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INTRODUCTION: Early-life exposure to antibiotics (ABX) has been linked to increases in asthma severity and prevalence in both children and laboratory animals. We explored the immunologic mechanisms behind this association using a mouse model of house dust mite (HDM)-induced asthma and early-life ABX exposure. METHODS: Mice were exposed to three short courses of ABX following weaning and experimental asthma was thereafter induced. Airway cell counts and differentials; serum immunoglobulin E (IgE); pulmonary function; lung histopathology; pulmonary regulatory T cells (Tregs); and the fecal microbiome were characterized following ABX exposure and induction of experimental asthma. RESULTS: Asthma severity was increased in mice exposed to ABX, including: airway eosinophilia, airway hyper-reactivity, serum HDM-specific IgE, and lung histopathology. ABX treatment led to sharp reduction in fecal microbiome diversity, including the loss of pro-regulatory organisms such as Lachnospira. Pulmonary Tregs were reduced with ABX treatment, and this reduction was directly proportional to diminished microbiome diversity. CONCLUSION: Intermittent exposure to ABX early in life worsened the severity of experimental asthma and reduced pulmonary Tregs; the latter change correlated with decreased microbiome diversity. These data may suggest targets for immunologic or probiotic therapy to counteract the harmful effects of childhood ABX.
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Antibacterianos/efeitos adversos , Asma/epidemiologia , Asma/etiologia , Pyroglyphidae , Linfócitos T Reguladores/citologia , Remodelação das Vias Aéreas , Alérgenos/imunologia , Animais , Antibacterianos/farmacologia , Citocinas/metabolismo , Modelos Animais de Doenças , Fezes/microbiologia , Feminino , Imunoglobulina E/sangue , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microbiota , Prevalência , RNA Ribossômico 16S/genética , Testes de Função Respiratória , Células Th2/citologiaRESUMO
Epidemiologic evidence suggests that N-acetyl-para-aminophenol (APAP) may play a role in the pathogenesis of asthma, likely through pro-oxidant mechanisms. However, no studies have investigated the direct effects of APAP on the development of allergic inflammation. To determine the likelihood of a causal relationship between APAP and asthma pathogenesis, we explored the effects of APAP on inflammatory responses in a murine house dust mite (HDM) model of allergic airway disease. We hypothesized that APAP would enhance the development of HDM-induced allergic inflammation. The HDM model consisted of once daily intranasal instillations for up to 2 weeks with APAP or vehicle administration 1 hour prior to HDM during either week 1 or 2. Primary assessment of inflammation included bronchoalveolar lavage (BAL), cytokine expression in lung tissue, and histopathology. Contrary to our hypothesis, the effects of HDM treatment were substantially diminished in APAP-treated groups compared with controls. APAP-treated groups had markedly reduced airway inflammation: including decreased inflammatory cells in the BAL fluid, lower cytokine expression in lung tissue, and less perivascular and peribronchiolar immune cell infiltration. The anti-inflammatory effect of APAP was not abrogated by an inhibitor of cytochrome P450 (P450) metabolism, suggesting that the effect was due to the parent compound or a non-P450 generated metabolite. Taken together, our studies do not support the biologic plausibility of the APAP hypothesis that APAP use may contribute to the causation of asthma. Importantly, we suggest the mechanism by which APAP modulates airway inflammation may provide novel therapeutic targets for asthma.
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Acetaminofen/farmacologia , Asma/tratamento farmacológico , Pyroglyphidae/fisiologia , Acetaminofen/efeitos adversos , Acetaminofen/uso terapêutico , Animais , Asma/induzido quimicamente , Asma/imunologia , Asma/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Citocinas/genética , Relação Dose-Resposta a Droga , Feminino , Imunoglobulinas/sangue , Imunoglobulinas/imunologia , Camundongos , Pyroglyphidae/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade da Espécie , Fatores de TempoRESUMO
BACKGROUND: Allergic asthma is a major cause of worldwide morbidity and results from inadequate immune regulation in response to innocuous, environmental antigens. The need exists to understand the mechanisms that promote nonreactivity to human-relevant allergens such as house dust mite (HDM) in order to develop curative therapies for asthma. The aim of our study was to compare the effects of short-, intermediate- and long-term HDM administration in a murine asthma model and determine the ability of long-term HDM exposure to suppress allergic inflammation. METHODS: C57BL/6 mice were intranasally instilled with HDM for short-term (2 weeks), intermediate-term (5 weeks) and long-term (11 weeks) periods to induce allergic airway disease (AAD). The severity of AAD was compared across all stages of the model via both immunological and pulmonary parameters. RESULTS: Short- and intermediate-term HDM exposure stimulated the development of AAD that included eosinophilia in the bronchoalveolar lavage fluid (BALF), pronounced airway hyperreactivity (AHR) and evidence of lung inflammation. Long-term HDM exposure promoted the suppression of AAD, with a loss of BALF eosinophilia and AHR despite persistent mononuclear inflammation in the lungs. Suppression of AAD with long-term HDM exposure was associated with an increase in both Foxp3+ regulatory T cells and IL-10-positive alveolar macrophages at the site of inflammation. CONCLUSIONS: This model recapitulates the key features of human asthma and may facilitate investigation into the mechanisms that promote immunological tolerance against clinically relevant aeroallergens.
