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Dentoalveolar bacterial infections cause localized tissue and bone destruction, but usually remain well-localized within teeth in immunocompetent hosts. However, in certain cases these infections may invade head and neck tissues, resulting in orofacial abscesses, cellulitis and sepsis, with resultant high morbidity and even mortality. In the present studies, we developed a novel model of spreading dentoalveolar infections in mice by treatment with neutralizing antibodies against both interleukin-1α (IL-1α) and IL-1β. Surprisingly male but not female mice given anti-IL-1 antibodies developed orofacial abscesses, weight loss, splenomegaly and sepsis. Female mice developed abscesses and sepsis comparable to males following ovariectomy (OVX), which was reversed by estrogen supplementation. Anti-IL-1 blockade inhibited IL-12, interferon γ (IFNγ) and IL-6 but not IL-10 expression in infrabony lesions, suggestive of a local anti-inflammatory response. There was greater infiltration of neutrophils and other inflammatory cells into lesions in anti-IL-1-treated animals; however, blood leukocytes had reduced bacterial phagocytic and killing activity ex vivo. Estrogen directly stimulated IL-1 production by macrophages, suggesting that the resistance of females to disseminating dentoalveolar infections may be due to their heightened pro-inflammatory responses following bacterial challenge, leading to enhanced localization of these infections.

To develop a systems-level understanding of host-microbial interactions that lead to the pathogenesis of periodontitis. Such an understanding may identify therapeutic targets for developing efficacious treatments for periodontitis.

ABSTRACTDental caries is one of the most prevalent infectious diseases in the United States, affecting approximately 80% of children and the majority of adults. Dental caries may lead to endodontic disease, where the bacterial infection progresses to the root canal system of the tooth, leading to periapical inflammation, bone erosion, severe pain, and tooth loss. Periapical inflammation may also exacerbate inflammation in other parts of the body. Although conventional clinical therapies for this disease are successful in approximately 80% of cases, there is still an urgent need for increased efficacy of treatment. In this study, we applied a novel gene-therapeutic approach using recombinant adeno-associated virus (AAV)-mediated Atp6i RNA interference (RNAi) knockdown of Atp6i/TIRC7gene expression to simultaneously target periapical bone resorption and periapical inflammation. We found that Atp6iinhibition impaired osteoclast function in vitroand in vivoand decreased the number of T cells in the periapical lesion. Notably, AAV-mediated Atp6i/TIRC7knockdown gene therapy reduced bacterial infection-stimulated bone resorption by 80% in the mouse model of endodontic disease. Importantly, Atp6i+/-mice with haploinsufficiency of Atp6iexhibited protection similar to that in mice with bacterial infection-stimulated bone erosion and periapical inflammation, which confirms the potential therapeutic effect of AAV-small hairpin RNA (shRNA)-Atp6i/TIRC7. Our results demonstrate that AAV-mediated Atp6i/TIRC7knockdown in periapical tissues can inhibit endodontic disease development, bone resorption, and inflammation, indicating for the first time that this potential gene therapy may significantly improve the health of those who suffer from endodontic disease.

Bacterial infections of the dental pulp result in soft tissue and alveolar bone destruction. It has been suggested that Th1 responses promote disease, whereas Th2 responses are protective. However, other studies have challenged this notion. To address this question, bone destruction was evaluated in mice immunized to develop strong and polarized Th1- or Th2-biased responses to the oral pathogen Porphyromonas gingivalis. Th1 bias was confirmed by the presence of high titers of serum IgG2a and the production of high levels of interferon (IFN)-γ and no interleukin (IL)-4 by lymph node cells stimulated with P. gingivalisantigens. In contrast, Th2-biased animals had high titer IgG1 and no IgG2a, and their lymph node cells produced high levels of IL-4 but no IFN-γ. Subsequent infection of the dental pulp with P. gingivaliscaused extensive inflammation and alveolar bone destruction in Th1-biased mice, whereas Th2-biased mice and controls developed minimal lesions. Inflammatory granulomas in Th1-biased mice were heavily infiltrated with osteoclasts and had high local expression of IFN-γ, IL-1α, and IL-1β. Little or no IFN-γ/IL-1α/IL-1β and no obvious osteoclasts were detected in lesions of Th2-biased and control groups. These results directly demonstrate that specific Th1 responses promote severe infection-stimulated alveolar bone loss.

