RESUMO
Our previous work demonstrated that basophils regulate a suite of malaria phenotypes, including intestinal mastocytosis and permeability, the immune response to infection, gametocytemia, and parasite transmission to the malaria mosquito Anopheles stephensi. Given that activated basophils are primary sources of the regulatory cytokines IL-4 and IL-13, we sought to examine the contributions of these mediators to basophil-dependent phenotypes in malaria. We generated mice with basophils depleted for IL-4 and IL-13 (baso IL-4/IL-13 (-)) and genotype controls (baso IL-4/IL-13 (+)) by crossing mcpt8-Cre and Il4/Il13fl/fl mice and infected them with Plasmodium yoelii yoelii 17XNL. Conditional deletion was associated with ileal mastocytosis and mast cell (MC) activation, increased intestinal permeability, and increased bacterial 16S levels in blood, but it had no effect on neutrophil activation, parasitemia, or transmission to A. stephensi. Increased intestinal permeability in baso IL-4/IL-13 (-) mice was correlated with elevated plasma eotaxin (CCL11), a potent eosinophil chemoattractant, and increased ileal MCs, proinflammatory IL-17A, and the chemokines MIP-1α (CCL3) and MIP-1ß (CCL4). Blood bacterial 16S copies were positively but weakly correlated with plasma proinflammatory cytokines IFN-γ and IL-12p40, suggesting that baso IL-4/IL-13 (-) mice failed to control bacterial translocation into the blood during malaria infection. These observations suggest that basophil-derived IL-4 and IL-13 do not contribute to basophil-dependent regulation of parasite transmission, but these cytokines do orchestrate protection of intestinal barrier integrity after P. yoelii infection. Specifically, basophil-dependent IL-4/IL-13 control MC activation and prevent infection-induced intestinal barrier damage and bacteremia, perhaps via regulation of eosinophils, macrophages, and Th17-mediated inflammation.
Assuntos
Translocação Bacteriana , Basófilos , Interleucina-13 , Interleucina-4 , Malária , Plasmodium yoelii , Animais , Interleucina-13/metabolismo , Basófilos/imunologia , Basófilos/metabolismo , Malária/imunologia , Camundongos , Plasmodium yoelii/imunologia , Interleucina-4/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos Endogâmicos C57BL , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/parasitologia , Camundongos Knockout , Feminino , Anopheles/parasitologia , Anopheles/imunologia , Anopheles/microbiologiaRESUMO
Malaria is strongly predisposed to bacteremia, which is associated with increased gastrointestinal permeability and a poor clinical prognosis. We previously identified mast cells (MCs) as mediators of intestinal permeability in malaria and described multiple cytokines that rise with parasitemia, including interleukin (IL)-10, which could protect the host from an inflammatory response and alter parasite transmission to Anopheles mosquitoes. Here, we used the Cre-loxP system and non-lethal Plasmodium yoelii yoelii 17XNL to study the roles of MC-derived IL-10 in malaria immunity and transmission. Our data suggest a sex-biased and local inflammatory response mediated by MC-derived IL-10, supported by early increased number and activation of MCs in females relative to males. Increased parasitemia in female MC IL-10 (-) mice was associated with increased ileal levels of chemokines and plasma myeloperoxidase (MPO). We also observed increased intestinal permeability in female and male MC IL-10 (-) mice relative to MC IL-10 (+) mice but no differences in blood bacterial 16S DNA levels. Transmission success of P. yoelii to A. stephensi was higher in female relative to male mice and from female and male MC IL-10 (-) mice relative to MC IL-10 (+) mice. These patterns were associated with increased plasma levels of pro-inflammatory cytokines in female MC IL-10 (-) mice and increased plasma levels of chemokines and markers of neutrophil activation in male MC IL-10 (-) mice. Overall, these data suggest that MC-derived IL-10 protects intestinal barrier integrity, regulates parasite transmission, and controls local and systemic host immune responses during malaria, with a female bias.
