RESUMO
Loss-of-function mutations in the gene (CSTB) encoding human cystatin B, a widely expressed cysteine protease inhibitor, are responsible for a severe neurological disorder known as Unverricht-Lundborg disease (EPM1). The primary cellular events and mechanisms underlying the disease are unknown. We found that mice lacking cystatin B develop myoclonic seizures and ataxia, similar to symptoms seen in the human disease. The principal cytopathology appears to be a loss of cerebellar granule cells, which frequently display condensed nuclei, fragmented DNA and other cellular changes characteristic of apoptosis. This mouse model of EPM1 provides evidence that cystatin B, a non-caspase cysteine protease inhibitor, has a role in preventing cerebellar apoptosis.
Assuntos
Apoptose/genética , Ataxia/genética , Cerebelo/patologia , Cistatinas/deficiência , Cistatinas/genética , Inibidores de Cisteína Proteinase/deficiência , Inibidores de Cisteína Proteinase/genética , Epilepsias Mioclônicas/genética , Sequência de Aminoácidos , Animais , Ataxia/patologia , Sequência de Bases , Opacidade da Córnea/genética , Cistatina B , Cistatinas/fisiologia , Inibidores de Cisteína Proteinase/fisiologia , Primers do DNA/genética , Modelos Animais de Doenças , Epilepsias Mioclônicas/patologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Modelos Genéticos , Mutação , FenótipoRESUMO
During thymic maturation, CD4-CD8-TCR- immature thymocytes differentiate through a CD4+CD8+TCRlo intermediate into two functionally distinct mature T cell subsets: helper T cells expressing CD4 and a major histocompatibility complex (MHC) class II-restricted T cell receptor (TCR), and cytotoxic T cells expressing CD8 and and MHC class I-restricted TCR. The mutually exclusive expression of CD4 and CD8 is maintained in the periphery during expansion of these mature T cell subsets. To elucidate the mechanisms controlling CD4 and CD8 expression on differentiating thymocytes and mature peripheral T cells, we have examined the expression of human CD4 gene constructs in the lymphoid tissues of transgenic mice. Our analyses demonstrate that sequences contained within or closely linked to the human CD4 gene are sufficient to reconstitute the appropriate regulation of human CD4 expression on all thymocyte and mature peripheral T cell subsets. Specifically, appropriate developmental regulation was dependent on two sets of sequences, one contained within a 1.3-kb restriction fragment located 6.5 kb upstream of the human CD4 gene, and the other present within or immediately flanking the gene. Nucleotide sequence analysis identified the 1.3-kb restriction fragment as the likely human homologue of an enhancer found 13 kb upstream of the mouse CD4 transcription initiation site. The human CD4 transgenic mice provide a useful system for the identification and characterization of additional sequence elements that participate in human CD4 gene regulation and for the elucidation of regulatory mechanisms governing the developmental program mediating the maturation of the CD4+ and CD8+ peripheral T cell subsets.
Assuntos
Antígenos CD4/genética , Linfócitos T CD4-Positivos/imunologia , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Timo/imunologia , Animais , Linfócitos B/imunologia , Sequência de Bases , Antígenos CD4/biossíntese , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular , DNA , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Baço/citologia , Baço/imunologia , Timo/citologiaRESUMO
The PATCHED (PTC) gene encodes a Sonic hedgehog (Shh) receptor and a tumor suppressor protein that is defective in basal cell nevus syndrome (BCNS). Functions of PTC were investigated by inactivating the mouse gene. Mice homozygous for the ptc mutation died during embryogenesis and were found to have open and overgrown neural tubes. Two Shh target genes, ptc itself and Gli, were derepressed in the ectoderm and mesoderm but not in the endoderm. Shh targets that are, under normal conditions, transcribed ventrally were aberrantly expressed in dorsal and lateral neural tube cells. Thus Ptc appears to be essential for repression of genes that are locally activated by Shh. Mice heterozygous for the ptc mutation were larger than normal, and a subset of them developed hindlimb defects or cerebellar medulloblastomas, abnormalities also seen in BCNS patients.
