University of Ouro Preto (LIMP). A molecular diagnostic method (PCR-restriction fragment length polymorphism) and a structured questionnaire were also used. To identify the factors associated with seroconversion, two time-dependent Cox regression models were performed with different sensitivities (model 1, seroconversion by ELISA/LZOON; model 2, seroconversion by ELISA/LIMP). The overall incidences of seroconversion were 6.5/1000 dogs-months and 11.2/1000 dogs-months for ELISA/LZOON and ELISA/LIMP, respectively. Increased risk of seroconversion was associated with short fur (model 1: hazard ratio [HR] 1.9), the presence of dry leaves (model 1: HR 2.8) or manure (model 1: HR 3.5) in the backyard, dogs sleeping predominantly in the backyard (model 2: HR 2.1), the presence of symptoms (model 2: HR 2.0), and positive molecular results during follow-up (model 2: HR 1.5). Decreased risk was associated with insecticide spraying in the house (model 2: HR 0.5). These results indicate that more-vulnerable domiciles, certain dog behaviors, lack of vector control measures, and positive molecular results were associated with the occurrence of canine VL. Furthermore, it is important to emphasize that PCR-positive dogs should be monitored, owing to the possibility of seroconversion. Identifying risk factors for seroconversion in dogs is crucial for developing adequate strategies for VL prevention and control.
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Bilateral nonarteritic anterior ischemic neuropathy following acute angle-closure glaucoma in a patient with iridoschisis: case report.
Arq Bras Oftalmol
PUBLISHED: 06-15-2011
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A 55-year-old woman was referred to our clinic because of a one-week history of visual loss and raised intraocular pressure in the left eye followed 4 days later by visual loss in the right eye. Slit-lamp examination showed bilateral conjunctival hyperemia, slight diffuse corneal edema, shallow anterior chamber and fixed and dilated pupil in both eyes. Splitting of the anterior layers of the iris with fibrillar degeneration extending for approximately one quadrant inferiorly was presented in each eye. Fundus examination showed optic disc edema with no vascular tortuosity and no cup in both eyes. The condition was treated as bilateral acute angle-closure glaucoma in a patient with irisdoschisis. After medical treatment and improvement of visual acuity, perimetry revealed a significant visual field defect especially in left eye; this case represents a rare concurrence of acute angle-closure glaucoma and bilateral nonarteritic ischemic optic neuropathy. Although most cases of elevated intraocular pressure, including acute angle-closure glaucoma, do not result in optic disc edema and irreversible vision loss, variations in the vascular supply of the nerve optic head along with others ocular systemic risk factors, may predispose certain individuals to nonarteritic ischemic optic neuropathy during periods of elevated intraocular pressure.
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Influence of clinical status and parasite load on erythropoiesis and leucopoiesis in dogs naturally infected with leishmania (Leishmania) chagasi.
PLoS ONE
PUBLISHED: 03-20-2011
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The bone marrow is considered to be an important storage of parasites in Leishmania-infected dogs, although little is known about cellular genesis in this organ during canine visceral leishmaniasis (CVL).
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Staphylococcus spp., Enterobacteriaceae and Pseudomonadaceae oral isolates from Brazilian HIV-positive patients. Correlation with CD4 cell counts and viral load.
Arch. Oral Biol.
PUBLISHED: 02-21-2011
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The aim was to evaluate the presence of Staphylococcus spp., Enterobacteriaceae and Pseudomonadaceae in the oral cavities of HIV-positive patients. Forty-five individuals diagnosed as HIV-positive by ELISA and Western-blot, and under anti-retroviral therapy for at least 1 year, were included in the study. The control group constituted 45 systemically healthy individuals matched to the HIV patients to gender, age and oral conditions. Oral rinses were collected and isolates were identified by API system. Counts of microorganisms from HIV and control groups were compared statistically by a Mann-Whitney test (?=5%). The percentages of individuals positive for staphylococci were similar between the groups (p=0.764), whereas for Gram-negative rods, a higher percentage was observed amongst HIV-positive (p=0.001). There was no difference in Staphylococcus counts between HIV and control groups (p=0.1008). Counts were lower in the oral cavities of patients with low viral load (p=0.021), and no difference was observed in relation to CD4 counts (p=0.929). Staphylococcus aureus was the most frequently isolated species in HIV group, and Staphylococcus epidermidis was the prevalent species in the control group. Significantly higher numbers of enteric bacteria and pseudomonas were detected in the oral cavities of the HIV group than in the control (p=0.0001). Enterobacter cloacae was the most frequently isolated species in both groups. Counts of enteric bacteria and pseudomonas were significantly lower in patients with low CD4 counts (p=0.011); however, there was no difference relating to viral load. It may be concluded that HIV group showed greater species diversity and a higher prevalence of Enterobacteriaceae/Pseudomonadaceae.