Assuntos
Anopheles , Malária , Parasitos , Plasmodium yoelii , Animais , Masculino , Feminino , Camundongos , Interleucina-10/genética , Anopheles/parasitologia , Mastócitos , Parasitemia , Citocinas , Quimiocinas , ImunidadeRESUMO
Environmental and genetic factors contribute to the etiology of autism spectrum disorder (ASD), but their interaction is less well understood. Mothers that are genetically more stress-susceptible have been found to be at increased risk of having a child with ASD after exposure to stress during pregnancy. Additionally, the presence of maternal antibodies for the fetal brain is associated with a diagnosis of ASD in children. However, the relationship between prenatal stress exposure and maternal antibodies in the mothers of children diagnosed with ASD has not yet been addressed. This exploratory study examined the association of maternal antibody response with prenatal stress and a diagnosis of ASD in children. Blood samples from 53 mothers with at least one child diagnosed with ASD were examined by ELISA. Maternal antibody presence, perceived stress levels during pregnancy (high or low), and maternal 5-HTTLPR polymorphisms were examined for their interrelationship in ASD. While high incidences of prenatal stress and maternal antibodies were found in the sample, they were not associated with each other (p = 0.709, Cramér's V = 0.051). Furthermore, the results revealed no significant association between maternal antibody presence and the interaction between 5-HTTLPR genotype and stress (p = 0.729, Cramér's V = 0.157). Prenatal stress was not found to be associated with the presence of maternal antibodies in the context of ASD, at least in this initial exploratory sample. Despite the known relationship between stress and changes in immune function, these results suggest that prenatal stress and immune dysregulation are independently associated with a diagnosis of ASD in this study population, rather than acting through a convergent mechanism. However, this would need to be confirmed in a larger sample.
RESUMO
Almonds (Prunus dulcis) are one of the most consumed tree nuts worldwide and have been recognized as a healthy and nutritious food. Nevertheless, almonds are also a source of allergenic proteins that can trigger several mild to life-threatening allergic reactions. The effects of selected extraction conditions (aqueous vs. protease-assisted aqueous extraction) on the protein profile determined by proteomics analysis of excised SDS-PAGE gel bands, in vitro protein digestibility, and immunoreactivity of almond protein extracts, were evaluated. Proteolysis altered almond protein sequential and conformational characteristics thus affecting digestibility and antigenicity. Proteomics analysis revealed that enzymatic extraction resulted in the reduction of allergen proteins and epitopes. While complete hydrolysis of Prunin 1 and 2 α-chain was observed, Prunin 1 and 2 ß-chains were more resistant to hydrolysis. Protein in vitro digestibility increased from 79.1 to 88.5% after proteolysis, as determined by a static digestion model. The degree of hydrolysis (DH) and peptide content of enzymatically extracted proteins during gastric and duodenal digestion were significantly higher than the ones from unhydrolyzed proteins. Proteolysis resulted in a 75% reduction in almond protein immunoreactivity as determined by a sandwich enzyme-linked immunosorbent assay and a reduction in IgE and IgG reactivities using human sera. The present study shows that moderated hydrolysis (7% DH) using protease can be used as a strategy to improve almond protein digestibility and reduce antigenicity. This study's findings could further enhance the potential use of almond protein hydrolysates in the formulation of hypoallergenic food products with improved nutritional quality and safety.
RESUMO
OBJECTIVE: The objective of this study was to investigate the presence of maternal autoantibody-related autism spectrum disorder (MAR-ASD) in 2 geographically distinct DBPNet clinical sites (Pennsylvania and Arkansas). MAR-ASD is a biologically defined subtype of ASD that is defined by the presence of autoantibodies specific to proteins in the fetal brain and present in approximately 20% of a Northern California sample but has not been studied in other states. METHODS: Sixty-eight mothers of children with ASD were recruited from 2 DBPNet clinics and provided blood samples. Mothers also completed behavioral questionnaires about their children, and data from the child's clinical diagnostic assessment were abstracted. RESULTS: The mean age of mothers was 38.5 ± 6.1 years, and the mean age of children was 8.3 ± 2.7 years. MAR-ASD was present in 24% of the sample and similar across sites. Children of +MAR mothers had more severe autism symptoms as measured by Autism Diagnostic Observation Schedule comparison scores (W = 3604; p < 0.001) and the Social Communication Questionnaire (W = 4556; p < 0.001). There were no differences in IQ, adaptive function, or aberrant behavior. CONCLUSION: MAR-ASD is a subtype of autism that is present in similar frequencies across 3 states and related to autism severity.
Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Adulto , Transtorno do Espectro Autista/diagnóstico , Transtorno do Espectro Autista/epidemiologia , Autoanticorpos , Criança , Pré-Escolar , Feminino , Humanos , Mães , Projetos PilotoRESUMO
We have recently demonstrated that basophils are protective against intestinal permeability during malaria and contribute to reduced parasite transmission to mosquitoes. Given that IL-18 is an early cytokine/alarmin in malaria and has been shown to activate basophils, we sought to determine the role of the basophil IL-18R in this protective phenotype. To address this, we infected control [IL18r flox/flox or basoIL-18R (+)] mice and mice with basophils lacking the IL-18R [IL18r flox/flox × Basoph8 or basoIL-18R (-)] with Plasmodium yoelii yoelii 17XNL, a nonlethal strain of mouse malaria. Postinfection (PI), intestinal permeability, ileal mastocytosis, bacteremia, and levels of ileal and plasma cytokines and chemokines were measured through 10 d PI. BasoIL-18R (-) mice exhibited greater intestinal permeability relative to basoIL-18R (+) mice, along with increased plasma levels of proinflammatory cytokines at a single time point PI, day 4 PI, a pattern not observed in basoIL-18R (+) mice. Surprisingly, mosquitoes fed on basoIL-18R (-) mice became infected less frequently than mosquitoes fed on basoIL-18R (+) mice, with no difference in gametocytemia, a pattern that was distinct from that observed previously with basophil-depleted mice. These findings suggest that early basophil-dependent protection of the intestinal barrier in malaria is mediated by IL-18, and that basophil IL-18R-dependent signaling differentially regulates the inflammatory response to infection and parasite transmission.
Assuntos
Culicidae , Mucosa Intestinal , Malária , Parasitos , Receptores de Interleucina-18 , Animais , Basófilos , Permeabilidade da Membrana Celular , Culicidae/parasitologia , Citocinas , Imunidade , Interleucina-18 , Mucosa Intestinal/parasitologia , Malária/parasitologia , Camundongos , Receptores de Interleucina-18/metabolismo , Receptores de Interleucina-18/fisiologiaRESUMO
Malaria-induced bacteremia has been shown to result from intestinal mast cell (MC) activation. The appearance of MCs in the ileum and increased intestinal permeability to enteric bacteria are preceded by an early Th2-biased host immune response to infection, characterized by the appearance of IL-4, IL-10, mast cell protease (Mcpt)1 and Mcpt4, and increased circulating basophils and eosinophils. Given the functional similarities of basophils and MCs in the context of allergic inflammation and the capacity of basophils to produce large amounts of IL-4, we sought to define the role of basophils in increased intestinal permeability, in MC influx, and in the development of bacteremia in the context of malaria. Upon infection with nonlethal Plasmodium yoelii yoelii 17XNL, Basoph8 × ROSA-DTα mice or baso (-) mice that lack basophils exhibited increased intestinal permeability and increased ileal MC numbers, without any increase in bacterial 16S ribosomal DNA copy numbers in the blood, relative to baso (+) mice. Analysis of cytokines, chemokines, and MC-associated factors in the ileum revealed significantly increased TNF-α and IL-13 at day 6 postinfection in baso (-) mice compared with baso (+) mice. Moreover, network analysis of significantly correlated host immune factors revealed profound differences between baso (-) and baso (+) mice following infection in both systemic and ileal responses to parasites and translocated bacteria. Finally, basophil depletion was associated with significantly increased gametocytemia and parasite transmission to Anopheles mosquitoes, suggesting that basophils play a previously undescribed role in controlling gametocytemia and, in turn, mammalian host-to-mosquito parasite transmission.