Assuntos
Sistema Nervoso Central/embriologia , Neoplasias Cerebelares/genética , Regulação da Expressão Gênica no Desenvolvimento , Meduloblastoma/genética , Proteínas de Membrana/genética , Anormalidades Múltiplas/genética , Animais , Padronização Corporal , Linhagem da Célula , Sistema Nervoso Central/citologia , Neoplasias Cerebelares/patologia , Ectoderma/metabolismo , Endoderma/metabolismo , Genes Supressores de Tumor , Heterozigoto , Homozigoto , Peptídeos e Proteínas de Sinalização Intracelular , Meduloblastoma/patologia , Proteínas de Membrana/fisiologia , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteínas Oncogênicas/genética , Receptores Patched , Receptor Patched-1 , Receptores de Superfície Celular , Transativadores , Fatores de Transcrição/genética , Proteína GLI1 em Dedos de ZincoRESUMO
One action of cyclosporin A thought to be central to many of its immunosuppressive effects is its ability to inhibit the early events of T lymphocyte activation such as lymphokine gene transcription in response to signals initiated at the antigen receptor. Cyclosporin A was found to specifically inhibit the appearance of DNA binding activity of NF-AT, AP-3, and to a lesser extent NF-kappa B, nuclear proteins that appear to be important in the transcriptional activation of the genes for interleukin-2 and its receptor, as well as several other lymphokines. In addition, cyclosporin A abolished the ability of the NF-AT binding site to activate a linked promoter in transfected mitogen-stimulated T lymphocytes and in lymphocytes from transgenic mice. These results indicate that cyclosporin A either directly inhibits the function of nuclear proteins critical to T lymphocyte activation or inhibits the action of a more proximal member of the signal transmission cascade leading from the antigen receptor to the nucleus.
Assuntos
Ciclosporinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Proteínas Nucleares/antagonistas & inibidores , Linfócitos T/imunologia , Sequência de Bases , Linhagem Celular , Deleção Cromossômica , Elementos Facilitadores Genéticos , Genes/efeitos dos fármacos , Humanos , Interleucina-2/genética , Dados de Sequência Molecular , Mutação , Sondas de Oligonucleotídeos , Receptores de Interleucina-2/genética , Sequências Repetitivas de Ácido Nucleico , Linfócitos T/efeitos dos fármacos , Transcrição GênicaRESUMO
Mutations in the tumor suppressor gene PATCHED (PTC) are found in human patients with the basal cell nevus syndrome, a disease causing developmental defects and tumors, including basal cell carcinomas. Gene regulatory relationships defined in the fruit fly Drosophila suggest that overproduction of Sonic hedgehog (SHH), the ligand for PTC, will mimic loss of ptc function. It is shown here that transgenic mice overexpressing SHH in the skin develop many features of basal cell nevus syndrome, demonstrating that SHH is sufficient to induce basal cell carcinomas in mice. These data suggest that SHH may have a role in human tumorigenesis.
Assuntos
Síndrome do Nevo Basocelular/genética , Carcinoma Basocelular/genética , Regulação Neoplásica da Expressão Gênica , Proteínas/genética , Neoplasias Cutâneas/genética , Transativadores , Animais , Síndrome do Nevo Basocelular/metabolismo , Síndrome do Nevo Basocelular/patologia , Carcinoma Basocelular/metabolismo , Carcinoma Basocelular/patologia , Embrião de Mamíferos , Proteínas Hedgehog , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Queratinócitos/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos SCID , Camundongos Transgênicos , Mutação , Transplante de Neoplasias , Receptores Patched , Receptor Patched-1 , Biossíntese de Proteínas , Proteínas/metabolismo , Receptores de Superfície Celular , Pele/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Transplante de PeleRESUMO
Human immunoglobulin transgenic mice provide a method of obtaining human monoclonal antibodies (Mabs) using conventional hybridoma technology. We describe a novel strain of human immunoglobulin transgenic mice and the use of this strain to generate multiple high-avidity human sequence IgG kappa Mabs directed against a human antigen. The light chain transgene is derived in part from a yeast artificial chromosome clone that includes nearly half of the germline human V kappa region. In addition, the heavy-chain transgene encodes both human mu and human gamma 1 constant regions, the latter of which is expressed via intratransgene class switching. We have used these animals to isolate human IgG kappa Mabs that are specific for the human T-cell marker CD4, have high binding avidities, and are immunosuppressive in vitro. The human Mab-secreting hybridomas display properties similar to those of wild-type mice including stability, growth, and secretion levels. Mabs with four distinct specificities were derived from a single transgenic mouse, consistent with an extensive diversity in the primary repertoire encoded by the transgenes.
