Intra-articular administration of an antibody against CSF-1 receptor reduces pain-related behaviors and inflammation in CFA-induced knee arthritis.

Several studies have shown that blockade of colony stimulating factor-1 (CSF-1) or its receptor (CSF-1R) inhibits disease progression in rodent models of rheumatoid arthritis (RA); however, the role of the CSF-1/CSF-1R pathway in RA-induced pain and functional deficits has not been studied. Thus, we examined the effect of chronic intra-articular administration of a monoclonal anti-CSF-1R antibody (AFS98) on spontaneous pain, knee edema and functional disabilities in mice with arthritis. Unilateral arthritis was produced by multiple injections of complete Freund's adjuvant (CFA) into the right knee joint of adult male ICR mice. CFA-injected mice were then treated twice weekly from day 10 until day 25 with anti-CSF-1R antibody (3 and 10 µg/5 µL per joint), isotype control (rat IgG 10 µg/5 µL per joint) or PBS (5 µl/joint). Knee edema, spontaneous flinching, vertical rearing and horizontal exploratory activity were assessed at different days. Additionally, counts of peripheral leukocytes and body weight were measured to evaluate general health status. Intra-articular treatment with anti-CSF-1R antibody significantly increased horizontal exploratory activity and vertical rearing as well as reduced spontaneous flinching behavior and knee edema as compared to CFA-induced arthritis mice treated with PBS. Treatment with this antibody neither significantly affect mouse body weight nor the number of peripheral leukocytes. These results suggest that blockade of CSF-1R at the initial injury site (joint) could represent a therapeutic alternative for improving the functional disabilities and attenuating pain and inflammation in patients with RA.

An anti-c-Fms antibody inhibits osteoclastogenesis in a mouse periodontitis model.

OBJECTIVE: Bacterial lipopolysaccharide (LPS) can induce inflammatory bone loss such as periodontal disease. The formation of osteoclasts depends on macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kb ligand (RANKL). It has recently been reported that administration of an antibody of the M-CSF receptor c-Fms completely blocked osteoclastogenesis and bone erosion induced by LPS in mouse calvaria. In this study, the effect of antibody against c-Fms in the mouse periodontitis model by injection of LPS was investigated. MATERIALS AND METHODS: C57BL6/J mice were injected with LPS and anti-c-Fms antibody into the mesial gingiva of the first molar in the left mandible. Histological sections of periodontal tissue were stained for tartrate-resistant acid phosphatase, and osteoclast numbers and ratio of alveolar bone resorption determined. RESULTS: The number of osteoclasts and ratio of alveolar bone resorption in mice administered both LPS and anti-c-Fms antibody was lower than those in mice administered LPS alone. The expression of RANKL receptor, RANK, was inhibited by the anti-c-Fms antibody in periodontal tissue. CONCLUSION: M-CSF and/or its receptor are potential therapeutic targets for the treatment of bone resorption, caused by LPS, in periodontitis. Injection of an anti-c-Fms antibody might be useful for inhibition of pathological bone resorption in periodontitis.

An anti-c-Fms antibody inhibits orthodontic tooth movement.

Orthodontic force induces osteoclastogenesis in vivo. It has recently been reported that administration of an antibody against the macrophage-colony-stimulating factor (M-CSF) receptor c-Fms blocks osteoclastogenesis and bone erosion induced by tumor necrosis factor-alpha (TNF-alpha) administration. This study aimed to examine the effect of an anti-c-Fms antibody on mechanical loading-induced osteoclastogenesis and osteolysis in an orthodontic tooth movement model in mice. Using TNF receptor 1- and 2-deficient mice, we showed that orthodontic tooth movement was mediated by TNF-alpha. We injected anti-c-Fms antibody daily into a local site, for 12 days, during mechanical loading. The anti-c-Fms antibody significantly inhibited orthodontic tooth movement, markedly reduced the number of osteoclasts in vivo, and inhibited TNF-alpha-induced osteoclastogenesis in vitro. These findings suggest that M-CSF plays an important role in mechanical loading-induced osteoclastogenesis and bone resorption during orthodontic tooth movement mediated by TNF-alpha.

Induced sensitization to nickel in guinea pigs immunized with mycobacteria by injection of purified protein derivative with nickel.