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Bacteriemia , Basófilos , Culicidae , Malária , Animais , Bacteriemia/etiologia , Interleucina-4 , Malária/complicações , Malária/transmissão , Camundongos , PermeabilidadeRESUMO
Maternal autoantibody-related ASD (MAR ASD) is a subtype of autism in which pathogenic maternal autoantibodies (IgG) cross the placenta, access the developing brain, and cause neurodevelopmental alterations and behaviors associated with autism in the exposed offspring. We previously reported maternal IgG response to eight proteins (CRMP1, CRMP2, GDA LDHA, LDHB, NSE, STIP1, and YBOX) and that reactivity to nine specific combinations of these proteins (MAR ASD patterns) was predictive of ASD risk. The aim of the current study was to validate the previously identified MAR ASD patterns (CRMP1 + GDA, CRMP1 + CRMP2, NSE + STIP1, CRMP2 + STIP1, LDHA + YBOX, LDHB + YBOX, GDA + YBOX, STIP1 + YBOX, and CRMP1 + STIP1) and their accuracy in predicting ASD risk in a prospective cohort employing maternal samples collected prior to parturition. We used prenatal plasma from mothers of autistic children with or without co-occurring intellectual disability (ASD = 540), intellectual disability without autism (ID = 184) and general population controls (GP = 420) collected by the Early Markers for Autism (EMA) study. We found reactivity to one or more of the nine previously identified MAR ASD patterns in 10% of the ASD group compared with 4% of the ID group and 1% of the GP controls (ASD vs GP: Odds Ratio (OR) = 7.81, 95% Confidence Interval (CI) 3.32 to 22.43; ASD vs ID: OR = 2.77, 95% CI (1.19-7.47)) demonstrating that the MAR ASD patterns are strongly associated with the ASD group and could be used to assess ASD risk prior to symptom onset. The pattern most strongly associated with ASD was CRMP1 + CRMP2 and increased the odds for an ASD diagnosis 16-fold (3.32 to >999.99). In addition, we found that several of these specific MAR ASD patterns were strongly associated with ASD with intellectual disability (ASD + ID) and others associated with ASD without ID (ASD-no ID). Prenatal screening for these MAR patterns may lead to earlier identification of ASD and facilitate access to the appropriate early intervention services based on each child's needs.
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Transtorno do Espectro Autista , Deficiência Intelectual , Criança , Gravidez , Feminino , Humanos , Deficiência Intelectual/etiologia , Estudos Prospectivos , Transtorno do Espectro Autista/etiologia , Autoanticorpos , Biomarcadores , Imunoglobulina GRESUMO
An increase in mast cells (MCs) and MCs mediators has been observed in malaria-associated bacteremia, however, the role of these granulocytes in malarial immunity is poorly understood. Herein, we studied the role of mouse MC protease (Mcpt) 4, an ortholog of human MC chymase, in malaria-induced bacteremia using Mcpt4 knockout (Mcpt4-/-) mice and Mcpt4+/+ C57BL/6J controls, and the non-lethal mouse parasite Plasmodium yoelii yoelii 17XNL. Significantly lower parasitemia was observed in Mcpt4-/- mice compared with Mcpt4+/+ controls by day 10 post infection (PI). Although bacterial 16S DNA levels in blood were not different between groups, increased intestinal permeability to FITC-dextran and altered ileal adherens junction E-cadherin were observed in Mcpt4-/- mice. Relative to infected Mcpt4+/+ mice, ileal MC accumulation in Mcpt4-/- mice occurred two days earlier and IgE levels were higher by days 8-10 PI. Increased levels of circulating myeloperoxidase were observed at 6 and 10 days PI in Mcpt4+/+ but not Mcpt4-/- mice, affirming a role for neutrophil activation that was not predictive of parasitemia or bacterial 16S copies in blood. In contrast, early increased plasma levels of TNF-α, IL-12p40 and IL-3 were observed in Mcpt4-/- mice, while levels of IL-2, IL-10 and MIP1ß (CCL4) were increased over the same period in Mcpt4+/+ mice, suggesting that the host response to infection was skewed toward a type-1 immune response in Mcpt4-/- mice and type-2 response in Mcpt4+/+ mice. Spearman analysis revealed an early (day 4 PI) correlation of Mcpt4-/- parasitemia with TNF-α and IFN-γ, inflammatory cytokines known for their roles in pathogen clearance, a pattern that was observed in Mcpt4+/+ mice much later (day 10 PI). Transmission success of P. y. yoelii 17XNL to Anopheles stephensi was significantly higher from infected Mcpt4-/- mice compared with infected Mcpt4+/+ mice, suggesting that Mcpt4 also impacts transmissibility of sexual stage parasites. Together, these results suggest that early MCs activation and release of Mcpt4 suppresses the host immune response to P. y. yoelii 17XNL, perhaps via degradation of TNF-α and promotion of a type-2 immune response that concordantly protects epithelial barrier integrity, while limiting the systemic response to bacteremia and parasite transmissibility.