ABSTRACTExposure of the mouse oral cavity to Porphyromonas gingivalisresults in the development of gingivitis and periapical bone loss, which apparently are associated with a Th1 response to bacterial antigens. We have used this infection model in conjunction with direct T-cell expression cloning to identify bacterial antigens that induce a preferential or biased T helper response during the infectious process. A P. gingivalis-specific CD4 T-cell line derived from mice at 3 weeks postchallenge was used to directly screen a P. gingivalisgenomic expression library. This screen resulted in the identification of five genes coding for previously identified proteins and three other putative protein antigens. One of the identified proteins, P. gingivalisthiol peroxidase, was studied in detail because this molecule belongs to a protein family that is apparently involved in microbial pathogenesis. Infection of mice with P. gingivalis, either via the subcutaneous route or after exposure of the animal's oral cavity to viable bacteria, resulted in the induction of a strong thiol peroxidase-specific immune response characterized by the production of high titers of specific serum immunoglobulin G2a antibody and the production of gamma interferon by antigen-stimulated lymphoid cells, a typical Th1-biased response. Thus, the use of a proven T-cell expression cloning approach and a mouse model of periodontal disease resulted in the identification and characterization of P. gingivalisproteins that might be involved in pathogenesis.

5‐HTT mediates antidepressant‐sensitive clearance of 5‐HT after its release into neural synapses. We found increased expression of 5‐HTT in RANKL‐induced osteoclast‐like cells. Fluoxetine, an inhibitor of 5‐HTT, reduced osteoclast differentiation but not activation. Reserpine, an inhibitor of 5‐HT intracellular transport, potentiated differentiation. These results indicate a role for 5‐HTT in osteoclast function and suggest that commonly used antidepressive agents may affect bone mass.

Periapical granulomas are induced by bacterial infection of the dental pulp and result in destruction of the surrounding alveolar bone. In previous studies we have reported that the bone resorption in this model is primarily mediated by macrophage-expressed interleukin-1 (IL-1). The expression and activity of IL-1 is in turn modulated by a network of Th1 and Th2 regulatory cytokines. In the present study, the functional roles of the Th1 cytokine gamma interferon (IFN-gamma) and IFN-gamma-inducing cytokines IL-12 and IL-18 were determined in a murine model of periapical bone destruction. IL-12-/-, IL-18-/-, and IFN-gamma-/- mice were subjected to surgical pulp exposure and infection with a mixture of four endodontic pathogens, and bone destruction was determined by microcomputed tomography on day 21. The results indicated that all IL-12-/-, IL-18-/-, and IFN-gamma-/- mice had similar infection-stimulated bone resorption in vivo as wild-type control mice. Mice infused with recombinant IL-12 also had resorption similar to controls. IFN-gamma-/- mice exhibited significant elevations in IL-6, IL-10, IL-12, and tumor necrosis factor alpha in lesions compared to wild-type mice, but these modulations had no net effect on IL-1alpha levels. Recombinant IL-12, IL-18, and IFN-gamma individually failed to consistently modulate macrophage IL-1alpha production in vitro. We conclude that, at least individually, endogenous IL-12, IL-18, and IFN-gamma do not have a significant effect on the pathogenesis of infection-stimulated bone resorption in vivo, suggesting possible functional redundancy in proinflammatory pathways.