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Asma/imunologia , Tolerância Imunológica/imunologia , Pneumonia/imunologia , Pyroglyphidae/imunologia , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/sangue , Citocinas/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Hipersensibilidade/imunologia , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: A successful transition from pediatric to adult sickle cell disease (SCD) care is paramount to continued improvements in survival. In order to enhance transition success, our pediatric SCD transition process was modified to include combined adult and pediatric provider clinics that incorporated participation by our local SCD community-based organization. All children ages 16 and over participated in this newly-formed transition program. PROCEDURE: After 5 years of implementation of the modified SCD transition program, we retrospectively studied clinical and non-clinical risk factors for an unsuccessful transition. Risk factor categories studied included patient demographics, transition clinic attendance, and disease severity. RESULTS: Thirty-two percent of patients did not transition successfully. Demographic factors such as gender, race, and type of insurance did not influence transition outcome, although travel distance to the adult SCD center was an identifiable risk factor for an unsuccessful transition. While transition clinic attendance rate did not affect transition outcomes, older age at first modified combined transition clinic visit was a significant risk factor for lack of transition. Patients with clinical markers of milder disease severity (SC and Sß(+) genotypes and no chronic transfusion therapy) were at higher risk for an unsuccessful transition than patients with severe disease. CONCLUSIONS: We have identified several risk factors for lack of transition success which will allow us to modify our transition efforts going forward to capture this highest risk subset.
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Anemia Falciforme/terapia , Acidente Vascular Cerebral/etiologia , Transição para Assistência do Adulto/estatística & dados numéricos , Adolescente , Adulto , Anemia Falciforme/complicações , Feminino , Seguimentos , Humanos , Masculino , Pediatria , Prognóstico , Estudos Retrospectivos , Fatores de Risco , Índice de Gravidade de Doença , Taxa de Sobrevida , Adulto JovemRESUMO
The role of CD8(+) T cells in the pathogenesis of asthma remains controversial, as both pro- and anti-inflammatory functions have been suggested. This study was designed to examine the endogenous CD8(+) T cell response in a biphasic ovalbumin (OVA)-induced model of allergic airway disease (AAD) and its subsequent resolution with the development of local inhalational tolerance (LIT). We observed increases in OVA-specific CD8(+) T cell numbers in the local lung compartments (bronchoalveolar lavage, lung tissue, hilar lymph node) at AAD and LIT; systemic compartments (spleen, inguinal lymph node) displayed no such increases in CD8(+) T cell numbers. OVA-specific CD8(+) T cells appeared to exhibit plasticity both phenotypically and functionally. They possessed pro-inflammatory characteristics at AAD, with high phenotypic expression of CD11a and increased functional expression of granzyme B and interferon-γ. In contrast, at LIT they showed increased phenotypic expression of the inhibitory marker NKG2A and functionally did not produce granzyme B or interferon-γ. In addition, in a discontinuous model the OVA-specific CD8(+) T cells could be recalled on re-exposure to OVA, demonstrating memory. Finally, confocal microscopy results showed that OVA-specific CD8(+) T cells at AAD are associated with B cell aggregates in lung tissue. These B cell aggregates resembled tertiary ectopic lymphoid tissue and may thus provide a local environment for the salient cellular interactions that contribute to the development of LIT.