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Immunological changes in canine peripheral blood leukocytes triggered by immunization with first or second generation vaccines against canine visceral leishmaniasis.
Vet. Immunol. Immunopathol.
PUBLISHED: 02-03-2011
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In this study, we summarized the major phenotypic/functional aspects of circulating leukocytes following canine immunization with Leishvaccine and Leishmune®. Our findings showed that Leishvaccine triggered early changes in the innate immunity (neutrophils and eosinophils) with late alterations on monocytes. Conversely, Leishmune(®) induced early phenotypic changes in both, neutrophils and monocytes. Moreover, Leishvaccine triggered mixed activation-related phenotypic changes on T-cells (CD4+ and CD8+ and B-lymphocytes, whereas Leishmune(®) promoted a selective response, mainly associated with CD8+ T-cell activation. Mixed cytokine profile (IFN-?/IL-4) was observed in Leishvaccine immunized dogs whereas a selective pro-inflammatory pattern (IFN-?/NO) was induced by Leishmune® vaccination. The distinct immunological profile triggered by Leishvaccine and Leishmune® may be a direct consequence of the distinct biochemical composition of these immunobiological, i.e. complex versus purified Leishmania antigen along with Bacillus Calmette-Guérin (BCG) versus saponin adjuvant. Both immunobiologicals are able to activate phagocytes and CD8+ T-cells and therefore could be considered as a putative vaccines against canine visceral leishmaniasis (CVL).
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Prevalence and factors associated with Leishmania infantum infection of dogs from an urban area of Brazil as identified by molecular methods.
PLoS Negl Trop Dis
PUBLISHED: 01-21-2011
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Various factors contribute to the urbanization of the visceral leishmaniasis (VL), including the difficulties of implementing control measures relating to the domestic reservoir. The aim of this study was to determine the prevalence of canine visceral leishmaniasis in an urban endemic area in Brazil and the factors associated with Leishmania infantum infection among seronegative and PCR-positive dogs.
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Rates of change in the visual field and optic disc in patients with distinct patterns of glaucomatous optic disc damage.
Ophthalmology
PUBLISHED: 01-12-2011
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To investigate the rate of visual field and optic disc change in patients with distinct patterns of glaucomatous optic disc damage.
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Cytokine and transcription factor profiles in the skin of dogs naturally infected by Leishmania (Leishmania) chagasi presenting distinct cutaneous parasite density and clinical status.
Vet. Parasitol.
PUBLISHED: 11-07-2010
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The immune response in the skin of dogs infected with Leishmania chagasi and its association with distinct levels of tissue parasitism and clinical progression of canine visceral leishmaniasis (CVL) are poorly understood and limited studies are available. A detailed analysis of the profiles of cytokines (IFN-?, IL-4, IL-5, IL-10, IL-12, IL-13, TGF-?1 and TNF-?) and transcription factors (T-bet, GATA-3 and FOXP3) in the skin of 35 naturally infected dogs was carried out using real-time PCR alongside determinations of skin parasite density and the clinical status of CVL. A mixed cytokine profile with high levels of expression of IFN-?, TNF-? and IL-13 was determined in asymptomatic dogs. Additionally, the levels of transcription factors GATA-3 and FOXP3 were correlated with the asymptomatic disease. A mixed cytokine profile was also observed during active CVL. Moreover, high levels of IL-10 and TGF-?1, concomitant with the low expression of IL-12, may represent a key condition that allows persistence of parasite replication in the skin. The results obtained indicate that in asymptomatic disease or lower levels of skin parasite density, a mixed inflammatory, regulatory immune response profile may be of major relevance for both the maintenance of the clinical status of the dogs as well as for parasite persistence and replication at low levels.
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Qualitative and quantitative immunohistochemical evaluation of iNOS expression in the spleen of dogs naturally infected with Leishmania chagasi.
Parasitol. Res.
PUBLISHED: 10-04-2010
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Nitric oxide (NO), the product of the nitric oxide synthase enzymes has been detected in Leishmania-infected animals. Besides its role on the immunity to infection, the role of NO and the inducible nitric oxide synthase (iNOS) in the pathogenesis of canine visceral leishmaniasis (CVL) is not well understood. This study aimed at evaluating immunohistochemically the iNOS expression in the spleen of dogs naturally infected (ID) with Leishmania (L.) chagasi compared with non-infected dogs (NID). The ID was grouped according to the clinical form and the parasite load. Symptomatic dogs (SD) presented higher parasite load in relation to oligosymptomatic (OD) and asymptomatic (AD). The qualitative expression of iNOS was observed only in ID. SD presented strong and prominent labeling of iNOS, followed by OD and AD. Quantitatively, the results showed that the median expression of iNOS was higher in SD and OD compared to NID. Also, dog spleens with high parasitism load showed marked iNOS expression. Taken together, the results suggest that the expression of iNOS in the spleen of infected dogs with CVL was associated with clinical worsening of the disease and with high parasitism.