ABSTRACTPeriapical granulomas are induced by bacterial infection of the dental pulp and result in destruction of the surrounding alveolar bone. In previous studies we have reported that the bone resorption in this model is primarily mediated by macrophage-expressed interleukin-1 (IL-1). The expression and activity of IL-1 is in turn modulated by a network of Th1 and Th2 regulatory cytokines. In the present study, the functional roles of the Th1 cytokine gamma interferon (IFN-?) and IFN-?-inducing cytokines IL-12 and IL-18 were determined in a murine model of periapical bone destruction. IL-12-/-, IL-18-/-, and IFN-?-/-mice were subjected to surgical pulp exposure and infection with a mixture of four endodontic pathogens, and bone destruction was determined by microcomputed tomography on day 21. The results indicated that all IL-12-/-, IL-18-/-, and IFN-?-/-mice had similar infection-stimulated bone resorption in vivo as wild-type control mice. Mice infused with recombinant IL-12 also had resorption similar to controls. IFN-?-/-mice exhibited significant elevations in IL-6, IL-10, IL-12, and tumor necrosis factor alpha in lesions compared to wild-type mice, but these modulations had no net effect on IL-1a levels. Recombinant IL-12, IL-18, and IFN-? individually failed to consistently modulate macrophage IL-1a production in vitro. We conclude that, at least individually, endogenous IL-12, IL-18, and IFN-? do not have a significant effect on the pathogenesis of infection-stimulated bone resorption in vivo, suggesting possible functional redundancy in proinflammatory pathways.

Solubilization of bone mineral by osteoclasts depends on the formation of an acidic extracellular compartment through the action of a V‐type ATPase. We previously cloned a gene encoding a putative osteoclast‐specific proton pump subunit, termed OC‐116 kDa, approved mouse Atp6i(ATPase, H+transporting, [vacuolar proton pump] member I). The function of Atp6ias osteoclast‐specific proton pump subunit was confirmed in our mouse knockout study. However, the transcription regulation of Atp6iremains largely unknown. In this study, the gene encoding mouse Atp6iand the promoter have been isolated and completely sequenced. In addition, the temporal and spatial expressions of Atp6ihave been characterized. Intrachromosomal mapping studies revealed that the gene contains 20 exons and 19 introns spanning ∼11 kilobases (kb) of genomic DNA. Alignment of the mouse Atp6igene exon sequence and predicted amino acid sequence to that of the human reveals a strong homology at both the nucleotide (82%) and the amino acid (80%) levels. Primer extension assay indicates that there is one transcription start site at 48 base pairs (bp) upstream of the initiator Metcodon. Analysis of 4 kb of the putative promoter region indicates that this gene lacks canonical TATA and CAAT boxes and contains multiple putative transcription regulatory elements. Northern blot analysis of RNAs from a number of mouse tissues reveals that Atp6iis expressed predominantly in osteoclasts, and this predominant expression was confirmed by reverse‐transcription polymerase chain reaction (RT‐PCR) assay and immunohistochemical analysis. Whole‐mount in situ hybridization shows that Atp6iexpression is detected initially in the headfold region and posterior region in the somite stage of mouse embryonic development (E8.5) and becomes progressively restricted to anterior regions and the limb bud by E9.5. The expression level of Atp6iis largely reduced after E10.5. This is the first report of the characterizations of Atp6igene, its promoter, and its gene expression patterns during mouse development. This study may provide valuable insights into the function of Atp6i, its osteoclast‐selective expression, regulation, and the molecular mechanisms responsible for osteoclast activation.