Assuntos
Anopheles/parasitologia , Permeabilidade da Membrana Celular/imunologia , Quimases/genética , Quimases/imunologia , Interações Hospedeiro-Parasita/imunologia , Malária/imunologia , Mastócitos/enzimologia , Plasmodium yoelii/imunologia , Animais , Feminino , Íleo/citologia , Íleo/patologia , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The incidence of autism spectrum disorder (ASD) has been rising, however ASD-risk biomarkers remain lacking. We previously identified the presence of maternal autoantibodies to fetal brain proteins specific to ASD, now termed maternal autoantibody-related (MAR) ASD. The current study aimed to create and validate a serological assay to identify ASD-specific maternal autoantibody patterns of reactivity against eight previously identified proteins (CRMP1, CRMP2, GDA, NSE, LDHA, LDHB, STIP1, and YBOX) that are highly expressed in developing brain, and determine the relationship of these reactivity patterns with ASD outcome severity. We used plasma from mothers of children diagnosed with ASD (n = 450) and from typically developing children (TD, n = 342) to develop an ELISA test for each of the protein antigens. We then determined patterns of reactivity a highly significant association with ASD, and discovered several patterns that were ASD-specific (18% in the training set and 10% in the validation set vs. 0% TD). The three main patterns associated with MAR ASD are CRMP1 + GDA (ASD% = 4.2 vs. TD% = 0, OR 31.04, p = <0.0001), CRMP1 + CRMP2 (ASD% = 3.6 vs. TD% = 0, OR 26.08, p = 0.0005) and NSE + STIP1 (ASD% = 3.1 vs. TD% = 0, OR 22.82, p = 0.0001). Additionally, we found that maternal autoantibody reactivity to CRMP1 significantly increases the odds of a child having a higher Autism Diagnostic Observation Schedule (ADOS) severity score (OR 2.3; 95% CI: 1.358-3.987, p = 0.0021). This is the first report that uses machine learning subgroup discovery to identify with 100% accuracy MAR ASD-specific patterns as potential biomarkers of risk for a subset of up to 18% of ASD cases in this study population.
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Transtorno do Espectro Autista , Transtorno Autístico , Autoanticorpos , Encéfalo , Criança , Feminino , Humanos , Medição de RiscoRESUMO
Despite the potential of rodent models of maternal immune activation (MIA) to identify new biomarkers and therapeutic interventions for a range of psychiatric disorders, current approaches using these models ignore two of the most important aspects of this risk factor for human disease: (i) most pregnancies are resilient to maternal viral infection and (ii) susceptible pregnancies can lead to different combinations of phenotypes in offspring. Here, we report two new sources of variability-the baseline immunoreactivity (BIR) of isogenic females prior to pregnancy and differences in immune responses in C57BL/6 dams across vendors-that contribute to resilience and susceptibility to distinct combinations of behavioral and biological outcomes in offspring. Similar to the variable effects of human maternal infection, MIA in mice does not cause disease-related phenotypes in all pregnancies and a combination of poly(I:C) dose and BIR predicts susceptibility and resilience of pregnancies to aberrant repetitive behaviors and alterations in striatal protein levels in offspring. Even more surprising is that the intermediate levels of BIR and poly(I:C) dose are most detrimental to offspring, with higher BIR and poly(I:C) doses conferring resilience to measured phenotypes in offspring. Importantly, we identify the BIR of female mice as a biomarker before pregnancy that predicts which dams will be most at risk as well as biomarkers in the brains of newborn offspring that correlate with changes in repetitive behaviors. Together, our results highlight considerations for optimizing MIA protocols to enhance rigor and reproducibility and reveal new factors that drive susceptibility of some pregnancies and resilience of others to MIA-induced abnormalities in offspring.
