RESUMO
A third-generation adenoviral vector containing recombinant human cystic fibrosis transmembrane conductance regulator (CFTR) gene was delivered by bronchoscope in escalating doses to the conducting airway of 11 volunteers with cystic fibrosis. Assessments of dose-limiting toxicity (DLT), efficiency of gene transfer, and cell-mediated and humoral immune responses to vector administration were performed. DLT, manifest by flulike symptoms and transient radiographic infiltrates, was seen at 2.1 x 10(11) total viral particles. A highly specific assay for gene transfer was developed using in situ hybridization with an oligoprobe against unique vector sequence. Detectable gene transfer was observed in harvested bronchial epithelial cells (<1%) 4 days after vector instillation, which diminished to undetectable levels by day 43. Adenovirus-specific cell-mediated T cells were induced in most subjects, although only mild increases in systemic humoral immune response were observed. These results demonstrate that gene transfer to epithelium of the lower respiratory tract can be achieved in humans with adenoviral vectors but that efficiency is low and of short duration in the native CF airway.
Assuntos
Adenoviridae/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/terapia , Terapia Genética , Pulmão/metabolismo , Sequência de Bases , Fibrose Cística/imunologia , Sondas de DNA , Técnicas de Transferência de Genes , Terapia Genética/efeitos adversos , Humanos , Testes de NeutralizaçãoRESUMO
Cystic fibrosis (CF) remains an attractive target for cure by gene therapy. Results from several trials are reviewed in this article and have shown that mature airway epithelial cells are relatively resistant to gene transfer, that host immune responses determine the duration of transgene expression and define the toxicity, and that the efficiency of transfection remains low. Significant hurdles to the development of gene therapy remain, including the definition of efficacy endpoints, the ability to produce enough material, and the ability to dose the entire lung. Nonetheless, invaluable insights into CF and pulmonary biology have been gained in the gene therapy research effort.
Assuntos
Fibrose Cística/terapia , Terapia Genética , Adenovírus Humanos , Ensaios Clínicos como Assunto , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Vetores Genéticos , HumanosRESUMO
We have previously demonstrated that 10-formyl-7,8-dihydrofolic acid (10-HCO-H2folate) is a better substrate for mammalian aminoimidazolecarboxamide ribotide transformylase (EC 2.1.2.3) than is 10-formyl-5,6,7,8-tetrahydrofolic acid (10-HCO-H4folate) (J.E. Baggott, G.L. Johanning, K.E. Branham, C.W. Prince, S.L. Morgan, I. Eto, W.H. Vaughn, Biochem. J. 308, 1995, 1031-1036). Therefore, the possible metabolism of 10-HCO-H4folate to 10-HCO-H2folate was investigated. A spectrophotometric assay for the oxidation of 10-HCO-H4folate to 10-HCO-H2folate which measures the disappearance of reactant (decrease in absorbance at 356 nm after acidification of aliquots of the reaction solution), is used to demonstrate that iron compounds catalyze the oxidation of 10-HCO-H4folate to 10-HCO-H2folate in the presence and absence of ascorbate. Chromatographic separation of the 10-HCO-H2folate product from the reaction mixture, its UV spectra, a microbiological assay and an enzymatic assay established that the iron-catalyzed oxidation product of 10-HCO-H4folate was 10-HCO-H2folate; without substantial side reactions. The inhibition of this iron-catalyzed oxidation by deferoxamine, apotransferrin and mannitol and the stimulation by citrate and EDTA indicated of a mechanism involving a reaction of 10-HCO-H4folate with hydroxyl radicals (*OH) generated by Fenton chemistry. The presence of "free iron" (e.g., Fe3+ citrate) in bile, cerebrospinal fluid and intracellularly suggest that this oxidation could occur in vivo and that 10-HCO-H4folate may be a *OH scavenger.
