Osteoclast polarization and orthodontic tooth movement.

INTRODUCTION: Osteoclasts polarize when they contact activation signals that are associated with bone. Polarization is required for bone resorption and involves highly specialized mechanisms that represent attractive targets for the development of osteoclast-specific therapeutic agents. One potential use of such agents is to block tooth movement in spatially discrete locations to provide orthodontic anchorage. MATERIALS AND METHODS: Our group's research was directed toward the development of agents that inhibited the polarization of osteoclasts, and efforts were underway to develop means to experimentally modulate orthodontic tooth movement. We performed 'proof-in-principle' experiments demonstrating pharmacological blockades of orthodontic tooth movement using integrin and matrix metalloproteinase inhibitors in a rat model. RESULTS: We identified novel mechanisms underlying osteoclast bone resorption. Interactions between vacuolar H(+)-ATPase and the microfilament cytoskeleton that were unique to osteoclasts were described and characterized. Our group is now seeking to make use of this new knowledge, coupled with an emerging technique, supercomputer-based molecular modeling for the rational development of novel, osteoclast-specific therapeutic agents. CONCLUSION: Fresh insight into the molecular details of osteoclastic bone resorption provides new opportunities for identifying agents to selectively modulate osteoclast activity. Such agents may contribute to evolution of the practice of orthodontics.

Characterization of HKE2: an ancient antigen encoded in the major histocompatibility complex.

Genes at the centromeric end of the human leukocyte antigen region influence adaptive autoimmune diseases and cancer. In this study, we characterized protein expression of HKE2, a gene located in the centromeric portion of the class II region of the major histocompatibility complex encoding subunit 6 of prefoldin. Immunohistochemical analysis using an anti-HKE2 antibody indicated that HKE2 protein expression is dramatically upregulated as a consequence of activation. In a tissue microarray and in several tumors, HKE2 was overexpressed in certain cancers compared with normal counterparts. The localization of the HKE2 gene to the class II region, its cytoplasmic expression and putative protein-binding domain suggest that HKE2 may function in adaptive immunity and cancer.

Amsacta moorei entomopoxvirus expresses an active superoxide dismutase.

The entomopoxvirus from Amsacta moorei serves as the prototype of the group B entomopoxviruses. One of the interesting genes found in Amsacta moorei entomopoxvirus (AmEPV) is a superoxide dismutase (sod) (open reading frame AMV255). Superoxide dismutases (SODs) catalyze the conversion of superoxide radicals to hydrogen peroxide and oxygen. Many vertebrate poxviruses contain a sod gene, but to date, none have been demonstrated to be active. There are three families of SODs, characterized by their metal ion-binding partners, Fe, Mn, or Cu and Zn. Poxvirus enzymes belong to the Cu-Zn SOD family. Unlike inactive vertebrate poxvirus SODs, AMVSOD contains all the amino acids necessary for function. We expressed and purified a 6X-His-tagged version of the AMVSOD in Escherichia coli. The recombinant AMVSOD demonstrates superoxide dismutase activity both in an in situ gel assay and by stopped flow spectrophotometry. The k(cat)/K(m) for AMVSOD is 4 x 10(7) M(-1)s(-1). In infected cells, the AMVSOD protein behaves as a dimer and is catalytically active; however, disruption of the gene in AMEPV has little or no effect on growth of the virus in cell culture. An analysis of mRNA expression indicates that AMVsod is expressed late during infection of Lymantria dispar (Ld652) cells and produces a discrete nonpolydisperse transcript. Characterization of protein expression with a monoclonal antibody generated against AMVSOD confirms that the AMVSOD protein can be classified as a late, postreplicative gene. Therefore, AMVSOD is the first example of an active poxvirus SOD.

Immunobiological analysis of TCR single-chain transgenic mice reveals new possibilities for interaction between CDR3alpha and an antigenic peptide bound to MHC class I.

The interaction between TCRs and peptides presented by MHC molecules determines the specificity of the T cell-mediated immune response. To elucidate the biologically important structural features of this interaction, we generated TCR beta-chain transgenic mice using a TCR derived from a T cell clone specific for the immunodominant peptide of vesicular stomatitis virus (RGYVYQGL, VSV8) presented by H-2K(b). We immunized these mice with VSV8 or analogs substituted at TCR contact residues (positions 1, 4, and 6) and analyzed the CDR3alpha sequences of the elicited T cells. In VSV8-specific CTLs, we observed a highly conserved residue at position 93 of CDR3alpha and preferred Jalpha usage, indicating that multiple residues of CDR3alpha are critical for recognition of the peptide. Certain substitutions at peptide position 4 induced changes at position 93 and in Jalpha usage, suggesting a potential interaction between CDR3alpha and position 4. Cross-reactivity data revealed the foremost importance of the Jalpha region in determining Ag specificity. Surprisingly, substitution at position 6 of VSV8 to a negatively charged residue induced a change at position 93 of CDR3alpha to a positively charged residue, suggesting that CDR3alpha may interact with position 6 in certain circumstances. Analogous interactions between the TCR alpha-chain and residues in the C-terminal half of the peptide have not yet been revealed by the limited number of TCR/peptide-MHC crystal structures reported to date. The transgenic mouse approach allows hundreds of TCR/peptide-MHC interactions to be examined comparatively easily, thus permitting a wide-ranging analysis of the possibilities for Ag recognition in vivo.

Structure of murine CTLA-4 and its role in modulating T cell responsiveness.

The effective regulation of T cell responses is dependent on opposing signals transmitted through two related cell-surface receptors, CD28 and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). Dimerization of CTLA-4 is required for the formation of high-avidity complexes with B7 ligands and for transmission of signals that attenuate T cell activation. We determined the crystal structure of the extracellular portion of CTLA-4 to 2.0 angstrom resolution. CTLA-4 belongs to the immunoglobulin superfamily and displays a strand topology similar to Valpha domains, with an unusual mode of dimerization that places the B7 binding sites distal to the dimerization interface. This organization allows each CTLA-4 dimer to bind two bivalent B7 molecules and suggests that a periodic arrangement of these components within the immunological synapse may contribute to the regulation of T cell responsiveness.