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Linfócitos T CD8-Positivos/imunologia , Hipersensibilidade Respiratória/imunologia , Administração por Inalação , Animais , Linfócitos B/imunologia , Brônquios/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Agregação Celular/imunologia , Feminino , Granzimas/metabolismo , Tolerância Imunológica , Memória Imunológica , Imunofenotipagem , Interferon gama/metabolismo , Pulmão/imunologia , Linfonodos/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Subfamília C de Receptores Semelhantes a Lectina de Células NK/análise , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
Although functional asplenia from infarctions may be a major contributor to increased infectious mortality in sickle-cell disease (SCD), this relationship has not been fully defined. We used the transgenic Berkeley SCD mouse to define blood and splenic immunophenotypic differences in this model compared with C57BL/6 and hemizygous controls. In the serum of SCD mice, we found increased IgG2a and suppressed IgM, IgG2b, and IgA levels. Serum IL-6 levels in SCD mice were elevated, whereas IL-1α, CXCL10, and CCL5 levels were decreased. The blood of SCD mice had higher white blood cell counts, with an increased percentage of lymphocytes and decreases in other leukocytes. Immunophenotyping of lymphocytes revealed higher percentages of CD8(+) and T-regulatory cells and lower percentages of B cells. SCD mouse spleens exhibited histological disorganization, with reduction of defined lymphoid follicles and expansion of red pulp, a greater than fourfold increase in splenic mononuclear cells, marked expansion of the nucleated red blood cell fraction, and B-cell and CD8(+) T-cell lymphopenia. Within the splenic B-cell population, there was a significant decrease in B-1a B cells, with a corresponding decrease in IgA secreting plasma cells in the gut. Confocal microscopy of spleens demonstrated complete disruption of the normal lymphofollicular structure in the white pulp of SCD mice without distinct B, T, and marginal zones. Our findings suggest that altered SCD splenic morphological characteristics result in an impaired systemic immune response.
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Anemia Falciforme/imunologia , Anemia Falciforme/patologia , Imunidade/imunologia , Baço/imunologia , Baço/patologia , Anemia Falciforme/sangue , Anemia Falciforme/complicações , Animais , Linfócitos B/imunologia , Quimiocinas/sangue , Modelos Animais de Doenças , Feminino , Hemizigoto , Imunoglobulinas/sangue , Interleucina-1alfa/sangue , Interleucina-6/sangue , Contagem de Leucócitos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Soro , Linfócitos T/imunologiaRESUMO
BACKGROUND: Children with sickle cell disease (SCD) are susceptible to recurrent infections, which are often life threatening and necessitate frequent vaccinations. Given the altered baseline immunity and proinflammatory state associated with SCD, we sought to determine the relative safety and efficacy of vaccination in transgenic SCD mice. METHODS: Eight-week-old SCD mice were vaccinated with ovalbumin and aluminum hydroxide weekly for 3 wk by the intraperitoneal or intramuscular route. One week after the third vaccination, serum cytokines/chemokines, immunoglobulins, and bronchoalveolar lavage fluid cytokines were measured. RESULTS: Only SCD mice were prone to mortality associated with vaccination, as 40% of the animals died after the intraperitoneal vaccinations and 50% died after the intramuscular vaccinations. Serum IgG2b and IgM were significantly lower in SCD mice than in C57BL/6 mice after vaccination, but ovalbumin-specific IgE was significantly higher. Serum interleukin (IL)-1α, IL-2, IL-5, macrophage inflammatory protein 1α, and granulocyte macrophage-colony stimulating factor were significantly lower in SCD mice than in C57BL/6 mice after vaccination, whereas bronchoalveolar lavage fluid IL-1ß and IL-6 were increased. CONCLUSION: Mice with SCD appear to have a dysregulated immune response to vaccination. Thus, the relative safety and immunogenicity of vaccination should be studied in greater detail in the context of SCD.
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Anemia Falciforme/imunologia , Vacinação/efeitos adversos , Vacinação/mortalidade , Hidróxido de Alumínio/administração & dosagem , Hidróxido de Alumínio/efeitos adversos , Análise de Variância , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/sangue , Citocinas/imunologia , Feminino , Imunoglobulinas/sangue , Imunoglobulinas/imunologia , Injeções Intramusculares , Camundongos , Camundongos Transgênicos , Ovalbumina/administração & dosagem , Ovalbumina/efeitos adversosRESUMO
Bromelain (Br) is a cysteine peptidase (GenBank AEH26024.1) from pineapple, with over 40 years of clinical use. The constituents mediating its anti-inflammatory activity are not thoroughly characterized and no peptide biomarker exists. Our objective is to characterize Br raw material and identify peptides in the plasma of Br treated mice. After SDS-PAGE in-gel digestion, Br (VN#3507; Middletown, CT, USA) peptides were analyzed via LC/MS/MS using 95% protein probability, 95% peptide probability, and a minimum peptide number = 5. Br spiked mouse plasma (1 ug/ul) and plasma from i.p. treated mice (12 mg/kg) were assessed using SRM. In Br raw material, we identified seven proteins: four proteases, one jacalin-like lectin, and two protease inhibitors. In Br spiked mouse plasma, six proteins (ananain, bromelain inhibitor, cysteine proteinase AN11, FB1035 precursor, FBSB precursor, and jacalin-like lectin) were identified. Using LC/MS/MS, we identified the unique peptide, DYGAVNEVK, derived from FB1035, in the plasma of i.p. Br treated mice. The spectral count of this peptide peaked at 6 hrs and was undetectable by 24 hrs. In this study, a novel Br peptide was identified in the plasma of treated mice for the first time. This Br peptide could serve as a biomarker to standardize the therapeutic dose and maximize clinical utility.