Assuntos
Ácido Fólico/análogos & derivados , Compostos de Ferro/metabolismo , Leucovorina/análogos & derivados , Animais , Apoproteínas/metabolismo , Ácido Ascórbico/metabolismo , Bovinos , Ácido Cítrico/metabolismo , Desferroxamina/metabolismo , Ácido Fólico/química , Ácido Fólico/metabolismo , Técnicas In Vitro , Quelantes de Ferro/metabolismo , Leucovorina/química , Leucovorina/metabolismo , Oxirredução , Espectrofotometria Ultravioleta , Transferrina/metabolismoRESUMO
We previously demonstrated that human tracheobronchial epithelial (TBE) cells synthesize mucin and form mucous granules in culture when they are maintained on a collagen gel (CG) substratum, but not on a plastic tissue culture surface or a thin collagen-coated surface (Wu et al., Am. J. Respir. Cell Mol. Biol., 3:467-478; 1990). This observation led us to examine the effects of CG thickness on cell growth and differentiation in primary human/monkey TBE cell cultures. Using the same CG preparation, culture dishes with different thicknesses of CG substratum were prepared. In general, equivalent degrees of cell attachment and proliferation were observed in all cultures maintained on a collagen gel, independent of the thicknesses of CG substratum. However, a greater degree of mucin synthesis and secretion by the cells was observed as the thickness of the CG substratum was increased. Cultures maintained on a thick collagen gel (1 mm) exhibited greater apical membrane complexity, more pseudostratification, and more mucous granules than did cultures maintained on a thin CG substratum. The optimal culture surface for airway mucous cell differentiation contains more than 1-mm thickness of collagen gel substratum.
Assuntos
Brônquios/citologia , Colágeno , Mucinas/biossíntese , Traqueia/citologia , Animais , Brônquios/metabolismo , Adesão Celular/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Células Epiteliais , Géis , Haplorrinos , Humanos , Mucinas/metabolismo , Traqueia/metabolismoRESUMO
We previously demonstrated that human tracheobronchial epithelial (TBE) cells synthesize mucin and form mucous granules in culture when they are maintained on a collagen gel (CG) substratum, but not on a plastic tissue culture surface or a thin collagen-coated surface (Wu et al., Am. J. Respir. Cell Mol. Biol., 3:467-478; 1990). This observation led us to examine the effects of CG thickness on cell growth and differentiation in primary human/monkey TBE cell cultures. Using the same CG preparation, culture dishes with different thicknesses of CG substratum were prepared. In general, equivalent degrees of cell attachment and proliferation were observed in all cultures maintained on a collagen gel, independent of the thicknesses of CG substratum. However, a greater degree of mucin synthesis and secretion by the cells was observed as the thickness of the CG substratum was increased. Cultures maintained on a thick collagen gel (1 mm) exhibited greater apical membrane complexity, more pseudostratification, and more mucous granules than did cultures maintained on a thin CG substratum. The optimal culture surface for airway mucous cell differentiation contains more than 1-mm thickness of collagen gel substratum.
RESUMO
Opioids are the major class of analgesics used in the management of moderate to severe cancer pain, and constipation is a common side effect of opioid administration. While monitoring for quality-assurance, nurses found that 95% of patients interviewed on a 28-bed oncology unit of a Midwestern hospital reported constipation as the major side effect of their opioid regimen for pain control. Through the efforts of a nursing research utilization committee, a protocol to prevent opioid-induced constipation in patients with cancer was developed and implemented.
Assuntos
Analgésicos Opioides/efeitos adversos , Pesquisa em Enfermagem Clínica/organização & administração , Constipação Intestinal/induzido quimicamente , Constipação Intestinal/prevenção & controle , Neoplasias/complicações , Enfermagem Oncológica/métodos , Dor/tratamento farmacológico , Planejamento de Assistência ao Paciente/normas , Algoritmos , Constipação Intestinal/enfermagem , Árvores de Decisões , Humanos , Avaliação em Enfermagem , Dor/etiologia , Desenvolvimento de Programas , Avaliação de Programas e Projetos de Saúde , Garantia da Qualidade dos Cuidados de SaúdeRESUMO
Clinical and anecdotal findings suggest that the completion of cancer treatment may be marked by heightened distress and disrupted adjustment. The present study examined psychological adjustment during the 3 months following treatment among 89 women with stages 0-III breast cancer. Participants completed measures of depression, cancer-related anxiety, cancer concerns, and quality of life at three time points: during treatment, 3 weeks following the end of treatment, and 3 months post-treatment. Post-treatment scores were suggestive of good psychological adjustment among the majority of women. Moreover, distress did not increase following treatment; longitudinal analyses showed no significant changes in depression or recurrence worry, while intrusive thoughts decreased, and quality of life improved. Younger age predicted greater distress across measures. A history of depression or anxiety predicted greater depressive symptomatology, while more extensive treatment predicted greater cancer-related anxiety. Despite the lack of distress endorsed on general depression and anxiety indices, participants reported moderate distress associated with cancer-related concerns, including physical problems, fear of cancer recurrence, and resuming normal life. In sum, while breast cancer survivors demonstrate good adjustment on general distress indices following treatment, some women are at risk for sustained distress. Moreover, significant cancer-related concerns are prevalent and may be important intervention targets.
