Characterization of agglutinating antibodies detected by the direct agglutination test for visceral leishmaniasis diagnosis.

This study aimed to characterize agglutinating antibodies detected by the direct agglutination test (DAT-LPC) for the diagnosis of visceral leishmaniasis (VL). The DAT-LPC antigen/antibodies complex was recovered, washed, and used as antigenic substrate in a modified enzyme-linked immunosorbent assay (modified ELISA), revealed with anti-human IgM, IgG, and IgG subtype conjugates, and in the immunofluorescent antibodies test (IFAT), revealed with anti-human IgG and IgG1 conjugates. IgM antibodies were detected in 50%, IgG and IgG1 in 100%, and IgG3 in 52.8% of the 36 samples from VL patients. IFAT showed that agglutinating IgG and IgG1 antibodies recognized more intensely antigens located in the membrane and kinetoplast of the parasite. No antibodies were detected in the 15 samples from healthy individuals. This study shows for the first time that the antibodies responsible for agglutination in DAT-LPC are mostly of the IgG1 subtype.

Subgrupos A1, A2 y AINT en donantes efectivos del banco de sangre materno infantil CNS La Paz ­ Bolivia / Subgroups A1, A2 and AINT in effective donors of the maternal and child blood bank CNS La Paz - Bolivia

Además del sistema ABO, los subgrupos del mismo revisten gran importancia en inmunohematología, Los subgrupos A difieren tanto en el número de sitios antigénicos como en la configuración del antígeno eritrocitario. Los principales, A1 y A2 se diferencian en que los eritrocitos A1 son aglutinados por el anticuerpo Anti-A1 humano o por la Lectina Anti-A1 (Dolichos biflorus), y los eritrocitos A2 son aglutinados por la Lectina Anti-H (Ulex europaeus). MATERIALES Y MÉTODOS: se realizó un estudio descriptivo, de Corte Transversal, Se analizó los registros tanto físico y electrónico del Banco de Sangre, se incluyeron todos los donadores efectivos, mismos que fueron tipificados por el laboratorio de inmunohematología en el periodo de mayo a julio del 2018. Método empleado, aglutinación en tubo y en micro placa. RESULTADOS: en un total de 1599 donantes, se determinó que el grupo O tiene mayor frecuencia con un 84% y el menos frecuente fue el AB con un 0,66%. Según el grupo sanguíneo A y AB tenemos las siguientes frecuencias: A1 que representa el (73.3%), A2 el (15.9%), Aint el (5.65%), A1 B el (3.60%) y A2 B el (1.55%). La importancia clínica se basa en que algunas personas del grupo A2 transfundidas con A1 , pueden producir Anti-A1 que es un anticuerpo natural irregular activo a 22 ºC, pero en ocasiones está activo a 37ºC causando una reacción transfusional extravascular, por lo que, si no se cuenta con eritrocitos A2 , se recomienda transfundir eritrocitos grupo O.

In addition to the ABO system, its subgroups review great importance in Immunohematology. Subgroups A differ both in the number of antigenic sites and in the configuration of the erythrocyte antigen. The main ones, A1 and A2 differ in that A1 erythrocytes are agglutinated by human Anti-A1 antibody or by Anti-A1 Lectin (Dolichos biflorus), and A2 erythrocytes are agglutinated by Anti-H Lectin (Ulex europaeus). MATERIALS AND METHODS: a descriptive, cross-sectional study was conducted. The physical and electronic records of the Blood Bank were analyzed, all effective donors were included, which were typified by the Immunohematology Laboratory in the period of May. to July 2018. Method used, agglutination in tube and in microplate. RESULTS: in a total of 1599 protocols, it was determined that group O has the highest frequency with 84% and the least frequent was the AB with 0.66%. According to blood group A and AB we have the following frequencies: A1 representing (73.3%), A2 (15.9%), Aint (5.65%), A1B (3.60%) and A2B (1.55%). The clinical importance is based on the fact that some people in group A2 transfused with A1, can produce Anti-A1 which is an irregular natural antibody active at 22 ° C but sometimes it is active at 37 °C causing an extravascular transfusion reaction, so if A2 erythrocytes are not available, it is recommended to transfuse group O erythrocytes.

The Zebrafish Perivitelline Fluid Provides Maternally-Inherited Defensive Immunity.

In the teleost egg, the embryo is immersed in an extraembryonic fluid that fills the space between the embryo and the chorion and partially isolates it from the external environment, called the perivitelline fluid (PVF). The exact composition of the PVF remains unknown in vertebrate animals. The PVF allows the embryo to avoid dehydration, to maintain a safe osmotic balance and provides mechanical protection; however, its potential defensive properties against bacterial pathogens has not been reported. In this work, we determined the global proteomic profile of PVF in zebrafish eggs and embryos, and the maternal or zygotic origin of the identified proteins was studied. In silico analysis of PVF protein composition revealed an enrichment of protein classes associated with non-specific humoral innate immunity. We found lectins, protease inhibitors, transferrin, and glucosidases present from early embryogenesis until hatching. Finally, in vitro and in vivo experiments done with this fluid demonstrated that the PVF possessed a strong agglutinating capacity on bacterial cells and protected the embryos when challenged with the pathogenic bacteria Edwardsiella tarda. Our results suggest that the PVF is a primitive inherited immune extraembryonic system that protects the embryos from external biological threats prior to hatching.