Nickel has been reported to be one of the most common causes of allergic contact dermatitis. Despite the fact that nickel is a frequent sensitizer in humans, establishing animal models for nickel allergy has met with considerable difficulties. In clinical cases, allergic contact hypersensitivity to nickel develops much more readily in inflamed skin than normal skin. In this study, we tried to induce nickel sensitization when inflammation has been evoked in guinea pigs immunized with mycobacteria followed by co-administration of a mycobacterial component with nickel. We first examined the delayed-type hypersensitivity (DTH) reaction of mycobacterial components such as the cell wall, cell membrane, 70S ribosomal fraction, cytoplasm, tuberculin purified protein derivative (PPD), RNA and DNA from Mycobacterium bovis BCG in guinea pigs immunized with live M. bovis BCG or heat killed M. tuberculosis. When PPD was used, the hypersensitivity reaction was strongest. Next, we tested whether PPD with nickel could induce nickel sensitivity in guinea pigs immunized with mycobacteria. Strong sensitization to nickel was achieved by injecting PPD with nickel. However, if too large an amount of PPD or nickel salts was used, sensitization to nickel decreased. In this way, sensitization of nickel developed much more easily in guinea pigs immunized with mycobacteria by injection of an appropriate amount of nickel at the inflammation site induced by a suitable amount of PPD.

Changed activation of HIV-1 LTR in monocytoid cells by mycobacteria with temporal progression of infection.

Coincubation of monocytoid cell line U937 cells cotransfected with HIV-1 LTR CAT plasmid and Tat expression plasmid, with Mycobacterium smegmatis, M. avium, M. bovis BCG and M. tuberculosis enhanced chloramphenicol acetyltransferase (CAT) production, indicating that these mycobacteria could activate the LTR in this cell line. The amount of CAT in the cells coincubated with M. smegmatis was higher than that infected with the other mycobacteria after 12, 24 and 48 hour time periods. However, the amount of CAT production in the cells cocultured with M. tuberculosis was higher than those coincubated with the other mycobacteria at 72 hours. These findings indicated that avirulent mycobacteria such as M. smegmatis may activate HIV replication at an early time and its effects are gradually decreased, while the effect of virulent M. tuberculosis increased gradually, and lasted for a long time resulting in an acceleration of HIV disease in patients.

TNF-alpha-mediated multiplication of human immunodeficiency virus in chronically infected monocytoid cells by mycobacterial infection.

Mycobacterial infection is a common occurrence in patients with acquired immune deficiency syndrome. Incubation of U1, a chronically HIV-1-infected human promonocytic cell line, with Mycobacterium smegmatis, M. avium, M. bovis BCG and M. tuberculosis resulted in enhancement of p24 antigen release in the supernatant, indicating that these mycobacteria could activate HIV replication from this cell line. The amount of p24 in the culture infected with M. smegmatis was higher than in cultures infected with other mycobacteria. The amounts of p24 release in cultures infected with M. avium and M. bovis BCG were intermediate. M. tuberculosis slightly stimulated HIV replication. The amount of TNF-alpha produced by U1 cells was correlated with the amount of p24 antigen release. The IL-1beta and IL-6 levels in the supernatant from cultures infected with all species were the same. The antibody to TNF-alpha inhibited p24 release induced by mycobacterial infections. The anti-IL-1beta and anti-IL-6 antibodies, however, scarcely influenced stimulation of HIV replication by mycobacterial infection. These data suggested that activation of HIV replication by mycobacteria mainly occurred by secondary release of cytokine TNF-alpha.

TNF-alpha-mediated activation of HIV-1 LTR in monocytoid cells by mycobacteria.

Mycobacterial infection occurs commonly in patients with acquired immune deficiency syndrome. Incubation of monocytoid cell line U937 cells, which was cotransfected HIV-1 long terminal repeat sequence (LTR) chloramphenicol acetyltransferase (CAT) plasmid and Tat expression plasmid, with Mycobacterium smegmatis, Mycobacterium avium, Mycobacterium bovis BCG and Mycobacterium tuberculosis resulted in enhancement of CAT production, indicating that these mycobacteria could activate LTR in this cell line. The amount of CAT in the cells coexisting with M. smegmatis was higher than that infected with other mycobacteria. The amounts of CAT production in the cells coculturing with M. avium and M. bovis BCG were intermediate. M. tuberculosis slightly stimulated CAT production. The amount of tumor necrosis factor (TNF)-alpha produced by transfected U937 cells was correlated with the amount of CAT production. The interleukin (IL)-1beta and IL-6 levels in the supernatant from coculturing with all species were similar. The antibody to TNF-alpha inhibited CAT production induced by mycobacterial infections. The anti-IL-1beta and anti-IL-6 antibodies, however, scarcely influenced stimulation of LTR by mycobacteria. In addition, U937 cells transfected with full length LTR CAT plasmid showed increased CAT production by activation with mycobacteria, but the cells transfected with mutant LTR CAT constructs from which the nuclear factor (NF)-kappaB binding site was deleted did not show activation. These findings indicated that activation of Mycobacterium-induced LTR CAT is NF-kappaB dependent. These findings suggested that activation of HIV-1 LTR by mycobacteria was mainly mediated by NF-kappaB-induced secondary release of cytokine TNF-alpha.

