RESUMO
Due to large outbreaks observed worldwide, Candida auris has emerged as a major threat to healthcare facilities. To prevent these phenomena, a systematic screening should be performed in patients transferred from regions where the pathogen is highly endemic. In this study, we recorded and analyzed French mycologists' current knowledge and practice regarding C. auris screening and diagnosis. Thirty-six centers answered an online questionnaire. Only 11 (30.6 %) participants were aware of any systematic screening for C. auris for patients admitted to their hospital. In the case of post-admission screening, axillae/groins (n = 21), nares (n = 7), rectum (n = 9), and mouth (n = 6) alone or various combinations were the body sites the most frequently sampled. Only six centers (8.3 %) reported using a commercially available plate allowing the differentiation of C. auris colonies from that of other Candida species, while five laboratories (13.8 %) had implemented a C. auris-specific qPCR. Considering the potential impact on infected patients and the risk of disorganization in the care of patients, it is crucial to remember to biologists and clinicians the utmost importance of systematic screening on admission.
RESUMO
A survey of mycology laboratories for antifungal susceptibility testing (AFST) was undertaken in France in 2018, to better understand the difference in practices between the participating centers and to identify the difficulties they may encounter as well as eventual gaps with published standards and guidelines. The survey captured information from 45 mycology laboratories in France on how they perform AFST (number of strains tested, preferred method, technical and quality aspects, interpretation of the MIC values, reading and interpretation difficulties). Results indicated that 86% of respondents used Etest as AFST method, with a combination of one to seven antifungal agents tested. Most of the participating laboratories used similar technical parameters to perform their AFST method and a large majority used, as recommended, internal and external quality assessments. Almost all the participating mycology laboratories (98%) reported difficulties to interpret the MIC values, especially when no clinical breakpoints are available. The survey highlighted that the current AFST practices in France need homogenization, particularly for MIC reading and interpretation.
Assuntos
Antifúngicos/uso terapêutico , Laboratórios , Testes de Sensibilidade Microbiana , Micologia , Prática Profissional/estatística & dados numéricos , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão/métodos , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão/normas , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão/estatística & dados numéricos , Farmacorresistência Fúngica , França , História do Século XXI , Humanos , Laboratórios/normas , Laboratórios/estatística & dados numéricos , Ensaio de Proficiência Laboratorial/métodos , Ensaio de Proficiência Laboratorial/estatística & dados numéricos , Testes de Sensibilidade Microbiana/métodos , Testes de Sensibilidade Microbiana/normas , Testes de Sensibilidade Microbiana/estatística & dados numéricos , Micologia/história , Micologia/métodos , Micologia/normas , Micologia/estatística & dados numéricos , Prática Profissional/normas , Controle de Qualidade , Inquéritos e QuestionáriosRESUMO
Actinomucor elegans is a fungus belonging to mucormycetes and is still probably underdiagnosed due to misidentification. Based on a recent first case of Actinomucor elegans sinusitis in Europe, in an immunocompromised patient under voriconazole treatment, this paper aims to summarize knowledge about A. elegans mucormycoses. Even if the diagnosis of mucormycosis was made using traditional mycology techniques, precise identification of the fungus could only be achieved using molecular tools. In this observation, the galactomannan dosage was positive until the introduction of treatment and surgical debridement. The patient experienced no relapse after one year. By reviewing the four previous A. elegans reported cases and describing the mycological characteristics of this species, we highlight the need to use a combination of tools to improve the diagnostic strategy in such rare and life-threatening clinical situations.