Assuntos
Anticorpos Monoclonais/biossíntese , Afinidade de Anticorpos , Imunoglobulina G/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antígenos CD4/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Hibridomas , Teste de Cultura Mista de Linfócitos , Camundongos , Camundongos Transgênicos , Linfócitos T/imunologiaRESUMO
BACKGROUND: The literature reports various treatment methodologies, such as trans-oral laser microsurgery, radiation therapy, total/partial laryngectomies, and concurrent radiation chemotherapy for patients with early larynx cancer. However, at the forefront of early glottis treatment is trans-oral laser microsurgery and radiation therapy, likely due to better functional and survival outcomes. Here we conduct the largest Canadian head-to-head comparison of consecutive patients treated with either radiation therapy or trans-oral laser microsurgery. Additionally, we compare these two treatments and their 5-year survival rates post treatment to add to the existing literature. METHODS: Charts of patients who were diagnosed with early glottic cancer between 2006 and 2013 were reviewed. Seventy-five patients were identified, and split into 2 groups based on their primary treatment, trans-oral laser microsurgery and radiation therapy. Kaplan-Meier survival curves, life-tables, and the log-rank statistic were reported to determine if there was a difference between the two treatment groups and their disease-specific survival, disease-free survival, and total laryngectomy-free survival. Additionally, each different survival analysis was stratified by potential confounding variables, to help conclude which treatment is more efficacious in this population. RESULTS: The 5-year disease-specific survival rate is 93.3 % σ = 0.063 and 90.8 % σ = 0.056 for patients treated with trans-oral laser microsurgery and radiation therapy, respectively (χ (2) < 0.001, p = 0.983). The disease free survival rate is 60.0 % (σ =0.121) for patients treated with trans-oral laser microsurgery, and 67.2 % (σ = 0.074) for those who received RT (χ (2) = 0.19, p = 0.663). Additionally, the total laryngectomy-free survival rate is 84.1 % (σ = 0.1) and 79.1 % (σ = 0.072) for patients' early glottic cancer treated by trans-oral laser microsurgery and radiation therapy, respectively (χ (2) = 0.235, p = 0.628). Chi-square analysis of age-group versus treatment group (χ (2) = 6.455, p = 0.04) and T-stage versus treatment group (χ (2) = 11.3, p = 0.001) revealed a statistically significant relationship, suggesting survival analysis should be stratified by these variables. However, after stratification, there was no statistically significant difference between the trans-oral laser microsurgery and radiation therapy groups in any of the survival analyses. CONCLUSION: No difference was demonstrated in the 5-year disease-specific survival, disease-free survival, and total laryngectomy-free survival, between the RT and TLM treatment groups. Additionally, both groups showed similar 5-year survival after stratifying by confounding variables.
Assuntos
Glote/cirurgia , Neoplasias Laríngeas/radioterapia , Neoplasias Laríngeas/cirurgia , Terapia a Laser/métodos , Microcirurgia/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Neoplasias Laríngeas/mortalidade , Laringectomia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Análise de Sobrevida , Taxa de SobrevidaRESUMO
BACKGROUND: Total laryngectomy (TL) is an appropriate oncologic operation for many patients with laryngeal cancer delivering excellent oncologic outcomes, however it remains beset with significant functional consequences. Following TL, the upper and lower airways are permanently disconnected, which causes unfiltered, cold air with reduced humidity to enter the tracheobronchial tree, resulting in mucus overproduction and an increase in the viscosity of the mucus. In response to this, Heat and moisture exchangers were developed to compensate for the lost functions of the upper respiratory tract and their effect on the patients' respiratory performance in addition to their quality of life. METHODS: The case records of 48 patients undergoing total laryngectomy were reviewed and data concerning demographics, surgical details, post-operative care requirements and adverse events was retrieved. Post hoc analysis of the case patients was undertaken to identify any benefit of using a heat and moisture exchanger (HME) system with particular reference to post-operative respiratory outcomes. RESULTS: There was no significant difference between case and control subjects based on demographics, extent of surgery or need for flap repair. 16 patients had used a HME and 32 patients had used external humidification (EH). Of those experiencing mucous plugging, only 3/24 (12.5 %) had used a HME system, in contrast to 21/24 (87.5 %) who used EH (Chi square = 9.375, p = 0.002). The odds ratio of having an adverse event if not using HME was 8.27 (CI = 1.94 - 35.71). Use of HME also significantly reduced the number of days requiring physiotherapy (1.75 days vs. 3.20 days, p = 0.034). CONCLUSION: Use of an HME system can reduce in-hospital complications, in particular episodes of mucus plugging, and post-operative care requirements. Furthermore, there is a cost benefit to using HME systems that warrants more widespread introduction of these devices in head and neck surgery centers.