Fibronectin-binding proteins secreted by Mycobacterium avium.

Mycobacterium avium is an intracellular pathogen and a major opportunistic infectious agent observed in patients with acquired immune deficiency syndrome (AIDS). Fibronectin is an extracellular matrix protein and is a virulence factor for several extracellular pathogenic bacteria binding to mucosal surfaces. We investigated the fibronectin (FN)-binding proteins in the culture filtrate of M. avium by two-dimensional electrophoresis (2DE). Proteins in Sauton medium of M. avium after 3 weeks were separated by 2DE. The proteins were blotted onto polyvinylidene difluoride membrane and incubated with FN. FN-binding proteins were detected by Western blotting using anti-FN antibody. FN bound to five spots (33 kDa, 32 kDa, 31 kDa, 30 kDa and 25 kDa). N-terminal amino acids of these were determined. The 33 kDa spot corresponded to antigen 85 (Ag 85) C. The 32 and 31 kDa spots were either Ag 85 A or Ag 85 B. The 30 kDa spot corresponded to Ag 85 B of M. avium. The 25 kDa spot corresponded to MPA51 (M. avium MPB51). Thus, FN bound exclusively to the Ag 85 complex and MPA51.

PAP-1, a novel target protein of phosphorylation by pim-1 kinase.

Protooncogene, pim-1, has been reported to be a predisposition for lymphomagenesis along with myc, and its protein product, Pim-1, has been shown to be a serine/threonine protein kinase, whose activity is involved in proliferation and differentiation of blood cells. The signal transduction pathways neither to nor from Pim-1, however, have been clarified. We have cloned a cDNA encoding a novel Pim-1 binding protein, PAP-1, comprising 213 amino acids with a basic amino-acid cluster near the C-terminus. PAP-1 was colocalized with Pim-1 in human HeLa cell nuclei. The in vitro binding assays using GST fusion proteins of the wild-type and various deletion mutants revealed that the whole molecule of Pim-1 is required for the binding activity to PAP-1 and that Pim-1 binds to the region from amino-acid numbers 1-147 of PAP-1, or to two segments in the region. The association of PAP-1 with Pim-1 was also shown in vivo in transfected cells. Furthermore, PAP-1 was phosphorylated in vitro by Pim-1, but not a kinase-negative Pim-1 mutant. The two serine residues of PAP-1 at amino acids 204 and 206 near the C-terminus were phosphorylated by Pim-1. PAP-1 is thus thought to be a target protein for Pim-1 kinase.

Standardization of 3-D CT measurements for length and angles by matrix transformation in the 3-D coordinate system.

OBJECTIVE: We attempted to establish a new 3-D cephalometry with helical computed tomography (CT) by introducing the matrix transformation of the 3-D coordinate system. SUBJECTS AND METHODS: Three-dimensional cephalometric landmarks on the craniofacial bones were expressed as 3-D vectors originating from the center of the sella. These vectors were standardized by the matrix transformation so that the midsagittal plane and cranial base line coincided with the XZ plane and X axis of the 3-D coordinate system, respectively. We also applied this new method to trace the normal growth of the craniofacial bones in 44 patients with head and neck cancer (age range, 5 to 26 years; 19 women and 25 men). RESULTS: The accuracy for length measurements was less than 3% of standard error of means with a slice thickness of 1 or 3 mm for 28 out of the 29 length measurements recorded. The precision errors by repeated measurements were 0.8% to 1.0% coefficients of variation for intra- and interobserver variability. Standardization of 3-D vectors representing the cephalometric landmarks allowed us to assess successfully the age-related transition of these landmarks of the patients' craniofacial bones. CONCLUSION: A new assessment method for 3-D CT cephalometry has been developed by standardizing cephalometric landmarks using a matrix transformation of the 3-D coordinate system. This new assessment method may offer potential in planning plastic and reconstructive surgery.