Comparison of a Brucella Enzyme Immunoassay and the Standard Agglutination with 2-Mercaptoethanol Test in the Diagnosis and Monitoring of Brucellosis in Mexican Patients.

BACKGROUND: The diagnosis of human brucellosis is difficult based on clinical grounds alone. Thus, the diagnosis is based on microbiological and serological tests. Therefore, the diagnosis relies predominantly on laboratory testing. The objective of this study was to determine the most efficient test for the diagnosis and monitoring of patients treated for brucellosis by comparing the standard agglutination test in a tube with 2-mercaptoethanol (SAT-2Me) to an enzyme-linked immunosorbent assay for the detection of antibodies against Brucella IgM (IgM ELISA). METHODS: A retrospective chart review was performed. A total of 108 patients with brucellosis were analyzed at diagnosis and at the first and second follow-ups after treatment. The data were captured and analyzed using the SPSS 18.0 program. Frequencies, percentages, the Pearson's chi-square test, the kappa coefficient, sensitivity, specificity, predictive values, odds ratio, and conditional odds ratio (OR and COR) were calculated. RESULTS: Diagnostic test: the IgM ELISA showed 96.3% sensitivity vs. 73.1% sensitivity for the SAT-2Me (p < 0.001). First follow-up: the IgM ELISA presented significant differences vs. the SAT-2Me in sensitivity (97.2% vs. 72.2%) and specificity (89.7% vs. 44.1%). Additionally, the second follow-up data showed significant differences in the sensitivity (85.7% vs. 71.4%) and specificity (82.8% vs. 41.4%) for the IgM ELISA vs. the SAT-2Me, re-spectively. In addition, the IgM ELISA showed significant concordance (0.836, p < 0.001 and 0.563, p < 0.001) at the first and second follow-ups, respectively, vs. the SAT-2Me. CONCLUSIONS: The IgM ELISA is a more reliable and useful assay for the diagnosis and monitoring of brucellosis patients than the SAT-2 Me, avoiding up to 45.6% of unnecessary treatments. The SAT-2Me showed lower efficiency for diagnosis than the IgM ELISA and limited relevance for monitoring.

Neutropenia aloimune neonatal em gêmeas idênticas / Neonatal alloimmune neutropenia in identical twins

A neutropenia aloimune neonatal (NAN) é uma patologia causada pelo antagonismo imunológico, como a doença hemolítica do recém-nascido ou a trombocitopenia aloimune neonatal, mas relacionada aos neutrófilos, em vez de glóbulos vermelhos ou plaquetas. Descreveremos um caso clínico de duas gêmeas idênticas nascidas a termo, com Apgar de 8 e 9, sendo que após algumas horas do nascimento apresentaram febre. Um exame de sangue revelou neutropenia grave que resultou em sepse. O diagnóstico da NAN foi realizado clinicamente e por testes de histocompatibilidade. A prova cruzada por citometria de fluxo foi positiva, usando soro da mãe e suspensões celulares (granulócitos e linfócitos) das gêmeas e do pai. Este teste não fornece informações sobre para qual sistema genético os anticorpos foram positivos, se contra os antígenos específicos de neutrófilos humanos (HNA) ou contra os antígenos leucocitários humanos (HLA). Para o esclarecimento, realizamos o teste de aglutinação de granulócitos (GAT) com um painel de doadores fidelizados e com antígenos HNA1-5 conhecidos, utilizando o soro materno como reagente. Foi também realizada a pesquisa de anticorpos anti-HLA e anti-HNA no soro materno. Os genótipos HLA e HNA foram identificados, permitindo conhecer as especificidades dos anticorpos maternos contra os antígenos dos neutrófilos do marido e das filhas. O diagnóstico de NAN não é realizado na maioria dos hospitais de nosso país e do exterior, devido à dificuldade de execução dos testes de histocompatibilidade, no entanto a prova cruzada por citometria de fluxo pode facilmente ser implantada nos laboratórios clínicos, sendo que está descrita detalhadamente nesse caso clínico.

Neonatal alloimmune neutropenia (NAN) is a disease caused by immunological antagonism, such as hemolytic disease of the newborn or neonatal alloimmune thrombocytopenia, but related to neutrophils rather than to red blood cells or platelets. We will describe a clinical case of two identical twins born with Apgar 8 and 9 that started with fever few hours after delivery. A blood test revealed severe neutropenia, which was followed by sepsis. The diagnosis of NAN was done clinically and by histocompatibility testing. Flow cytometry crossmatch was positive, using mother serum and cell suspensions (granulocytes and lymphocytes) from the twin girls and from the father. This test did not provide information about the genetic system for which the antibodies are positive, if against human neutrophil antigens (HNA) or human leucocyte antigens (HLA). To clear this, the granulocyte agglutination test (GAT) was performed with a panel of control donors with known HNA1-5 antigens, using the maternal serum as a reagent. We did also a Luminex screening assay for detection of anti-HLA and anti-HNA antibodies in the mother serum. The HLA and HNA genotypes were identified, which allowed to define specificities in mother's antibodies against the neutrophil surface antigens from her husband and from the twins. The diagnosis of NAN diagnose is not done in most hospitals worldwide, mainly by the difficulty in executing the histocompatibility test. However, the crossmatch by flow cytometry could be easily done in clinical laboratories following the method described in this article.