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Leishmania (Viannia) braziliensis: immunoblotting analysis for the detection of IgG subclasses in the diagnosis of symptomatic and asymptomatic dogs.
Vet. Parasitol.
PUBLISHED: 05-19-2010
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During a seroepidemiological survey 2004-2006 from areas in Brazil endemic for American cutaneous leishmaniasis (ACL), serum samples from 10 dogs with ulcerated cutaneous lesions (S-ACL) and 52 asymptomatic dogs (AS-ACL) of unknown age and breed living in areas endemic for ACL were monitored for 1 year for ulcerated cutaneous lesions and immunoblotting using peroxidase-conjugated secondary anti-IgG, anti-IgG1 and anti-IgG2 dog antibodies. We reported that antibodies against Leishmania (Viannia) braziliensis in the sera of 22/52 dogs with asymptomatic disease showed intense reactivity to peptides larger than 66 kDa. We believe that dogs harboring subclinical amastigotes show an immunoblotting profile similar to that of symptomatic animals because a dog with self-healing presented antigens greater than 66 kDa. Such patterns can be exploited for diagnostic and epidemiological research for leishmaniasis.
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Immunity to Leishmania and the rational search for vaccines against canine leishmaniasis.
Trends Parasitol.
PUBLISHED: 04-05-2010
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The control of infection by Leishmania infantum (syn. Leishmania chagasi) in dogs is essential to stop the current spread of zoonotic visceral leishmaniasis. The past few years have seen significant advances in achieving efficient immunization of dogs and, more than ever before, an effective vaccine against canine leishmaniasis can now be considered a feasible goal. This article summarizes experimental data gathered from recent dog trials aimed at identifying immunological mechanisms implicated in protection against canine infection to discuss their potential to serve as quantitative surrogate markers of immunization and, more importantly, its usefulness to evaluate whether the immunity induced by the vaccine candidate is strong enough to protect against canine leishmaniasis.
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Relationship of Leishmania-specific IgG levels and IgG avidity with parasite density and clinical signs in canine leishmaniasis.
Vet. Parasitol.
PUBLISHED: 01-14-2010
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The clinical status and tissue parasite burden of the skin and spleen of 40 dogs naturally infected with Leishmania chagasi (syn. Leishmania infantum), together with 5 uninfected control dogs, were assessed. On the basis of the clinical evaluation, infected dogs were classified as asymptomatic (AD) or symptomatic (SD). Infected animals were also grouped according to their parasite load as exhibiting low (LP), medium (MP) and high (HP) parasitism. The results indicated a high parasite load in the skin samples of SD animals in relation to the AD group. The serum immunoglobin isotype profiles of the studied animals revealed increased levels of IgG(1) in the AD and LP dogs, whereas high levels of IgG(2) were correlated with SD and HP dogs. The avidity index (AI) of IgG(total) in the SD group was high in comparison of that of the AD group. Moreover, animals with a larger parasite burden either in the spleen or skin showed higher AI values than animals with lower parasitism. Based on these findings, it is suggested that CVL commences with an asymptomatic clinical form with low parasitism, high production of IgG(1) and low affinity of IgG(total) molecules, and evolves into a symptomatic clinical form with higher parasitism intensity, higher IgG(2) levels, and high affinity of IgG(total).
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A comparison of bimatoprost 0.03% versus the fixed-combination of latanoprost 0.005% and timolol 0.5% in adult patients with elevated intraocular pressure: an eight-week, randomized, open-label trial.
J Ocul Pharmacol Ther
PUBLISHED: 10-29-2009
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Bimatoprost and the fixed combination of latanoprost with timolol maleate are 2 medications widely used to treat glaucoma and ocular hypertension (OHT). The aim of the study is to compare the efficacy of these 2 drugs in reducing intraocular pressure (IOP) after 8 weeks of treatment in patients with primary open angle glaucoma (POAG) or OHT.
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Systemic and compartmentalized immune response in canine visceral leishmaniasis.
Vet. Immunol. Immunopathol.