TOK-1, a novel p21Cip1-binding protein that cooperatively enhances p21-dependent inhibitory activity toward CDK2 kinase.

A p21(Cip1/Waf1/Sdi1) is known to act as a negative cell-cycle regulator by inhibiting kinase activity of a variety of cyclin-dependent kinases. In addition to binding of the cyclin-dependent kinase to the N-terminal region of p21, p21 is also bound at its C-terminal region by proliferating cell nuclear antigen (PCNA), SET/TAF1, and calmodulin, indicating the versatile function of p21. In this study, we cloned cDNA encoding a novel protein named TOK-1 as a p21 C-terminal-binding protein by a two-hybrid system. Two splicing isoforms of TOK-1, TOK-1alpha and TOK-1beta, comprising 322 and 314 amino acids, respectively, were co-localized with p21 in nuclei and showed a similar expression profile to that of p21 in human tissues. TOK-1alpha, but not TOK-1beta, directly bound to the C-terminal proximal region of p21, and both were expressed at the G(1)/S boundary of the cell cycle. TOK-1alpha also preferentially bound to an active form of cyclin-dependent kinase 2 (CDK2) via p21, and these made a ternary complex in human cells. Furthermore, the results of three different types of experiments showed that TOK-1alpha enhanced the inhibitory activity of p21 toward histone H1 kinase activity of CDK2. TOK-1alpha is thus thought to be a new type of CDK2 modulator.

Reciprocal regulation via protein-protein interaction between c-Myc and p21(cip1/waf1/sdi1) in DNA replication and transcription.

The c-myc protooncogene product (c-Myc) is a transcription factor and is rapidly induced in resting cells following various mitogenic stimuli. c-Myc is thus suggested to play an important role in the transition from quiescence to proliferation. Despite numerous studies, including those on the connection between cyclin E/cyclin-dependent kinase 2 and c-Myc, little has been clarified about c-Myc in terms of the cell cycle regulation. Here we show that c-Myc can directly bind to the carboxyl-terminal region of the cyclin-dependent kinase inhibitor p21(cip1/waf1/sdi1) and thus partially relieves the p21 of the inhibitory effect on DNA synthesis directed by the proliferating cell nuclear antigen-dependent DNA polymerase delta. As for transcription, on the other hand, the p21 binding to the Myc box II region of c-Myc blocks c-Myc-Max complex formation on the E-box and thereby suppresses the transcriptional activation from the E-box by c-Myc. These results suggest that c-Myc activates DNA replication via inactivation of p21 and that p21, vice versa, represses the transcriptional activity of c-Myc. The balance of the reciprocal inactivation between c-Myc and p21 may determine the course of cellular processes such as cell proliferation, differentiation, and apoptosis.

Ribosomal protein L7 included in tuberculin purified protein derivative (PPD) is a major heat-resistant protein inducing strong delayed-type hypersensitivity.

The tuberculin purified protein derivative (PPD) is a widely used diagnostic antigen for tuberculosis. It consists of more than 100 denatured proteins in a culture filtrate of a heated culture of Mycobacterium tuberculosis. In two-dimensional electrophoretic analysis of PPDs from M. tuberculosis and M. bovis BCG, most proteins were diffusely separated and could not be seen as spots because of denaturation, whereas a few proteins showed relatively clear spots, indicating heat resistance. Two such proteins corresponded to ribosomal proteins L7 and L12. The mixture of these proteins L7/L2 induced a strong delayed-type hypersensitivity reaction. Another protein showing a clear spot was a GroES analogue, but this did not induce delayed-type hypersensitivity. There were a few other unidentified proteins. It is well known that L7 and L12 are encoded by the same gene and that they differ from each other only by an acetylic post-translational modification that occurs at the N-terminus of L12 converting it to L7 in Escherichia coli. L12, but not L7, was found in two-dimensional electrophoresis of BCG ribosomes, although we found two proteins corresponding to L7 and L12 in PPDs and a native culture filtrate of BCG. We compared the delayed-type hypersensitivity reaction elicited by L7/L12 derived from a culture filtrate of BCG and L12 derived from BCG ribosomes. L7/L12 from the culture filtrate could induce delayed-type hypersensitivity, but L12 from ribosomes could not, indicating that L7 was attributable to the induction of delayed-type hypersensitivity. The activity of L7/L12 was heat resistant. Neither glycosylation nor phosphorylation of L7/L12 from a culture filtrate could be detected. The acetylation at N-terminal of L12 was essential for the delayed-type hypersensitivity activity.