Avaliação das reações de Hemaglutinação Indireta (HI) e Aglutinação Modificada (MAT) na detecção de anticorpos IgG anti-T. gondii em exsudatos cárneos bovinos / Evaluation of indirect Hemagglutination (HI) and Modified Agglutination (MAT) reactions in the detection of anti-T. gondiiIgG antibodies in bovine meat exudates

Toxoplasmose é uma zoonose parasitária com ampla distribuição mundial provocada pelo Toxoplasma gondii, considerado um dos protozoários mais bem sucedidos do planeta, pois infecta cerca de um terço da população mundial. Dentre as formas de transmissão, o consumo de carne mal cozida, contendo cistos, tem sido considerado um fator de risco para aquisição desta zoonose. Uma abordagem alternativa para o controle da toxoplasmose pela ingestão de carne bovina seria a sorologia dos bovinos, já que animais soropositivos albergam cistos teciduais. Contudo, a obtenção de soro para esta avaliação, nem sempre é factível, dada a dificuldade de coleta de sangue durante a linha de abate e sua ausência em cortes comerciais. O exsudato cárneo é uma alternativa para detecção de anticorpos anti - T. gondii em cortes comerciais de carne, que foi a proposta deste estudo para avaliar o desempenho dos testes de Hemaglutinação Indireta (HI) e Aglutinação Modificada (MAT) quando comparados ao ELISA usando exsudato cárneo. Este estudo mostrou que a acurácia dos testes de aglutinação não foi viável devido aos baixos índices de sensibilidade e especificidade quando comparados ao ELISA. Estes dados demonstram a importância da escolha de testes eficientes como ELISA para aplicação no controle da qualidade e inocuidade de cortes comerciais de carne bovina. (AU)

Toxoplasmosis is a parasitic zoonosis with a wide worldwide distribution caused by Toxoplasmagondii, which is considered one of the most successful protozoa on the planet, since it can infect a third of the world population. Among the forms of transmission, consumption of undercooked meat has been considered as a risk factor for the acquisition of this zoonosis. An alternative approach to toxoplasmosis control by beef ingestion could be the serological diagnosis in cattle, since seropositives animals harbor tissue cysts. However, the use of serum for this evaluation is not always feasible due to the difficulty of blood collection during slaughter and its absence in commercial beef cuts. Meat exudate is an alternative for the detection of anti-T. gondii antibodies in commercial beef cuts, which was the propose of this study to evaluate the performance of Indirect Hemagglutination (HI) and Agglutination Modified (MAT) tests compared to ELISA using meat exudates. This study showed that the agglutination tests accuracy was not viable due to low sensitivity and specificity indexes when compared to ELISA. These data demonstrate the importance of choosing accurate tests such as ELISA for application in quality control and safety of commercial beef cuts. (AU)

Correlation among automated scores of agglutination, antigen density by flow cytometry and genetics of D variants.

BACKGROUND: Laboratory testing to identify the molecular basis of serologic weak D phenotypes is recommended to determine whether a pregnant woman or potential transfusion recipient should be managed as RhD-positive or RhD-negative. The variation in D antigen expression on RBCs, different potencies of anti-D typing reagents, lack of standardized test methods, and the subjectivity of interpreting agglutination reactions complicate the detection of D variants. We evaluated the correlation of agglutination scores by an automated immunoassay analyzer with D antigen densities determined by flow cytometry, and D variant types identified by molecular analysis. MATERIALS AND METHODS: We selected 273 blood donor samples with agglutination scores of less than 92 (4+), measured by an automated analyzer (NEO®, Immucor, Norcross, GA, USA). D antigen densities were measured by flow cytometry for 89 samples. Samples were classified as molecularly-determined weak D or partial D variants by multiplex PCR, PCR RFLP and DNA sequencing. RESULTS: All samples with a D antigen density ≥15% had an agglutination score >80 (4+). Agglutination scores for weak D types varied from 10 to 90. Agglutination scores for partial D antigens were graded with scores varying from 60 to 99. D antigen densities varied from 0.55% to 10.67% for weak Ds and 4.1% to 30.5% for partial Ds. DISCUSSION: Our results showed that score values follow a pattern among D variants that could be related to antigen density and to the RhD variant classification.

Actinobacillus seminis GroEL-homologous protein agglutinates sheep erythrocytes.