PUBLISHED: 08-15-2009
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Human visceral leishmaniasis (VL) and canine visceral leishmaniasis (CVL) are the most important emerging diseases with high prevalence in Latin American countries and are mainly caused by Leishmania (L.) chagasi (Syn=L. infantum). CVL has a great impact on Brazilian public health because domestic dogs are the most important VL peri-domicile reservoirs in both urban and peri-urban areas. Our findings highlight the complexity of cellular immunological events related to the natural infection from dogs by L. chagasi, additionally correlating major peripheral blood phenotypic markers with clinical status and tissues parasite density. Our main results demonstrated that lower frequency of circulating B cells and monocytes are important markers of severe CVL, whereas increased levels of CD8+ lymphocytes appear to be the major phenotypic feature of asymptomatic disease. Determination of the isotypes patterns during CVL demonstrated that asymptomatic dogs and those with low parasitism are associated with an increase of IgG1, while the symptomatic dogs and those with high parasitism are associated with an increase of IgG, IgG2, IgM, IgA and IgE immunoglobulins. Pioneer findings obtained by our group showed a correlation between clinical status of CVL with degree of tissue parasite density. This data demonstrated that asymptomatic dogs presented low parasitism while symptomatic dogs are associated with high parasite load in various tissues such as skin, bone marrow and spleen. We have also investigated the association between tissue parasitism and CVL clinical forms. Regardless of clinical status, skin and spleen are the major sites of high parasite density during ongoing CVL. Furthermore, we demonstrated that bone marrow and spleen parasite density are the most reliable parasitological markers to decode the clinical status of CVL. In this article, we have reviewed some aspects of the histopathological and immunological events occurring in natural and experimental L. chagasi/L. infantum infection, pointing out the main L. chagasi-parasitized tissue. We have discussed the importance of the association between parasite density, immunological/histopathological aspects and clinical status of the CVL, their current applications, challenges for the future and potential opportunities in CVL research.
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Advances in flow cytometric serology for canine visceral leishmaniasis: diagnostic applications when distinct clinical forms, vaccination and other canine pathogens become a challenge.
Vet. Immunol. Immunopathol.
PUBLISHED: 08-15-2009
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We have previously reported the applicability of flow cytometry anti-fixed Leishmania infantum chagasi promastigotes IgG (FC-AFPA-IgG) as a novel serological device for laboratorial diagnosis of CVL. Herein, we validate throughout a blind study applied into a broader range of coded sera samples that FC-AFPA-IgG at serum dilution 1:8192 have an outstanding performance to discriminate the serological reactivity of canine visceral leishmaniasis (CVL, n=64) and Leishmune vaccines (VAC, n=62) and non-infected controls (NI, n=25). Moreover, we have evaluated the performance of indirect immunofluorescence antibody test (IFAT) and the crude-antigen enzyme-linked immunosorbent assay (ELISA) in parallel with FC-AFPA-IgG, to discriminate the seroreactivity of NI, CVL and VAC. Our data demonstrated that both ELISA and FC-AFPA-IgG showed similar performance to detect the seronegativity in 100% of NI, whereas FC-AFPA-IgG displayed better performance to exclude seropositivity in 100% of VAC. The high kappa agreement indexes observed suggested similar performance between these two serological testes when distinct clinical forms of CVL become a challenge. Furthermore, the FC-AFPA-IgG applied at sera dilution 1:8192 showed a remarkable performance to discriminate CVL from other co-endemic canine infections with high co-negativity in dogs infected with Trypanosoma cruzi and Leishmania braziliensis (86% and 84%, respectively). In conclusion, the data presented here re-emphasize the applicability of FC-AFPA-IgG as an innovative methodology able to discriminate post-infection imunomediated seroreactivity from that triggered by prophylactic immunization with minor cross-reactivity with other relevant canine pathogens, which may contribute as a supplementary assay for the CVL immunodiagnosis.
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Agreement between stress intraocular pressure and long-term intraocular pressure measurements in primary open angle glaucoma.
Clin. Experiment. Ophthalmol.
PUBLISHED: 05-29-2009
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The aim of this paper is to study the correlation between the intraocular pressure peaks and fluctuation detected during the water drinking test and the same parameters observed during long-term follow up.
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T-cell-derived cytokines, nitric oxide production by peripheral blood monocytes and seric anti-Leishmania (Leishmania) chagasi IgG subclass patterns following immunization against canine visceral leishmaniasis using Leishvaccine and Leishmune.
