Primary cell culture of human type II pneumonocytes: maintenance of a differentiated phenotype and transfection with recombinant adenoviruses

Studies of the regulation of surfactant lipoprotein metabolism and secretion and surfactant protein gene expression have been hampered by the lack of a cell culture system in which the phenotypic properties of type II cells are maintained. We have developed a primary culture system that facilitates the maintenance of a number of morphologic and biochemical properties of type II pneumonocytes for up to 2 wk. Cells were isolated by collagenase digestion of midgestation human fetal lung tissue that had been maintained in organ culture in the presence of dibutyryl cyclic AMP (Bt2cAMP) for 5 days. The isolated cells were enriched for epithelial components by treatment with DEAE-dextran, plated on an extracellular matrix (ECM) derived from Madin-Darby canine kidney (MDCK) cells, and incubated at an air/liquid interface in a minimal amount of culture medium containing Bt2cAMP. The cell cultures were comprised of islands of round epithelial-like cells containing numerous dense osmiophilic granules, surrounded by sparse spindle-shaped cells with the appearance of fibroblasts. Ultrastructural examination revealed that the osmiophilic granules had the appearance of lamellar bodies, the distinguishing feature of type II pneumonocytes. Additionally, the cultures maintained elevated levels of SP-A gene expression for up to 2 wk. The expression of mRNAs encoding SP-A, SP-B, and SP-C were regulated in the cultured cells by glucocorticoids and cyclic AMP in a manner similar to that observed in fetal lung tissue in organ culture. The differentiated phenotype was most apparent when the cells were cultured at an air/liquid interface. In order to utilize the cultured type II cells for study of the effects of overexpression of various proteins and for promoter analysis, it is of essence to transfect DNA constructs into these cells with high efficiency. Unfortunately, we found the cells to be refractory to efficient transfer of DNA using conventional methods (i.e., lipofection, electroporation, or calcium phosphate-mediated transfection). However, replication-defective recombinant human adenoviruses were found to provide a highly efficient means of introducing DNA into the type II pneumonocytes. Furthermore, we observed in type II cell-enriched cultures infected with recombinant adenoviruses containing the lacZ gene under control of a cytomegalovirus promoter, that beta-galactosidase was expressed uniformly in the islands of type II cells and surrounding fibroblasts. By contrast, in cultures infected with recombinant adenoviruses containing the human growth hormone (hGH) gene under control of the SP-A gene promoter and 5'-flanking region, hGH was expressed only in the type II cells. Thus, this culture system provides an excellent means for identifying genomic elements that mediate type II cell-specific gene expression.     

Prevalence of Staphylococcus aureus among food handlers from a metropolitan university in Chile

BACKGROUND: An important agent of food intoxication is Staphylococcus aureus, that is able to produce enterotoxins. AIM: To detect Staphylococcus aureus contamination in cafeteria food handlers of a Chilean University. SUBJECTS AND METHODS: Nose, throat, hands and nail samples from 87 food handlers were obtained for microbiological examination. RESULTS: Fifty seven subjects (65.5%) were carriers of Staphylococcus aureus. Enterotoxigenic Staphylococcus aureus was found in 36 subjects (41%). The most frequently found enterotoxin was type B (18 samples) followed by type D (12 samples). Men bad a higher frequency of contamination than women (83 and 57% of positive samples respectively). CONCLUSIONS: The frequency of Staphylococcus aureus contamination among food handlers is high and should prompt personal and environmental hygienic measures.     

Deletions in one domain of the Friend virus-encoded membrane glycoprotein overcome host range restrictions for erythroleukemia

Although the Friend virus-encoded membrane glycoprotein (gp55) activates erythropoietin receptors (EpoR) to cause erythroblastosis only in certain inbred strains of mice but not in other species, mutant viruses can overcome aspects of mouse resistance. Thus, mice homozygous for the resistance allele of the Fv-2 gene are unaffected by gp55 but are susceptible to mutant glycoproteins that have partial deletions in their ecotropic domains. These and other results have suggested that proteins coded for by polymorphic Fv-2 alleles might directly or indirectly interact with EpoR and that changes in gp55 can overcome this defense. A new viral mutant with an exceptionally large deletion in its ecotropic domain is now also shown to overcome Fv-2rr resistance. In all cases, the glycoproteins that activate EpoR are processed to cell surfaces as disulfide-bonded dimers. To initiate analysis of nonmurine resistances, we expressed human EpoR and mouse EpoR in the interleukin 3-dependent mouse cell line BaF3 and compared the abilities of Friend virus-encoded glycoproteins to convert these cells to growth factor independence. Human EpoR was activated in these cells by erythropoietin but was resistant to gp55. However, human EpoR was efficiently activated in these cells by the same viral mutants that overcome Fv-2rr resistance in mice. By construction and analysis of human-mouse EpoR chimeras, we obtained evidence that the cytosolic domain of human EpoR contributes to its resistance to gp55 and that this resistance is mediated by accessory cellular factors. Aspects of host resistance in both murine and nonmurine species are targeted specifically against the ecotropic domain of gp55.     

