Transfer of the active form of transforming growth factor-beta 1 gene to newborn rat lung induces changes consistent with bronchopulmonary dysplasia

Bronchopulmonary dysplasia is a chronic lung disease of premature human infancy that shows pathological features comprising varying sized areas of interstitial fibrosis in association with distorted large alveolar spaces. We have previously shown that transfer of active transforming growth factor (TGF)-beta 1 (AdTGF beta 1(223/225)) genes by adenovirus vector to embryonic lungs results in inhibition of branching morphogenesis and primitive peripheral lung development, whereas transfer to adult lungs results in progressive interstitial fibrosis. Herein we show that transfer of TGF-beta1 to newborn rat pups results in patchy areas of interstitial fibrosis developing throughout a period of 28 days after transfer. These areas of fibrosis appear alongside areas of enlarged alveolar spaces similar to the prealveoli seen at birth, suggesting that postnatal lung development and alveolarization has been inhibited. In rats treated with AdTGF beta 1(223/225), enlarged alveolar spaces were evident by day 21, and by 28 days, the mean alveolar cord length was nearly twice that in control vector or untreated rats. Hydroxyproline measurements confirmed the presence of fibrosis. These data suggest that overexpression of TGF-beta 1 during the critical period of postnatal rat lung alveolarization gives rise to pathological, biochemical, and morphological changes consistent with those seen in human bronchopulmonary dysplasia, thus inferring a pathogenic role for TGF-beta in this disorder.     

Tumor genes and their proteins in cytologic and surgical specimens: Relevance and detection systems

Oncogenesis is the consequence of a series of genetic alterations that allow unrestrained cellular growth, tissue invasion, and eventual metastases. Tumor-related genes can be classified into functional categories. Proto-oncogenes/oncogenes have a stimulatory role in cell growth, and the inactivation of cancer-suppressor genes/antioncogenes results in the loss of cell cycle regulation. More recently, three other groups of tumor-related genes have been recognized. They include the antiapoptosis genes which protect from programmed cell death, the antimetastasis genes, and multidrug resistance genes. Besides aiding in tumor diagnosis, the detection of such tumor-associated genes and their products allows the identification of individuals with an inherited predisposition to neoplastic growths, and the overexpression of many of these oncogene products has been shown to be a potential marker of tumor behavior and a predictor of treatment outcome and response. The ability to utilize DNA and RNA probes for nucleic acid hybridization and polymerase chain reaction procedures in cell and tissue preparations of solid tumors and lymphoid proliferations expands and complements the information provided by immunohistochemical techniques. These probes allow direct visualization and correlation of specific genes and their protein products with cytomorphologic features, and form a powerful addition to the armamentarium of the cytopathologist and surgical pathologist. © 1995 Wiley-Liss, Inc.     

Cellular immunity in Guamanians with amyotrophic lateral sclerosis and Parkinsonism-dementia.

To test the hypothesis that host resistance factors may be abnormal in Guamanians in whom amyotrophic lateral sclerosis and Parkinsonism-dementia develop, cellular immunity was evaluated in both diseases and compared to that of Guamanians with other nervous-system diseases, normal adult Guamanians and non-Guamanians with amyotrophic lateral sclerosis and Parkinsonism. Diminished responses to skin-test antigens, lymphopenia, diminished per cent and total T cells and, less frequently, decreased mitogen responses were seen in Guamanian patients with amytorophic lateral sclerosis and Parkinsonism-dementia but not in the other patient or normal groups. Guamanian patients with amyotrophic lateral sclerosis and diminished cellular immunity had an increased frequency of HLA-Bw35 (P less than 0.005) and shorter mean duration of disease (P less than 0.05) than those with normal cellular immunity. In Parkinsonism dementia diminished cellular immunity was less strongly associated with HLA-BW35 (P less than 0.05) and was not associated with differences in duration of disease. Normal Guamanians and those with other nervous-system diseases showed no association of diminished cellular immunity with HLA-Bw35. The association appeared disease-related, with onset concomitant with the neurologic expression of Guamanian amyotrophic lateral sclerosis and Parkinsonism-dementia.     