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CONTEXT: Allergic asthma continues to increase despite new pharmacological advances for both acute treatment and chronic-disease management. Asthma is a multifactorial disease process with genetic, allergic, infectious, environmental, and dietary origins. Researchers are investigating the benefits of lifestyle changes and alternative asthma treatments, including the ability of bromelain to inhibit inflammation. Bromelain is a commonly used, proteolytically active pineapple extract. OBJECTIVE: The present study intended to determine the ability of bromelain to reduce the inflammation of preexisting asthma via an ovalbumin (OVA)-induced murine model of allergic airway disease (AAD). DESIGN: The research team designed a study examining the effects of bromelain in a control group of mice that received phosphate buffered saline (PBS) only and in an intervention group that received bromelain in PBS. Setting The study took place in the Department of Immunology at the University of Connecticut's School of Medicine, Farmington. Intervention The research team sensitized female C57BL/6J mice with intraperitoneal OVA/alum and then challenged them with OVA aerosolization for 10 consecutive days. On day 4, the team began administering daily doses of PBS to the control group (n = 10) and bromelain (6mg/kg) in PBS to the bromelain (intervention) group (n = 10). OUTCOME MEASURES: The primary measures included bronchoalveolar lavage (BAL) cellular differential, cellular phenotype via flow cytometry, and lung histology. Additional outcomes included testing for serum cytokines and immunoglobulin. RESULTS: Bromelain treatment of AAD mice (bromelain group) resulted in significant anti-inflammatory activity as indicated by reduced BAL total leukocytes (P < .05), eosinophils (P < .05), and cellular infiltrates via lung pathology (P < .005), as compared to the control group. In addition, bromelain significantly reduced BAL CD4+ and CD8+ T cells without affecting cell numbers in the spleen or hilar lymph node. The study found decreased interleukins IL-4, IL-12, IL-17, as well as IFN-α in the serum of bromelain-treated animals. CONCLUSIONS: The results suggest that bromelain has a therapeutic effect in established AAD, which may translate into an effective adjunctive therapy in patients with similar conditions, such as allergic asthma, who have chosen to initiate treatment after the onset of symptoms.
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Anti-Inflamatórios não Esteroides/farmacologia , Asma/tratamento farmacológico , Asma/imunologia , Bromelaínas/farmacologia , Alérgenos/imunologia , Animais , Asma/prevenção & controle , Líquido da Lavagem Broncoalveolar/imunologia , Linfócitos T CD4-Positivos/imunologia , Modelos Animais de Doenças , Feminino , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina , Receptores de Fator de Crescimento Neural/imunologia , Receptores do Fator de Necrose Tumoral/imunologiaRESUMO
Children and youth with special health care needs require more health care and related services and consequently incur more costs than other individuals. Implementation of the "medical home" concept has benefitted children with special needs, resulting in fewer unmet medical needs and more consistent health care delivery. As advances in health care have enabled an increasingly higher percentage of children with special needs to live far into adulthood, the transition from adolescence to adulthood poses new challenges in obtaining medical care, education, job training, and employment opportunities. A more comprehensive medical home paradigm for children with special needs is composed of three fundamental components: 1) home/community, 2) education, and 3) medical/dental care. These components should be developed equally and in parallel, emphasizing consumer advocacy, care coordination, education, life skills, and career development, to attain independent or minimally dependent living. This new model has been initiated at Hospital for Special Care in New Britain, Connecticut, in its Special Care Family Academy.