ABSTRACTPeriapical bone destruction occurs as a consequence of pulpal infection. In previous studies, we showed that interleukin-1 (IL-1) is the primary stimulator of bone destruction in this model. IL-6 is a pleiotropic cytokine that is induced in these infections and has both pro- and anti-inflammatory activities. In the present study, we determined the role of IL-6 in regulating IL-1 expression and bone resorption. The first molars of IL-6 knockouts (IL-6−/−) and wild-type mice were subjected to surgical pulp exposure and infection with a mixture of four common pulpal pathogens, includingPrevotella intermedia, Fusobacterium nucleatum,Peptostreptococcus micros, and Streptococcus intermedius. Mice were killed after 21 days, and bone destruction and cytokine expression were determined. Surprisingly, bone destruction was significantly increased in IL-6−/−mice versus that in wild-type mice (by 30%; P< 0.001). In a second experiment, the effects of chronic (IL-6−/−) IL-6 deficiency and short-term IL-6 deficiency induced by in vivo antibody neutralization were determined. Both IL-6−/−(30%;P< 0.001) and anti-IL-6 antibody-treated mice (40%;P< 0.05) exhibited increased periapical bone resorption, compared to wild-type controls. The increased bone resorption in IL-6-deficient animals correlated with increases in osteoclast numbers, as well as with elevated expression of bone-resorptive cytokines IL-1α and IL-1β, in periapical lesions and with decreased expression of the anti-inflammatory cytokine IL-10. These data demonstrate that endogenous IL-6 expression has significant anti-inflammatory effects in modulating infection-stimulated bone destruction in vivo.

ABSTRACTWe have previously demonstrated that a high proportion of RAG-2 SCID knockout mice, which lack T and B cells, develop orofacial abscesses and disseminated infections following pulpal infection, whereas immunocompetent control mice do not. In the present study, we sought to identify the components of the adaptive immune response which contribute to protection against disseminating anaerobic infections and sepsis. For this purpose, various genetically engineered immunodeficient mice were employed, including RAG-2 SCID, Igh-6 (B-cell deficient), Tcrb Tcrd (T-cell deficient) and Hc0(C5 deficient). For abscess induction, the mandibular first molars were subjected to pulp exposure on day 0. Teeth were infected with a mixture of four anaerobic pathogens, including Prevotella intermedia, Streptococcus intermedius,Fusobacterium nucleatum, and Peptostreptococcus micros, and teeth were sealed to prevent communication with the oral cavity. The findings demonstrate that both RAG-2 SCID and B-cell-deficient mice, but not T-cell- or C5-deficient mice, have increased susceptibility to the development of disseminating anaerobic infections. Abscess-susceptible RAG-2 SCID and B-cell-deficient mice also showed a significant loss of body weight, splenomegaly, and absent antibacterial antibody production. Furthermore, dissemination was significantly reduced, from 74 to 25%, in susceptible RAG-2 mice by passively transferred antibody, predominantly immunoglobulin G2b (IgG2b) and IgM, against the infecting bacterial innoculum. Fractionated IgG-enriched preparations were more efficient in transferring protection than IgM preparations. We conclude that an antibody-mediated mechanism(s), most likely bacterial opsonization, is of importance in localizing anaerobic root canal infections and in preventing their systemic spread.

ABSTRACTC3H/HeJ mice have an impaired ability to respond to lipopolysaccharide (LPS) due to a mutation in the gene that encodesToll-like receptor 4 (TLR4). The effect of TLR4 deficiency on host responses to endodontic infections is unknown. In the present study, we compared periapical bone destruction, sepsis, and inflammatory cytokine production in LPS-hyporesponsive C3H/HeJ and wild-type control C3H/HeOuJ mice. The mandibular first molars of both strains were subjected to pulpal exposure and infection with a mixture of four anaerobic pathogens, Prevotella intermedia,Fusobacterium nucleatum, Streptococcus intermedius, and Peptostreptococcus micros. At sacrifice on day 21, TLR4-deficient C3H/HeJ mice had significantly reduced periapical bone destruction compared to wild-type C3H/HeOuJ mice (P< 0.001). The decreased bone destruction in C3H/HeJ correlated with reduced expression of the bone resorptive cytokines interleukin 1α (IL-1α) (P< 0.01) and IL-1β (P< 0.05) as well as the proinflammatory cytokine IL-12 (P< 0.05). No significant differences were seen in the levels of gamma interferon, tumor necrosis factor alpha (TNF-α), or IL-10 between the two strains. The expression of IL-1α, IL-1β, TNF-α, IL-10, and IL-12 were all significantly reduced in vitro in macrophages from both TLR4-deficient C3H/HeJ and C57BL/10ScNCr strains, compared to wild-type controls. Notably, the responses of TLR4-deficient macrophages to both gram-positive and gram-negative bacteria were similarly reduced. Neither C3H/HeJ nor C3H/HeOuJ mice exhibited orofacial abscess development or infection dissemination as determined by splenomegaly or cachexia. We conclude that intact TLR function mediates increased proinflammatory responses and bone destruction in response to mixed anaerobic infections.

