Inhibition of FcepsilonRI-dependent mediator release and calcium flux from human mast cells by sialic acid-binding immunoglobulin-like lectin 8 engagement

BACKGROUND: Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of glycan-binding inhibitory receptors, and among them, Siglec-8 is selectively expressed on human eosinophils, basophils, and mast cells. On eosinophils, Siglec-8 engagement induces apoptosis, but its function on mast cells is unknown. OBJECTIVE: We sought to study the effect of Siglec-8 engagement on human mast cell survival and mediator release responses. METHODS: Human mast cells were generated from CD34+ precursors. Apoptosis was studied by using flow cytometry. Mast cell mediator release or human lung airway smooth muscle contraction was initiated by FcepsilonRI cross-linking with or without preincubation with Siglec-8 or control antibodies, and release of mediators was analyzed along with Ca++ flux. RBL-2H3 cells transfected with normal and mutated forms of Siglec-8 were used to study how Siglec-8 engagement alters mediator release. RESULTS: Siglec-8 engagement failed to induce human mast cell apoptosis. However, preincubation with Siglec-8 mAbs significantly (P < .05) inhibited FcepsilonRI-dependent histamine and prostaglandin D(2) release, Ca++ flux, and anti-IgE-evoked contractions of human bronchial rings. In contrast, release of IL-8 was not inhibited. Siglec-8 ligation was also shown to inhibit beta-hexosaminidase release and Ca++ flux triggered through FcepsilonRI in RBL-2H3 cells transfected with full-length human Siglec-8 but not in cells transfected with Siglec-8 containing a tyrosine to phenylalanine point mutation in the membrane-proximal immunoreceptor tyrosine-based inhibitory motif domain. CONCLUSION: These data represent the first reported inhibitory effects of Siglec engagement on human mast cells.

Longterm survey (7 years) in a population at risk for Lyme borreliosis: what happens to the seropositive individuals?

In 1986, a 26% seroprevalence of IgG- anti-Borrelia burgdorferi antibodies was observed among 950 orienteers and the incidence of new clinical infections was 0.8%. In 1993, a total of 305 seropositive orienteers were reexamined. During that time, 15 cases (4.9%) of definite/probable Lyme disease occurred in this seropositive group (12 skin manifestations and 3 monoarticular joint manifestations). Among the 12 definite cases, 9 showed new clinical infections (7 EM, 1 acrodermatitis chronica atrophicans, 1 arthritis), and 3 were recurrent (2 EM, 1 arthritis). The annual incidence (0.8%) in this seropositive group was identical to the incidence observed among the whole population in 1986. The individual antibody titer decreased slightly but the seroreversion rate was low (7%). Serology was not very helpful in identifying clinical cases and evolutions, and it can be stated, that a positive serology is much more frequent in this risk group than clinical disease.

Antibody Responses of Vaccinated and Nonvaccinated Calves to Haemophilus somnus

Respiratory disease resulting from infection of calves with Haemophilus somnus (H. somnus) is an annual occurrence in fall calves at the McNay Farm. Previous observations of skin test reactivity to H. somnus antigens suggested a role for this phenomenon in the pathogenesis of the disease. Groups of calves, about 90 days of age, were vaccinated with four different commercial H. somnus vaccines, and serum levels of H. somnus antibodies were determined. Antibodies of the IgG and IgE classes were detected with ELISA procedures conducted on sera collected before and after vaccination. Most of the calves had detectable H. somnus IgE class antibodies at the start of the experimentation but IgG class antibodies were minimal. Antibodies of both classes increased in nonvaccinated and vaccinated calves during the 30 day period of experimentation. However, the level of IgE class antibodies in vaccinates was lower than in controls suggesting that vaccination may limit the IgE response.

A highly conserved interspecies V(H) in the human genome.