Nutrition support affects the distribution and organ uptake of cachectin/tumor necrosis factor in rats

BACKGROUND: We have previously observed a potentiation of the metabolic response to cachectin/tumor necrosis factor (TNF) by total parenteral nutrition (TPN) but not in anorexic orally fed animals. We hypothesized that nutritional status might affect TNF clearance kinetics. METHODS: We compared the clearance of a bolus of labeled TNF in TPN-fed animals given sufficient nutrients to grow called weight-gaining rats (WGR) with those given 50% of the WGR called weight-losing rats (WLR) and with orally fed rats (OFR). Data were analyzed using a two-compartment open system model and by linear systems analysis. RESULTS: The data from both types of analysis indicator that although metabolic clearance was similar, WGR had a slower fractional TNF clearance rate (FCR) as well as a larger volume of distribution than WLR or OFR. Further analysis showed that an increased proportion of the total mass of TNF resided in a plasma-associated compartment in WGR compared with WLR and OFR. In addition, WGR had reduced uptake of labeled TNF by the kidney. CONCLUSION: The data suggest that nutrition support influences either the distribution of TNF or the FCR, resulting in a greater retention in the plasma-associated compartment with intact absolute removal rates. This study has important implications concerning the type of nutrition support provided to the critically ill patient because our data suggest that clinical states with increased circulating TNF levels may be adversely affected by currently available nutritional practices.     

Preoperative pulmonary evaluation of the thoracic surgical patient

A test designed to separate those undergoing thoracic surgery without complications and those with complications must be both highly specific and sensitive. Clearly, the difference between patients at opposite ends of the population curves is easy to identify. Spirometry can be helpful for screening, although it is not a very discriminating test. If patients fall in the overlap region between the populations, however, it is impossible to discern the risks with any certainty using low-yield tests. A test with higher sensitivity, specificity, and predictive values is necessary to ascertain such marginal differences. With this kind of analysis at hand, preoperative testing can be divided into three predictive value groups. Calculating the predictive value of each preoperative test can provide a comparative measure of usefulness of discriminative power (Table 1). In this way, spirometry, blood gas analysis, and stair climbing tolerance are shown to be poor predictors of outcome. An intermediate predictive value can be achieved using diffusion capacity, exercise-induced decreases in O2 saturation, and exercise PVR. High predictive value can be accomplished with combination indexes (PPP, possibly PRQ), measurement of VO2 at 40 watts of exercise, or VO2max. Logic dictates a step-wise preoperative evaluation using prediction value analysis (Fig.4). A flow decision chart for the preoperative evaluation of patients for pulmonary resection begins with exercise oximetry, spirometry, and blood gas analysis as general screening tests to separate those patients at minimal or no risks for complications from those patients that require further evaluation. Functional indexes (PPP, PRQ) or exercise testing can aid further in the selection of those patients in whom a nonsurgical option should be considered. Flow decision chart for the preoperative evaluation of patients for pulmonary resection should continue to evolve as new information about outcome studies is gathered. Examination of outcome data will provide us with reduction of the size of the nonoperable population, so that we can deny only those patients who truly pose a prohibitive risk.     

The neu-oncogene product in serum and tissue of patients with breast carcinoma

Patients with non-cardiac chest pain (NCCP) (n = 387) and cardiac chest pain (CCP) (n = 93) were compared with community controls (n = 81), using a symptom questionnaire that assessed the presence of irritable bowel syndrome (IBS), functional dyspepsia, and oesophageal dysfunction and chest pain characteristics. A significantly (p < 0.05) increased prevalence of symptoms compatible with IBS occurred in NCCP patients when compared with those with CCP and with controls. Dysphagia was more frequent in both those with non-cardiac and cardiac chest pain than in controls; this was not apparent, however, when patients with concomitant IBS were excluded. The presence of oesophageal or gastrointestinal symptoms did not enable discrimination with regard to the chest pain characteristics. We conclude that unselected referred patients with documented NCCP are more likely to have IBS and that the presence of oesophageal symptoms such as dysphagia may merely reflect the spectrum of the 'irritable gut'.     

Evaluation of aging pilots: evidence, policy, and future directions

A male patient presented with complaints of fever, cough with expectoration, burning micturition and 5-6 semisolid motions per day for the past 6 days. Skiagram chest (PA view) revealed lung abscess in the left mid zone. There was no improvement, symptomatically and radiologically, after an empirical course of antibiotics (IV ampicillin and gentamycin). Sputum, urine and stool cultures grew salmonella group E organisms sensitive only to cefotaxime. The patient was treated with IV cefotaxime and responded well clinically, radiologically and bacteriologically.