Behavioral, clinical, and physiologic analysis of mice used for ascites monoclonal antibody production.

BACKGROUND AND PURPOSE: The effects of pristane inoculation, ascites accumulation, peritoneocentesis, and analgesics on the well-being of mice used in monoclonal antibody (MAb) production protocols were investigated. METHODS: Four experiments, each containing 17 to 21, 6- to 8-week-old male Balb/c mice, were conducted. Each experiment involved a period in which baseline data were collected, followed by intraperitoneal injections of pristane or phosphate-buffered saline (PBS) inoculations into each mouse. One week later mice received intraperitoneal inoculations of either hybridoma cells or PBS. Parameters used to assess well-being throughout each of these periods included: wheel-running activity, food and water consumption, open-field box activity, clinical observation, and plasma corticosterone concentration. RESULTS: Compared to controls, pristane inoculation had slight to no affect on mice. There was no evidence of distress in cell-inoculated mice prior to their gaining 25% of their baseline body weight. The number of times (up to three) that peritoneocentesis was performed did not have a significant impact on mice's well-being, but ascites yields were greater when multiple harvests were performed. Cell-inoculated mice that gained weight slowly or developed high-particulate ascites were at higher risk of being distressed. CONCLUSION: Ascites yields can be maximized by performing multiple harvests; however, the well-being of mice used in such protocols should be closely monitored, as suggested here.

Bacterial expression and characterization of recombinant biologically active anti-tyrosine kinase receptor antibody forms.

Homomeric and heteromeric interactions among cell-surface tyrosine kinase receptors belonging to the ErbB family lead to intracellular signaling cascades which are involved in cell activation, cytoskeletal interactions, and cellular transformation leading to neoplasia. Monoclonal antibodies which specifically bind to p185neu or epidermal growth factor receptor (EGFR), such as 7.16.4 and 225, respectively, can elicit tumor growth-inhibitory effects on transformed cells which overexpress either or both of these receptors. In order to better understand these receptor-receptor and receptor-antibody interactions and to gain insights that may be useful in the production and design of an antibody-based anticancer therapeutic, novel small recombinant 7.16.4 and 225 single-chain Fv fragments (scFv) were constructed, expressed, and characterized. We showed that these recombinant antibody fragments, which retain binding affinity, can be produced and purified from bacterial cell lysates. Our analyses further demonstrate that fusion of a 61 amino-acid dimerization domain with 7.16.4 and 225 scFv (7.16.4hth and 225hth) is sufficient to restore biological activity to these recombinant proteins.

Recombinant antibodies: alternative strategies for developing and manipulating murine-derived monoclonal antibodies.

Since the introduction of hybridoma technology 20 years ago, numerous monoclonal antibodies with specificity to cellular, bacterial, and viral proteins have been developed. Application of monoclonal antibodies in biomedical research has substantially contributed to our understanding of the structural and physiologic components of intra- and extracellular protein interactions. Monoclonal antibodies that target antigens specific to infective agents or tumor cells may also be used as therapeutic agents. Despite the versatility of these molecules, monoclonal antibody/hybridoma production is labor-intensive and requires the use of live animals. The fact that monoclonal antibodies are derived from animals limits their use as systemic therapeutic agents in humans. This can be attributed to the human anti-mouse antibody response that is mounted against these therapeutically administered foreign proteins. Recent advances in our understanding of immunoglobulin structure through three-dimensional studies--using nuclear magnetic resonance and X-ray crystallography and increased computer-assisted molecular modeling capabilities, combined with the application of recombinant approaches--has led to the evolution of a new class of antibody-like molecules or man-made antibodies. The potential of recombinant antibodies has been realized globally by academic and industrial institutions; the efficacy, toxicity, and pharmacokinetics of these antibody-derived compounds are being tested in a variety of animal models. This review summarizes various approaches for producing recombinant antibodies and discusses their potential as anti-cancer compounds so that those who are involved in relevant experimental animal protocols may gain a better understanding of this rapidly growing area. Additionally, by mimicking the affinity maturation of antibodies in vitro, phage display strategies have the potential to reduce or eliminate the use of animals in antibody production protocols.

A helical polymer with a cooperative response to chiral information.

