A549 lung epithelial cells grown as three-dimensional aggregates: alternative tissue culture model for Pseudomonas aeruginosa pathogenesis

A three-dimensional (3-D) lung aggregate model was developed from A549 human lung epithelial cells by using a rotating-wall vessel bioreactor to study the interactions between Pseudomonas aeruginosa and lung epithelial cells. The suitability of the 3-D aggregates as an infection model was examined by immunohistochemistry, adherence and invasion assays, scanning electron microscopy, and cytokine and mucoglycoprotein production. Immunohistochemical characterization of the 3-D A549 aggregates showed increased expression of epithelial cell-specific markers and decreased expression of cancer-specific markers compared to their monolayer counterparts. Immunohistochemistry of junctional markers on A549 3-D cells revealed that these cells formed tight junctions and polarity, in contrast to the cells grown as monolayers. Additionally, the 3-D aggregates stained positively for the production of mucoglycoprotein while the monolayers showed no indication of staining. Moreover, mucin-specific antibodies to MUC1 and MUC5A bound with greater affinity to 3-D aggregates than to the monolayers. P. aeruginosa attached to and penetrated A549 monolayers significantly more than the same cells grown as 3-D aggregates. Scanning electron microscopy of A549 cells grown as monolayers and 3-D aggregates infected with P. aeruginosa showed that monolayers detached from the surface of the culture plate postinfection, in contrast to the 3-D aggregates, which remained attached to the microcarrier beads. In response to infection, proinflammatory cytokine levels were elevated for the 3-D A549 aggregates compared to monolayer controls. These findings suggest that A549 lung cells grown as 3-D aggregates may represent a more physiologically relevant model to examine the interactions between P. aeruginosa and the lung epithelium during infection.     

Selective targeting of antibody-conjugated nanoparticles to leukemic cells and primary T-lymphocytes

In the present study, surface-modified nanoparticles based on biodegradable material were used for antibody coupling in order to get a selective drug carrier systems. Gelatin nanoparticles were prepared by a desolvation process. Sulfhydryl groups were introduced which enabled the linkage of NeutrAvidin (NAv). Antibodies specific for the CD3 antigen on lymphocytic cells were conjugated to the nanoparticles surface. The binding of biotinylated anti-CD3 antibody was achieved by NAv-biotin-complex formation. Cellular binding and uptake were determined by flow cytometry and confocal laser scanning microscopy (CLSM). Cell-type-specific targeting of anti-CD3-conjugated nanoparticles into CD3-positive human T-cell leukemia cells and primary T-lymphocytes could be shown. Celluar uptake and effective internalization of antibody-conjugated nanoparticles into CD3 expressing cells were demonstrated. Uptake rates of about 84% into T-cell leukemia cells were observed. To confirm selectivity of T-cell targeting, competition experiments were carried out adding excessive free anti-CD3 prior to nanoparticle incubation leading to significantly reduced cellular uptake of antibody-conjugated nanoparticles. Further analysis on the mechanism of uptake confirmed a receptor-mediated endocytotic process. Protein-based nanoparticles conjugated with an antibody against a specific cellular antigen hold promise as selective drug delivery systems for specific cell types.     

c-Fos-dependent induction of the small ras-related GTPase Rab11a in skin carcinogenesis

Malignant transformation of mouse skin by tumor promoters and chemical carcinogens, such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), is a multistage process leading to the formation of squamous cell carcinomas. It has been shown that mice lacking the AP-1 family member c-Fos exhibit an impaired transition from benign to malignant skin tumors. Here, we demonstrate enhanced expression of the small Ras-related GTPase Rab11a after short-term TPA treatment of mouse back skin. Expression of Rab11a in vivo and in vitro critically depended on c-Fos, because TPA application to the back skin of c-Fos-deficient mice and to mouse embryonic fibroblasts did not induce Rab11a mRNA or protein expression. Moreover, dexamethasone, which is a potent inhibitor of AP-1-mediated transactivation that exhibits anti-inflammatory and anti-tumor promoting activities, inhibited TPA-induced expression of Rab11a. Within the Rab11a gene promoter, we identified a functional AP-1 binding element that exhibited elevated c-Fos binding activity after TPA treatment of keratinocytes. Enhanced expression was not restricted to chemically induced mouse skin tumors but was also found in tumor specimens derived from patients with epithelial skin tumors. These data identify Rab11a as a novel, tumor-associated c-Fos/AP-1 target and may point to an as yet unrecognized function of Rab11a in the development of skin cancer.     