Idiotype conservation between human and mouse antibodies has been observed in association with various infectious and autoimmune diseases. We have isolated a human anti-idiotypic antibody to a mouse monoclonal anti-IgE antibody (BSW17) suggesting a conserved interspecies idiotype associated with an anti-IgE response. To find the homologue of BSW17 in the human genome we applied the guided selection strategy. Combining V(H) of BSW17 with a human V(L) repertoire resulted in three light chains. The three V(L) chains were then combined with a human V(H) repertoire resulting in three clones specific for human IgE. Surprisingly, one clone, Hu41, had the same epitope specificity and functional in vitro activity as BSW17 and V(H) complementarity-determining regions identical with that of BSW17. Real-time PCR analysis confirmed the presence of the Hu41 V(H) sequence in the human genome. These data document the first example of the isolation of a human antibody where high sequence similarity to the original murine V(H) sequence is associated with common antigen and epitope specificity.

The clinical utility of basophil activation testing in diagnosis and monitoring of allergic disease

The basophil activation test (BAT) has become a pervasive test for allergic response through the development of flow cytometry, discovery of activation markers such as CD63 and unique markers identifying basophil granulocytes. Basophil activation test measures basophil response to allergen cross-linking IgE on between 150 and 2000 basophil granulocytes in <0.1 ml fresh blood. Dichotomous activation is assessed as the fraction of reacting basophils. In addition to clinical history, skin prick test, and specific IgE determination, BAT can be a part of the diagnostic evaluation of patients with food-, insect venom-, and drug allergy and chronic urticaria. It may be helpful in determining the clinically relevant allergen. Basophil sensitivity may be used to monitor patients on allergen immunotherapy, anti-IgE treatment or in the natural resolution of allergy. Basophil activation test may use fewer resources and be more reproducible than challenge testing. As it is less stressful for the patient and avoids severe allergic reactions, BAT ought to precede challenge testing. An important next step is to standardize BAT and make it available in diagnostic laboratories. The nature of basophil activation as an ex vivo challenge makes it a multifaceted and promising tool for the allergist. In this EAACI task force position paper, we provide an overview of the practical and technical details as well as the clinical utility of BAT in diagnosis and management of allergic diseases.

Precursor B cells for autoantibody production in genomically Fas-intact autoimmune disease are not subject to Fas-mediated immune elimination

The Fas/Fas ligand (FasL) system participates in regulation of the immune system through the apoptotic process. However, the extent to which abnormalities in this system are involved in the loss of self-tolerance and development of autoimmune disease not associated with Fas/FasL mutations remains unknown. The present study addresses this issue in Fas/FasL-intact, systemic lupus erythematosus (SLE)-prone (NZB × NZW) (NZB/W) F1 mice. While splenic B cells from 2-month-old mice before overt SLE expressed Fas poorly, in vitro stimulation with an agonistic anti-CD40 mAb up-regulated their Fas expression, thus revealing the existence of two populations: one was Fashigh and highly susceptible to anti-Fas mAb-induced apoptosis, and the other was Faslow and apoptosis-resistant. The Faslow cells were included in the CD5+ B cell subpopulation and contained most of the cells that produced IgM anti-DNA antibodies. The isotype of anti-DNA antibodies switches from IgM to IgG in NZB/W F1 mice at ages beginning at about 6 months. These IgG anti-DNA antibodies were produced almost exclusively by a subpopulation of splenic B cells that spontaneously expressed low levels of Fas in vivo and were apoptosis-resistant. The findings indicate that precursor B cells for autoantibody production and presumably autoantibody-secreting cells in these mice are relatively resistant to Fas-mediated apoptosis, a finding supporting the concept that abnormalities of Fas-mediated apoptotic process are involved in the development of autoreactive B cells in Fas/FasL-intact autoimmune disease.