Species-specific B-cell epitope on the C-terminal region of the alpha antigen from Mycobacterium intracellulare in mice.

The alpha antigen, which is an immunodominant antigen, is a 30 kDa protein secreted by mycobacterial species. The C-terminal regions of alpha antigens are quite divergent. We investigated the question of whether the C-terminal regions of Mycobacterium avium alpha antigen (A-alpha), M. intracellulare alpha antigen (I-alpha) and M. bovis BCG alpha antigen (B-alpha) contained species-specific B-cell epitopes. We investigated the reactions of these peptides with anti-A-alpha, anti-I-alpha and anti-B-alpha sera prepared from BALB/c in a Western blot assay and ELISA. The C-terminal regions of I-alpha reacted exclusively with anti-I-alpha serum. The results of the inhibition assay of antibodies binding to I-alpha by peptides of C-A-alpha, C-I-alpha, and C-B-alpha are that only C-I-alpha inhibited the binding of antibodies to C-I-alpha. We found that the C-terminal region was B-cell epitope-specific to I-alpha in BALB/c mice.

AMY-1, a novel C-MYC binding protein that stimulates transcription activity of C-MYC.

BACKGROUND: The c-myc proto-oncogene has been suggested to play key roles in cell proliferation, differentiation, transformation and apoptosis. A variety of functions of C-MYC, the product of c-myc, are attributed to protein-protein interactions with various cellular factors including Max, YY1, p107, Bin1 and TBP. Max and YY1 bind to the C-terminal region of C-MYC, while p107, Bin1 and TBP bind to the N-terminal region covering myc boxes. The N-terminal region is involved in all the biological functions of C-MYC, and different proteins are therefore thought to interact with the N-terminal region of C-MYC to display different functions. RESULTS: We cloned two cDNAs which encode a novel C-MYC-binding protein of 11 kDa, designated AMY-1 (Associate of C-MYC). The two cDNAs, AMY-1L and AMY-1S, derived from alternative usage of polyadenylation signals, code for the same protein of 11 kDa. AMY-1 was bound via its C-terminal region to the N-terminal region of C-MYC (amino acids nos 58-148) corresponding to the transactivation domain. AMY-1 was localized in the cytoplasm in cells expressing c-myc at low levels, but in the nucleus in the cells of a high c-myc expression in transiently transfected cells. A similar difference in endogenous AMY-1 localization was observed during the cell cycle: AMY-1 translocated from cytoplasm to nucleus during the S phase when c-myc expression was increased. AMY-1 by itself did not recognize the E-box element, the MYC/Max binding sequence, nor did it transactivate via the element, but stimulated the activation of E-box-regulated transcription by MYC/Max. FISH analyses revealed that the amy-1 gene was located at 1p32.2-1p33 in human genome. CONCLUSIONS: AMY-1 is a 11 kDa protein which binds to the N-terminal region of C-MYC and stimulates the activation of E-box-dependent transcription by C-MYC. AMY-1, which mostly localizes in the cytoplasm, translocates into the nucleus in the S phase of the cell cycle upon an increase of c-myc expression, and may thus control the transcriptional activity of C-MYC.

Serological analysis of C-terminal region of alpha antigen from Mycobacterium avium-intracellulare complex and Mycobacterium tuberculosis.

The alpha antigen, which is a 30 kDa protein secreted by mycobacterial species, is an immunodominant antigen. The C-terminal regions of alpha antigens are highly divergent, though there are regions where the amino acid sequence of alpha antigen is conserved. We investigated whether the C-terminal regions of the Mycobacterium avium alpha antigen, M. intracellulare alpha antigen and M. tuberculosis alpha antigen contain sequence-specific B-cell epitopes. The C-terminal regions of M. avium alpha antigen and M. intracelluare alpha antigen reacted to anti-M. avium alpha antigen but not to anti-M. tuberculosis alpha antigen derived from rabbits. Thus, M. avium and M. intracellulare have an antigenic determinant in common with rabbit. The C-terminal region of M. tuberculosis alpha antigen did not react to anti-M. avium alpha antigen or anti-M. tuberculosis alpha antigen. An enzyme-linked immunosorbent assay revealed that only the C-terminal region of M. avium alpha antigen reacted to the sera of two of six patients with M. avium-intracellulare (MAC) but not to the sera of patients with M. tuberculosis. In contrast, the C-terminal regions of M. intracellulare alpha antigen and M. tuberculosis alpha antigen were not recognized by the sera from patients with MAC or M. tuberculosis. This region of M. avium alpha antigen can produce a sequence-specific B-cell epitope in humans.