Assuntos
Adaptação Psicológica , Neoplasias da Mama/psicologia , Qualidade de Vida , Adulto , Idoso , Idoso de 80 Anos ou mais , Ansiedade , Neoplasias da Mama/terapia , Depressão , Feminino , Previsões , Humanos , Pessoa de Meia-Idade , Fatores de Risco , Fatores de TempoRESUMO
The literature documents the importance of using a conceptual model to explain and guide nursing practice (Fawcett, 1989; Riehl-Sisca, 1988; Meleis, 1991). However, can a single model describe and guide practice for an entire nursing service department? In this article, the author describes and analyzes the process of developing an eclectic conceptual nursing practice model as experienced by the nursing division of a 250-bed community hospital in the midwest.
Assuntos
Hospitais Comunitários , Modelos de Enfermagem , Serviço Hospitalar de Enfermagem/organização & administração , Humanos , Filosofia em Enfermagem , Desenvolvimento de ProgramasRESUMO
Use of single photon emission computerized tomography (SPECT) with Tc 99m HDP for localization of an active sequestrum in a case of chronic head and neck osteomyelitis is described. Correlation with x-ray computed tomography (CT) for anatomy was helpful for surgical debridement. The potential of the complementary roles of SPECT and CT in chronic head and neck osteomyelitis for assessing sequestrum activity and anatomy prior to surgery is discussed.
Assuntos
Osteomielite/diagnóstico , Crânio , Tomografia Computadorizada de Emissão , Tomografia Computadorizada por Raios X , Adulto , Doença Crônica , Desbridamento , Feminino , Humanos , Maxila/cirurgia , Doenças Maxilares/diagnóstico , Pescoço , Medronato de Tecnécio Tc 99m/análogos & derivadosRESUMO
The bio-inactive C-6 isomer, [6R]-5-formyl-tetrahydrofolate (5-HCO-H(4)F), is not found in Nature. An oral dose of 13.5 micromol of [6R]-5-HCO-H(4)F in humans results in the appearance of the naturally occurring [6S]-5-methyl-tetrahydrofolate and relatively large amounts of other bioactive folates in plasma. The removal of the asymmetry at C-6 could account for these results. Two oxidized cytochrome c [cyt c (Fe3+)] molecules oxidize one 10-formyl-tetrahydrofolate (10-HCO-H(4)F) with second-order kinetics and a rate constant of 1.3 x 10(4) M(-1) x s(-1). The folate product of this oxidation reaction is 10-formyl-dihydrofolate (10-HCO-H(2)F), which has no C-6 asymmetric centre and is therefore bioactive. The folate-requiring bacterium, Enterococcus hirae, does not normally biosynthesize cytochromes but does so when given an exogenous source of haem (e.g. haemin). E. hirae grown in haemin-supplemented media for 3 days utilizes both [6R]- and [6S]-5-HCO-H(4)F in contrast to that grown in control medium, which utilizes only the [6S] isomer. Since known chemical reactions form 10-HCO-H(4)F from 5-HCO-H(4)F, the unusually large rate constant for the oxidation of 10-HCO-H(4)F by cyt c (Fe3+) may account for the unexpected bioactivity of [6R]-5-HCO-H(4)F in humans and in E. hirae grown in haemin-containing media. We used an unnatural C-6 folate isomer as a tool to reveal the possible in vivo oxidation of 10-HCO-H(4)F to 10-HCO-H(2)F; however, nothing precludes this oxidation from occurring in vivo with the natural C-6 isomer.
Assuntos
Grupo dos Citocromos c/metabolismo , Enterococcus/metabolismo , Ácido Fólico/análogos & derivados , Ácido Fólico/metabolismo , Leucovorina/metabolismo , Hemina/metabolismo , Humanos , Leucovorina/análogos & derivados , OxirreduçãoRESUMO
Acid hydrolases are synthesized as precursors that undergo several posttranslational modifications including proteolytic processing to a smaller mature enzyme. The amount of proteolytic processing varies for different acid hydrolases, and many details of the intracellular pathways are not known. The processing of alpha-L-fucosidase was distinguished from that of other acid hydrolases reported when studied in systematic pulse-chase labeling experiments. Only one form of alpha-L-fucosidase, Mr 56,000-57,000, was demonstrated intra- and extracellularly. Under the same conditions, N-acetyl-beta-D-glucosaminidase was shown to be processed with several forms, as previously reported by Hasilik and Neufeld (1980a). To obtain these results, human skin fibroblasts were labeled metabolically with L-[3H]leucine for periods of 20 min to 8 hr with varying periods of chase from 1 to 96 hr with nonradioactive L-leucine. alpha-L-Fucosidase was immunoprecipitated by a polyclonal antibody from material extracted from cells and ammonium sulfate precipitated medium and was examined by polyacrylamide gel electrophoresis under denaturing conditions. N-Acetyl-beta-D-glucosaminidase was examined with similar procedures and served as a control for the methods. Tunicamycin treatment of the cells was used to show that glycosylation did not obscure proteolytic processing because, again, only one form of the intra- and extracellular enzyme was observed, although of smaller size, Mr 52,000-53,000. In addition, separation of the cells into prelysosomal and lysosomal fractions showed only one form of the enzyme. It is concluded that alpha-L-fucosidase does not undergo proteolytic processing in human skin fibroblasts in the usual manner described for other acid hydrolases.