ER-27319, an acridone-related compound, inhibits release of antigen-induced allergic mediators from mast cells by selective inhibition of Fcɛ receptor I-mediated activation of Syk

Engagement of the mast cell high-affinity receptor for immunoglobulin E (IgE), FcɛRI, induces tyrosine phosphorylation of Syk, a non-receptor tyrosine kinase, that has been demonstrated as critical for degranulation. Herein we describe a synthetic compound, ER-27319, as a potent and selective inhibitor of antigen or anti-IgE-mediated degranulation of rodent and human mast cells. ER-27319 affected neither Lyn kinase activity nor the antigen-induced phosphorylation of the FcɛRI but did effectively inhibit the tyrosine phosphorylation of Syk and thus its activity. As a consequence, tyrosine phosphorylation of phospholipase C-γ1, generation of inositol phosphates, release of arachidonic acid, and secretion of histamine and tumor necrosis factor α were also inhibited. ER-27319 did not inhibit the anti-CD3-induced tyrosine phosphorylation of phospholipase C-γ1 in Jurkat T cells, demonstrating a specificity for Syk-induced signals. In contrast the tyrosine phosphorylation and activation of Syk, induced by in vitro incubation with the phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) of FcɛRI γ subunit or by antigen activation of RBL-2H3 cells, was specifically inhibited by ER-27319. However, when ER-27319 was added to immunoprecipitated Syk, derived from activated cells, no effect was seen on Syk activity. ER-27319 did not inhibit the tyrosine phosphorylation of Syk induced by activation in the presence of Igβ ITAM or the anti-IgM-induced phosphorylation of Syk in human peripheral B cells. Therefore, ER-27319 selectively interferes with the FcɛRI γ phospho-ITAM activation of Syk in vitro and in intact cells. These results confirm the importance of Syk in FcɛRI-mediated responses in mast cells and demonstrate the mast cell selectivity and therapeutic potential of ER-27319 in the treatment of allergic disease.

The killing of Leishmania major by human macrophages is mediated by nitric oxide induced after ligation of the Fc epsilon RII/CD23 surface antigen.

Serum IgE concentrations and the expression of the low-affinity receptor for IgE (Fc epsilon RII/CD23) are increased in cutaneous leishmaniasis or after immune challenge with Leishmania antigens. In vitro, the ligation of CD23 by IgE-anti-IgE immune complexes (IgE-IC) or by anti-CD23 monoclonal antibody (mAb) induces nitric oxide (NO) synthase and the generation of various cytokines by human monocytes/macrophages. The present study shows that IgE-IC, via CD23 binding, induce intracellular killing of Leishmania major in human monocyte-derived macrophages through the induction of the L-arginine:NO pathway. This was demonstrated by increased generation of nitrite (NO2-), the stable oxidation product of NO, and by the ability of NG-monomethyl-L-arginine to block both NO generation and parasite killing. A similar NO-dependent effect was observed with interferon gamma-treated cells. Tumor necrosis factor alpha is involved in this process, since both the induction of NO synthase and the killing of parasites caused by anti-CD23 mAb were inhibited by an anti-tumor necrosis factor alpha mAb. Treatment of noninfected CD23+ macrophages with IgE-IC provided protection against subsequent in vitro infection of these cells by Leishmania major promastigotes. Thus, IgE-IC promote killing of L. major by inducing NO synthase in human macrophages.

Activation of Ca2+ signaling in neutrophils by the mast cell-released immunophilin FKBP12.

The immunophilins of the FK506-binding protein (FKBP) family are intracellular proteins that bind the immunosuppresants FK506 and rapamycin. In this study we show that HMC-1 mast cells sensitized with IgE release FKBP12 upon stimulation with anti-IgE. The release is rapid and not affected by actinomycin D or cycloheximide, suggesting that it is due to exocytosis from a storage compartment. FKBP12 from HMC-1 mast cells exhibits biological activity. When applied extracellularly to human neutrophils, it induces transient changes in the intracellular Ca2+ concentration ([Ca2+]i) due to Ca2+ release from intracellular stores. Inhibition of [Ca2+]i changes by ruthenium red and ryanodine indicates that ryanodine receptor/Ca2+ release channels are involved in FKBP12-induced Ca2+ signaling. Neutrophil activation by mast cell-derived FKBP12 is prevented by complexing FKBP12 with FK506 or rapamycin. These results demonstrate that extracellular FKBP12 functions as a cytokine in cell-to-cell communication. They further suggest a pathophysiological role for FKBP12 as a mediator in immediate or type I hypersensitivity and may have implications for novel therapeutic strategies in the treatment of allergic disorders with FK506 and rapamycin.