Vaccine
PUBLISHED: 04-17-2009
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It is generally accepted that distinct cytokine expression by the cellular immune response plays a critical role during the outcome of experimental as well as natural canine visceral Leishmaniasis (CVL). Despite the fact that immunoprophylaxis of CVL has become an important control strategy and protective immunity has been reported upon immunization with whole as well as purified Leishmania antigens, the cytokine profile of T-cells triggered by anti-CVL vaccines still remain to be determined. Herein, we have developed a cross-sectional analysis of German Shepherd dogs submitted to vaccination protocols with Leishvaccine (n=6) and Leishmune (n=6). Our data identified distinct immunological profiles elicited by Leishvaccine and Leishmune, with the Leishvaccine triggering a mixed, IFN-gamma and IL-4, cytokine pattern in addition to high levels of anti-Leishmania IgG1, whereas the Leishmune induced an immunological pattern characterized by enhanced levels of IFN-gamma, NO and anti-Leishmania chagasi IgG2. It was important to notice that despite the distinct immunological patterns triggered by Leishvaccine and Leishmune, the ability of both immunobiologicals to activate T-cell-derived IFN-gamma synthesis further suggesting their immunogenic potential against CVL. These findings added support to our hypothesis that both antigenic composition (whole antigen in Leishvaccine versus purified antigen in Leishmune) as well as the adjuvant nature (BGC and saponin) used for the vaccine formulation may count for the distinct activation pattern observed.
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Topography-guided (NIDEK customized aspheric treatment zone) photorefractive keratectomy with mitomycin C after penetrating keratoplasty for keratoconus: case report.
J Refract Surg
PUBLISHED: 03-03-2009
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To report topography-guided photorefractive keratectomy (PRK) with mitomycin C (MMC) after penetrating keratoplasty for keratoconus.
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Intraocular pressure profile during the modified diurnal tension curve using Goldman applanation tonometry and dynamic contour tonometry.
J Ocul Biol Dis Infor
PUBLISHED: 02-03-2009
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The aim of this study was to compare the intraocular pressure (IOP) profile during the modified diurnal tension curve (mDTC) using Goldman applanation tonometry (GAT) and dynamic contour tonometry (DCT) in treated glaucomatous eyes. Eligible subjects were submitted to the mDTC using GAT and DCT in this sequence. IOP measurements were performed at 8 a.m., 10 a.m., 2 p.m., and 4 p.m.. Central corneal thickness was measured using ultrasound pachymetry in the morning. Statistical analysis was performed using paired Students t test and Bland-Altman plot. The mean difference between DCT and GAT measurements was 0.9 mmHg. The mean +/- SD IOP measurements during the mDTC were 19.68 +/- 4.68, 17.63 +/- 4.44, 17.25 +/- 5.41, and 17.32 +/- 4.25 mmHg using GAT and 19.97 +/- 4.75, 18.79 +/- 4.61, 19.53 +/- 5.30, and 19.43 +/- 5.45 mmHg using DCT. IOP measurements were higher in the morning (8 a.m.) and decreased throughout the day using both tonometers. The difference between IOP measurements using GAT and DCT was smaller in the morning and increased throughout the day. The IOP variability using GAT was higher than using DCT. Corneal biomechanical properties might help explain our findings.
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Histological study of cell migration in the dermis of hamsters after immunisation with two different vaccines against visceral leishmaniasis.
Vet. Immunol. Immunopathol.
PUBLISHED: 01-17-2009
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Vaccine candidates, including live and/or killed parasites, Leishmania-purified fractions, defined recombinant antigens and antigen-encoding DNA-plasmids have been proposed to use as vaccine anti-Leishmania. More recently, the hamsters have been used to pre-selection of antigens candidate to apply in further experiments using canine model. In this report we evaluated the kinetics of cell migration in dermal inflammatory infiltrate, circulating leukocytes and the presence of nitric oxide (NO)/induced nitric oxide synthase during the early (1-24h) and late (48-168h) periods following inoculation of hamsters with antigenic components of anti-canine visceral leishmaniasis vaccines Leishmune and Leishmania braziliensis antigen (LB) with and without saponin (Sap) adjuvant. Our results show that LB caused an early reduction of lymphocytes in the dermis while Sap and LBSap triggered a late recruitment, suggesting the role of the adjuvant in the traffic of antigen-presenting cells and the induction of lymphocyte migration. In that manner our results suggest that the kinetics of cell migration on hamster model may be of value in the selection of vaccine antigens prior the tests in dogs particularly in respect of the toxicity of the preparations.
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Trypanosoma cruzi: Serum levels of nitric oxide and expression of inducible nitric oxide synthase in myocardium and spleen of dogs in the acute stage of infection with metacyclic or blood trypomastigotes.
Exp. Parasitol.