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Efeitos Tardios da Exposição Pré-Natal , Animais , Comportamento Animal , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Poli I-C , Gravidez , Reprodutibilidade dos TestesRESUMO
Autism spectrum disorder (ASD) is an important health issue and affects 1 in 59 children in the US. Prior studies determined that maternal autoantibody related (MAR) autism is thought to be associated with ~23% of ASD cases. We previously identified seven MAR-specific autoantigens including CRMP1, CRMP2, GDA, LDHA, LDHB, STIP1, and YBX1. We subsequently described the epitope peptide sequences recognized by maternal autoantibodies for each of the seven ASD-specific autoantigens. The aim of the current study was to expand upon our previous work and identify additional antigens recognized by the ASD-specific maternal autoantibodies, as well as to map the unique ASD-specific epitopes using microarray technology. Fetal Rhesus macaque brain tissues were separated by molecular weight and a fraction containing bands between 37 and 45 kDa was analyzed using 2-D gel electrophoresis, followed by peptide mass mapping using MALDI-TOF MS and TOF/TOF tandem MS/MS. Using this methodology, Neuron specific enolase (NSE) was identified as a target autoantigen and selected for epitope mapping. The full NSE sequence was translated into 15-mer peptides with an overlap of 14 amino acids onto microarray slides and probed with maternal plasma from mothers with an ASD child and from mothers with a Typically Developing child (TD) (ASD = 27 and TD = 21). The resulting data were analyzed by T-test. We found 16 ASD-specific NSE-peptide sequences for which four sequences were statistically significant (p < 0.05) using both the t-test and SAM t-test: DVAASEFYRDGKYDL (p = 0.047; SAM score 1.49), IEDPFDQDDWAAWSK (p = 0.049; SAM score 1.49), ERLAKYNQLMRIEEE (p = 0.045; SAM score 1.57), and RLAKYNQLMRIEEEL (p = 0.017; SAM score 1.82). We further identified 5 sequences that were recognized by both ASD and TD antibodies suggesting a large immunodominant epitope (DYPVVSIEDPFDQDDWAAW). While maternal autoantibodies against the NSE protein are present both in mothers with ASD and mothers of TD children, there are several ASD-specific epitopes that can potentially be used as MAR ASD biomarkers. Further, studies including analysis of NSE as a target protein in combination with the previously identified MAR ASD autoantigens are currently underway.
Assuntos
Transtorno do Espectro Autista , Biomarcadores , Peptídeos , Fosfopiruvato Hidratase , Animais , Transtorno do Espectro Autista/sangue , Transtorno do Espectro Autista/diagnóstico , Autoanticorpos , Autoantígenos/análise , Biomarcadores/sangue , Criança , Mapeamento de Epitopos , Feminino , Humanos , Macaca mulatta , Peptídeos/análise , Fosfopiruvato Hidratase/sangue , Fosfopiruvato Hidratase/imunologia , Espectrometria de Massas em TandemRESUMO
Mouse DC-SIGN CD209a is a type II transmembrane protein, one of a family of C-type lectin genes syntenic and homologous to human DC-SIGN. Current anti-mouse DC-SIGN monoclonal antibodies (MAbs) are unable to react with DC-SIGN in acetone-fixed cells, limiting the chance to visualize DC-SIGN in tissue sections. We first produced rabbit polyclonal PAb-DSCYT14 against a 14-aa peptide in the cytosolic domain of mouse DC-SIGN, and it specifically detected DC-SIGN and not the related lectins, SIGN-R1 and SIGN-R3 expressed in transfected CHO cells. MAbs were generated by immunizing rats and DC-SIGN knockout mice with the extracellular region of mouse DC-SIGN. Five rat IgG2a or IgM MAbs, named BMD10, 11, 24, 25, and 30, were selected and each MAb specifically detected DC-SIGN by FACS and Western blots, although BMD25 was cross-reactive to SIGN-R1. Two mouse IgG2c MAbs MMD2 and MMD3 interestingly bound mouse DC-SIGN but at 10 fold higher levels than the rat MAbs. When the binding epitopes of the new BMD and two other commercial rat anti-DC-SIGN MAbs, 5H10 and LWC06, were examined by competition assays, the epitopes of BMD11, 24, and LWC06 were identical or closely overlapping while BMD10, 30, and 5H10 were shown to bind different epitopes. MMD2 and MMD3 epitopes were on a 3rd noncompeting region of mouse DC-SIGN. DC-SIGN expressed on the cell surface was sensitive to collagenase treatment, as monitored by polyclonal and MAb. These new reagents should be helpful to probe the biology of DC-SIGN in vivo.