Descubriendo el virus de la Hepatitis E en Córdoba: implicancias clínicas, zoonóticas y ambientales.

La infección por el virus Hepatitis E (HEV) es un importante problema de salud pública en regiones endémicas, causando grandes brotes epidémicos. Sin embargo, en los últimos años se ha reportado la ocurrencia de casos de hepatitis E esporádicos autóctonos en países industrializados y/o zonas no endémicas. Las cepas humanas de HEV se clasifican en 4 genotipos, 1-4, los cuales a su vez se subclasifican en subtipos. Estos genotipos poseen incidencia sobre la clínica y han sido asociados con áreas geográficas y modos específicos de transmisión. Si bien el virus se transmite fundamentalmente por vía fecal-oral, la trasmisión zoonótica desde reservorios animales (principalmente porcinos) también se ha reportado, así como también su transmisión a través de consumo de alimentos derivados de carne porcina (salchichas, salames, entre otros). Durante los últimos años la actividad humana ha provocado la contaminación de los recursos hídricos en una magnitud históricamente sin precedentes, relacionado, entre otras causas, con un manejo inadecuado de las aguas residuales. En este marco se ve favorecida la diseminación de virus entéricos (entre los que se encuentra HEV) en matrices acuosas superficiales provocando contaminación de espacios acuáticos destinados a recreación, constituyendo un riesgo de infección para la población expuesta. Si bien en Argentina se han reportado casos de HEV en humanos, poco se conoce acerca de este virus en Córdoba. Actualmente, la detección del virus no está incorporada al algoritmo diagnóstico de las hepatitis virales. Hasta hace 2 meses los equipos diagnósticos para detección de IgM/IgG anti-HEV no habían sido aprobados por ANMAT en nuestro país, por lo que no había posibilidad de acceso al diagnóstico ni a estudios seroepidemiológicos específicos que evidencien su circulación. Además, el monitoreo de HEV ambiental y en alimentos es desconocido en nuestro país. El presente proyecto propone investigar el estado de situación de la circulación de HEV en Córdoba, Argentina, mediante estudios moleculares (por RT-Nested PCR), serológicos (Elisa e inmunofluorescencia) y aislamiento viral en cultivo celular, a partir de diferentes fuentes de transmisión (humanos, cerdos, alimentos y matrices acuosas) a fin de identificar su potencial de diseminación en nuestra población, su impacto en salud pública y para evaluar su posible incorporación en el algoritmo diagnóstico de hepatitis virales. El acceso a los diagnósticos serológicos permitirá concretar estudios epidemiológicos en población inmunocompetente e inmunosuprimida aportando datos inéditos en nuestra región. Además, las técnicas moleculares implementadas y optimizadas en el presente estudio podrán ser transferidas a los centros de salud que así lo requieran. El monitoreo ambiental y en alimentos, sumado al aislamiento viral permitirá detectar las fuentes de transmisión de HEV y profundizar en el perfil molecular de las cepas circulantes locales. Este trabajo aportará datos originales sobre la presencia viral en escenarios de procesamiento de carne porcina para consumo humano. Datos generados en este estudio podrán ser utilizados para evaluar la calidad sanitaria de la carne para consumo humano. Asimismo, este proyecto intenta aportar información a los programas sanitarios de la región sobre la circulación de contaminación viral de aguas recreacionales a fin de reforzar el sistema de saneamiento ambiental, mejorar el diagnóstico de calidad microbiológica de aguas superficiales e impulsar a programas de control para atenuar la diseminación de virus entéricos en nuestro medio. Estudios preliminares recientes de vigilancia viral ambiental en aguas residuales mostraron la presencia de HEV, mediante detección molecular, lo que muestra los primeros indicios de la circulación de este virus en la población de Córdoba enfatizando la necesidad de profundizar su estudio y caracterización.