Assuntos
Mucorales/classificação , Mucorales/isolamento & purificação , Mucormicose/diagnóstico , Mucormicose/microbiologia , Adulto , Antifúngicos/administração & dosagem , Desbridamento , Europa (Continente) , Galactose/análogos & derivados , Humanos , Hospedeiro Imunocomprometido , Masculino , Mananas/sangue , Mucormicose/patologia , Mucormicose/terapiaRESUMO
OBJECTIVES: Fungi belonging to the Metarhizium anisopliae complex comprise ubiquitous arthropod pathogenic moulds used as mycopesticides. Rare cases of human infections due to M. anisopliae have been reported. We hypothesize misidentifications of fungal strains implicated in these cases or used in mycopesticides. METHODS: A review of the literature was conducted to identify previously published cases. We collected some of these previous described strains and reported new cases, and a French mycopesticide containing M. anisopliae. All identifications were performed based on elongation factor-1α gene sequencing. RESULTS: We report eight new cases of Metarhizium infection in humans (three from France and five from Australia). The strains isolated from these cases, and three others from already published cases and reported as M. anisopliae, were molecularly identified based on elongation factor-1α (Ef1-α) gene sequencing as follows: Metarhizium robertsii (six), Metarhizium guizhouense (three), Metarhizium brunneum (one) and Metarhizium pingshaense (one). CONCLUSIONS: In this study, we report new human cases of Metarhizium infections, and, based on Ef-1α gene sequencing, we demonstrate the misidentification of species in case reports. We also correct the species identification of a strain reported as M. anisopliae used in a commercially available mycopesticide. According to our results, none of the strains from the human infection reports reviewed belongs to the species M. anisopliae.
Assuntos
Metarhizium , Micoses/microbiologia , Adolescente , Adulto , Idoso , Antifúngicos/uso terapêutico , Criança , Pré-Escolar , Erros de Diagnóstico , Feminino , Genes Fúngicos/genética , Humanos , Masculino , Metarhizium/genética , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Micoses/diagnóstico , Micoses/tratamento farmacológico , Filogenia , Estudos Retrospectivos , Análise de Sequência de DNARESUMO
Dermatophytes are responsible for widespread superficial fungal infections, currently representing a real public health problem. Some of the fungi involved in these mycoses are transmitted by pets, illustrating great host specificity within this fungal group. Thus, a new variety of zoophilic dermatophyte has been described in recent years by the Mycology Laboratory of the University Hospital of Nancy, within the complex T. mentagrophytes. This variant was named T. mentagrophytes var. porcellae, following the observation of a significant number of patients with dermatomycoses of exposed parts of the body and having had contact with a guinea pig. The current work follows this first description and aims to assess the frequency of T. mentagrophytes var. porcellae in guinea pigs within three pet shops in the region of Nancy (France). In total, almost two thirds of collected guinea pigs were carriers of this new dermatophyte. This study highlights the risks associated with the adaptation of dermatophytes to potential new hosts that may spread to new species. Thus, in this context, sanitary measures could be proposed to the pet shops, usually not informed of the risks facing the growing enthusiasm of the population for new pets, in order to limit contamination.
Assuntos
Cobaias/microbiologia , Doenças dos Roedores/epidemiologia , Doenças dos Roedores/microbiologia , Tinha/epidemiologia , Tinha/microbiologia , Trichophyton/isolamento & purificação , Criação de Animais Domésticos/normas , Criação de Animais Domésticos/estatística & dados numéricos , Animais , Dermatomicoses/epidemiologia , Dermatomicoses/microbiologia , Dermatomicoses/veterinária , Surtos de Doenças/estatística & dados numéricos , Surtos de Doenças/veterinária , Feminino , França/epidemiologia , Humanos , Masculino , Tinha/veterináriaRESUMO
We have examined the evolution of the genes at the major human beta-defensin locus and the orthologous loci in a range of other primates and mammals. For the first time, these data allow us to examine selective episodes in the more recent evolutionary history of this locus as well as in the ancient past. We have used a combination of maximum-likelihood-based tests and a maximum-parsimony-based sliding window approach to give a detailed view of the varying modes of selection operating at this locus. We provide evidence for strong positive selection soon after the duplication of these genes within an ancestral mammalian genome. During the divergence of primates, however, variable selective pressures have acted on beta-defensin genes in different evolutionary lineages, with episodes of both negative and, more rarely, positive selection. Positive selection appears to have been more common in the rodent lineage, accompanying the birth of novel rodent-specific beta-defensin gene clades. Sites in the second exon have been subject to positive selection and, by implication, are important in functional diversity. A small number of sites in the mature human peptides were found to have undergone repeated episodes of selection in different primate lineages. Particular sites were consistently implicated by multiple methods at positions throughout the mature peptides. These sites are clustered at positions that are predicted to be important for the function of beta-defensins.