Genes for immunodominant protein antigens are highly homologous in Mycobacterium tuberculosis, Mycobacterium africanum, and the vaccine strain Mycobacterium bovis BCG.

The relatedness of immunodominant protein antigens in Mycobacterium tuberculosis, M. africanum, and M. bovis BCG was investigated by comparing the genes that encode major protein antigens in M. tuberculosis with their counterparts in the other two mycobacteria. Genes encoding homologs of M. tuberculosis major protein antigens were isolated from M. africanum and M. bovis BCG by constructing lambda gt11 recombinant DNA expression libraries and screening them with murine monoclonal antibodies and DNA probes. The antibodies were directed against four major protein antigens of M. tuberculosis with molecular masses of 71, 65, 19, and 14 kilodaltons. The isolated M. africanum and M. bovis BCG DNA clones were mapped with restriction endonucleases, and the maps of the mycobacterial genes were confirmed by Southern analysis of mycobacterial genomic DNA. The restriction maps of DNA containing the four genes in M. tuberculosis, M. africanum, and M. bovis BCG are identical, indicating that the immunodominant proteins that they encode are highly homologous in the three mycobacteria. Thus, the immunity against tuberculosis engendered by M. bovis BCG vaccination could be provided, at least in part, by the immune response to these homologous antigens.     

Differential control over cocaine-seeking behavior by nucleus accumbens core and shell

Nucleus accumbens (NAc) dopamine is widely implicated in mediating the reinforcing effects of drugs of abuse. However, the precise function of the NAc itself in drug self-administration has been difficult to establish. Here we show a neural double-dissociation of the behavioral processes that underlie cocaine self-administration in rats. Whereas selective excitotoxic lesions of the NAc core had only a minor effect on the acquisition of responding for cocaine under a standard schedule of continuous reinforcement, these lesions profoundly impaired the acquisition of drug-seeking behavior that was maintained by drug-associated conditioned reinforcers and assessed using a second-order schedule of cocaine reinforcement. In contrast, selective excitotoxic lesions of the NAc shell did not impair drug self-administration or the acquisition of cocaine-seeking, but they did attenuate the psychostimulant effects of cocaine. These results further our understanding of how the NAc controls drug-seeking and drug-taking behavior.     

Protection against pneumococcal infection in mice conferred by phosphocholine-binding antibodies: specificity of the phosphocholine binding and relation to several types.

Some anti-phosphocholine antibodies protect mice against challenge with certain, but not all, pneumococcal types. We found that both their isotype and reactivity with the cell wall C-polysaccharide of encapsulated pneumococci, as measured by immunodiffusion, were important in predicting the protective activity of anti-phosphocholine antibodies. We propose that the specificity of the protective antibodies includes the backbone of the phosphocholine-containing structure.     

Prevalence of neutralizing antibodies to adenoviral serotypes 5 and 35 in the adult populations of The Gambia, South Africa, and the United States

One of the major limitations of the use of adenoviruses as gene therapy vectors is the existence of preformed immunity in various populations. Recent studies have linked failure of adenoviral gene therapy trials to the presence of antiadenoviral neutralizing antibodies (NAb). Understanding the distribution and specificity of such antibodies will assist in the design of successful recombinant adenoviral gene therapies and vaccines. To assess the prevalence of NAb to adenovirus serotypes 5 and 35 (Ad5 and Ad35), we analyzed serum samples from adult immunocompetent individuals living in The Gambia, South Africa, and the United States by using a neutralization assay. Serum samples were incubated with A549 lung carcinoma cells and adenoviruses encoding enhanced green or yellow fluorescent proteins; results were analyzed by fluorescence microscopy and flow cytometry. Using this technique, we found a high prevalence of NAb against Ad5 in Gambian, South African, and U.S. subjects at both low and high titers. Conversely, all subjects displayed a low prevalence of NAb to Ad35; when present, anti-Ad35 NAb were seen at low titers. Because of the ability of adenoviruses to elicit systemic and mucosal immune responses, Ad35 with its low NAb prevalence appears to be an attractive candidate vector for gene therapy applications.     