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Acetona/metabolismo , Anticorpos Monoclonais/metabolismo , Moléculas de Adesão Celular/imunologia , Lectinas Tipo C/imunologia , Receptores de Superfície Celular/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos CD/metabolismo , Células CHO , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Cricetinae , Cricetulus , Reações Cruzadas/imunologia , Mapeamento de Epitopos , Epitopos/metabolismo , Feminino , Hibridomas , Imunização , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Camundongos , Camundongos Knockout , Engenharia de Proteínas , Estrutura Terciária de Proteína/genética , Coelhos , Ratos , Ratos Endogâmicos WF , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/genética , Fixação de TecidosRESUMO
DC present exogenous proteins to MHC class I-restricted CD8+ T cells. This function does not require endogenous antigen synthesis within DC, providing the potential to elicit CD8+ T-cell responses to immune complexes, inactivated microbes, dying cells, and proteins such as OVA. In mice, the CD8+ or DEC-205+ DC are specialized for cross-presentation, and this subset can be increased 10-fold in numbers following Fms-like tyrosine kinase 3 ligand (Flt3L) treatment in vivo. Therefore, we studied cross-presentation by abundant Flt3L DC using HIV gag protein. When enriched by positive selection with anti-CD11c beads, cells from Flt3L mice are not only more abundant but are also more highly enriched in CD11chigh DC, particularly the DEC-205+ subset. DC cross-present HIV gag to primed CD8+ T cells, but when the antigen is delivered within an antibody to DEC-205 receptor, cross-presentation becomes 100-fold more efficient than non-targeted antigen. This finding requires gag to be engineered into anti-DEC antibody, not just mixed with antibody. Flt3L DC are a valuable tool to study cross-presentation, since their use overcomes the obstacle posed by the low number of cross-presenting DC in the steady state. These findings support future experiments to use Flt3L to enhance presentation of DC-targeted vaccines.
Assuntos
Anticorpos/imunologia , Antígenos CD/imunologia , Células Dendríticas/imunologia , HIV/imunologia , Lectinas Tipo C/imunologia , Proteínas de Membrana/imunologia , Receptores de Superfície Celular/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Cricetinae , Reações Cruzadas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade MenorRESUMO
Autism spectrum disorders (ASD) are complex neurodevelopmental disorders that manifest in childhood. Immune dysregulation and autoimmune reactivity may contribute to the etiology of ASD and are likely the result of both genetic and environmental susceptibilities. A common environmental contaminant, 2,2',4,4'-tetrabrominated biphenyl (BDE-47), was tested for differential effects on the immune response of peripheral blood mononuclear cells (PBMC) isolated from children with ASD (n=19) and age-matched typically developing controls (TD, n=18). PBMC were exposed in vitro to either 100 nM or 500 nM BDE-47, before challenge with bacterial lipopolysaccharide (LPS), an innate immune activator, with resultant cytokine production measured using the Luminex multiplex platform. The cytokine responses of LPS stimulated PBMC from ASD and TD subjects diverged in the presence of 100 nM BDE. For example, cells cultured from the TD group demonstrated significantly decreased levels of the cytokines IL-12p40, GM-CSF, IL-6, TNFalpha, and the chemokines MIP-1alpha and MIP-1beta following LPS stimulation of PBMC pretreated with 100 nM BDE-47 compared with samples treated with vehicle control (p<0.05). In contrast, cells cultured from subjects with ASD demonstrated an increased IL-1beta response to LPS (p=0.033) when pretreated with 100 nM BDE-47 compared with vehicle control. Preincubation with 500 nM BDE-47 significantly increased the stimulated release of the inflammatory chemokine IL-8 (p<0.04) in cells cultured from subjects with ASD but not in cells from TD controls. These data suggest that in vitro exposure of PBMC to BDE-47 affects cell cytokine production in a pediatric population. Moreover, PBMC from the ASD subjects were differentially affected when compared with the TD controls suggesting a biological basis for altered sensitivity to BDE-47 in the ASD population.
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Transtorno Autístico/imunologia , Imunidade Inata/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Bifenil Polibromatos/farmacologia , Células Cultivadas , Pré-Escolar , Feminino , Éteres Difenil Halogenados , Humanos , Leucócitos Mononucleares/imunologia , MasculinoRESUMO
SIGN-R1, or CD209b, is a mouse C-type lectin receptor that is expressed at high levels on macrophages in lymphoid tissues, especially within the marginal zone of the spleen. SIGN-R1 can bind and mediate the uptake of various microbial polysaccharides, including dextrans, lipopolysaccharides and pneumococcal capsular polysaccharides. It has been shown that SIGN-R1 mediates the clearance of encapsulated pneumococcus, complement fixation via binding C1q independent of antibody and innate resistance to pneumococcal infection. Recently, SIGN-R1 has also been demonstrated to bind sialylated antibody and mediate its activity to suppress autoimmunity. The carbohydrate-recognition domain (CRD) of SIGN-R1 has been cloned and overexpressed in a soluble secretory form in mammalian Chinese hamster ovary (CHO) cells. The CRD protein of SIGN-R1 was purified from CHO cell-culture supernatant and concentrated for crystallization using the hanging-drop vapour-diffusion method at 291 K. Crystals grew from a mixture of 2 M ammonium sulfate in 0.1 M bis-tris pH 5.5. Single crystals, which belonged to the monoclinic space group C2 with unit-cell parameters a = 146.72, b = 92.77, c = 77.06 A, beta = 121.66 degrees , allowed the collection of a full X-ray data set to a maximum resolution of 1.87 A.