Parasitos de importância para a caprinocultura e a ovinocultura potiguar

The goat and sheep industry shows up as an agricultural activity of great importance for the semiarid Northeast. However, the sheep and goats production is made with various difficulties. Among them, parasitic infections, particularly helminth infections of the gastrointestinal tract, the eimeriosis and toxoplasmosis; this one related to problems in reproduction. For this reason, the aim of this study is to to make a survey of the occurrence and some determinants of parasitic diseases that affect small ruminant flocks of the microregions Natal, Macaíba, Litoral Sul, Angicos, Vale do Açu and Borborema Potiguar. Thereunto, epidemiological tools were applied with producers, keepers or guardians of herds and also held collections of blood and feces of animals in eight properties located in seven municipalities of these microregions. The parasite load of the animals was determined through eggs and oocysts counting per gram of feces EPG and OPG, respectively. In addition, the recovery of infective larvae was made. Blood samples were used to measure the globular cell volume and the search for anti-Toxoplasma gondii IgG in sheep serum, by Enzyme Linked Immune Sorbent Assay (ELISA). For categorical variables, the statistical analysis was performed using Poisson regression, with significance level of 0.05. The analysis of the instruments showed that ivermectin is the anthelmintic used in 85,71% of properties. From the total of feces samples of the sheep (n = 179), 53,07% were positive for helminth eggs and 48,04% were positive for oocysts of Eimeria. From the samples of faeces of goats (n = 133), 72,18% were positive for helminth eggs and 96,99% for oocysts of Eimeria. The lowest EPG and OPG count was observed in the micro region of Angicos. Most of the EPG count was found in the micro region Litoral Sul and the OPG count in the micro-region Borborema Potiguar. Both cases the difference was statistically significant(p- value0,000)The most prevalent helminth genus found was Haemonchus, present in 49,87% of the sheep and 80,42% of goats. The average of hematocrit ranged from 22,91 to 33,25 in sheep and from 22,62 to 28,25 in goats. The prevalence of anti-Toxoplasma gondii IgG ranged from 63,33% to 100,00%. The goats showed to be more susceptible to infections by parasites of the gastrointestinal tract than the sheep. In all the properties was observed high prevalence of infection by T. gondii, with the lowest percentages recorded in the micro regions Angicos and Borborema Potiguar.

Uma Lectina D-lactose específica da esponja marinha Cinachyrella apion: purificação, caracterização e interação com Leishmania chagasi

Four different sponge species were screened using Ouchterlony agarose gel and immunodiffusion tests to identify cross-reactivity with the polyclonal antibody IgG anti-deglicosilated CvL, a lectin from Cliona varians. Crude extract from the sponge Cinachyrella apion showed cross-reactivity and also a strong haemmaglutinating activity towards human erythrocytes of all ABO groups. Thus, it was submitted to acetone fractionation, IgG anti-deglicosilated CvL Sepharose affinity chromatography, and Fast Protein Liquid Chromatography (FPLC-AKTA) gel filtration on a Superose 6 10 300 column to purify a novel lectin. C. apion lectin (CaL) agglutinated all types of human erythrocytes with preference for papainized type A and O erythrocytes. The haemagglutinating activity is independent of Ca2+, Mg2+ and Mn2+ ions, and it was strongly inhibited by the disaccharide D-lactose, up to a minimum concentration of 6.25 mM. CaL molecular mass determined by FPLC-AKTA gel filtration on a Superose 12 10 300 column and SDS gel electrophoresis was approximately 124 kDa, consisting of eight subunits of 15.5 kDa, assembled by hydrophobic interactions. The lectin was relatively heat- and pH-stable. Leishmania chagasi romastigotes were agglutinated by CaL, indicating that lactose receptors could be presented in this parasite stage. These findings are indicative of the physiological defense roles of CaL and its possible use in the antibiosis of pathogenic protozoa

