Variant antibody identification by peptide mapping

During development of CGP56901, a monoclonal antibody (MAb) specific for a unique epitope on human IgE, the protein A-purified IgG from one of the candidate production cell lines, showed an additional minor heavy chain (H-chain) band with a molecular weight slightly lower than that of the principal H-chain band on SDS-PAGE. The N-terminal amino acid sequence of this minor H-chain species indicated that at least the first 30 amino acids were identical to those of the antibody light-chain (L-chain) variable domain. More detailed studies using peptide mapping and amino acid sequencing analysis confirmed a crossover event between the V genes of the antibody. The position is between Arg108 of the L chain and Ala124 of the H chain. This crossover resulted in a variant H chain, which had 16 fewer amino acid residues than the normal CGP56901 H chain. These results show that peptide mapping is a useful "first-line" analytical tool in the characterization of the quality of the monoclonal antibody.     

An ecological approach to landscape planning using geographic information system technology

This paper describes two research programs that are aimed at developing ecologically-based landscape planning models. The first of these models was developed as part of a more comprehensive landscape planning research program at the University of Massachusetts. The second is an on-going program in forest land planning at the University of Vermont. In addition to an emphasis on ecological values, both projects make extensive use of computerized geographic information systems (GIS) for data storage, analysis and display. The emphasis has been to develop a synthesis of planning procedures, ecological theories and quantitative techniques. Both research efforts were guided by a three-part methodological framework adapted from the work of Eugene Odum. The Massachusetts work generated a two-part classification system. The first part of the classification uses the statistical technique of discriminant analysis to place land uses into five groups, each having similar ecological characteristics. The second part of the classification is based on the physical and environmental characteristics of a site. The biological and denudational potentials of a site are determined from soil characteristics and slope. A comparison of the two classification schemes was made to determine ecological compatibility. The procedures were applied to two communities in Western Massachusetts. The Vermont study was aimed at integrating ecological values into forest land management and planning processes. The research utilized soils and topographic information in the development of an ecologically based forest land suitability model. Forest site index and soil erosion potential were determined using data stored in a computerized geographic information system. Forest site index estimates were made by utilizing an existing, well documented soil-site index regression equation applicable to the Green Mountains of Vermont. Soil erosion potential estimates were made by combining a soil erodability factor (K) with slope. The site index and soil erosion estimates were combined to produce land suitability classes for resource protection, forest management, multiple-use and trade-off. The procedure was applied in the Mad River Valley in Central Vermont. Both the Massachusetts and the Vermont studies are seen as significant contributions toward the integration of ecological factors into processes for landscape planning and resource management.     

Basis of pulmonary toxicity associated with cationic lipid-mediated gene transfer to the mammalian lung

Studies have indicated that although abundant levels of transgene expression could be achieved in the lungs of mice instilled with cationic lipid:pDNA complexes, the efficiency of gene transfer is low. As a consequence, a relatively large amount of the complex will need to be administered to the human lungs to achieve therapeutic efficacy for indications such as cystic fibrosis. Because all cationic lipids exhibit some level of cytotoxicity in vitro, we assessed the safety profile of one such cationic lipid, GL-67, following administration into the lungs of BALB/c mice. Dose-dependent pulmonary inflammation was observed that was characterized by infiltrates of neutrophils, and, to a lesser extent, macrophages and lymphocytes. The lesions in the lung were multifocal in nature and were manifested primarily at the junction of the terminal bronchioles and alveolar ducts. The degree of inflammation abated with time and there were no apparent permanent fibrotic lesions, even in animals that were treated at the highest doses. Analysis of the individual components of the complex revealed that the pulmonary inflammation was primarily cationic lipid-mediated with a minor contribution from the neutral co-lipid DOPE. Associated with the lesions in the lungs were elevated levels of the pro-inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) that peaked at days 1-2 post-instillation but resolved to normal limits by day 14. Total cell counts, primarily of neutrophils, were also significantly elevated in the bronchoalveolar lavage fluids of GL-67:pDNA-treated mice between days 1 and 3 but returned to normal limits by day 14. No specific immune responses were detected against the cationic lipid or plasmid DNA in mice that had been either instilled or immunized with the individual components or complex, nor was there any evidence of complement activation. These studies indicate that a significant improvement in the potency of cationic lipid:pDNA formulations is desirable to minimize the toxicity associated with cationic lipids.