RESUMO
BACKGROUND: In order to study inter-individual differences in bacterial adhesion/invasion of periodontal tissues, an in vitro model for culturing multi-layered pocket epithelium without feeder layers or stromal equivalents (including the evaluation of their cytokeratin profiles) was developed. METHODS: Pocket epithelium was collected and grown until confluent in Falcon flasks using keratinocyte-serum free medium (KSFM), without a feeder layer. In the second passage, oral keratinocytes were re-grown in a 2 compartment system using either a clear polyester (transwell-clear [TCL]) or a collagen (transwell-col [TCO]) membrane as culture surface. After the first week, the calcium concentration was raised to 1.2 mM and in half the wells, the KSFM was supplemented with 10% fetal calf serum (FCS). Histology and immunohistochemistry were performed after 1, 2, and 3 weeks of additional growth. RESULTS: In general, all conditions resulted in a structured epithelium consisting of 3 to 5 layers, but important differences were observed between the membrane types and between the media. CK4 was rarely and only lightly expressed while CK18 and 19 (characteristic of junctional epithelium) were very strongly expressed in the older (2 and 3 weeks) cultures. CK13 and 14 (characteristic of any stratifiable epithelial cell) also tended to increase over time; CK13 seemed to be stronger in KSFM with FCS while the contrary was true for CK14. The multi-layer created by the combination TCL/KSFM + 10% FCS resembled a junctional epithelium most, while that grown on TCO without FCS mimicked the sulcular epithelium. CONCLUSIONS: It seems possible to create a histiotypic culture resembling either periodontal pocket or junctional epithelium without the use of stromal equivalents or feeder layers which make this approach more cumbersome. This multi-layered culture offers a model to investigate the permeability of pocket epithelium and the adhesion and penetration of bacteria under well-defined environmental conditions.
Assuntos
Técnicas de Cultura de Células/métodos , Células Epiteliais/patologia , Bolsa Periodontal/patologia , Animais , Bovinos , Cultura em Câmaras de Difusão , Inserção Epitelial/citologia , Células Epiteliais/química , Células Epiteliais/ultraestrutura , Humanos , Imuno-Histoquímica , Queratinas/análise , Microscopia , Microscopia Eletrônica , Modelos BiológicosAssuntos
Processamento Alternativo/genética , Hidrocefalia/genética , Glicoproteínas de Membrana/genética , Moléculas de Adesão de Célula Nervosa/genética , Paraplegia/genética , Criança , Análise Mutacional de DNA , DNA Complementar/química , DNA Complementar/genética , Saúde da Família , Feminino , Humanos , Complexo Antígeno L1 Leucocitário , Masculino , Linhagem , Mutação Puntual , Deleção de Sequência , VenezuelaRESUMO
The combination of X-linked mental retardation (XLMR) and neurological disorders occurs in a number of syndromes. Differential diagnosis mostly depends on clinical data and mapping of responsible genes by linkage analysis. We present a Belgian family with severe XLMR and a progressive neurological disorder with ataxia, spasticity and convulsions. Biochemical investigations, neuroimaging and neuropathology were normal. Linkage analysis pointed to region Xq27-28 as the probable locus for the genetic defect. The sequence of the L1CAM cDNA, a possible candidate gene, proved to be normal in the patients. This suggests the presence of a genetic factor on Xq27-28, different from L1CAM, which can lead to severe XLMR and a progressive neurological disorder.
Assuntos
Sistema Nervoso Central/patologia , Ligação Genética , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Cromossomo X/genética , Adolescente , Adulto , Bélgica , Criança , Pré-Escolar , Mapeamento Cromossômico , Análise Mutacional de DNA , DNA Complementar/análise , DNA Complementar/química , Progressão da Doença , Saúde da Família , Genes/genética , Marcadores Genéticos/genética , Humanos , Lactente , Cariotipagem , Complexo Antígeno L1 Leucocitário , Masculino , Moléculas de Adesão de Célula Nervosa/genética , Linhagem , Aberrações dos Cromossomos SexuaisRESUMO
A neonate presented with the fetal hypokinesia sequence and signs of spinal muscular atrophy (SMA). Severe pathological changes including ballooned neurons and neuronophagia were found not only in the motor nerve nuclei but also in the thalamic, cerebellar, and brainstem nuclei as well as in the dorsal root ganglia. Direct DNA analysis showed the presence of a chimeric SMN gene, with a rearrangement occurring between exon 7 of the centromeric SMN gene and exon 8 of the telomeric SMN gene. Circumstantial evidence suggests that only a single copy of this gene is present, with transcriptional characteristics of a centromeric SMN gene. In addition, a homozygous deletion in the NAIP genes was demonstrated. This observation demonstrates that at least some cases with fetal hypokinesia and SMA may represent the severe end of a spectrum of disorders caused by deletions in the SMA locus on chromosome 5q13. In addition, these findings are compatible with a modifying role for the centromeric SMN genes and the NAIP genes in the severity of the SMA phenotype.