PUBLISHED: 01-06-2009
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The participation of nitric oxide (NO) in the control of blood parasitemia and parasitism during the acute phase of infection in dogs inoculated with blood trypomastigotes (BT) or metacyclic trypomastigotes (MT group) of Berenice-78 Trypanosoma cruzi strain has been evaluated. Animals of the MT group (n=4) presented increased levels of serum NO throughout the infection when compared with the BT (n=4) or control (n=4) groups, and a delay in parasitemia peak compared with the BT group. In spleen fragments, tissue parasitism was not observed but the MT group presented larger areas associated with inducible NO synthase (iNOS) in relation to BT and control groups. Heart fragments of MT-infected animals exhibited comparatively low tissue parasitism and high iNOS expression, while animals of the BT group presented high inflammatory infiltrate, high tissue parasitism and low iNOS expression. These results indicate that the source of inoculum can interfere with the development of the acute phase of Chagas disease, and may also trigger a distinct parasite-host interaction during this phase.
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First description of autochthonous canine visceral leishmaniasis in the metropolitan region of Vitória, State of Espírito Santo, Brazil.
Rev. Soc. Bras. Med. Trop.
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We investigated autochthonous canine visceral leishmaniasis (CVL) in the metropolitan region of Vitória (MRV), an area in which a human case was previously reported.
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Enhanced detection of open-angle glaucoma with an anatomically accurate optical coherence tomography-derived neuroretinal rim parameter.
Ophthalmology
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Neuroretinal rim assessment based on the clinical optic disc margin (DM) lacks a sound anatomic basis for 2 reasons: (1) The DM is not reliable as the outer border of rim tissue because of clinically and photographically invisible extensions of Bruchs membrane (BM) inside the DM and (2) nonaccountability of rim tissue orientation in the optic nerve head (ONH). The BM opening-minimum rim width (BMO-MRW) is a parameter that quantifies the rim from its true anatomic outer border, BMO, and accounts for its variable orientation. We report the diagnostic capability of BMO-MRW.
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Performance of LBSap vaccine after intradermal challenge with L. infantum and saliva of Lu. longipalpis: immunogenicity and parasitological evaluation.
PLoS ONE
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In the last decade, the search for new vaccines against canine visceral leishmaniasis has intensified. However, the pattern related to immune protection during long periods after experimental infection in vaccine trials is still not fully understood. Herein, we investigated the immunogenicity and parasitological levels after intradermal challenge with Leishmania infantum plus salivary gland extract in dogs immunized with a vaccine composed of L. braziliensis antigens plus saponin as an adjuvant (LBSap vaccine). The LBSap vaccine elicited higher levels of total anti-Leishmania IgG as well as both IgG1 and IgG2. Furthermore, dogs vaccinated had increased levels of lymphocytes, particularly circulating B cells (CD21(+)) and both CD4(+) and CD8(+) T lymphocytes. LBSap also elicited an intense in vitro cell proliferation associated with higher levels of CD4(+) T lymphocytes specific for vaccine soluble antigen and soluble lysate of L. infantum antigen even 885 days after experimental challenge. Furthermore, LBSap vaccinated dogs presented high IFN-? and low IL-10 and TGF-?1 expression in spleen with significant reduction of parasite load in this tissue. Overall, our results validate the potential of LBSap vaccine to protect against L. infantum experimental infection and strongly support further evaluation of efficiency of LBSap against CVL in natural infection conditions.
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An assessment on epitope prediction methods for protozoa genomes.
BMC Bioinformatics
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Epitope prediction using computational methods represents one of the most promising approaches to vaccine development. Reduction of time, cost, and the availability of completely sequenced genomes are key points and highly motivating regarding the use of reverse vaccinology. Parasites of genus Leishmania are widely spread and they are the etiologic agents of leishmaniasis. Currently, there is no efficient vaccine against this pathogen and the drug treatment is highly toxic. The lack of sufficiently large datasets of experimentally validated parasites epitopes represents a serious limitation, especially for trypanomatids genomes. In this work we highlight the predictive performances of several algorithms that were evaluated through the development of a MySQL database built with the purpose of: a) evaluating individual algorithms prediction performances and their combination for CD8+ T cell epitopes, B-cell epitopes and subcellular localization by means of AUC (Area Under Curve) performance and a threshold dependent method that employs a confusion matrix; b) integrating data from experimentally validated and in silico predicted epitopes; and c) integrating the subcellular localization predictions and experimental data. NetCTL, NetMHC, BepiPred, BCPred12, and AAP12 algorithms were used for in silico epitope prediction and WoLF PSORT, Sigcleave and TargetP for in silico subcellular localization prediction against trypanosomatid genomes.
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Parasite burden in hamsters infected with two different strains of leishmania (Leishmania) infantum: "Leishman Donovan units" versus real-time PCR.