Assuntos
Calefação , Umidade , Laringectomia/efeitos adversos , Cuidados Pós-Operatórios/instrumentação , Complicações Pós-Operatórias/prevenção & controle , Idoso , Estudos de Casos e Controles , Feminino , Calefação/instrumentação , Temperatura Alta , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
A new purification protocol has been developed for the rapid isolation to physical homogeneity of T4 endonuclease V. The enzyme was purified from an Escherichia coli strain which harbors a plasmid containing the T4 denV structural gene downstream of the lambda rightward promoter. The purification of the enzyme was monitored by pyrimidine dimer-specific nicking activity, Western blot analysis and silver or Coomassie Blue staining of SDS-polyacrylamide gels. Milligram quantities of the enzyme have been purified by the following procedure. After sonication of cells and removal of major cell debris, total protein and nucleic acids were passed over a single-stranded DNA agarose column. Endonuclease V was eluted at 650 mM KCl with a linear salt gradient yielding enzyme of approximately 20% purity and following dialysis, was applied to a chromatofocusing column. The enzyme elutes at pH 9.4 and is greater than 90% homogeneous at this step. The final purification step is CM-Sephadex chromatography which attains greater than 98% homogeneity.
Assuntos
Endodesoxirribonucleases/isolamento & purificação , Escherichia coli/enzimologia , Fagos T/enzimologia , Proteínas Virais , Desoxirribonuclease (Dímero de Pirimidina) , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Genes , Genes Virais , Cinética , Peso Molecular , Fagos T/genéticaRESUMO
Mesenchymal patterning is an active process whereby genetic commands coordinate cell adhesion, sorting and condensation, and thereby direct the formation of morphological structures. In mice that lack the Hoxa13 gene, the mesenchymal condensations that form the autopod skeletal elements are poorly resolved, resulting in missing digit, carpal and tarsal elements. In addition, mesenchymal and endothelial cell layers of the umbilical arteries (UAs) are disorganized, resulting in their stenosis and in embryonic death. To further investigate the role of Hoxa13 in these phenotypes, we generated a loss-of-function allele in which the GFP gene was targeted into the Hoxa13 locus. This allele allowed FACS isolation of mesenchymal cells from Hoxa13 heterozygous and mutant homozygous limb buds. Hoxa13(GFP) expressing mesenchymal cells from Hoxa13 mutant homozygous embryos are defective in forming chondrogenic condensations in vitro. Analysis of pro-adhesion molecules in the autopod of Hoxa13 mutants revealed a marked reduction in EphA7 expression in affected digits, as well as in micromass cell cultures prepared from mutant mesenchymal cells. Finally, antibody blocking of the EphA7 extracellular domain severely inhibits the capacity of Hoxa13(GFP) heterozygous cells to condense and form chondrogenic nodules in vitro, which is consistent with the hypothesis that reduction in EphA7 expression affects the capacity of Hoxa13(-/-) mesenchymal cells to form chondrogenic condensations in vivo and in vitro. EphA7 and EphA4 expression were also decreased in the mesenchymal and endothelial cells that form the umbilical arteries in Hoxa13 mutant homozygous embryos. These results suggest that an important role for Hoxa13 during limb and UA development is to regulate genes whose products are required for mesenchymal cell adhesion, sorting and boundary formation.