Assuntos
Anticorpos/metabolismo , Moléculas de Adesão Celular/química , Lectinas Tipo C/química , Macrófagos/metabolismo , Polissacarídeos/metabolismo , Receptores de Superfície Celular/química , Baço/metabolismo , Animais , Células CHO , Moléculas de Adesão Celular/metabolismo , Cricetinae , Cricetulus , Cristalografia por Raios X , Lectinas Tipo C/metabolismo , Macrófagos/imunologia , Camundongos , Conformação Proteica , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Baço/imunologiaRESUMO
OBJECTIVES: To assay if plasma antibody levels in children with autism or developmental delays (DD) differ from those with typical development as an indicator of immune function and to correlate antibody levels with severity of behavioral symptoms. METHODS: Plasma was collected from children with autistic disorder (AU; n=116), DD but not autism (n=32), autism spectrum disorder but not full autism (n=27), and age-matched typically developing (TD) controls (n=96). Samples were assayed for systemic levels of immunoglobulin (IgG, IgM, IgA, and IgE) by enzyme-linked immunosorbent assay. Subjects with autism were evaluated using the Autism Diagnostic Observation Schedule and the Autism Diagnostic Interview-Revised, and all subjects were scored on the Aberrant Behavior Checklist (ABC) by the parents. Numerical scores for each of the ABC subscales as well as the total scores were then correlated with Ig levels. RESULTS: Children with AU have a significantly reduced level of plasma IgG (5.39+/-0.29 mg/mL) compared to the TD (7.72+/-0.28 mg/mL; P<0.001) and DD children (8.23+/-0.49 mg/mL; P<0.001). Children with autism also had a reduced level of plasma IgM (0.670.06 mg/mL) compared to TD (0.79+/-0.05 mg/mL; P<0.05). Ig levels were negatively correlated with ABC scores for all children (IgG: r=-0.334, P<0.0001; IgM: r=-0.167, P=0.0285). CONCLUSION: Children with AU have significantly reduced levels of plasma IgG and IgM compared to both DD and TD controls, suggesting an underlying defect in immune function. This reduction in specific Ig levels correlates with behavioral severity, where those patients with the highest scores in the behavioral battery have the most reduced levels of IgG and IgM.
Assuntos
Transtorno Autístico/epidemiologia , Transtorno Autístico/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Transtornos Mentais/epidemiologia , Criança , Pré-Escolar , Feminino , Humanos , MasculinoRESUMO
Epidemiological data suggest that environmental factors may trigger autoimmunity in genetically susceptible individuals. In primary biliary cirrhosis (PBC), it has been postulated that halogenated xenobiotics can modify self-molecules, facilitating the breakdown of tolerance to mitochondrial antigens. The transport and metabolism of xenobiotics is highly dependent on key genetic polymorphisms that alter enzymatic phenotype. We analyzed genomic DNA from 169 patients with PBC and 225 geographically and sex-matched healthy subjects for polymorphisms of genes coding for cytochromes P450 (CYPs) 2D6 (CYP2D6*4, CYP2D6*3, CYP2D6*5, and CYP2D6*6) and 2E1 (cl/c2), multidrug resistance 1 (MDR1 C3435T) P-glycoprotein, and pregnane X receptor (PXR C-25385T, C8055T, and A7635G). We compared the genotype frequencies in patients and controls and also correlated polymorphisms with PBC severity. The distributions of the studied genotypes did not significantly differ between patients and controls. However, when clinical characteristics of patients with PBC were compared according to genotype, the CYP2E1 c2 allele was associated with signs of more severe disease. In conclusion, genetic polymorphisms of CYP 2D6 and 2E1, PXR, and MDR1 do not appear to play a role in the onset of PBC.