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Doença Crônica/reabilitação , Prestação Integrada de Cuidados de Saúde/organização & administração , Crianças com Deficiência/reabilitação , Promoção da Saúde/organização & administração , Vida Independente , Assistência Centrada no Paciente/organização & administração , Adolescente , Criança , Connecticut , Emprego , Humanos , Modelos Organizacionais , Defesa do Paciente , Apoio Social , Transição para Assistência do AdultoRESUMO
Staphylococcus aureus, a primary source of bacterial superantigen (SAg), is known to colonize the human respiratory tract and has been implicated in airway inflammation. Studies have documented a role for SAgs in respiratory disorders, such as nasal polyps, chronic obstructive pulmonary disease, chronic rhinosinusitis, and asthma. However, cellular and molecular mediators involved in SAg-mediated pulmonary disease have not been clearly identified. In this study, we investigated the effect of intranasal staphylococcal enterotoxin A (SEA) exposure on murine lung. The pathological features in the lung resulting from SEA exposure had characteristics of both obstructive and restrictive pulmonary disorders. There was also an increase in bronchoalveolar lavage protein concentration and cellularity following SEA challenge. Massive CD8(+)Vbeta3(+) T cell accumulation observed in the lung was dependent on CD4 T cell help, both for recruitment and for IFN-gamma synthesis. The primary source of IFN-gamma synthesis was CD8 T cells, and depletion of these cells abrogated disease. IFN-gamma deficiency also prevented SEA-mediated disease, and this was by enhancing early recruitment of neutrophils as detected in the bronchoalveolar lavage. Thus, IFN-gamma appeared to selectively aid the recruitment of T cells to the lungs while preventing the neutrophil accumulation. Therefore, our results show that IFN-gamma-producing CD8 T cells mediated pulmonary alveolitis and inflammation, which were dependent upon CD4 T cells for their recruitment to the lung.
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Linfócitos T CD8-Positivos/metabolismo , Enterotoxinas/farmacologia , Interferon gama/biossíntese , Pneumopatias/etiologia , Animais , Linfócitos T CD4-Positivos , Quimiotaxia de Leucócito , Enterotoxinas/administração & dosagem , Inalação , Pulmão , Camundongos , NeutrófilosRESUMO
Mucosal immunity is an important mechanism in the response to injury. Our hypothesis is that surfactant protein A (SP-A) is an autocrine factor that stimulates alveolar type II epithelial cell release of neutrophil chemotactic factors by binding to the SP-A receptor expressed by these cells. We examined (1) the effect of SP-A (20 µg/ml) or IL-1ß (10 ng/ml) on release of neutrophil chemotactic factors by primary cultures of type II cells or alveolar macrophages, and (2) the effect of intratracheal instillation of the blocking antibody to the SP-A receptor on the response to oleic acid-induced lung injury in vivo. All media and cell culture supernates were assayed for neutrophil chemotactic activity, and bronchoalveolar lavage fluid from the in vivo experiments was analyzed for inflammatory cell counts. While SP-A and media used for the cell cultures has no intrinsic neutrophil chemotactic activity, supernates from primary cultures of type II cells incubated in either SP-A or IL-1ß had twofold higher neutrophil chemotactic factor activity compared to supernates from controls. SP-A had no effect on release of neutrophil chemotactic factor by alveolar macrophages. Oleic acid-induced lung injury resulted in a marked influx of neutrophils into BAL, and this influx was reduced by 70% by pretreatment with the antibody to SP-A receptor. We conclude that SP-A stimulates the release of neutrophil chemotactic factor by alveolar type II cells, and this effect is mediated by the receptor for SP-A specifically expressed by these cells.
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Células Epiteliais Alveolares/imunologia , Fatores Quimiotáticos/metabolismo , Quimiotaxia , Imunidade nas Mucosas , Lesão Pulmonar/imunologia , Neutrófilos/imunologia , Proteína A Associada a Surfactante Pulmonar/metabolismo , Células Epiteliais Alveolares/metabolismo , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Células Cultivadas , Meios de Cultura/metabolismo , Modelos Animais de Doenças , Imunoglobulina G/administração & dosagem , Interleucina-1beta/metabolismo , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/metabolismo , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Masculino , Neutrófilos/metabolismo , Ácido Oleico , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismoRESUMO
BACKGROUND: The mechanism(s) responsible for the reduced risk of allergic disease in breastfed infants are not fully understood. Using an established murine model of asthma, we demonstrated previously that resistance to allergic airway disease transmitted from allergic mothers to breastfed offspring requires maternal B cell-derived factors. OBJECTIVE: The aim of this study was to investigate the role of offspring neonatal Fc receptor for IgG uptake by intestinal epithelial cells (FcRn) in this breast milk transferred protection from allergy. METHODS: Allergic airway disease was induced during pregnancy in C57BL/6 female mice. These allergic mothers foster nursed naive FcRn+/- or FcRn-/- progeny born to FcRn+/- females that were mated to C57BL/6J-FcRn-/- male mice. In offspring deficient in FcRn, we expected reduced levels of systemic allergen-specific IgG1, a consequence of decreased absorption of maternal IgG from the lumen of the neonatal gastrointestinal tract. Using this model, we were able to investigate how breast milk IgG affected offspring responses to allergic sensitization. RESULTS: Levels of maternal antibodies absorbed from the breast milk of allergic foster mothers were determined in weanling FcRn-sufficient or -deficient mice. Maternal transmission of allergen-specific IgG1 to breastfed FcRn-/- offspring was at levels 103-104 lower than observed in FcRn+/- or FcRn+/+ mice. Five weeks after weaning, when offspring were 8 wk old, mice were sensitized and challenged to evaluate their susceptibility to develop allergic airway disease. Protection, indicated by reduced parameters of disease (allergen-specific IgE in serum, eosinophilic inflammation in the airways and lung) were evident in FcRn-sufficient mice nursed as neonates by allergic mothers. In contrast, FcRn-deficient mice breastfed by the same mothers acquired limited, if any, protection from development of allergen-specific IgE and associated pathology. CONCLUSIONS: FcRn expression was a major factor in determining how breastfed offspring of allergic mothers acquired levels of systemic allergen-specific IgG1 sufficient to inhibit allergic sensitization in this model.