Assuntos
Proteínas de Homeodomínio/genética , Botões de Extremidades , Receptores Proteína Tirosina Quinases/genética , Artérias Umbilicais/embriologia , Animais , Padronização Corporal/genética , Adesão Celular/genética , Indução Embrionária , Efrina-A3 , Efrina-A4 , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Heterozigoto , Proteínas de Homeodomínio/metabolismo , Masculino , Proteínas de Membrana/genética , Mesoderma , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Mutação , Receptores Proteína Tirosina Quinases/imunologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptor EphA7RESUMO
A common practice in immunoassay is the use of sequential dilutions of an initial stock solution of the antigen of interest to obtain standard samples in a desired concentration range. Nonlinear, heteroscedastic regression models are a common framework for analysis, and the usual methods for fitting the model assume that measured responses on the standards are independent. However, the dilution procedure introduces a propagation of random measurement error that may invalidate this assumption. We demonstrate that failure to account for serial dilution error in calibration inference on unknown samples leads to serious inaccuracy of assessments of assay precision such as confidence intervals and precision profiles. Techniques for taking serial dilution error into account based on data from multiple assay runs are discussed and are shown to yield valid calibration inferences.
Assuntos
Imunoensaio/normas , Algoritmos , Alérgenos/análise , Animais , Antígenos de Dermatophagoides , Asma/etiologia , Biometria , Criança , Poeira/efeitos adversos , Poeira/análise , Ensaio de Imunoadsorção Enzimática/normas , Ensaio de Imunoadsorção Enzimática/estatística & dados numéricos , Glicoproteínas/análise , Glicoproteínas/imunologia , Humanos , Imunoensaio/estatística & dados numéricos , Ácaros/imunologia , Modelos Estatísticos , Método de Monte Carlo , Radioimunoensaio/normas , Radioimunoensaio/estatística & dados numéricos , Padrões de ReferênciaRESUMO
BACKGROUND: Chemical agents such as tannic acid and detergents have been shown to introduce non-random bias in allergen measurement. OBJECTIVE: We investigated how several substances that are commonly found in floor dust (carpet fresheners, powdered pesticides, and table salt) affected immunoassays of purified standard allergens. METHODS: Three sets of experiments were conducted to: (1) screen for interference with allergen enzyme-linked immunosorbent assay (ELISA); (2) test for concentration-response; and (3) assess the site-of-action of a given dust additive (i.e. the effect on allergen binding to primary or secondary antibody). The ELISAs are commercially available two-site monoclonal antibody assays for Der p 1, Der f 1, and Fel d 1, and a monoclonal/polyclonal assay for Bla g 1. Outcomes are reported in terms of reaction rate (colour change per unit time), which is directly proportional to the amount of bound allergen. RESULTS: In the initial screening experiments, carpet fresheners tended to decrease Der p 1 assay reaction rates, increase Der f 1 assay rates, and produce little change in Fel d 1 assay rates. Three carpet fresheners decreased Der p 1 assay rate responses in a concentration-dependent manner. Two carpet fresheners noticeably increased Der f 1 assay reaction rates in both the screening and the concentration-response tests. Powdered pesticides increased reaction rates in the Bla g 1 assays and increased the slope of the dilution curve compared with that of the purified allergen. Salt decreased the reaction rates of Bla g 1 assays at allergen concentrations greater than 0.01 U/mL. For each of the four allergens, the largest effects of dust additives occurred when secondary antibody binding was altered. CONCLUSIONS: Some common household dust components can introduce systematic error into immunoassays for arthropod allergens.
Assuntos
Alérgenos/análise , Pisos e Cobertura de Pisos , Antígenos de Dermatophagoides , Poeira , Ensaio de Imunoadsorção Enzimática , Glicoproteínas/análise , Praguicidas/farmacologia , Bicarbonato de Sódio/farmacologiaRESUMO
The expression of the T4 denV gene, which previously had been cloned in plasmid constructs downstream of the bacteriophage lambda hybrid promoter-operator oLpR, was analyzed under a variety of growth parameters. Expression of the denV gene product, endonuclease V, was confirmed in DNA repair-deficient Escherichia coli (uvrA recA) by Western blot analyses and by enhancements of resistance to UV irradiation.
