Anti-IgE efficacy in murine asthma models is dependent on the method of allergen sensitization.

BACKGROUND: Murine models used to delineate mechanisms and key mediators of asthma have yielded conflicting results and suggest that the dominant mechanism and mediators required for disease induction differ depending on the model and method of allergen sensitization used. OBJECTIVE: The goal of this study was to determine whether the mode of allergen sensitization influenced the role that IgE had in allergen-induced pulmonary eosinophilic inflammation. METHODS: Mice were exposed to dust mite extract in 2 models of allergic inflammation that differed in the method of sensitization. We compared sensitization by aerosol exposure with and without concomitant human respiratory syncytial virus infection with sensitization by means of systemic (intraperitoneal) exposure with adjuvant. After sensitization, animals were similarly challenged with aerosolized allergen. Animals were treated with anti-IgE mAb to deplete IgE and to determine its role in the induction of allergic inflammation and mucosa pathology in these models. RESULTS: Concomitant respiratory syncytial virus infection significantly enhanced allergen sensitization by aerosol exposure and exacerbated eosinophilic inflammation and airway mucosa pathology. Depletion of IgE in this model significantly reduced lung eosinophilic inflammation and airway mucosa pathology. However, in the model in which animals were sensitized by means of systemic allergen exposure with adjuvant, depletion of IgE had no ameliorative effect on lung inflammation or pathology. CONCLUSION: We demonstrated that the method of antigen sensitization can delineate the role of IgE in allergen-induced lung inflammation. In a murine model that more closely resembles ambient allergen exposure in human subjects, IgE had a critical role in the pathogenesis of allergic asthma and mucosa pathology. The results parallel the results reported with anti-IgE efficacy in allergic asthmatic human subjects.

Optimisation of the quantitative determination of chlormequat by matrix-assisted laser desorption/ionisation mass spectrometry.

The plant growth regulator chlormequat, an involatile quaternary ammonium salt, has been quantified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). Restrictions for quantitative MALDI-TOFMS analysis, such as irreproducible crystallisation and unsatisfactory laser stability, have been overcome by the application of two synthesised isotopically labelled standards and the optimisation of the measurement protocol. Data acquisition at constant laser power was compared to data acquisition at approximately constant ion abundance of the relevant ions (analyte and internal standards). Data acquisition at constant ion abundance performed better and enabled a high number of consecutive firings to the same sample deposition area. Furthermore an increased sample-to-sample repeatability and a high reproducibility over several weeks without re-calibration have been attained by this method. Linearity over three orders of magnitude (0.05 to 30 ng/microL chlormequat), with a correlation coefficient of 0.9997, was achieved using [13C3]-chlormequat as internal standard. Limit of detection and limit of determination were determined to be in the low pg/microL range for pure standard solutions. Thin-layer chromatography was applied for the removal of high amounts of choline, which is often present in plant tissue extracts and can adversely affect the ionisation and detection of chlormequat by MALDI-TOFMS. The use of two internal standards ([13C3]- and [2H9]-chlormequat) enabled direct quantification and simultaneous control of the recovery.

Humanization of an anti-lymphocyte function-associated antigen (LFA)-1 monoclonal antibody and reengineering of the humanized antibody for binding to rhesus LFA-1.

Lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18) is involved in leukocyte adhesion during cellular interactions essential for immunologic responses and inflammation. mAbs against LFA-1 have been shown to inhibit several T cell-dependent immune functions in vitro and prevent graft failure after bone marrow transplantation in vivo. A murine anti-human CD11a mAb, MHM24, has been humanized and shown to prevent adhesion of human T cells to human keratinocytes and the proliferation of T cells in response to nonautologous leukocytes in the mixed lymphocyte response assay. However, of the nonhuman primate CD11a that we tested, the murine and humanized mAbs cross-reacted only with chimpanzee CD11a. To have a mAb available for preclinical studies in rhesus monkeys, the humanized mAb was reengineered to bind to rhesus CD11a by changing four residues in one of the complementarity-determining regions, CDR-H2, in the variable heavy domain. Cloning and molecular modeling of rhesus CD11a I-domain suggested that the changes from Lys197 and/or Arg189 in human CD11a I-domain to Glu197 and Gln189 in rhesus CD11a I-domain may be the reason that rhesus CD11a does not bind to the murine and humanized mAbs.

X-ray structures of fragments from binding and nonbinding versions of a humanized anti-CD18 antibody: structural indications of the key role of VH residues 59 to 65.

X-ray crystal structures of fragments from two different humanized anti-CD18 antibodies are reported. The Fv fragment of the nonbinding version has been refined in space group C2 with a = 64.2 A, b = 61.3 A, c = 51.8 A, and beta = 99 degrees to an R-value of 18.0% at 1.9 A, and the Fab fragment of the tight-binding version has been refined in space group P3 with a = 101. A and c = 45.5 A to an R-value of 17.8% at 3.0 A resolution. The very large difference in their binding affinity (> 1000-fold) is attributed to large and local structural differences in the C-terminal part of CDR-H2, and from this we conclude there is direct contact between this region and antigen when they combine. X-ray structures of antibody-antigen complexes available in the literature have yet to show this part of CDR-H2 in contact with antigen, despite its hypervariable sequence. Implications of this result for antibody humanization are discussed.

Engineering a humanized bispecific F(ab')2 fragment for improved binding to T cells.

We recently constructed a humanized bispecific antibody (BsF(ab')2v1) by separate E. coli expression of each Fab' arm followed by directed chemical coupling in vitro. BsF(ab')2 v1 (anti-CD3/anti-p185HER2) was demonstrated to retarget the cytotoxic activity of human CD3+ CTL in vitro against the human breast-tumor cell line, SK-BR-3, which over-expresses the p185HER2 product of the proto-oncogene HER2. Our minimalistic humanization strategy is to install as few murine residues as possible into a human antibody in order to recruit antigen-binding affinity and biological properties comparable to that of the murine parent antibody. This strategy proved very successful for the anti-p185HER2 arm of BsF(ab')2 v1. In contrast BsF(ab')2 v1 binds to T cells via its anti-CD3 arm much less efficiently than does the chimeric BsF(ab')2 which contains the variable domains of the murine parent anti-CD3 antibody. Here we have constructed additional BsF(ab')2 fragments containing variant anti-CD3 arms with selected amino acid replacements in an attempt to improve antibody binding to T cells. One such variant, BsF(ab')2 v9, was created by replacing 6 residues in the second hypervariable loop of the anti-CD3 heavy chain variable domain of BsF(ab')2 v1 with their counterparts from the murine parent anti-CD3 antibody. BsF(ab')2 v9 binds to T cells (Jurkat) much more efficiently than does BsF(ab')2 v1 and almost as efficiently as the chimeric BsF(ab')2. This improvement in the efficiency of T-cell binding of the humanized BsF(ab')2 is an important step in its development as a potential therapeutic agent for the treatment of p185HER2 over-expressing cancers.

Utilization of part-time employees can enhance operating efficiency.

Part-time employees offer some distinct advantages in improving hospital efficiency. Part-time employees provide significant scheduling benefits that will enable perceptive supervisors to enhance their department's performance. Desperately needed skills to staff laboratories, nursing units, admissions offices, clinics, and other divisions can be found among housewives and retirees. These benefits can be achieved while cutting the hospital's total labor costs--the most promising dimension of all. It is becoming increasingly important for administrators to discover new methods of achieving their organizations' objectives. With labor costs representing a significant part of operating expenditures, creative manpower innovations hold the potential for major improvements, especially in hospitals.