Isolation and characterization of additional genes influencing resistance to various mutagens in the yeast Saccharomyces cerevisiae

Summary Screening of a multi-copy vector-based yeast genomic library in haploid cells of wild-type Saccharomyces cerevisiae yielded transformants hyper-resistant to various chemical mutagens. Genetical analysis of the yeast insert DNAs revealed three genes SNG1, SNQ2, and SNQ3 that confer the phenotype hyper-resistance to MNNG, to 4-NQO and triaziquone, and to mutagens 4-NQO, MNNG, and triaziquone, respectively. Integration of the gene disruption-constructs into the haploid yeast genome yielded viable null-mutants with a mutagen-sensitive phenotype. Thus, copy number of these non-essential yeast genes determines the relative resistance to certain chemical mutagens, with zero copies yielding a phenotype of mutagen sensitivity and multiple copies one of mutagen hyper-resistance, respectively.Dedicated to Professor Dr. R. W. Kaplan on the occasion of his 80th birthday     

Improvement in histological diagnosis of primary hyperparathyroidism with a monoclonal antiparathyroid antibody

The monoclonal antiparathyroid antibody E11 reacts with a glycoprotein of high molecular weight, which acts as a calcium receptor on the surface of parathyroid cells and mediates calcium regulation of parathyroid hormone (PTH) release. Reduced expression of the calcium receptor has been implicated as a cause of the defect in PTH regulation in the pathological parathyroid parenchyma of patients with hyperparathyroidism (HPT). The present study evaluated the efficacy of immunostainings with the E11 antibody in comparison with routine histopathological methods including staining by the oil red O technique for histological discrimination between normal and pathological parathyroid glands. Parathyroid tissue from euparathyroid individuals invariably presented intense and homogeneous surface staining, with the antibody on virtually all chief cells, while the pathological glands from patients with HPT consistently showed heterogeneous and reduced immunostaining. Even minimally enlarged pathological glands from individuals with mild hypercalcemia and the normal-sized glands associated with adenomas displayed parathyroid chief cells with reduced antibody reactivity. The monoclonal antiparathyroid antibody should constitute a useful tool in parathyroid histopathology not only by its ability to identify the parathyroid tissue, but also by directly demonstrating the functionally normal and abnormal cells within the parathyroid tissue.     

Characterization of an IgA receptor from group B streptococci: specificity for serum IgA

Some strains of group B streptococci express a cell surface protein which binds IgA. This report describes some properties of such an IgA receptor and compares it with a previously described IgA receptor from group A streptococci. The group B receptor was released in an almost pure form from bacteria incubated at elevated pH, and could be isolated by IgA-Sepharose affinity chromatography. The sequence of the N-terminal 19 amino acid residues was unique. The receptor preferentially binds IgA of human origin, as shown in immunoblotting experiments with purified IgA from nine different species. The affinity constant of the purified receptor for serum IgA was determined to be 3.5 x 10(8) M-1, but for secretory IgA it was too low to allow determination. This result indicates that secretory component and/or J chain interferes with the binding of IgA to this type of bacterial receptor, which may be one of the physiological functions of these polypeptides. A reduction in affinity was also observed for another complexed form of IgA, alpha 1-microglobulin-IgA. The group B receptor is antigenically unrelated to the IgA receptor from group A streptococci (protein Arp), but competitive inhibition experiments indicate that they bind to the same region in IgA. The implications of these findings, and the biological role of bacterial IgA receptors, are discussed.     

Mucosal barrier mechanisms. Interplay between secretory IgA (SIgA), IgG and mucins on the surface properties and association of salmonellae with intestine and granulocytes.

Rough Salmonella typhimurium 395 MR 10 bacteria sensitized with SIgA were used to assess the effect of secretory IgA (SIgA) on bacterial association with the intestine of rat and with a column of hog gastric mucin, and on IgG-mediated surface properties and interaction with polymorphonuclear leucocytes. It was found that SIgA increased the affinity for mucus belt of the intestine and for the mucin column, but reduced IgG-enhanced phagocytosis and surface hydrophobicity and charge of the bacteria. It is suggested that the ability of SIgA to render bacteria mucophilic and to modify IgG-mediated reactions serve the purpose of secluding bacteria from contact with mucosal membranes and depress inflammatory reactions at the site of infection.