Assuntos
Endodesoxirribonucleases/genética , Escherichia coli/genética , Genes Virais , Genes , Fagos T/genética , Clonagem Molecular , DNA Recombinante , Desoxirribonuclease (Dímero de Pirimidina) , Endodesoxirribonucleases/biossíntese , Escherichia coli/enzimologia , Escherichia coli/efeitos da radiação , Plasmídeos , Fagos T/enzimologia , Raios UltravioletaRESUMO
Patched (Ptc) is a human tumor suppressor protein and a candidate receptor for Hedgehog (Hh) proteins, which regulate growth and patterning in embryos. Ptc represses expression of Hh target genes such as Gli1 and ptc1 itself. Localized secretion of Hh appears to induce transcription of target genes in specific patterns by binding to Ptc and preventing it from functioning in recipient cells. People who are heterozygous for PTC1 exhibit a range of developmental defects, suggesting that some genes are inappropriately expressed when there is not enough Ptc protein. To test the idea that a balance between Hh and Ptc activities is essential for normal development, we overexpressed Ptc in the neural tube. We find that excess Ptc is sufficient to inhibit expression of Gli1 and ptc1, suggesting that Sonic hedgehog (Shh) cannot signal effectively. This leads to partial dorsalization of the neural tube and a wide spectrum of neural defects, ranging from embryonic lethality to hydrocephaly.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/genética , Sistema Nervoso/embriologia , Proteínas/genética , Transativadores , Animais , Indução Embrionária/genética , Marcação de Genes , Proteínas Hedgehog , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Transgênicos , Fenômenos Fisiológicos do Sistema Nervoso , Receptores Patched , Receptor Patched-1 , Receptores de Superfície CelularRESUMO
Hedgehog (Hh) proteins control many developmental events by inducing specific cell fates or regulating cell proliferation. The Patched1 (Ptc1) protein, a binding protein for Hh molecules, appears to oppose Hh signals by repressing transcription of genes that can be activated by Hh. Sonic hedgehog (Shh), one of the vertebrate homologs of Hh, controls patterning and growth of the limb but the early embryonic lethality of ptc1(-)(/)(-) mice obscures the roles of ptc1 in later stages of development. We partially rescued ptc1 homozygous mutant embryos using a metallothionein promoter driving ptc1. In a wild-type background, the transgene causes a marked decrease in animal size starting during embryogenesis, and loss of anterior digits. In ptc1 homozygotes, a potent transgenic insert allowed survival to E14 and largely normal morphology except for midbrain overgrowth. A less potent transgene gave rise to partially rescued embryos with massive exencephaly, and polydactyly and branched digits in the limbs. The polydactyly was preceded by unexpected anterior limb bud transcription of Shh, so one function of ptc1 is to repress Shh expression in the anterior limb bud.
Assuntos
Constituição Corporal/genética , Padronização Corporal/genética , Extremidades/embriologia , Proteínas de Membrana/genética , Transativadores , Animais , Sequência de Bases , Primers do DNA/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog , Homozigoto , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Defeitos do Tubo Neural/embriologia , Defeitos do Tubo Neural/genética , Receptores Patched , Receptor Patched-1 , Fenótipo , Polidactilia/embriologia , Polidactilia/genética , Gravidez , Proteínas/genética , Receptores de Superfície CelularRESUMO
Fungal exposure inside homes has been associated with adverse respiratory symptoms in children and adults. While fungal assessment has traditionally relied upon questionnaires, fungal growth on culture plates and spore counts, new immunoassays for extracellular polysaccharides (EPS) and beta (1-->3)-glucans have enabled quantitation of fungal agents in house dust in a more timely and cost-effective manner, possibly providing a better measure of fungal exposure. We investigated associations among measurements of EPS, beta (1-->3)-glucans and culturable fungi obtained from 23 Dutch homes. From each home, dust samples were vacuumed from the living room floor twice during the Fall, Winter and Spring seasons for a total of six collections (every 6 weeks from October 1997 to May 1998). Samples were sieved and fine dust was analyzed for EPS from Aspergillus and Penicillium spp. combined, beta (1-->3)-glucans and culturable fungi. EPS was positively associated with glucan; an increase from the 25th to the 75th percentile of glucan concentration was associated with a 1.6-fold increase in EPS concentration (95% CI = 1.3 to 2.0; p < 0.01). The most significant variables associated with EPS and glucan concentrations were the surface type that was vacuumed and the concentration of total culturable fungi (in colony forming units (CFU)/g dust), with an increase in CFU/g from the 25th to the 75th percentile associated with a 1.3 (1.1-1.6)-fold increase in glucan and a 1.7 (1.3-2.2)-fold increase in EPS concentrations. In addition, the within-home variation of EPS levels were smaller than those between homes (25,646 U/g vs. 50,635 U/g), whereas the variation of glucan levels was similar within and between homes (1,300 vs. 1,205 micrograms/g). These positive associations suggest that house dust concentrations of beta (1-->3)-glucan, and particularly those of EPS, are good markers for the overall levels of fungal concentrations in floor dust which is a surrogate for estimating airborne fungal exposure.