Assuntos
Processamento de Proteína Pós-Traducional , alfa-L-Fucosidase/metabolismo , Acetilglucosaminidase/metabolismo , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Fibroblastos , Humanos , Peso Molecular , Testes de Precipitina , Tunicamicina/farmacologia , alfa-L-Fucosidase/análise , alfa-L-Fucosidase/biossínteseRESUMO
Previous studies using the CA 19-9 antibody have demonstrated that serum mucin levels in patients with cystic fibrosis (CF) are elevated and that the degree of elevation relates to the age of the patient and possibly to his or her clinical status. However, CA 19-9 only recognizes the mucin-associated blood group sialyl Le(a+) antigen, so mucin levels cannot be measured in patients without Lewis antigens. The present study used the 17B1 monoclonal antibody to measure serum mucin levels in normal subjects, and in patients with CF, patients with chronic obstructive pulmonary disease (COPD), and patients with lung transplants. Serum mucin levels were 25 ng/ml (+/- 1 SEM, n = 8) in normal subjects, 13,853 ng/ml (+/- 1,281, n = 25) in patients with CF, and 25.5 ng/ml (+/- 1.9, n = 17) in patients with COPD. Patients with CF who were sialyl Le(a-b-) also had elevated serum mucin levels (715 +/- 152, n = 2). Serum mucin levels of six lung transplant recipients with CF were elevated compared with those in normal subjects (4,621 +/- 765 ng/ml), but they were not different from serum mucin levels in six lung transplant recipients without CF (5,307 +/- 1.677 ng/ml). Preliminary characterization of the serum mucin antigen showed that: (1) in CF sera, the antigen is polydisperse and smaller than the antigen in normal sera; (2) the mucin antigen is distinct from ABO blood group antigens. Serum mucin levels may be a useful marker to follow a specific patient's response to therapy.
Assuntos
Antígenos/sangue , Fibrose Cística/sangue , Mucinas/imunologia , Sistema ABO de Grupos Sanguíneos/imunologia , Adulto , Idoso , Anticorpos Monoclonais , Biomarcadores/sangue , Bronquite/sangue , Bronquite/imunologia , Fibrose Cística/imunologia , Feminino , Humanos , Isoantígenos/sangue , Antígenos do Grupo Sanguíneo de Lewis/imunologia , Pneumopatias Obstrutivas/sangue , Pneumopatias Obstrutivas/imunologia , Transplante de Pulmão/imunologia , Masculino , Pessoa de Meia-Idade , Mucinas/sangueRESUMO
The effects of culture conditions on growth and differentiation of human tracheobronchial epithelial (HTBE) cells have been defined. Epithelial cells were dissociated from tissues by protease treatment and were plated on tissue culture dishes in F12 medium supplemented with insulin, transferrin, epidermal growth factor, hydrocortisone, cholera toxin, bovine hypothalamus extract, and retinol. HTBE cells did not express any mucociliary function (ciliogenesis or mucin secretion) on tissue culture plastic, but they could be passaged 3 to 5 times with a total of 10 to 25 population doublings. Cells from early passages re-express both these functions when transplanted to tracheal grafts. When tissue culture plates were coated with collagen film or collagen gel substrata, cell attachment and proliferation were stimulated. However, the expression of mucous cell function in culture occurred only when cells were plated on collagen gel substrata and vitamin A (retinol) was present in the medium. Mucous cell differentiation under optimal conditions was defined by ultrastructural studies, by immunologic studies with mucin-specific monoclonal antibodies, and by carbohydrate and amino acid compositional analyses of mucin-like glycoproteins purified from culture medium. These results demonstrate for the first time that HTBE cells can express mucin synthesis and secretion under appropriate culture conditions.