Assuntos
Evolução Molecular , beta-Defensinas/genética , beta-Defensinas/metabolismo , Animais , Humanos , Filogenia , Processamento de Proteína Pós-Traducional , Seleção GenéticaRESUMO
Staphylococcus aureus is a pathogen often found in pneumonia and sepsis. In the context of the resistance of this organism to conventional antibiotics, an understanding of the regulation of natural endogenous antimicrobial molecules is of paramount importance. Previous studies have shown that both human and mouse airways express a variety of these molecules, including defensins, cathelicidins, and the four-disulfide core protein secretory leukocyte protease inhibitor. We demonstrate here by culturing mouse tracheal epithelial cells at an air-liquid interface that, despite the production of Defb1, Defb14, and Defr1 in this system, these cells are unable to clear S. aureus when exposed to this respiratory pathogen. Using an adenovirus (Ad)-mediated gene transfer strategy, we show that overexpression of elafin, an anti-elastase/antimicrobial molecule (also a member of the four-disulfide core protein family), dramatically improves the clearance of S. aureus. In addition, we also demonstrate that this overexpression is efficient in vivo and that intratracheal instillation of Ad-elafin significantly reduced the lung bacterial load and demonstrates concomitant anti-inflammatory activity by reducing neutrophil numbers and markers of lung inflammation, such as bronchoalveolar lavage levels of tumor necrosis factor and myeloperoxidase. These findings show that an increased antimicrobial activity phenotype is provided by the elafin molecule and have implications for its use in S. aureus-associated local and systemic infections.
Assuntos
Terapia Genética , Pulmão/imunologia , Proteínas/genética , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Adenoviridae/genética , Animais , Feminino , Transferência Genética Horizontal , Imunidade Inata , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Secretadas Inibidoras de ProteinasesRESUMO
beta-Defensin genes encode broad spectrum antimicrobial cationic peptides. We have analysed the largest murine and human clusters of these genes, which localise to mouse and human chromosome 8. Using hidden Markov models, we identified novel mouse and human beta-defensin genes. We subsequently found full-length expressed transcripts for these novel genes. Expression in the mouse was high in brain and reproductive tissues. Fourteen murine beta-defensins could be grouped into two clear sub-groups by virtue of their position and high signal sequence (exon 1 encoded) identity. In contrast, there was a very low level of sequence conservation in the exon 2 region encoding the mature antimicrobial peptide. Evolutionary analysis revealed strong evidence that following gene duplication, exon 1 and surrounding non-coding DNA show little divergence within subfamilies. The focus for rapid sequence divergence is localised in the DNA encoding the mature peptide and this is driven by accelerated positive selection. In the human we also conclude that the locus has evolved by successive rounds of duplication followed by substantial divergence involving positive selection, to produce a diverse cluster of paralogous genes prior to human-baboon divergence. This mechanism of adaptive evolution is consistent with the role of this gene family as defence against bacterial pathogens. In order to look at function of these rapidly evolving genes, we characterised one of the novel mouse beta-defensin genes. This gene deviates from the canonical six cysteine motif present in the mature functional peptide of all other beta defensins. This defensin related gene (Defr1) is most highly expressed in testis and heart and the genomic organisation is highly similar to Defb3-6. A synthetic Defr1 peptide was shown to exist as a dimer and yet displayed both antimicrobial and chemotactic activity. The antimicrobial activity of Defr1 against S. aureus, E. coli and B. cepacia was found to be reduced in raised concentration of NaCl, but its action against P. aeruginosa was independent of NaCl concentration. These data have major implications on the structure and functions of these important host defence molecules.