Assuntos
Poluição do Ar em Ambientes Fechados/análise , Antígenos de Fungos/análise , Poeira/análise , Fungos/isolamento & purificação , Glucanos/análise , Habitação , Hipersensibilidade Respiratória/epidemiologia , Adulto , Aspergillus/química , Aspergillus/isolamento & purificação , Criança , Relação Dose-Resposta a Droga , Humanos , Umidade , Países Baixos , Penicillium/química , Penicillium/isolamento & purificação , Hipersensibilidade Respiratória/etiologia , Estações do Ano , Esporos Fúngicos/isolamento & purificaçãoRESUMO
BACKGROUND: We examined seasonal variation of dust-mite (Der f 1 and Der p 1), cat (Fel d 1), and cockroach (Bla g 1) allergens in Boston, while adjusting for other covariates. Limited data are available on seasonal patterns of indoor allergen concentrations for different geographic regions in the USA. Understanding within-home seasonal variation of allergens is important epidemiologically and clinically. METHODS: From June 1995 to June 1996, dust samples were vacuumed monthly from the bed, bedroom floor, and kitchen of 20 homes. Indoor temperatures were measured monthly and used in calculating relative and absolute humidity. Monthly home characteristics questionnaires were completed by an adult resident of each home. Dust samples were assayed by enzyme-linked immunosorbent assays. RESULTS: Der f 1 and Der p 1 in beds and floors peaked in the autumn months, Fel d 1 peaked in winter and spring, and Bla g 1 was highest in summer. Dust-mite allergen concentrations were 1.9-2.4 times higher in autumn than spring, but the levels in beds were 19-31 times higher in houses than those in apartments. Although Fel d 1 levels in beds were 2.4 times higher in spring than summer, homes with cats had levels 224 times higher than those without cats. Similarly, Bla g 1 levels in kitchens were 2.1 times higher in summer than winter, but apartments had levels five times higher than those of houses. CONCLUSIONS: Sampling season is a source of within-home dust-mite, cat, and cockroach allergen variation in the northeastern USA. However, the influence of housing type and owning a cat far outweighed the seasonal variation of these indoor allergens.
Assuntos
Poluição do Ar em Ambientes Fechados/análise , Alérgenos/análise , Habitação , Adulto , Poluição do Ar , Animais , Antígenos de Dermatophagoides , Antígenos de Plantas , Boston/epidemiologia , Gatos , Baratas/imunologia , Ambiente Controlado , Glicoproteínas , Humanos , Umidade , Ácaros/imunologia , Estações do Ano , TemperaturaRESUMO
The proto-oncogene c-kit encodes a transmembrane tyrosine kinase receptor that is allelic with the murine white-spotting locus (W). W mutations affect melanogenesis, gametogenesis, and hematopoiesis during development and adult life, and they result from the partial or complete loss of c-kit function. The W42 allele is a W mutation with severe effects in both the homozygous and the heterozygous states. Previous analysis of the W42 allele identified a missense mutation in an essential amino acid of the c-kitW42 kinase domain that abolishes the in vitro kinase activity of the c-kitW42 protein but does not affect its normal expression. These results suggested that the c-kitW42 allele was a dominant negative mutation within the context of c-kit-mediated signal transduction. To further explore the dominant negative characteristics of the W42 mutation, we have generated transgenic mice in which ectopic expression is driven by the human beta-actin promoter (hAP). Two mouse lines carrying the hAP-c-kitW42 transgene show an effect on pigmentation and the number of tissue mast cells. The patchy coat color pattern of the line 695 mice may reflect variable expression of the transgene in melanoblast progenitors and their descendants and, consequently, is indicative of a function for c-kit in early melanoblasts. Germ cell development and erythropoiesis, however, do not appear to be affected by the transgene. Mice expressing the c-kitW42 transgene therefore recapitulate some of the phenotypes of mice with W mutations. These results are therefore in agreement with the molecular basis of the W42 mutation and the dominant-negative characteristics of the c-kitW42 protein product.