Use of an antineoepitope antibody for identification of type-II collagen degradation in equine articular cartilage.

OBJECTIVE: To develop an antibody that specifically recognizes collagenase-cleaved type-II collagen in equine articular cartilage. SAMPLE POPULATION: Cartilage specimens from horses euthanatized for problems unrelated to the musculoskeletal system. PROCEDURE: A peptide was synthesized representing the carboxy- (C-) terminus (neoepitope) of the equine type-II collagen fragment created by mammalian collagenases. This peptide was used to produce a polyclonal antibody, characterized by western analysis for reactivity to native and collagenase-cleaved equine collagens. The antibody was evaluated as an antineoepitope antibody by ELISA, using peptides +/- an amino acid at the C-terminus of the immunizing peptide. Collagen cleavage was assayed from equine articular cartilage cultured with interleukin-1 (IL-1), +/- a synthetic MMP inhibitor, BAY 12-9566. Cartilage specimens from osteoarthritic and nonarthritic joints were compared for antibody staining. RESULTS: An antibody, 234CEQ, recognized only collagenase-generated 3/4-length fragments of equine type-II collagen. This was a true antineoepitope antibody, as altering the C-terminus of the immunizing peptide significantly decreased competition for binding in an inhibition ELISA. The IL-1-induced release of type-II collagen fragments from articular cartilage was prevented with the MMP inhibitor. Cartilage from an osteoarthritic joint of a horse had increased staining with the 234CEQ antibody, compared with normal articular cartilage. CONCLUSIONS AND CLINICAL RELEVANCE: We generated an antineoepitope antibody recognizing collagenase-cleaved type-II collagen of horses. This antibody detects increases in type-II collagen cleavage in diseased equine articular cartilage. The 234CEQ antibody has the potential to aid in the early diagnosis of arthritis and to monitor treatment responses.

Morphologically docked synaptic vesicles are reduced in synaptotagmin mutants of Drosophila.

Nerve terminal specializations include mechanisms for maintaining a subpopulation of vesicles in a docked, fusion-ready state. We have investigated the relationship between synaptotagmin and the number of morphologically docked vesicles by an electron microscopic analysis of Drosophila synaptotagmin (syt) mutants. The overall number of synaptic vesicles in a terminal was reduced, although each active zone continued to have a cluster of vesicles in its vicinity. In addition, there was an increase in the number of large vesicles near synapses. Examining the clusters, we found that the pool of synaptic vesicles immediately adjacent to the presynaptic membrane, the pool that includes the docked population, was reduced to 24 +/- 5% (means +/- SEM) of control in the sytnull mutation. To separate contributions of overall vesicle depletion and increased spontaneous release from direct effects of synaptotagmin on morphological docking, we examined syt mutants in an altered genetic background. Recombining syt alleles onto a second chromosome bearing an as yet uncharacterized mutation resulted in the expected decrease in evoked release but suppressed the increase in spontaneous release frequency. Motor nerve terminals in this genotype contained more synaptic vesicles than control, yet the number of vesicles immediately adjacent to the presynaptic membrane near active zones was still reduced (33 +/- 4% of control). Our findings demonstrate that there is a decrease in the number of morphologically docked vesicles seen in syt mutants. The decreases in docking and evoked release are independent of the increase in spontaneous release. These results support the hypothesis that synaptotagmin stabilizes the docked state.