Assuntos
Adaptação Fisiológica , Evolução Molecular , beta-Defensinas/genética , Sequência de Aminoácidos , Animais , Humanos , Camundongos , Dados de Sequência Molecular , Família Multigênica , FilogeniaRESUMO
Mouse models for cystic fibrosis (CF) mimic intestinal manifestations of the human disease, but the lung disease phenotypes are lacking in most strains. In this work, the issue was addressed whether aging of the respiratory tract leads to lung pathophysiology in the exon 10 insertional mutant cftr(tm1Hgu) mouse. Weight gain, body weight and life-span of cftr(tm1Hgu) mice were significantly reduced compared with control mice. cftr(tm1Hgu) mice expressed 20, 21 or 37% (median) of wild-type cystic fibrosis conductance transmembrane regulator (cftr) mRNA transcript in lungs, intestine and kidney. Wild-type cftr mRNA in renal and respiratory epithelia varied with age from levels similar to Ztm:MF1 controls at the age of 2 and 4 months to levels seen in patients with CFTR splice mutations beyond the age of 6 months. The morphology of the bronchi and more distal airways was apparently normal in cftr(tm1Hgu) mice during their first year of life. The alveolar surfactant phospholipid pool was increased in cftr(tm1Hgu) mice by 1.5- to 2-fold compared with Ztm:MF1 controls. Alveolar clearance of gamma-labelled scandium oxide - the first report of lung clearance measurement in living mice - was reduced in cftr(tm1Hgu) mice compared with littermate controls. Although no progressive lung pathology was seen in the cftr expression of cftr(tm1Hgu) mice, surfactant phospholipid homeostasis, and alveolar and mucociliary clearance were abnormal. Therefore, the described model is useful for studying the initial CF lung pathophysiology.
Assuntos
Envelhecimento , Regulador de Condutância Transmembrana em Fibrose Cística/biossíntese , Fibrose Cística/genética , Fibrose Cística/fisiopatologia , Camundongos Endogâmicos CFTR , Animais , Peso Corporal , Líquido da Lavagem Broncoalveolar/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Mucosa Intestinal/metabolismo , Rim/metabolismo , Pulmão/química , Pulmão/metabolismo , Pulmão/patologia , Pulmão/fisiopatologia , Camundongos , Modelos Animais , Depuração Mucociliar/genética , Depuração Mucociliar/fisiologia , Mutação , Fosfolipídeos/análise , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The airway surface liquid (ASL) that lines the airway surface epithelium plays a major role in airway antibacterial defense and mucociliary transport efficiency, two key factors in cystic fibrosis (CF) disease. A major difficulty is to collect ASL in native conditions without stimulation or alteration of the underlying airway epithelium. Using a cryoprobe specifically adapted to collect native ASL from the tracheal mouse surface, we analyzed by X-ray microanalysis the complete ASL and plasma ion content in Cftr(tm1Hgu)/Cftr(tm1Hgu) mice compared with that in control littermates. ASL ion content from eight Cftr(tm1Hgu)/Cftr(tm1Hgu) mice and eight control littermates did not appear significantly different. The mean (+/-SE) concentrations were 2,352 +/- 367 and 2,058 +/- 401 mmol/kg dry weight for Na, 1,659 +/- 272 and 1,448 +/- 281 mmol/kg dry weight for Cl, 357 +/- 57 and 337 +/- 38 mmol/kg dry weight for S, 1,066 +/- 220 and 787 +/- 182 mmol/kg dry weight for K, 400 +/- 82 and 301 +/- 58 mmol/kg dry weight for Ca, 105 +/- 31 and 105 +/- 20 mmol/kg dry weight for Mg, 33 +/- 15 and 29 +/- 9 mmol/kg dry weight for P in non-CF and CF mice, respectively. This cryotechnique appears to be a promising technique for analyzing the complete elemental composition of native ASL in CF and non-CF tissues.