Actinobacillus seminis, a commensal of ovine and caprine reproductive organs, is able to induce epididymitis in the small ruminants that it infects. In this work, we characterised two protein bands of approximately 150 kDa and 65 kDa. These proteins cross-reacted with a polyclonal serum against Gallibacterium anatis hemagglutinin and with a polyclonal serum from sheep with epididymitis, indicating that the proteins are expressed in vivo; the two proteins also interacted with biotin-labeled sheep fibrinogen and fibronectin, suggesting that they may function as adhesins. The participation of these proteins as adhesins was confirmed by a cultured human bladder cell-A. seminis adhesion assay and adherence inhibition by preincubation of A. seminis with polyclonal antiserum to the 150 kDa protein. Both proteins presented sequence identity with an A. seminis GroEL protein by mass spectrometry analysis and agglutinated glutaraldehyde-fixed sheep red blood cells. Immunogold labeling was observed by transmission electron microscopy on bacterial cells that were negatively stained, and a peroxidase reaction was detected in A. seminis biofilms, when an anti-A. seminis 150 kDa protein serum was used, indicating the presence of this protein on the surface of A. seminis and in biofilms. The A. seminis GroEL-homologue is a multifunctional protein that likely acts as a hemagglutinin.

Frecuencia y procedencia del antígeno Kell en mujeres donantes de sangre durante los años 2016-2017 / Frequency and origin of the Kell antigen in female blood donors during 2016-2017

Resumen Introducción: El sistema Kell está formado por dos antígenos principales: el Kell (K) y el Cellano (k), estos son capaces de causar reacciones graves, tales como reacción hemolítica postransfusional y la enfermedad hemolítica del recién nacido. Los antígenos de este sistema son altamente inmunogénicos lo que les confiere el tercer lugar en importancia clínica. Objetivo: Determinar la frecuencia del antígeno Kell y procedencia de las mujeres donantes de sangre con antígeno Kell positivo en el Hemocentro del Centro Oriente Colombiano (HCOC). Metodología: Estudio descriptivo de corte transversal que incluyó 186 donantes voluntarias de sangre del Hemocentro Centro Oriente Colombiano, se realizó la fenotipificación del antígeno Kell, utilizando la técnica Aglutinación en lámina, la cual se basa en enfrentar glóbulos rojos del donante con anticuerpo monoclonal anti K. Se calculó la frecuencia fenotípica del antígeno Kell, en porcentajes y para el procesamiento de la información se utilizó el paquete estadístico SPSS versión 21.0 en español donde se realizó todo el análisis de los datos de la población. Resultados: Se procesaron 177 muestras obtenidas en 9 campañas de donación de sangre realizadas en diferentes municipios del departamento de Boyacá, obteniéndose una frecuencia fenotípica del 7,5% para el antígeno Kell, en la población de mujeres donantes de sangre del HCOC, siendo esta similar con la frecuencia encontrada en Colombia y Latinoamérica. Conclusión: Se determinó que la frecuencia del antígeno Kell en las mujeres donantes de sangre del HCOC fue del 7,5%, y se logró identificar que no existe una relación estadísticamente entre la procedencia y la presencia del antígeno Kell en las donantes, lo anterior está relacionado con el mestizaje y los procesos de migración.

Abstract Introduction: The Kell system consists of two major antigens: Kell (K) and Cellano (K), which are capable of causing serious reactions, such as posttransfusion hemolytic reaction and hemolytic disease of the newborn. The antigens of this system are highly immunogenic which gives them the third place in clinical importance. Objective: To determine the frequency of Kell antigen and origin of blood donors in the Hemocenter of the Centro Oriente Colombiano (H.C.O.C). Methods: Cross-sectional descriptive study involving 186 blood donors from the Centro Oriente Colombian Hemocenter, phenotyping of the Kell antigen was carried out, using the technique Aglutination in lamina, which is based on facing donor red blood cells with anti-K monoclonal antibody. Calculated the phenotypic frequency of the Kell antigen in percentages and for the processing of the information was used the statistical package SPSS version 21.0 in Spanish where all the analysis of the data of the population was carried out. Results: 177 samples obtained in 9 blood donation campaigns were carried out in different municipalities of the department of Boyacá, obtaining a phenotypic frequency of 7.5% for the Kell antigen in the population of female HCOC blood donors. Similar to the frequency found in Colombia and Latin America. Conclusion: It was determined that the frequency of Kell antigen in the female HCOC donors was 7.5%, and it was possible to identify that there is no statistically relation between the origin and the presence of Kell antigen in the donors, Is related to mestizaje and migration processes.

Lipoproteins from vertebrate host blood plasma are involved in Trypanosoma cruzi epimastigote agglutination and participate in interaction with the vector insect, Rhodnius prolixus.

