Based on their mechanisms-of-action, CD20 monoclonal antibodies (mAbs) are grouped into Type I [complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)] and Type II [programmed cell death (PCD) and ADCC] mAbs. We generated 17 new hybridomas producing CD20 mAbs of different isotypes and determined unique heavy and light chain sequence pairs for 13 of them. We studied their epitope binding, binding kinetics and structural properties and investigated their predictive value for effector functions, i.e. PCD, CDC and ADCC. Peptide mapping and CD20 mutant screens revealed that 10 out of these 11 new mAbs have an overlapping epitope with the prototypic Type I mAb rituximab, albeit that distinct amino acids of the CD20 molecule contributed differently. Binding kinetics did not correlate with the striking differences in CDC activity among the mIgG2c mAbs. Interestingly, chimerization of mAb m1 resulted in a mAb displaying both Type I and II characteristics. PCD induction was lost upon introduction of a mutation in the framework of the heavy chain affecting the elbow angle, supporting that structural changes within this region can affect functional activities of CD20 mAbs. Together, these new CD20 mAbs provide further insights in the properties dictating the functional efficacy of CD20 mAbs.

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Abstract. Two cats with congenital hypothyroidism are described. In vivo discharge of accumulated labelled iodide by perchlorate administration revealed defective organification of iodide, which was complete in one cat and partial in the other. In the cat with the partial organification defect, thyroid tissue was obtained for biochemical studies. No membrane-bound peroxidase activity could be demonstrated. The activity was found in the 100000 × g supernatant. It is suggested that the loose enzyme anchoring caused decreased availability of peroxidase and as a consequence reduced capacity for organic binding of trapped iodide.

Interleukin 12 (IL-12) is a potent enhancer of interferon ␥ production by activated T cells. The high-affinity IL-12 receptor (IL-12R) is a heterodimer of a ␤1 and a ␤2 subunit. Expression of the signaling IL-12R␤2 chain is usually low, as compared with the more abundant ␤1 chain, and may be rate-limiting for IL-12 sensitivity. Little is known about the mechanisms controlling IL-12R␤2 gene expression. Reporter gene assays in IL-12R␤2-expressing Jurkat cells showed that truncation of the region from ؊151 to ؊61 abrogated promoter activity. The proximal promoter region does not contain a typical TATA box, suggesting a role for SP-1. Indeed, mutagenesis of the ؊63 SP-1 consensus site decreased transcription by 50%. Electrophoretic mobility shift experiments confirmed the binding of SP-1 and SP-3 at this site. In contrast, truncation of ؊252 to ؊192 increased promoter activity. Likewise, mutagenesis of the consensus nuclear factor of activated T cells site at ؊206 increased promoter activity by 70%, suggesting silencer activity of this element. Electrophoretic mobility shift experiments with primary Th (T helper) cells showed the formation of a specific, T-cell receptor-inducible complex at this site that is sensitive to cyclosporin A and supershifted with anti-NFATc2 in both Th1 and Th2 cells. Accordingly, cyclosporin A dose-dependently increased IL-12R␤2 mRNA expression. These first data on IL-12R␤2 gene regulation indicate a TATA-less promoter, depending on SP-1/SP-3 transcription factors, and a negative regulatory NFAT element at ؊206. This element may contribute to the overall low level of IL-12R␤2 expression on Th cells.

SUMMARYChronic periodontitis is a destructive inflammatory disease linked with unbalanced production between matrix metalloproteinases (MMPs), such as interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3) and their endogenous tissue inhibitors of MMPs (TIMPs). In addition to aberrant MMP-1 and MMP-3 expression, periodontal lesions are characterized by dense infiltrations of activated T lymphocytes which may interact with CD40-expressing gingival fibroblasts in the connective tissue via the CD40L-CD40 pathway. In this study we investigated whether CD40 cross-linking influenced MMP production by gingival fibroblasts. Therefore, we analysed the CD40L-induced MMP production by these fibroblasts in the presence of cytokines that are increased in periodontal lesions, such as IL-1b, tumour necrosis factor-alpha (TNF-a) and interferon-gamma (IFN-g). We show that CD40 ligation on gingival fibroblasts resulted in a decrease of their MMP-1 and MMP-3 production, while MMP-2 and TIMP-1 production were unaffected as determined by Western blot. This down-regulatory effect of CD40 engagement on MMP-1 and MMP-3 production by gingival fibroblasts was also present when MMP production was up-regulated by IL-1b and TNF-a or down-regulated by IFN-g. These results suggest that CD40 ligation on gingival fibroblasts leads to a restraining of MMP-1 and MMP-3 production by gingival fibroblasts and thereby may be an important mechanism in the retardation of further periodontal tissue damage.