First evidence of Schmallenberg virus introduction in Portugal, 2014

Objectives: Serra da Estrela native (SEN) breed of sheep is one of the most important in Portugal, standing responsible for the production of one of the most internationally recognized cheeses in the world, "Queijo Serra da Estrela” (protected designation of origin). One of the major risks to the sustainability of SEN dairy flocks if footrot, an infectious diseases that causes lameness, decrease in milk production, weight loss and decrease in fertility. The aim of this work was to determine which parameters become decisive for the appearance of footrot in SEN dairy flocks, while establishing associations with environmental and nutritional variables. Materials and Methods: The present study was carried out by performing an especially designed 27 multiple choice questionnaire, based on the underlying causes of lameness in livestock and in the clinical diagnosis performed by the vet techinichian of the clinical cases evaluated at the moment of the inquiry. The survey was performed during the execution of the oficial veterinary health program between February and September of 2014 by a veterinarian team from the Association of SEN Sheep Breeders (ANCOSE). The ovine producers (N=30, with a total of 1270 animals) were randomly selected from the extense area of production of “Queijo Serra da Estrela”. The parameters evaluated in the study were: season and consequent weather changes during the period of the study, floor types, hygiene conditions, bed types, the existence and use of footbaths, location of footbaths, foot trimming and foot hygiene procedures. After the construction of the database and using the Statistical Package for Social Sciences version 16.0 the frequency response for each item was calculated. Results: All SEN livestock producers are proactive in the treatment of lameness (70%). About 99.7% of lameness was related with footroot and most appeared in winter (36.7%). In some occasions there was not a specific season distribution (56.7% rainiest years). From the analyzed farms, 70% use straw as a floor bed, followed by bush (21%). 45.6% of animals were clinically diagnosed with footrot and were sheltered in straw floor. Bed quality is good in 40%, however frequent changes in the floor bed in the preceding 15 days had a higher rate of footroot diagnosed cases (33,23%) compared to monthly changes. Regarding sheepfold animal number, the distribution is proper in 36.7%, elevated in 30%, overcrowded in 6.7% and low in 23.3%. Clinically ill diagnosed animals in last category were the lowest observed (3%). Concerning hoof trimming, 76.7% trims while 23.3% reported not to perform that task. From those that perform trimming, 73.9% do it only when necessary, 21.7% at the time of clipping and 4.4% when the animal is affected. One curious result was that animals who perform trimming at clipping had higher footrot cases (52.6%), unlike those who trim only when necessary (40.2%) or even when animals are clinically ill (0.91%). Mostly all in the presence of footrot choose a local treatment (95.2%) using antibiotic sprays, instead of parenteral antibiotic treatment. Footbath is rarely used in the prevention (13.3%) of this disease and when it is misconceived (25%) and incorrectly formulated (100%). Conclusions: This study was the first performed in Portugal focusing footrot caracterization in native SEN flocks.Economic impact in SEN dairy flocks is atributed to factors such as direct decrease in milk and meat production,early slaughter of affected animals due to non responsive treatment and medical expenses.The most effective eradication method combines first of all the ability to understand the problems of SEN producers and implementation of preventive measures and treatment of footrot.Often linked to lack of formation, the advance age of portuguese producers the mentality and social factors block the advance of veterinarian intervention itself.

Frequency of specific anti-Toxoplasma gondii IgM, IgA and IgE in Colombian patients with acute and chronic ocular toxoplasmosis

We studied the frequency of specific anti-Toxoplasma IgM, IgA and IgE antibodies in serum of 28 immunocompetent Colombian patients, selected by ophthalmologists and with lesions that were compatible with ocular toxoplasmosis. Patients were classified in three groups: (i) group 1 consisted of ten patients with a first episode; (ii) group 2, with seven patients with a recurrence and (iii) group 3, consisted of eleven patients with chronic chorioretinal lesion without uveitis. We found that 10/28 (35%) of Colombian patients with ocular toxoplasmosis possessed at least one serological marker for Toxoplasma infection different from IgG. In group 1 (first episode), we found simultaneous presence of specific IgM plus IgA plus IgE in 1/10 (10%). In group 2 (recurrences) in 1/7 (14%) we found IgM and IgA test positives and in 1/7 (14%) we found IgM and IgE tests positives. In group 3 (toxoplasmic chorioretinal scar) the IgA serological test was positive in 2/11 (18%). These results show that serum IgM or IgA or IgE can be present during recurrences.