Assuntos
Líquidos Corporais/metabolismo , Fibrose Cística/metabolismo , Microanálise por Sonda Eletrônica , Traqueia/metabolismo , Animais , Íons , Camundongos , Camundongos Endogâmicos CFTR , Valores de ReferênciaRESUMO
It is generally important to elucidate airway epithelial cell lineages and to identify multipotent progenitors as targets for gene therapy. Stem (S) cells are typically present in specialized compartments spatially proximal to their differentiated progeny, but an equivalent paradigm has not been demonstrated in the airway. We discovered a distinct population of cells displaying high levels of keratin expression in murine tracheal submucosal gland ducts, and tested the hypothesis that bromodeoxyuridine (BrdU) label-retaining cells (LRCs), thought to represent the S-cells, were present in this compartment. Mice received weekly epithelial damage by intratracheal detergent or SO(2) inhalation for 4 wk and received intraperitoneal injections of BrdU every 48 h during the injury and repair period. At 3 and 6 d after injury, BrdU-positive epithelial cells were noted along the entire tracheal length in both basal and lumenal cell positions. At later time points (20 and 95 d) LRCs were localized to gland ducts in the upper trachea and to systematically arrayed foci in the lower trachea, typically near the cartilage-intercartilage junction. LRCs were not pulmonary neuroendocrine cells. Heterotopic tracheal grafts after surface epithelial removal demonstrated reconstitution of a surface-like epithelium from gland remnants. These results suggest that airway epithelial S cells are localized to specific niches.
Assuntos
Regeneração , Mucosa Respiratória/fisiologia , Células-Tronco/fisiologia , Traqueia/fisiologia , Animais , Queratinas/biossíntese , Camundongos , Camundongos Transgênicos , Mucosa Respiratória/citologia , Mucosa Respiratória/lesões , Mucosa Respiratória/transplante , Transplante de Células-Tronco , Células-Tronco/citologia , Traqueia/citologia , Traqueia/lesões , Traqueia/transplanteRESUMO
Submucosal glands (SMG) are important secretory glands that are present in the major airways and bronchioles of humans. In mice the structure, cellular composition, and density of SMG are similar to those seen in humans, but the glands are present only in the trachea. Characterization of SMG is important as they secrete bacteriocidal products such as lactoferrin, lysozyme, and defensins believed to be of importance in the innate defense system. Serous cells in SMG are the primary site of cystic fibrosis transmembrane conductance regulator (CFTR) gene expression and the initial site of histological abnormality in cystic fibrosis (CF) individuals. In this study, we examined four inbred strains of mice (A/J, C57BL/6N, FVB/N, and BALB/CAnN) and revealed that the extent to which glands descend in the mouse trachea varied between inbred strains. In particular, the A/J and C57BL/6N strains exhibited few SMG extending further than the first or second intercartilaginous space (mean depth of 0.4+/-0.11 and 1.5+/-0.32 tracheal rings respectively) in the trachea, whereas the FVB/N and BALB/CAnN strains had SMG extending beyond the fourth space (mean depths of 3.3+/-0.46 and 5.6+/-0.45 rings respectively). We have previously shown that in congenic C57Bl/ 6N Cftr mutant mice (CF mice), the SMG are distributed more distally than in wild-type C57Bl/6N but are indistinguishable from BALB/CAnN wild-type or CF mice. The implication that SMG distribution is influenced by Cftr gene expression (or a gene closely linked to Cftr) led us to investigate the genetic difference between C57Bl6/N and BALB/CAnN mice. In recombinant inbred strain (RIS) analysis (with BALB/CJ and C57BL/6J progenitors), two loci were identified as being linked to the SMG phenotype (peak likelihood statistic levels of 8.8 and 9.9 on Chrs 9 and 10 respectively, indicating suggestive linkage). A subsequent segregation analysis of an F2 intercross between the C57BL/6N and BALB/CAnN mice indicated that there were at least two major genetic factors responsible for SMG distribution. The loci indicated in the RI analysis were included in a targeted genome scan involving 235 F2 intercross animals (C57BL/6N and BALB/CAnN strain intercross). The genome scan confirmed the locus on Chr 9 (between genetic markers D9Mit11 and D9Mit182), designated Smgdl, as significantly linked to the SMG distribution phenotype (peak LOD score 5.8) within a 95% confidence interval of 12 cM.