The effect of the bone resprptive cytokines IL-1L, IL-1β, and TNF on bone formation was studied in an in vitro system. All three cytokines were profoundly inhibitory, with the rank order of potency IL-1β > IL-1α > TNF. Inhibition was mediated by a depression of differentiated osteoblast functions, including alkaline phosphatase expression and matrix synthesis. Osteoblast proliferation was not affected. Bone formation inhibition was independent of PGE2 production, indicating a direct effect of cytokines on osteoblasts. High concentrations of IL-1β (10 U/ml) abrogated IGF-1-stimulated bone formation, providing evidence for the hypothesis that cytokines act as 'uncoupling factors'. Conversely, high concentrations of IGF-1 circumvented inhibition by IL-1β (0.1-1.0 U/ml). The interaction of cytokines and bone growth factors with osteoblasts are likely to be of critical importance in the regulation of bone mass at local inflammatory sites.

The effect of interleukin‐1β, the major component of osteoclast‐activating factor (OAF), on bone formation by fetal rat osteoblast‐rich cells was investigated. An in vitroculture system developed by Ecarot‐Charrier et al.(1983) and Bellows et al. (1986) was utilized in which osteoblasts form mineralized nodules which closely resemble woven bone. Continuous exposure of cultures to homogenous IL‐1β resulted in potent inhibition of mineralized nodule formation, which was half maximal at 0.1 U/ml (7.5 × 10−13M). Bone formation may thus be considerably more sensitive to IL‐1β than is bone resorption (half maximal at 3.8 × 10−11M). Inhibition of bone formation occurred when cultures were exposed to IL‐1β at both early and late time periods and was unaffected by the presence of indomethacin or by the osteoclast inhibitors calcitonin and γ‐interferon. Instead, IL‐1β exerted multiple inhibitory effects on osteoblast functions, including inhibition of collagen and noncollagen protein synthesis, alkaline phosphatase expression, and cell replication. On a dose response basis, the inhibition of protein synthesis correlated most closely with inhibition of bone formation. IL‐1β is clearly inhibitory rather than stimulatory for bone formation as assessed in this system and is therefore unlikely to function as a coupling factor linking the processes of bone resorption and bone formation.

Here, we report the isolation and characterization of zebrafish activin receptor-like kinase-8 (zALK-8), a novel type I serine/threonine (ser/thr) kinase receptor of the transforming growth factor beta (TGF-β) family. zALK-8 is novel, in that it contains an extracellular domain that is quite distinct from that of previously identified ALK receptors 1 through 7. Analysis of the predicted amino acid sequence of the 506 amino acid zALK-8 receptor reveals an ser/thr kinase domain characteristic of type I TGF-β family member receptors. zALK-8, therefore, is a traditional type I ser/thr kinase receptor of the TGF-β family, but it may exhibit novel ligand-binding activities. The developmental expression of zALK-8 mRNA was examined by wholemount in situ hybridization analysis using a probe from the 3'-untranslated sequence of zALK-8, which does not cross react with other members of the highly conserved TGF-β receptor family. zALK-8 mRNA is present as a maternal message that is expressed ubiquitously before the start of zygotic transcription. By 16 hr postfertilization (hpf), zALK-8 mRNA is still expressed fairly evenly throughout the embryo. In 24-hpf embryos, zALK-8 mRNA is expressed predominantly in the developing eye and neural structures. By 48 hpf, zALK-8 mRNA is faintly detectable as a diffuse signal throughout the head. zALK-8 mRNA is not detectable by this method in 72-hpf or 96-hpf embryos. Northern analysis of zALK-8 mRNA in poly(A+) mRNA isolated from 6–9 hpf embryos detects a major transcript of 3.6 kb and a minor transcript of 4.3 kb. zALK-8 mRNA expression correlates well with known functions of TGF-β family members as early axial patterning and mesoderm-inducing growth factors and as potent growth and differentiation factors in craniofacial development. Dev. Dyn. 1998;211:352–361. © 1998 Wiley-Liss, Inc.