Hydrolytic stability of pneumococcal group 6 (type 6A and 6B) capsular polysaccharides.

The hydrolyses of the immunologically cross-reactive and constitutionally isomeric group 6 pneumococcal polysaccharides, types 6A and 6B, were investigated by 31P nuclear magnetic resonance spectroscopy, gel filtration through Sepharose 4B, reducing-sugar analysis, and rocket immunoelectrophoresis. Phosphorus nuclear magnetic resonance spectroscopy showed that cleavage of the repeating-unit phosphodiester linkages at pH 10, 60 degrees C was considerably faster (greater than 10(3) ) for the type 6A than the type 6B polysaccharide. Under these reaction conditions, 31P nuclear magnetic resonance kinetic measurements showed that the Na+ form of the type 6A polysaccharide underwent phosphodiester-linkage hydrolysis two times slower than the corresponding Ca+2 form; a stoichiometrically excess amount of Ca+2 caused a 30-fold enhancement of the latter hydrolysis rate. The spectroscopic characterization of phosphorus-containing end groups resulting from hydrolysis of the type 6A polymer provided additional mechanistic information. Heating the type 6A and 6B polysaccharides at 56 degrees C for various times led to gel filtration coefficients of distribution (Kd values) which indicated that the type 6A material underwent size reductions considerably faster than did the type 6B antigen; these increased Kd values qualitatively correlated with the loss of immunochemical reactivity measured by rocket immunoelectrophoresis. The application of a statistical theory to the depolymerization of the type 6A and 6B polysaccharides was consistent with random bond cleavage, as evidenced by the calculated versus measured gel filtration patterns. Although the molecular changes causing the size reductions were not fully elaborated, it was established that the acetal linkages of the type 6A and 6B polysaccharides were comparatively resistant to hydrolysis and that depolymerization by hydrolysis of the phosphodiester linkage was a major factor only in the type 6A structure. It was concluded that the hydrolytic stability of the type 6B antigen would favor its use in the polyvalent pneumococcal vaccine rather than the cross-reactive, but comparatively unstable, type 6A polysaccharide, if all other factors are equal.     

Synthesis and physicochemical and immunological characterization of pneumococcus type 12F polysaccharide-diphtheria toxoid conjugates.

A scheme for the synthesis and purification of conjugates, composed of the type 12F capsular polysaccharide of Streptococcus pneumoniae (Pn12F) and diphtheria toxoid, is described. The scheme is a modification of that described previously for the Vi capsular polysaccharide of Salmonella typhi, a linear homopolymer of N-acetylgalactoseaminouronic acid (S. C. Szu, A. L. Stone, J. D. Robbins, R. Schneerson, and J. B. Robbins, J. Exp. Med. 166:1510-1524, 1986). Pn12F is a branched-chain copolymer composed of a hexasaccharide repeating unit containing an aminouronic acid, N-acetylmannoseaminouronic acid (K. Leontein, B. Lindberg, and J. Lonngren, Can. J. Chem. 59:2081-2085, 1981). Sulfhydryl groups were introduced into Pn12F by forming an amide bond between cystamine and carboxyl groups of N-acetylmannoseaminouronic acid in the presence of a carbodiimide. The disulfide moiety of cystamine was reduced to form the cysteamine derivative of Pn12F which was, in turn, covalently bound to diphtheria toxoid by using the heterobifunctional linker N-succinimidyl-3-(2-pyridylthio)propionate. Unbound, high-molecular-weight Pn12F was removed from the conjugate by hydrophobic interaction chromatography through octyl Sepharose by using n-octyl-beta-D-glucopyranoside as the eluent. In young outbred mice, Pn12F did not elicit detectable serum antibodies. Pn12F-diphtheria toxoid, in contrast, elicited antibodies after two injections and had T-cell-dependent properties as evidenced by a response to priming and by its ability to elicit booster responses. This scheme seems applicable to the synthesis of conjugates with other capsular polysaccharides containing aminouronic acids. Clinical evaluation of Pn12F-diphtheria toxoid conjugates in healthy and in immunocompromised hosts is planned.