Assuntos
Mapeamento Cromossômico , Mucosa Respiratória/citologia , Animais , Cruzamentos Genéticos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CFTR , Fenótipo , Mucosa Respiratória/metabolismoRESUMO
The goal of this study was to develop a primary culture model of differentiated murine tracheal epithelium. When grown on semipermeable membranes at an air interface, dissociated murine tracheal epithelial cells formed confluent polarized epithelia with high transepithelial resistances ( approximately 12 kOmega. cm(2)) that remained viable for up to 80 days. Immunohistochemistry and light and electron microscopy demonstrated that the cells were epithelial in nature (cytokeratin positive, vimentin and alpha-smooth muscle actin negative) and differentiated to form ciliated and secretory cells from day 8 after seeding onward. With RT-PCR, expression of the cystic fibrosis transmembrane conductance regulator (Cftr) and murine beta-defensin (Defb) genes was detected (Defb-1 was constitutively expressed, whereas Defb-2 expression was induced by exposure to lipopolysaccharide). Finally, Ussing chamber experiments demonstrated an electrophysiological profile compatible with functional amiloride-sensitive sodium channels and cAMP-stimulated CFTR chloride channels. These data indicate that primary cultures of murine tracheal epithelium have many characteristics similar to those of murine tracheal epithelium in vivo. This method will facilitate the establishment of primary cultures of airway epithelium from transgenic mouse models of human diseases.
Assuntos
Diferenciação Celular , Mucosa Respiratória/citologia , Mucosa Respiratória/fisiologia , Amilorida/farmacologia , Animais , Bumetanida/farmacologia , Técnicas de Cultura de Células/métodos , Polaridade Celular , Separação Celular/métodos , Células Cultivadas , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Defensinas/genética , Feminino , Humanos , Queratinas/análise , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Mucosa Respiratória/ultraestrutura , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Traqueia , Vimentina/análiseRESUMO
The creation of precise clinical mutations by targeting is important in elucidating disease pathogenesis using mouse models. 'Hit and run' gene targeting is an elegant method to achieve this goal. This uses first a positive selection to introduce the targeting vector carrying the required mutation and then a negative selection to identify clones which have removed vector and wild-type sequences by intrachromosomal recombination. However, this approach has only been successfully used in a handful of cases. We used this procedure to introduce precise clinical mutations into the exon 10 region of the cystic fibrosis transmembrane conductance regulator (Cftr) gene. Using a CMV promoter driven hygromycin/thymidine kinase (hyg/tk) fusion gene as both our dominant and negative selectable marker, we targeted the Cftr locus very efficiently but only identified false runs after the negative selection step. This defect in thymidine kinase induced toxicity to gancyclovir correlated with methylation of the transgene. Consequently we devised a stringent screening procedure to select only true 'run' clones. Unfortunately these 'run' clones had lost the mutation so we altered the vector design to bias the run step to retain the mutation and used a different tk selection cassette with a HSVtk promoter sequence. This new vector design allowed both efficient 'hit and run' for two cystic fibrosis (CF) mutations with no false positives and successful germline transmission of the novel G480C missense mutation.
Assuntos
Cinamatos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Marcação de Genes/métodos , Animais , Fusão Gênica Artificial , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Citomegalovirus/genética , Resistência a Medicamentos , Ganciclovir/farmacologia , Vetores Genéticos , Higromicina B/análogos & derivados , Higromicina B/química , Metilação , Camundongos , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Simplexvirus/genética , Timidina Quinase/genéticaRESUMO
The mouse Bapx1 gene is homologous to the Drosophila homeobox containing bagpipe (bap) gene. A shared characteristic of the genes in these two organisms is expression in gut mesoderm. In Drosophila, bap functions to specify the formation of the musculature of the midgut. To determine the function of the mammalian cognate, we targeted a mutation into the Bapx1 locus. Bapx1, similar to Drosophila, does have a conspicuous role in gut mesoderm; however, this appears to be restricted to development of the spleen. In addition, Bapx1 has a major role in the development of the axial skeleton. Loss of Bapx1 affects the distribution of sclerotomal cells, markedly reducing the number that appear ventromedially around the notochord. Subsequently, the structures in the midaxial region, the intervertebral discs, and centra of the vertebral bodies, fail to form. Abnormalities are also found in those bones of the basal skull (basioccipital and basisphenoid bones) associated with the notochord. We postulate that Bapx1 confers the capacity of cells to interact with the notochord, effecting inductive interactions essential for development of the vertebral column and chondrocranium.