Chagas disease, infecting ca. 8 million people in Central and South America, is mediated by the protozoan parasite, Trypanosoma cruzi. The parasite is transmitted by the bite of blood sucking triatomine insects, such as Rhodnius prolixus, that had previously fed on parasite-infected vertebrate blood and voided their contaminated feces and urine into the wound. The stages of the parasite life cycle in both the insect vector and human host are well-known, but determinants of infection in the insect gut are complex and enigmatic. This paper examines the possible role of the R. prolixus gut agglutinins in the parasite life cycle. The results, derived from gut extracts made from R. prolixus fed on various diets with different vertebrate blood components, and cross adsorption experiments, showed for the first time that R. prolixus has two distinct gut agglutinins originating from their vertebrate blood meal, one for T. cruzi (the parasite agglutinin, PA) and the other for the erythrocytes (the hemagglutinin, HA). Again, uniquely, the results also demonstrate that these two agglutinins are derived, respectively, from the plasma and erythrocyte components of the vertebrate blood. Subsequent experiments, examining in more detail the nature of the plasma components forming the T. cruzi PA, used fractionated extracts of the vertebrate plasma (high density lipoprotein, HDL; low density lipoprotein, LDL, and delipidated plasma) in agglutination assays. The results confirmed the identity of the PA as a high density lipoprotein (HDL) in the plasma of the vertebrate blood meal which agglutinates parasites in the R. prolixus gut. In addition, the use of single or double labeled HDL in fluorescence and confocal microscopy showed the interaction of the labeled HDL with the parasite surface and its internalization at later times. Finally, results of T. cruzi parasitization of R. prolixus, incorporating various vertebrate blood components, resulted in highly significant increases in infectivity in the presence of HDL from the 2nd day of infection, thus confirming the important role of this molecule in T. cruzi infection of R. prolixus.

A lectin of a non-invasive apple snail as an egg defense against predation alters the rat gut morphophysiology.

The eggs of the freshwater Pomacea apple snails develop above the water level, exposed to varied physical and biological stressors. Their high hatching success seems to be linked to their proteins or perivitellins, which surround the developing embryo providing nutrients, sunscreens and varied defenses. The defensive mechanism has been unveiled in P. canaliculata and P. maculata eggs, where their major perivitellins are pigmented, non-digestible and provide a warning coloration while another perivitellin acts as a toxin. In P. scalaris, a species sympatric to the former, the defense strategy seems different, since no toxin was found and the major perivitellin, PsSC, while also colored and non-digestible, is a carbohydrate-binding protein. In this study we examine the structure and function of PsSC by sequencing its subunits, characterizing its carbohydrate binding profile and evaluating its effect on gut cells. Whereas cDNA sequencing and database search showed no lectin domain, glycan array carbohydrate binding profile revealed a strong specificity for glycosphingolipids and ABO group antigens. Moreover, PsSC agglutinated bacteria in a dose-dependent manner. Inspired on the defensive properties of seed lectins we evaluated the effects of PsSC on intestinal cells both in vitro (Caco-2 and IEC-6 cells) and in the gastrointestinal tract of rats. PsSC binds to Caco-2 cell membranes without reducing its viability, while a PsSC-containing diet temporarily induces large epithelium alterations and an increased absorptive surface. Based on these results, we propose that PsSC is involved in embryo defenses by altering the gut morphophysiology of potential predators, a convergent role to plant defensive lectins.

Morphological and functional characterization of the hemocytes from the pearl oyster Pteria hirundo and their immune responses against Vibrio infections.

Hemocyte populations of the pearl oyster Pteria hirundo were characterized at morphological, ultrastructural and functional levels. Three main hemocyte populations were identified: hyalinocytes, granulocytes and blast-like cells. Hyalinocytes were the most abundant population (88.2%) characterized by the presence of few or no granules in the cytoplasm and composed by two subpopulations, large and small hyalinocytes. Comparatively, granulocytes represented 2.2% of the hemocyte population and were characterized by the presence of numerous large electron-lucid granules in the cytoplasm. Finally, the blast-like cells (9.5%) were the smallest hemocytes, showing spherical shape and a high nucleus/cytoplasm ratio. Hemocytes exhibited a significant phagocytic capacity for inert particles (38.5%) and showed to be able to produce microbicidal molecules, such as reactive oxygen species (ROS) (ex vivo assays). The immune role of hemocytes was further investigated in the P. hirundo defense against the Gram-negative Vibrio alginolyticus. A significant decrease in the total number of hemocytes was observed at 24 h following injection of V. alginolyticus or sterile seawater (injury control) when compared to naïve (unchallenged) animals, indicating the migration of circulating hemocytes to the sites of infection and tissue damage. Bacterial agglutination was only observed against Gram-negative bacteria (Vibrio) but not against to marine Gram-positive-bacteria. Besides, an increase in the agglutination titer was observed against V. alginolyticus only in animals previously infected with this same bacterial strain. These results suggest that agglutinins or lectin-like molecules may have been produced in response to this particular microorganism promoting a specific recognition. The ultrastructural and functional characterization of P. hirundo hemocytes constitutes a new important piece of the molluscan immunity puzzle that can also contribute for the improvement of bivalve production sustainability.

Understanding the interaction of concanavalin a with mannosyl glycoliposomes: A surface plasmon resonance and fluorescence study.