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BACKGROUND: Mice sensitized to ovalbumin develop allergic airway disease (AAD) with short-term aerosol challenge; however, airway inflammation resolves with long-term aerosol challenge, referred to as local inhalational tolerance (LIT). METHODS: We sought to determine if resolution of airway inflammation correlated with increases in lymphocyte subsets in local lung compartments, including putative regulatory T cells. RESULTS: At the AAD stage, total numbers of T and B lymphocytes in bronchoalveolar lavage (BAL) were significantly increased above controls; however, at LIT, T and B lymphocytes were significantly reduced compared to AAD. In the lung tissue, the only alteration was a significant increase in CD4+ CD25+ T cells at AAD. In the hilar lymph node (HLN), CD4+ and CD4+ CD25+ T cells were significantly increased at AAD and LIT. In addition, CD8+ T cells were significantly elevated in the HLN at LIT, and CD19+ B cells were significantly increased at AAD. Adoptive transfer of HLN lymphocytes to lymphopenic mice confirmed that AAD lymphocytes could induce airway inflammation in response to aerosol challenge, whereas LIT lymphocytes were unable to do so. Depletion of CD4+ CD25+ T cells in vivo resulted in exacerbation of inflammation at AAD and LIT. CD4+ CD25+ T cells in the HLN also displayed suppressive activity in vitro. Additionally, T cells expressing Foxp3 were increased in the BAL and HLN during LIT. CONCLUSIONS: These results indicate that lymphocytes with regulatory functions are increased and sustained in local lung compartments at LIT and that their appearance correlates with the resolution of lung inflammation.
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Asma/imunologia , Tolerância Imunológica , Pulmão/imunologia , Linfonodos/imunologia , Hipersensibilidade Respiratória/imunologia , Linfócitos T Reguladores/imunologia , Aerossóis , Alérgenos/imunologia , Animais , Antígenos CD19/metabolismo , Asma/patologia , Linfócitos B/citologia , Linfócitos B/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Antígenos CD4/metabolismo , Contagem de Células , Proliferação de Células , Doença Crônica , Modelos Animais de Doenças , Feminino , Fatores de Transcrição Forkhead/metabolismo , Inflamação/patologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Hipersensibilidade Respiratória/patologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismoRESUMO
BACKGROUND: One of the most common causes of morbidity and mortality in children with sickle cell disease (SCD) is infection with the pneumococcal bacterium (Streptococcus pneumoniae). Unfortunately, the polysaccharide-conjugate vaccine appears to be less effective in individuals with SCD when compared to the general population. We sought to better understand the relative efficacy of pneumococcal vaccination in a SCD mouse challenge model. METHODS: Transgenic control and SCD mice were monitored for mortality after intranasal pneumococcal infection or pneumococcal vaccination with Prevnar-13 and type-matched challenge. Anti-pneumococcal antibody titers were measured by ELISA and opsonophagocytosis was measured in vitro. RESULTS: Mortality after pneumococcal infection was similar between control and SCD mice. However, after three intramuscular polysaccharide-conjugate vaccinations, all control mice were protected following high-dose intranasal infection, whereas 60% of SCD mice died. Anti-pneumococcal antibody titers showed initial IgG and IgM responses in both groups, but waning titers were observed in the SCD group, even after boosting. When functionally assayed in vitro, serum from SCD mice 13 weeks after a second booster shot maintained little to no ability to opsonize pneumococci, while serum from control mice sustained a significantly higher capacity opsonization. Thus, it appears that SCD mice do not maintain antibody responses to pneumococcal polysaccharides after Prevnar-13 vaccination, thereby leaving them susceptible to mortality after type-matched infection. CONCLUSION: Our results emphasize the need to better understand the correlates of immune protection in SCD so that pneumococcal vaccines can be improved and mortality reduced in this susceptible population.