A cDNA encoding a possible novel human 116-kDa polypeptide subunit of the osteoclastic proton pump (OC-116KDa) has been identified by differential screening of a human osteoclastoma cDNA library. The predicted sequence of OC-116KDa consists of 822 amino acids and is 46.9% and 47.2% identical at the amino acid level to the 116-KDa polypeptide of the vacuolar proton pump of rat and bovine brain respectively. OC-116KDa mRNA was found at high levels in osteoclastomas by Northern analysis but was not detected in tumor stromal cells or in other tissues including kidney, liver, skeletal muscle and brain. OC-116KDa mRNA was localized to multinucleated giant cells within the osteoclastoma tumor byin situhybridization.

The levels of3 bone resorptive cytokines, interleukin 1α (IL‐1α), IL‐1β, and tumor necrosis factor α (TNFα), were assessed in tissues from sites of periodontal disease. As determined by ELISA of tissue extracts, IL‐1β and TNFα were detected in all diseased sites, whereas IL‐1α was present in 8/22 sites. IL‐1β was present in highest concentration (mean ± SEM: 11,695 ± 2,888 pg/ml; 672 pM), followed by TNFα (434 ± 135 pg/ml; 26 pM), and IL‐1α (342 ± 160 pg/ml; 20 pM). The levels of all 3 mediators were significantly lower in clinically healthy tissues. There was a highly significant correlation between levels of IL‐1β and TNFα (rs= 0.61, P<0.001), suggesting coordinated expression of these 2 mediators. The numbers of cells containing each mediator was also determined by indirect immunofluorescence on frozen tissue sections. Consistent with findings from tissue extracts, IL‐1β‐containing cells were present in approximately 5‐fold higher numbers than TNFα‐containing cells, and 40‐fold higher numbers than IL‐1α‐containing cells. Taken together with previous findings, these results indicate that IL‐1β is likely to be an important mediator in the pathogenesis of periodontal disease. J Periodontol 1991; 62:504–509.

A gene encoding a possible novel human cathepsin, a cysteine proteinase that is distinct from previously characterized enzymes, has been identified by differential screening of a human osteoclastoma cDNA library. This molecule, termed cathepsin X, appears to represent the human homolog of the osteoclast‐expressed rabbit cathepsin OC‐2. Cathepsin X (GenBank accession number U20280) is 93.9% identical to OC‐2 at the amino acid level, and is 92% identical at the nucleotide level within the coding region. Cathepsin X is 52.2 and 46.9% identical to cathepsins S and L, respectively, and is therefore clearly distinct from these enzymes. Cathepsin X mRNA was localized to multinucleated giant cells within the osteoclastoma tumor by in situ hybridization. These data strongly support the hypothesis that cathepsin X represents a novel cysteine proteinase which is expressed at high levels in osteoclasts.