Polyisocyanates, long studied as theoretical models for wormlike chains in dilute solution and liquid crystals, differ from their biological helical analogs in the absence of a pre-determined helical sense. These polymers have an unusual sensitivity to chiral effects that arises from a structure in which alternating right- and left-handed long helical blocks are separated by infrequent and mobile helical reversals. Statistical thermodynamic methods yield an exact description of the polymer and the cooperative nature of its chiral properties. Minute energies that favor one of the helical senses drive easily measurable conformational changes, even though such energies may be extremely difficult to calculate from structural theory. In addition, the chiral nature of the polymer can be used to test theoretical ideas concerned with cholesteric liquid crystals, one of which solves the problem of assigning the helical sense.

Considerations in the design and production of small anti-receptor antibody forms: optimizing gains while reducing size.

Recent advances which have lead to the cloning of numerous immunoglobulin genes has resulted in the production of a variety of recombinant small antibody-like molecules. These smaller immunoglobulin derived molecules have potential for the development of diagnostic and pharmaceutical compounds. The considerations in the design and production of smaller antibody-like molecules and their clinical applications will be summarized in this review.

Antibody-structure-based design of pharmacological agents.

The well-studied antigen-combining sites of antibody molecules hold considerable promise as a model system for the design of bioactive peptides. These small, immunoglobulin-derived peptides can be used in the development of alternative treatments for disease and in diagnostic strategies. The general principles derived from the design of small pharmacological agents based on the structural features of antibodies may also be extended to the design of other bioactive peptides.

The neu-oncogene: signal transduction pathways, transformation mechanisms and evolving therapies.

A variety of pathways exist to transmit biological signals. One mechanism used for the regulated control of cell growth and differentiation is through the transduction of signals resulting from the binding of soluble polypeptide growth factors to their cognate receptors. The specificity of growth factor action is mediated by the interaction of ligand with cognate receptors which can lead to exquisite control in a tissue- and developmental-specific manner. In addition, individual receptors on the cell surface can form complex assemblies with other receptor/signal transduction molecules that potentially lead to additional levels of signal transmissions. Biological signaling by peptide ligands can be mediated through the enzymatic activation of the receptor resulting in the triggering of a defined biochemical pathway. Ultimately, a mitogenic or differentiation signal is delivered to the nucleus, completing the biological action of the growth factor. The biochemical mechanisms of signal transduction by the p185 neu/c-erbB-2 growth factor receptor and the subsequent physiological responses are the topics of this review. Study of the p185 growth factor receptor has helped to illustrate the functional role of receptor homo- (and hetero-) dimerization in enzyme activation and, in malignant cells, the detrimental results of structural mutations or aberrant gene expression which may effect this dimerization. The ability of one type of growth factor receptor to affect the activity of another (as illustrated by the p185/epidermal growth factor receptor heterodimeric complex) is likely to be a common regulatory feature of growth factor receptor action. The nomenclature to be used in this review will refer to the oncogenic mutated form of the rat protein as 'p185neu', the proto-oncogenic rat protein as 'p185c-neu' and the human form as 'p185c-erbB-2'. The term 'p185' will be used to refer to any type of protein, regardless of the source.

Concurrent validity of the Gardner Test of Visual-Motor Skills.

Scores on the Test of Visual-motor Skills, Developmental Test of Visual-motor Integration, and Bender-Gestalt test were compared for a sample of 44 elementary school children referred for evaluation of learning disorders. While the tests shared common variance, the mean standard score on the Test of Visual-motor Skills was significantly lower than the means of the other two tests, suggesting caution in the clinical use of the new scale.

TEA Laser Induced Multiphoton Dissociation of Ethylene in a Collisional Regime: Model and Experiment.

The focused beam of a pulsed CO2 TEA laser was used to photodissociate ethylene gas at total pressures of 100-300 torr. The laser pulse induces molecular hydrogen elimination to yield primarily C2H2 and H2 in an large excess of helium to eliminate heating. An invesigation was made of the dependence of product yield on wavelength, inert gas pressure, ethylene pressure and intensity. Photochemical yield was followed over a 10,000 fold range. Its dependence was investigated employing alternate methods of focusing the laser beam, one of which results in a constant volume geometry. A photometric method of characterizing this focal zone is described. A "structureless" computer model of the laser pumping process involving coupled rate equations and phenomenological absorption and stimulated emission coefficients is described. The model assumes rapid intramolecular energy transfer between the various vibrational modes throughout the entire vibrational manifold, and rapid equilibration of rotational states. The model includes stimulated emission and deactivating collisions, and predicts a product yield versus intensity dependence that does not exhibit threshold behavior in agreement with experiment. Calculated dependences of product yield on laser power for various parameters are given in order to relate the model to various photolysis experiments.