Assuntos
Encéfalo/patologia , Cromossomos Humanos Par 5 , Proteínas do Tecido Nervoso/genética , Polimorfismo Conformacional de Fita Simples , Ribonucleoproteínas Nucleares Pequenas , Atrofias Musculares Espinais da Infância/genética , Atrofias Musculares Espinais da Infância/patologia , Autoantígenos/biossíntese , Autoantígenos/genética , Tronco Encefálico/patologia , Centrômero , Cerebelo/patologia , Quimera , Mapeamento Cromossômico , Primers do DNA , Eletromiografia , Éxons , Feminino , Gânglios Espinais/patologia , Deleção de Genes , Humanos , Recém-Nascido , Masculino , Neurônios Motores/patologia , Proteína Inibidora de Apoptose Neuronal , Neurônios/patologia , Linhagem , Reação em Cadeia da Polimerase , Núcleos Talâmicos/patologia , Tálamo/patologia , Transcrição Gênica , Proteínas Centrais de snRNPRESUMO
An arrayed library of human heterogeneous nuclear complementary (hnc) DNA was constructed from a somatic cell hybrid (M28) containing an i(12p) marker as the sole human chromosome. Heterogeneous nuclear (hn) RNA of M28 was used to synthesize first-strand hncDNA with a primer (RT) containing a random hexanucleotide at its 3' end. Specific amplification of human sequences from this hncDNA was performed using Alu primers in combination with the RT primer. The products were directionally cloned and an arrayed library was constructed. Experiments indicated that all clones were derived from transcribed sequences. A number of randomly isolated clones were evaluated by Southern and Northern experiments, sequence analysis, and PCR. At least 80% of these clones were of human 12p origin. The number of independent clones in the library was estimated to be approximately 550. Using 60 hncDNA clones as probes, 6 showed positive signals on Northern blots. For 3 of these, the corresponding cDNAs were isolated: clone CD60A1 codes for the cation-dependent mannose 6-phosphate receptor, clone CC6 is a human homologue of the bovine 39-kDa nuclear-encoded NADH:ubiquinone oxidoreductase subunit, and CD18 belongs to the family of tumor necrosis factor receptor proteins. Southern experiments showed the 3 cDNAs to map to human chromosome 12p as expected. Taken together these results show that the generation of a hncDNA library is a useful tool for the isolation of unknown genes located on a human chromosome (fragment) present in a somatic cell hybrid.
Assuntos
Cromossomos Humanos Par 12 , DNA/genética , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Sondas de DNA , Biblioteca Gênica , Marcadores Genéticos , Humanos , Células Híbridas , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Sequências Repetitivas de Ácido Nucleico , Transcrição GênicaRESUMO
Dissociated human nasal epithelial cells from nasal polyps were cultured in Ham's F12-DME 1/1 supplemented with NU-serum 10%, choleratoxin (10 ng/ml), retinoic acid (10(-7) M) and antibiotics. In monolayer cultures, the epithelial cells grew to confluency on collagen gels, became squamous, and lost their cilia within 2-6 weeks. In suspension cultures, epithelial cell sheaths formed stable vesicles and aggregates. These maintained a respiratory-type morphology and normal ciliary activity for over 6 months. When deciliated, squamous cells from monolayer cultures were brought in suspension, a respiratory-type morphology with cilia reappeared. This in vitro ciliogenesis resulted in normal and coordinated ciliary activity observed for more than 5 months.
Assuntos
Cílios/ultraestrutura , Mucosa Nasal/citologia , Regeneração , Células Cultivadas , Células Epiteliais , Humanos , Microscopia Eletrônica de Varredura , Pólipos Nasais/ultraestruturaRESUMO
Human nasal epithelial cells, dissociated from nasal polyps, lost all their cilia when cultured as monolayers on 0.2% collagen gels in Ham's F12-DME 1/1, supplemented with NU-serum 10%, choleratoxin (10 ng/ml), retinoic acid (10(7) M) and antibiotics. These deciliated epithelial cell sheaths were then placed in a suspension culture system, and epithelial aggregates and vesicles formed. After 1 week in suspension, cilia progressively reappeared. This in vitro ciliogenesis resulted in ultrastructurally normal cilia with an intercellularly coordinated beating pattern. These epithelial aggregates and vesicles maintained this differentiated ciliary morphology and function for more than 6 months.