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Anemia Falciforme , Anticorpos Antibacterianos/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Vacinas Pneumocócicas , Streptococcus pneumoniae/imunologia , Anemia Falciforme/genética , Anemia Falciforme/imunologia , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Vacinas Pneumocócicas/imunologia , Vacinas Pneumocócicas/farmacologia , Fatores de TempoRESUMO
Abolishing the inhibitory signal of intracellular cAMP is a prerequisite for effector T (Teff) cell function. The regulation of cAMP within leukocytes critically depends on its degradation by cyclic nucleotide phosphodiesterases (PDEs). We have previously shown that PDE8A, a PDE isoform with 40-100-fold greater affinity for cAMP than PDE4, is selectively expressed in Teff vs. regulatory T (Treg) cells and controls CD4(+) Teff cell adhesion and chemotaxis. Here, we determined PDE8A expression and function in CD4(+) Teff cell populations in vivo. Using magnetic bead separation to purify leukocyte populations from the lung draining hilar lymph node (HLN) in a mouse model of ovalbumin-induced allergic airway disease (AAD), we found by Western immunoblot and quantitative (q)RT-PCR that PDE8A protein and gene expression are enhanced in the CD4(+) T cell fraction over the course of the acute inflammatory disease and recede at the late tolerant non-inflammatory stage. To evaluate PDE8A as a potential drug target, we compared the selective and combined effects of the recently characterized highly potent PDE8-selective inhibitor PF-04957325 with the PDE4-selective inhibitor piclamilast (PICL). As previously shown, PF-04957325 suppresses T cell adhesion to endothelial cells. In contrast, we found that PICL alone increased firm T cell adhesion to endothelial cells by ~20% and significantly abrogated the inhibitory effect of PF-04957325 on T cell adhesion by over 50% when cells were co-exposed to PICL and PF-04957325. Despite its robust effect on T cell adhesion, PF-04957325 was over two orders of magnitude less efficient than PICL in suppressing polyclonal Teff cell proliferation, and showed no effect on cytokine gene expression in these cells. More importantly, PDE8 inhibition did not suppress proliferation and cytokine production of myelin-antigen reactive proinflammatory Teff cells in vivo and in vitro. Thus, targeting PDE8 through PF-04957325 selectively regulates Teff cell interactions with endothelial cells without marked immunosuppression of proliferation, while PDE4 inhibition has partially opposing effects. Collectively, our data identify PF-04957325 as a novel function-specific tool for the suppression of Teff cell adhesion and indicate that PDE4 and PDE8 play unique and non-redundant roles in the control of Teff cell functions.
RESUMO
Multiphoton microscopy (MPM) imaging of intrinsic two-photon excited fluorescence (TPEF) is performed on humanized sickle cell disease (SCD) mouse model splenic tissue. Distinct morphological and spectral features associated with SCD are identified and discussed in terms of diagnostic relevance. Specifically, spectrally unique splenic iron-complex deposits are identified by MPM; this finding is supported by TPEF spectroscopy and object size to standard histopathological methods. Further, iron deposits are found at higher concentrations in diseased tissue than in healthy tissue by all imaging methods employed here including MPM, and therefore, may provide a useful biomarker related to the disease state. These newly characterized biomarkers allow for further investigations of SCD in live animals as a means to gain insight into the mechanisms impacting immune dysregulation and organ malfunction, which are currently not well understood.