PLoS ONE
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To develop and test new therapeutics and immune prophylaxis strategies for visceral leishmaniasis (VL), understanding tissue parasitism evolution after experimental infection with Leishmania infantum is important. Experimental infection in a hamster model (Mesocricetus auratus) reproduces several typical aspects of canine and human VL that are closely related to the inoculums route. We quantified the parasitism in the liver and spleen of hamsters experimentally infected by various routes (intradermal, intraperitoneal, and intracardiac [IC]) and different strains of L. infantum (MHOM/BR/74/PP75 and Wild) and compared two different methodologies to evaluate tissue parasitism (Leishman Donovan units [LDU] and real-time qPCR). In addition, the quantification of specific total-IgG in the serum of uninfected and infected hamsters was determined by ELISA. The animals were followed for 1, 3, 6 and 9 months post-infection for survival analysis. We found that infection with the Wild strain by the IC route resulted in higher mortality. Positive antibody (IgG) responses were detected with higher peaks at 6 and 9 months in the IC group inoculated with PP75 strain. However, in animals infected with the Wild strain the IgG levels were elevated in all infected groups during all the time evaluated. We also observed by LDU analysis that the IC route lead to higher parasitism in the liver and spleen with both strains. Furthermore, qPCR showed higher sensitivity for identifying animals with low parasitic burden. In conclusion, qPCR can be useful for assessing parasitism in the spleen and liver of a hamster model infected with L. infantum independent of the route of infection, and this technique may become an essential tool for assessing parasite density in the hamster model after experimental treatment or immunization with potential vaccine candidates.
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An antigenic domain within a catalytically active Leishmania infantum nucleoside triphosphate diphosphohydrolase (NTPDase 1) is a target of inhibitory antibodies.
Parasitol. Int.
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We identified a shared B domain within nucleoside triphosphate diphosphohydrolases (NTPDases) of plants and parasites. Now, an NTPDase activity not affected by inhibitors of adenylate kinase and ATPases was detected in Leishmania infantum promastigotes. By non-denaturing gel electrophoresis of detergent-homogenized promastigote preparation, an active band hydrolyzing nucleosides di- and triphosphate was visualized and, following SDS-PAGE and silver staining was identified as a single polypeptide of 50kDa. By Western blots, it was recognized by immune sera raised against potato apyrase (SA), r-pot B domain (SB), a recombinant polypeptide derived from the potato apyrase, and LbB1LJ (SC) or LbB2LJ (SD), synthetic peptides derived from the Leishmania NTPDase 1, and by serum samples from dogs with visceral leishmaniasis, identifying the antigenic L. infantum NTPDase 1 and, also, its conserved B domain (r83-122). By immunoprecipitation assays and Western blots, immune sera SA and SB identified the catalytically active NTPDase 1 in promastigote preparation. In addition, the immune sera SB (44%) and SC or SD (87-99%) inhibited its activity, suggesting a direct effect on the B domain. By ELISA, 37%, 45% or 50% of 38 infected dogs were seropositive for r-pot B domain, LbB1LJ and LbB2LJ, respectively, confirming the B domain antigenicity.
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Cell recruitment and cytokines in skin mice sensitized with the vaccine adjuvants: saponin, incomplete Freunds adjuvant, and monophosphoryl lipid A.
PLoS ONE
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Vaccine adjuvants are substances associated with antigens that are fundamental to the formation of an intense, durable, and fast immune response. In this context, the use of vaccine adjuvants to generate an effective cellular immune response is crucial for the design and development of vaccines against visceral leishmaniasis. The objective of this study was to evaluate innate inflammatory response induced by the vaccine adjuvants saponin (SAP), incomplete Freunds adjuvant (IFA), and monophosphoryl lipid A (MPL). After a single dose of adjuvant was injected into the skin of mice, we analyzed inflammatory reaction, selective cell migration, and cytokine production at the injection site, and inflammatory cell influx in the peripheral blood. We found that all vaccine adjuvants were able to promote cell recruitment to the site without tissue damage. In addition, they induced selective migration of neutrophils, macrophages, and lymphocytes. The influx of neutrophils was notable at 12 h in all groups, but at other time points it was most evident after inoculation with SAP. With regard to cytokines, the SAP led to production of interleukin (IL)-2, IL-6, and IL-4. IFA promoted production of tumor necrosis factor (TNF)-?, interferon (IFN)-?, IL-6, IL-17, IL-4, and IL-10. We also observed that MPL induced high production of IL-2, TNF-?, and IFN-?, in addition to IL-6, IL-17, and IL-10. In peripheral blood, values of certain cell populations in the local response changed after stimulation. Our data demonstrate that the three vaccine adjuvants stimulate the early events of innate immune response at the injection site, suggesting their ability to increase the immunogenicity of co-administered antigens. Moreover, this work provides relevant information about elements of innate and acquired immune response induced by vaccine adjuvants administered alone.