Assuntos
Padronização Corporal/genética , Proteínas de Drosophila , Proteínas de Homeodomínio/fisiologia , Osteogênese/genética , Baço/embriologia , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas de Homeodomínio/genética , Hibridização In Situ , Camundongos , Camundongos Knockout , Notocorda/anormalidades , Notocorda/embriologia , Fator de Transcrição PAX2 , Base do Crânio/embriologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
We present here an in vivo mouse model for intestinal stem cell function and differentiation that uses postnatal intestinal epithelial cell aggregates to generate a differentiated murine small intestinal mucosa with full crypt-villus architecture. The process of neomucosal formation is highly similar to that of intestinal regeneration. Both in vivo grafting and primary culture of these cells reveal two different epithelial cell populations, which display properties consistent with intestinal epithelial transit amplifying and stem cell populations. Using this model system with a mixture of wild-type and transgene marked cells, we have shown that neomucosae originally develop from single aggregates, but that over time the mucosae fuse to form chimaeric mucosae. Despite fusion, the chimaeric mucosae maintain crypt clonality and villus polyclonality, demonstrating that clonal segregation persists during intestinal epithelial regeneration.
Assuntos
Mucosa Intestinal/citologia , Mucosa Intestinal/fisiologia , Intestino Delgado/citologia , Modelos Biológicos , Células-Tronco/citologia , Células-Tronco/fisiologia , Animais , Animais Recém-Nascidos , Diferenciação Celular , Divisão Celular , Quimera , Técnicas de Cultura , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Células Epiteliais/transplante , Mucosa Intestinal/crescimento & desenvolvimento , Intestino Delgado/crescimento & desenvolvimento , Intestino Delgado/fisiologia , Camundongos , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Organoides , Regeneração , Transplante de Células-TroncoRESUMO
Submucosal glands (SMGs) are the major site of expression of the cystic fibrosis (CF) transmembrane conductance regulator gene (CFTR) in the human lung. As such, SMGs may be a critical component of CF lung disease pathogenesis and an important target for gene therapy. Gene-targeted mouse models exist for CF and these are used to validate gene therapy or other interventions and to dissect CF phenotypes. It is important, therefore, to compare human and mouse SMGs. We show that SMGs in the mouse are similar in structure, cell types, and Cftr expression to those in the human. Murine SMGs were found to be present in the proximal regions of the trachea at the same density as in humans but, unlike in humans, did not extend below the trachea. Upon investigation of homozygous Cftr tm1HGU and Cftr tm1G551D mutant mice, SMGs were found to extend more distally than those in wild-type control mice (P < 0.05). To investigate the development of SMGs we generated aggregation chimeric mice. Chimeric offspring contained a contribution of transgenic cells that were detectable either by DNA in situ hybridization (reiterated beta-globin transgene TgN[Hbb-bl]83Clo) or beta-galactosidase histochemistry (Lac Z reporter gene TgR[ROSA26]- 26Sor). Analysis of the distribution of transgenic cells in chimeric SMGs suggests that SMGs are clonally derived.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Mucosa/metabolismo , Traqueia/metabolismo , Animais , Células Clonais , Humanos , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Modelos Genéticos , Mucosa/fisiologia , Muco/metabolismo , Muramidase/biossíntese , Membrana Serosa/metabolismo , Células-Tronco/metabolismo , Traqueia/anatomia & histologia , Quimeras de TransplanteRESUMO
Studies have shown that beta defensins are present in the human airways and may be relevant to the pathogenesis of cystic fibrosis lung disease. Here we report the identification of a novel mouse gene, Defb2, which shows sequence similarity to previously described mouse and human airway beta defensins. Defb2 does not appear to be expressed in the airways of untreated mice but it is upregulated in response to lipopolysaccharide. The induced expression of this gene by an inflammatory stimulus strongly suggests that this defensin contributes to host defence at the mucosal surface of the airways.