The specificity of carbohydrate-protein interaction is a key factor in many biological processes and it is the foundation of technologies using glycoliposomes in drug delivery. The incorporation of glycolipids in vesicles is expected to increase their specificity toward particular targets such as lectins; however, the degree of exposure of the carbohydrate moiety at the liposome surface is a crucial parameter to be considered in the interaction. Herein we report the synthesis of mannose derivatives with one or two hydrophobic chains of different length, designed with the purpose of modifying the degree of exposure of the mannose when they were incorporated into liposomes. The interaction of glycovesicles with Con A was studied using: (i) agglutination assays; measured by dynamic laser light scattering (DLS); (ii) time resolved fluorescence methods and (iii) surface plasmon resonance (SPR) kinetic measurements. DLS data showed that an increase in hydrophobic chain length promotes a decrease of liposomes hydrodynamic radius. A longer hydrocarbon chain favors a deeper insertion into the bilayer and mannose moiety results less exposed at the surface to interact with lectin. Fluorescence experiments showed changes in the structure of glycovesicles due to the interaction with the protein. From SPR measurements the kinetic and equilibrium constants associated to the interaction of ConA with the different glycolipid synthetized were determined. The combination of SPR and fluorescence techniques allowed to study the interaction of Con A with mannosyl glycovesicles at three levels: at the surface, at the interface and deeper into the bilayer.

Anemia hemolítica imunomediada em cadela da raça teckel - relato de caso / Autoimmune hemolytic anemia in a dachshund bitch - a case report / Anemia hemolítica autoinmune en una perra Teckel - reporte de un caso

A anemia hemolítica imunomediada leva à remoção prematura dos eritrócitos. Considerada reação de hipersensibilidade tipo 2, nela os eritrócitos estão recobertos por anticorpos ou complemento e são fagocitados no baço ou lisados nos vasos sanguíneos. É primária, quando as causas são desconhecidas, ou secundária, quando decorre de antígenos externos. O diagnóstico é terapêutico ou pelo teste de Coombs direto positivo e achados laboratoriais, como anemia moderada a grave de regeneração variável, policromasia, esferocitose, aglutinação de hemácias, hiperbilirrubinemia e hemoglobinúria. O tratamento é feito com doses imunossupressoras de anti-inflamatórios esteroidais, como a prednisona. Relata-se o caso de uma cadela com anemia hemolítica imunomediada primária, apresentando quadro de início agudo, apatia, dispneia, dor à palpação abdominal, esplenomegalia e mucosas pálidas. O teste de Coombs direto foi positivo. Utilizou-se prednisona, com resposta desfavorável, seguida pela associação de outros fármacos. Com a evolução desfavorável e mau prognóstico, o proprietário optou pela eutanásia.

Immune-mediated hemolytic anemia leads to the premature removal of erythrocytes. It is a type 2 hypersensitivity reaction in which erythrocytes are covered by antibodies or complement and are phagocytized in the spleen or Iysed within the blood vessels. It is considered of primary origin when the causes of the onset of the disease are unknown, or regarded as secondary when resulting from external antigens. The diagnosis may be therapeutic or based on the direct positive Coombs test in conjunction with laboratory findings, such as moderate to severe variable regeneration anemia, polychromasia, spherocytosis, red blood cell agglutination, hyperbilirubinemia, and hemoglobinuria. Treatment is based on immunosuppressive doses of steroidal anti-inflammatory drugs such as prednisone. This paper reports the case of an acute onset in a dog diagnosed with primary immune-mediated hemolytic anemia, presenting apathy, dyspnea, pain upon abdominal palpation, splenomegaly and pale mucous membranes. Coombs direct test was positive. Treatment started with prednisone, but as the response was not favorable, other drugs were associated to it. Evolution was unfavorable and the owner opted for euthanasia due to poor prognosis.

La anemia hemolítica inmunomediada provoca una eliminación prematura de eritrocitos. Considerada como reacción de hipersensibilidad tipo 2, los eritrocitos están cubiertos por anticuerpos o complemento, que sufren un proceso de fagocitosis en el bazo, o bien pueden ser lisados en el interior de los vasos sanguíneos. Se dice que la enfermedad es de tipo primaria cuando sus causas son desconocidas, y secundaria, en aquellos casos provocados por antígenos externos. El diagnóstico puede ser terapéutico o a través de la prueba de Coombs y otros resultados de laboratorio, como una anemia moderada a grave de diferentes grados de regeneración, policromasia, esferocitosis, aglutinación de eritrocitos, hiperbilirrubinemia y hemoglobinuria. El tratamiento se realiza con dosis inmunosupresoras de antiinflamatorios esteroides, como la prednisona. Se relata el caso de una perra de un ano con un cuadro agudo de anemia hemolítica inmunomediada primaria, que presentaba apatía, disnea, dolor abdominal, esplenomegalia y mucosas pálidas. La prueba de Coombs directa dio positiva. La prednisona no provocó la respuesta esperada, 10 que llevó a la asociación con otros fármacos. Debido a la evolución desfavorable y el mal pronóstico, el propietario decidió por la eutanasia del paciente.