MM-1, a novel c-Myc-associating protein that represses transcriptional activity of c-Myc.

We have isolated the cDNA encoding a novel c-Myc-binding protein, MM-1, by the yeast two-hybrid screening of a human HeLa cell cDNA library. The protein deduced from the cDNA comprises 167 amino acids and was localized in the nucleus of introduced COS-I cells. The MM-1 mRNA was highly expressed in human pancreas and skeletal muscle and moderately in other tissues. As for the c-Myc binding, glutathione S-transferase MM-1 expressed in Escherichia coli bound in vitro to c-Myc translated in reticulocyte lysate, and almost whole, the MM-1 molecule was necessary for the binding in the yeast two-hybrid system. The mammalian two-hybrid assays in hamster CHO cells revealed that MM-1 interacts in vivo with the N-terminal domain covering the myc box 2, a transcription-activating domain, of c-Myc. Furthermore, MM-1 repressed the activation of E-box-dependent transcription by c-Myc.

The 16-kDa alpha-crystallin-like protein of Mycobacterium bovis BCG is produced under conditions of oxygen deficiency and is associated with ribosomes.

A 16-kDa protein, identical to the alpha-crystallin-like stress protein, was induced under O2-deficient culture conditions and bound principally to the 30S ribosomal subunits of Mycobacterium bovis BCG substrain Tokyo (BCG). The 16-kDa protein was shown to be tightly associated with the ribosome.

Analysis of the genes encoding the antigen 85 complex and MPT51 from Mycobacterium avium.

The components of the fibronectin-binding antigen 85 complex (85A, 85B, and 85C) and the related protein MPB/MPT51 are major secreted proteins in Mycobacterium tuberculosis and Mycobacterium bovis BCG. The fbpA, fbpC, and mpt51 genes encoding 85A, 85C, and MPT51, respectively, were isolated from Mycobacterium avium and sequenced in this study. The structures of these genes, and that of the fbpB gene encoding the 85B protein, were conserved in these three species. The secreted amounts of 85A, 85B, 85C, and MPB/MPT51 were compared for M. tuberculosis, BCG, and M. avium. These four proteins were found in large amounts in the culture filtrates from M. tuberculosis and BCG. In contrast, in the culture filtrate from M. avium, 85B and MPT51 were abundant whereas 85A and 85C were hardly found, in spite of the presence of the encoding genes. The difference in the secretion amounts might be regulated at the transcription level. These facts might reflect host immunopathogenesis, the protective immunities against infections, and the drug susceptibilities of these organisms.

Knowledge and attitudes of Japanese dental health care workers towards HIV-related disease.

OBJECTIVE: The present study was undertaken to investigate knowledge of AIDS and HIV infection among Japanese dental health care workers, the source of that knowledge and attitudes of dental workers towards infected patients. METHODS: The study population surveyed by means of a self-administered questionnaire consisted of 174 dental health workers at Nagasaki University Dental Hospital, including students and trainee hygienists. RESULTS: Most respondents (100% response) claimed their major source of AIDS knowledge to be derived from the media. Almost all considered their knowledge of AIDS and HIV infection to be more than moderate but still inadequate. The majority of respondents would be hesitant about performing dental treatment on HIV-positive patients. It was widely anticipated that dental patients infected with HIV would increase in the next few years and many were anxious about the increasing occupational risk of HIV infection. Only 22.4% of respondents had the same attitude towards treating HIV-positive and HIV-negative patients. Most also considered that they would be able to take care of the oral opportunistic diseases associated with HIV. Over 90.0% of respondents requested additional education about HIV, particularly information about the prevention and spread of the virus and cross-infection requirements. CONCLUSION: It is concluded that further training in the medical and psychological aspects of treating HIV-positive patients is indicated in Japan.