The digestion of type I collagen is an essential step in bone resorption. It is well established that osteoclasts solubilize the mineral phase of bone during the resorptive process, but the mechanism by which they degrade type I collagen, the major proteinaceous component of bone, is controversial. Differential screening of a human osteoclastoma cDNA library was performed to characterize genes specifically expressed in osteoclasts. A large number of cDNA clones obtained by this procedure were found to represent 92 kD type IV collagenase (gelatinase B; MMP‐9, EC 3.4.24.35), as well as tartrate‐resistant acid phosphatase. In situhybridization localized mRNA for gelatinase B to multinucleated giant cells in human osteoclastomas. Gelatinase B immunoreactivity was demonstrated in giant cells from eight of eight osteoclastomas, osteoclasts in normal bone, and osteoclasts of Paget's disease by use of a polyclonal antiserum raised against a synthetic gelatinase B peptide. In contrast, no immunoreactivity for 72 kD type IV collagenase (gelatinase A; MMP‐2, EC 3.4.24.24), which is the product of a separate gene, was detected in osteoclastomas or normal osteoclasts. We propose that the 92 kD type IV collagenase/gelatinase B plays an important role in the resorption of collagen during bone remodeling.

The interaction of interleukin 1 (IL‐1), a locally produced factor, and parathyroid hormone (PTH), a systemic factor, in stimulating bone resorption was examined using fetal rat long bone organ culture. Concentrations of IL‐1 and PTH, which stimulated little bone resorption when present singly, produced marked resorption when present simultaneously. This synergistic interaction of IL‐1 and PTH was not affected by the presence of the prostaglandin synthetase inhibitor indomethacin. Both interleukin 1α and interleukin 1β were capable of producing synergy. Synergy was not produced by sequential exposure of bone to IL‐1 and PTH, but required the simultaneous presence of both mediators. The leftward shift in the dose response curve of PTH produced by IL‐1 may be an important mechanism controlling localized bone resorption. A role for IL‐1 in stimulating bone resorption in pathologic conditions, such as arthritis and periodontal disease, is strengthened by the finding that even low concentrations of IL‐1 can produce resorptive effects by synergistic interaction with PTH.

The Ia antigens of the mouse are the basis for the genetic control of the immune response. The HLA-D/DR locus is considered to be the human counterpart of the Ia subregion of the murine major histocompatibility complex1–4. The HLA-D/DR antigens are polymorphic, and eight well defined alleles have been identified using alloantisera5. More recently, ‘supertypic’ antigens (MB and MT) have been defined which identify clusters of HLA-D/DR specificities6–9. Little is known about the molecular basis for the cellular and serological polymorphism of the HLA-D/DR antigens, as alloantisera are usually of very low titre and heteroantisera frequently lack monospecificity. We present here the preparation and characterization of a monoclonal antibody which defines a new polymorphic system of the HLA-D/DR region. This and similar antisera should now begin to provide the reagents with which to correlate molecular structure with the functional repertoire of the human Ia-like antigens.

Osteocalcin, the major non-collagenous protein In bone, is transcribed in osteoblasts at the onset of extracellular matrix mineralization. In this study it was demonstrated that sequences located in the first exon of the human osteocalcin gene possess a differentiation- related osteocalcin silencer element (OSE). Osteocalcin was rendered transcribable In UMR-106 cells and proliferating normal osteoblasts after deletion of the -3 to +51 region. Site-specific mutagenesis of this region revealed that a 7 bp sequence (TGGCCCT) (+29 to +35) is critical for silencing function. Mobility shift assays demonstrated that a nuclear factor bound to the OSE. The OSE binding protein was present in proliferating normal pre-osteoblasts and In UMR-106 and ROS 17/2.8 osteosarcoma cells, but was absent from post-proliferative normal osteoblasts. The binding protein was inhibited by fragments containing the +29/+35 sequence, but not by other promoter fragments or by the consensus oligomers of unrelated nuclear factors AP-1 and Sp1. DNase I footprinting demonstrated that the OSE binding-protein protected the +17 to +36 portion of the first exon, consistent with the results of mapping studies and competitive mobility shift assays. It is hypothesized that this silencer is activated by complexing of the OSE binding protein to the OSE during the osteoblast proliferation stage and that the OSE binding protein is down-regulated at the onset of extracellular matrix mineralization.