Anemia hemolítica imunomediada em cadela da raça teckel - relato de caso / Autoimmune hemolytic anemia in a dachshund bitch - a case report / Anemia hemolítica autoinmune en una perra Teckel - reporte de un caso

A anemia hemolítica imunomediada leva à remoção prematura dos eritrócitos. Considerada reação de hipersensibilidade tipo 2, nela os eritrócitos estão recobertos por anticorpos ou complemento e são fagocitados no baço ou lisados nos vasos sanguíneos. É primária, quando as causas são desconhecidas, ou secundária, quando decorre de antígenos externos. O diagnóstico é terapêutico ou pelo teste de Coombs direto positivo e achados laboratoriais, como anemia moderada a grave de regeneração variável, policromasia, esferocitose, aglutinação de hemácias, hiperbilirrubinemia e hemoglobinúria. O tratamento é feito com doses imunossupressoras de anti-inflamatórios esteroidais, como a prednisona. Relata-se o caso de uma cadela com anemia hemolítica imunomediada primária, apresentando quadro de início agudo, apatia, dispneia, dor à palpação abdominal, esplenomegalia e mucosas pálidas. O teste de Coombs direto foi positivo. Utilizou-se prednisona, com resposta desfavorável, seguida pela associação de outros fármacos. Com a evolução desfavorável e mau prognóstico, o proprietário optou pela eutanásia.(AU)

Immune-mediated hemolytic anemia leads to the premature removal of erythrocytes. It is a type 2 hypersensitivity reaction in which erythrocytes are covered by antibodies or complement and are phagocytized in the spleen or Iysed within the blood vessels. It is considered of primary origin when the causes of the onset of the disease are unknown, or regarded as secondary when resulting from external antigens. The diagnosis may be therapeutic or based on the direct positive Coombs test in conjunction with laboratory findings, such as moderate to severe variable regeneration anemia, polychromasia, spherocytosis, red blood cell agglutination, hyperbilirubinemia, and hemoglobinuria. Treatment is based on immunosuppressive doses of steroidal anti-inflammatory drugs such as prednisone. This paper reports the case of an acute onset in a dog diagnosed with primary immune-mediated hemolytic anemia, presenting apathy, dyspnea, pain upon abdominal palpation, splenomegaly and pale mucous membranes. Coombs direct test was positive. Treatment started with prednisone, but as the response was not favorable, other drugs were associated to it. Evolution was unfavorable and the owner opted for euthanasia due to poor prognosis.(AU)

La anemia hemolítica inmunomediada provoca una eliminación prematura de eritrocitos. Considerada como reacción de hipersensibilidad tipo 2, los eritrocitos están cubiertos por anticuerpos o complemento, que sufren un proceso de fagocitosis en el bazo, o bien pueden ser lisados en el interior de los vasos sanguíneos. Se dice que la enfermedad es de tipo primaria cuando sus causas son desconocidas, y secundaria, en aquellos casos provocados por antígenos externos. El diagnóstico puede ser terapéutico o a través de la prueba de Coombs y otros resultados de laboratorio, como una anemia moderada a grave de diferentes grados de regeneración, policromasia, esferocitosis, aglutinación de eritrocitos, hiperbilirrubinemia y hemoglobinuria. El tratamiento se realiza con dosis inmunosupresoras de antiinflamatorios esteroides, como la prednisona. Se relata el caso de una perra de un ano con un cuadro agudo de anemia hemolítica inmunomediada primaria, que presentaba apatía, disnea, dolor abdominal, esplenomegalia y mucosas pálidas. La prueba de Coombs directa dio positiva. La prednisona no provocó la respuesta esperada, 10 que llevó a la asociación con otros fármacos. Debido a la evolución desfavorable y el mal pronóstico, el propietario decidió por la eutanasia del paciente.(AU)

A Lectin Purified from Blood Red Bracket Mushroom, Pycnoporus sanguineus (Agaricomycetidae), Mycelium Displayed Affinity Toward Bovine Transferrin.

Fungal lectins constitute excellent ligands for development of affinity adsorbents useful in affinity chromatography. In this work, a lectin was purified from Pycnoporus sanguineus (PSL) mycelium using 3 procedures: by affinity chromatography, using magnetic galactosyl-nanoparticles or galactose coupled to Sepharose, and by ionic exchange chromatography (IEC). The highest lectin yield was achieved by IEC (55%); SDS-PAGE of PSL showed 2 bands with molecular mass of 68.7 and 55.2 kDa and IEC displayed 2 bands at pi 5.5 and 5.2. The lectin agglutinates rat erythrocytes, exhibiting broad specificity toward several monosaccharides, including galactose. The agglutination was also inhibited by the glycoproteins fetal calf fetuin, bovine lactoferrin, bovine transferrin, and horseradish peroxidase. The lectin was then used to synthesize an affinity adsorbent (PSL-Sepharose) and the interaction with glycoproteins was evaluated by analyzing their chromatographic behaviors. The strongest interaction with the PSL-derivative was observed with transferrin, although lower interactions were also displayed toward fetuin and lactoferrin. These results indicate that the purified PSL constitutes an interesting ligand for the design of affinity adsorbents to be used (i.e., in glycoprotein purification).

Sperm conjugation in mammal reproductive function: Different names for the same phenomenon?