Assuntos
Anemia Falciforme/patologia , Histocitoquímica/métodos , Processamento de Imagem Assistida por Computador/métodos , Ferro/química , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Baço/patologia , Animais , Biomarcadores , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Baço/químicaRESUMO
Mice sensitized to ovalbumin (OVA) develop allergic airway disease (AAD) with short-term daily OVA aerosol challenge; inflammation resolves with long-term OVA aerosol exposure, resulting in local inhalational tolerance (LIT). Cbl-b is an E3 ubiquitin ligase involved with CD28 signaling; Cbl-b(-/-) effector T cells are resistant to regulatory T cell-mediated suppression in vitro and in vivo. The present study utilized Cbl-b(-/-) mice to investigate the role of Cbl-b in the development of AAD and LIT. Cbl-b(-/-) mice exhibited increased airway inflammation during AAD, which failed to resolve with long-term OVA aerosol exposure. Exacerbation of inflammation in Cbl-b(-/-) mice correlated with increased proinflammatory cytokine levels and expansion of effector T cells in the BAL during AAD, but did not result in either a modulation of lymphocyte subsets in systemic tissues or in OVA-specific IgE in serum. These results implicate a role for Cbl-b in the resolution of allergic airway inflammation.
RESUMO
Comorbid asthma in sickle cell disease (SCD) confers higher rates of vaso-occlusive pain and mortality, yet the physiological link between these two distinct diseases remains puzzling. We used a mouse model of SCD to study pulmonary immunology and physiology before and after the induction of allergic airway disease (AAD). SCD mice were sensitized with ovalbumin (OVA) and aluminum hydroxide by the intraperitoneal route followed by daily, nose-only OVA-aerosol challenge to induce AAD. The lungs of naive SCD mice showed signs of inflammatory and immune processes: (1) histologic and cytochemical evidence of airway inflammation compared with naive wild-type mice; (2) bronchoalveolar lavage (BAL) fluid contained increased total lymphocytes, %CD8+ T cells, granulocyte-colony stimulating factor, interleukin 5 (IL-5), IL-7, and chemokine (C-X-C motif) ligand (CXCL)1; and (3) lung tissue and hilar lymph node (HLN) had increased CD4+, CD8+, and regulatory T (Treg) cells. Furthermore, SCD mice at AAD demonstrated significant changes compared with the naive state: (1) BAL fluid with increased %CD4+ T cells and Treg cells, lower %CD8+ T cells, and decreased interferon gamma, CXCL10, chemokine (C-C motif) ligand 2, and IL-17; (2) serum with increased OVA-specific immunoglobulin E, IL-6, and IL-13, and decreased IL-1α and CXCL10; (3) no increase in Treg cells in the lung tissue or HLN; and (4) hyporesponsiveness to methacholine challenge. In conclusion, SCD mice have an altered immunologic pulmonary milieu and physiological responsiveness. These findings suggest that the clinical phenotype of AAD in SCD mice differs from that of wild-type mice and that individuals with SCD may also have a unique, divergent phenotype perhaps amenable to a different therapeutic approach.
Assuntos
Anemia Falciforme/complicações , Anemia Falciforme/imunologia , Hipersensibilidade/complicações , Hipersensibilidade/imunologia , Inflamação/complicações , Inflamação/imunologia , Pulmão/patologia , Anemia Falciforme/sangue , Animais , Asma/sangue , Asma/complicações , Asma/imunologia , Hiper-Reatividade Brônquica/sangue , Hiper-Reatividade Brônquica/complicações , Hiper-Reatividade Brônquica/imunologia , Líquido da Lavagem Broncoalveolar , Citocinas/sangue , Modelos Animais de Doenças , Eosinófilos/metabolismo , Feminino , Hemizigoto , Hipersensibilidade/sangue , Inflamação/sangue , Contagem de Leucócitos , Pulmão/imunologia , Camundongos Endogâmicos C57BL , Muco/metabolismo , Ovalbumina/imunologia , Linfócitos T/imunologiaRESUMO
The immediate responses of the upper respiratory tract (URT) to the irritants acrolein and acetic acid were examined in healthy and allergic airway-diseased C57Bl/6J mice. Acrolein (1.1 ppm) and acetic acid (330 ppm) vapors induced an immediate increase in flow resistance, as measured in the surgically isolated URT of urethane-anesthetized healthy animals. Acrolein, but not acetic acid, induced a small URT vasodilatory response. In awake spontaneously breathing mice, both vapors induced a prolonged pause at the start of expiration (a response mediated via stimulation of nasal trigeminal nerves) and an increase in total respiratory specific airway flow resistance, the magnitude of which was similar to that observed in the isolated URT. Both responses were significantly reduced in animals pretreated with large doses of capsaicin to defunctionalize sensory nerves, strongly suggesting a role for sensory nerves in development of these responses. The breathing pattern and/or obstructive responses were enhanced in mice with ovalbumin-induced allergic airway disease. These results suggest that the primary responses to acrolein and acetic acid vapors are altered breathing patterns and airway obstruction, that sensory nerves play an important role in these responses, and that these responses are enhanced in animals with allergic airway disease.