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Laminar displacement and prelaminar tissue thickness change after glaucoma surgery imaged with optical coherence tomography.
Invest. Ophthalmol. Vis. Sci.
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To study changes in lamina cribrosa position and prelaminar tissue thickness (PTT) after surgical IOP reduction in glaucoma patients.
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Nerve fiber layer in glaucomatous hemifield loss: a case-control study with time- and spectral-domain optical coherence tomography.
Arq Bras Oftalmol
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To evaluate the retinal nerve fiber layer measurements with time-domain (TD) and spectral-domain (SD) optical coherence tomography (OCT), and to test the diagnostic ability of both technologies in glaucomatous patients with asymmetric visual hemifield loss.
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Higher expression of CCL2, CCL4, CCL5, CCL21, and CXCL8 chemokines in the skin associated with parasite density in canine visceral leishmaniasis.
PLoS Negl Trop Dis
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The immune response in the skin of dogs infected with Leishmania infantum is poorly understood, and limited studies have described the immunopathological profile with regard to distinct levels of tissue parasitism and the clinical progression of canine visceral leishmaniasis (CVL).
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Peripapillary choroidal thickness in healthy controls and patients with focal, diffuse, and sclerotic glaucomatous optic disc damage.
Arch. Ophthalmol.
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To examine peripapillary choroidal thickness in healthy controls and in patients with glaucoma who have focal, diffuse, and sclerotic optic disc damage.
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Different infective forms trigger distinct immune response in experimental Chagas disease.
PLoS ONE
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Although metacyclic and blood trypomastigotes are completely functional in relation to parasite-host interaction and/or target cell invasion, they differ in the molecules present on the surface. Thus, aspects related to the variability that the forms of T. cruzi interacts with host cells may lead to fundamental implications on the immune response against this parasite and, consequently, the clinical evolution of Chagas disease. We have shown that BT infected mice presented higher levels of parasitemia during all the acute phase of infection. Moreover, the infection with either MT or BT forms resulted in increased levels of total leukocytes, monocytes and lymphocytes, specifically later for MT and earlier for BT. The infection with BT forms presented earlier production of proinflammatory cytokine TNF-? and later of IFN-? by both T cells subpopulations. This event was accompanied by an early cardiac inflammation with an exacerbation of this process at the end of the acute phase. On the other hand, infection with MT forms result in an early production of IFN-?, with subsequent control in the production of this cytokine by IL-10, which provided to these animals an immunomodulatory profile in the end of the acute phase. These results are in agreement with what was found for cardiac inflammation where animals infected with MT forms showed intense cardiac inflammation later at infection, with a decrease in the same at the end of this phase. In summary, our findings emphasize the importance of taking into account the inoculums source of T. cruzi, since vectorial or transfusional routes of T. cruzi infection may trigger distinct parasite-host interactions during the acute phase that may influence relevant biological aspects of chronic Chagas disease.
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Influence of clinically invisible, but optical coherence tomography detected, optic disc margin anatomy on neuroretinal rim evaluation.
Invest. Ophthalmol. Vis. Sci.
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We previously demonstrated that most eyes have regionally variable extensions of Bruchs membrane (BM) inside the clinically identified disc margin (DM) that are clinically and photographically invisible. We studied the impact of these findings on DM- and BM opening (BMO)-derived neuroretinal rim parameters.
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Profile of anti-Leishmania antibodies related to clinical picture in canine visceral leishmaniasis.
Res. Vet. Sci.
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This research investigated the profile of anti-Leishmania antibodies in different clinical forms of canine visceral leishmaniasis (CVL). Naturally infected dogs were divided into two groups: subclinical dogs (SD, n=10) and clinical dogs (CD, n=68). Non-infected dogs (ND, n=7) comprised the negative control group. The humoral response was evaluated by the profile of total IgG, IgG1, IgG2, IgM, IgA and IgE, determined by ELISA. Infected animals showed increased levels of total IgG, IgA and IgE in addition to IgG1 and IgG2 in groups SD and CD, when compared with group ND. Furthermore, it was observed that IgG2 and IgM were correlated with symptomatology, while total IgG, IgG1 and IgA were negatively correlated and IgE showed no correlation. It follows that serum levels of IgG2 anti-Leishmania are correlated with typical clinical signs of disease. Furthermore the determination of specific anti-Leishmania antibodies could be an important tool in monitoring CVL clinical picture.
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Optic disc margin anatomy in patients with glaucoma and normal controls with spectral domain optical coherence tomography.
Ophthalmology
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To characterize optic nerve head (ONH) anatomy related to the clinical optic disc margin with spectral domain-optical coherence tomography (SD-OCT).
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.