In many mammalian and non-mammalian species, mature sperm interact within the female reproductive tract or inside the epididymal lumen using cohesive forces. This phenomenon, known as "sperm conjugation," is sometimes confused with sperm agglutination, which is the result of the interaction of epididymal or ejaculate spermatozoa upon release into culture medium. In addition to "agglutination," the terms "association," "rouleaux," or "rosettes" are employed interchangeably to describe the conjugation phenomenon, which inevitably causes confusion due to the non-unifying nomenclature. This variety of descriptions is likely due to a poor understanding of the molecular mechanisms involved in such conspicuous cell-cell interaction as well as the different morphologies that result from such interactions among species. Here, we summarize the published data regarding mammalian sperm conjugation, considering the organisms in which sperm interaction was observed; the particular terminology employed; findings regarding the components that enable sperm to adhere; sperm behavior when deposited in the female reproductive tract; and hypotheses formulated to clarify the biological function and, when known, the mechanisms for sperm interaction. We also propose a new classification system for this phenomenon that might clearly unify the criteria used to describe this behavior. Mol. Reprod. Dev. 83: 884-896, 2016 © 2016 Wiley Periodicals, Inc.

Quince años de estudio sobre las adherencias de labios menores / Fifteen-Year Study on the Labial Adhesions

Introducción: las adherencias de labios menores es un problema ginecológico frecuente en niñas prepuberales. De las niñas que lo padecen en algún momento de su vida (1,8 por ciento), el pico de mayor incidencia está entre los 13 y 23 meses de edad. Objetivo: mostrar los resultados de esta experiencia en el diagnóstico y manejo de esta entidad. Métodos: se realizó un estudio descriptivo, longitudinal y retrospectivo. Se revisó la totalidad de las historias clínicas de 804 niñas atendidas desde el 1º de enero de 1998 hasta el 31 de diciembre de 2012. La muestra quedó integrada por 54 casos con diagnóstico de adherencias de labios menores. Las variables estudiadas fueron: edad al diagnóstico, motivo de consulta, hallazgos clínicos, tipo de sinequia, tratamiento impuesto, entre otras. Resultados: hubo mayor incidencia de sinequias en niñas entre 3 meses y 3 años (74 por ciento), con predominio de la variedad incompleta y dentro de esta la del tercio inferior (66,6 por ciento). La distorsión anatómica de los genitales externos resultó el principal motivo de consulta (42,6 por ciento). Los mejores resultados del tratamiento se lograron con la combinación de medidas higiénicas y ejercicios de tracción lateral vulvar con el uso tópico de estrógenos. Las recidivas se presentaron en 12,9 por ciento de los casos a los seis meses de edad. Conclusiones: la adherencia de labios menores es una causa importante de consulta ginecológica en niñas. Se asocia a una gran ansiedad en padres y familiares. Se obtuvo buenos resultados con el tratamiento médico, sobre todo cuando se empleó estrógenos localmente(AU)

Introduction: Labial adhesions is a common gynecological problem in prepubertal adolescents. The highest peak incidence is between 13 and 23 months old of those girls sufferingfrom this problem at some point in their life (1.8 percent). Objective: Show the results of this experience in the diagnosis and management of this condition. Methods: A descriptive, longitudinal and retrospective study was conducted at revising all the medical records of 804 girls assisted from 1st January 1998 to 31st December, 2012. The sample was composed of 54 cases diagnosed with labial adhesions. The variables studied were age at diagnosis, presenting complaint, clinical findings, type of synechia, treatment, among others. Results: There was higher incidence of synechiae in children between 3 months and 3 years (74 percent), predominantly incomplete, andin the lower third (66.6 percent). The anatomical distortion of the external genitalia was the main reason for consultation (42.6 percent). Best treatment results were achieved with the combination of hygienic measures and vulvar lateral traction exercises with topical estrogen use. Recurrences occurred in 12.9 percent of cases at six months of age. Conclusions: Labialadhesion is a major cause of gynecological consultation in girls. It is associated with great anxiety in parents and relatives. Good results were obtainedwith medical treatment, especially when the topical use of estrogen(AU)

Hevea brasiliensis prohevein possesses a conserved C-terminal domain with amyloid-like properties in vitro.

Prohevein is a wound-induced protein and a main allergen from latex of Hevea brasiliensis (rubber tree). This 187 amino-acid protein is cleaved in two fragments: a N-terminal 43 amino-acids called hevein, a lectin bearing a chitin-binding motif with antifungal properties and a C-terminal domain (C-ter) far less characterized. We provide here new insights on the characteristics of prohevein, hevein and C-terminal domain. Using complementary biochemical (ThT/CR/chitin binding, agglutination) and structural (modeling, ATR-FTIR, TEM, WAXS) approaches, we show that this domain clearly displays all the characteristics of an amyloid-like proteins in vitro, that could confer agglutination activity in synergy with its chitin-binding activity. Additionally, this C-ter domain is highly conserved and present in numerous plant prohevein-like proteins or pathogenesis-related (PR and WIN) proteins. This could be the hallmark of the eventual presence of proteins with amyloid properties in plants, that could potentially play a role in defense through aggregation properties.