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Biomedical subjects

T Hirofuji

Publications and source records attributed to T Hirofuji.

16 recordsLinked to original sources

Gingipains in the culture supernatant of Porphyromonas gingivalis cleave CD4 and CD8 on human T cells.

Porphyromonas gingivalis has been shown to attack host defense systems through proteolytic cleavage of a wide variety of members of the systems. In this study, we examined the ability of P. gingivalis culture supernatant to alter the expression of human T cell surface proteins. As judged by flow cytometric analysis, detection of CD4 expression was completely eliminated by the supernatant, but CD8 was less sensitive. When the culture supernatant was added with reducing agents, proteolytic activity was enhanced, resulting in the cleavage of CD8. Mitogenic response of T cells to phytohemagglutinin or concanavalin A was decreased by the treatment of the cells with the culture supernatant of P. gingivalis. The three forms of gingipains (high molecular mass arginine-specific gingipain, arginine-specific gingipain 2 and lysine-specific gingipain) purified from the culture supernatant of P. gingivalis actively cleaved CD4 and CD8 on human T cells, indicating that proteolytic activity of the culture supernatant was due to gingipains. These results suggest that cysteine proteinases like gingipains released from P. gingivalis cleave T cell surface proteins and impede T cell function.

Adhesins, Bacterial↗

The presence of chemokine receptor (CCR5, CXCR3, CCR3)-positive cells and chemokine (MCP1, MIP-1alpha, MIP-1beta, IP-10)-positive cells in human periapical granulomas.

The infiltration of leukocytes into inflammation sites such as observed in human periapical granulomas is considered to be mediated by chemotactic factors. In this study, we examined the presence of chemokine- and chemokine receptor-positive cells in samples obtained from human subjects by means of immunohistochemical methods. Macrophage chemotactic protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and IFN-inducible protein 10 (IP-10)-producing cells were present in periapical granulomas. In addition, chemokine receptor CCR3-, CCR5-, and CXCR3-positive cells were also present. In contrast, no factor expression was observed in clinically healthy periodontal ligament, serving as a negative control. Our findings suggest that these chemokines are responsible for modulating the process of disease, such as human apical periodontitis.

Adolescent↗

Mixed infection of Porphyromonas gingivalis and Bacteroides forsythus in a murine abscess model: involvement of gingipains in a synergistic effect.

Several microorganisms including Porphyromonas gingivalis and Bacteroides forsythus have been implicated to be etiologically important agents of periodontal disease. In this study, we determined the ability of combinations of periodontopathogenic microorganisms to cause tissue destruction in a murine abscess model. Although all bacterial combinations used in this study produced larger abscesses than did monoinfection of each bacterium, the combination of P. gingivalis and B.forsythus showed a synergistic effect on abscess formation. Since these two bacteria have been frequently found together in lesions of periodontitis, these results suggest the significance of their co-infection in the progression of periodontitis. P. gingivalis produces extracellular and cell-associated cysteine proteinases (gingipains) which appear to be involved in its virulence. The rgpA rgpB double and kgp mutants induced significantly smaller abscesses than the wild type. Moreover, the rgpA rgpB kgp triple (gingipain-null) mutant hardly showed lesion formation at all with the experimental conditions used in this study, indicating that these genes encoding gingipains are important for virulence of P. gingivalis. Mixed infection of these P. gingivalis mutants with B. forsythus showed an additive effect on abscess formation, indicating that the gingipains of P. gingivalis may play an important role in the pathological synergism between P. gingivalis and B. forsythus.

Abscess↗

Development of mineralized nodules in fetal rat mandibular osteogenic precursor cells: requirement for dexamethasone but not for beta-glycerophosphate.

We have reported that a cell population obtained from fetal rat mandible with neutral protease (Pro I) has a unique differentiation sequence in which the elevation of alkaline phosphatase (ALPase), calcium accumulation, and collagen synthesis occurs simultaneously. In this report, we further characterized Pro I-released population of cells by studying the effect of dexamethasone (Dex) or beta-glycerophosphate (beta-GP) on the formation of bone nodules. The formation of bone nodules in Pro I-released population of cells (ProIRPC) was augmented by the addition of Dex (10(-7) M) from days 3 to 14, suggesting that Pro IRPC contained osteoprogenitor (OP) cells. A 24-hour pulse treatment of ProIRPC released population of cells with Dex on days 9 and 12 resulted in an increase in the number of nodules but treatment on days 3, 6, or 15 did not. The number of bone nodules formed in Pro IRPC pulse treated with Dex on day 9 was comparable with that in Pro IRPC treated with Dex from days 3 to 14. Dex caused an earlier elevation of ALPase, in which maximal expression was observed on day 10. beta-GP caused a prolonged elevation of ALPase, but did not affect the formation of bone nodules. Unlike Pro I-released population of cells, rat calvarial cells did not form mineralized nodules without beta-GP, and showed that a Dex-responsive period on bone nodule formation in rat calvarial cells was at preconfluency (days 0 and 1). Thus, it appeared that the Dex-induced differentiation of early OP cells in Pro IRPCs occurred during the limited period from day 9 to day 12. Pro IRPC was found to have an unique characteristic that bone nodule formation was not affected by beta-GP.

Alkaline Phosphatase↗

Colorimetric microtiter plate based assay for detection and quantification of amplified Actinobacillus actinomycetemcomitans DNA.

We developed a colorimetric microtiter plate-based assay for the detection and quantification of polymerase chain reaction-amplified DNA fragment specific for Actinobacillus actinomycetemcomitans. We amplified the 396-bp leukotoxin-specific DNA fragment by using two oligonucleotide primers, one carrying a biotin group at the 5' end and another one with a digoxigenin at the 5' end. Following amplification, the biotinylated polymerase chain reaction products were applied to a microtiter well precoated with avidin. The colorimetric detection and quantification were achieved by an enzyme-linked immunosorbent assay using alkaline phosphatase-conjugated anti-digoxigenin antibody. The detection limit of the colorimetric assay was found to be as little as 500 fg of purified A. actinomycetemcomitans DNA and as few as 50 A. actinomycetemcomitans. Therefore, this colorimetric assay was able to estimate the amount of A. actinomycetemcomitans in subgingival plaque samples. We concluded that the colorimetric assay of the PCR product is a very useful method not only to detect the presence of A. actinomycetemcomitans but also to quantify the amount of A. actinomycetemcomitans in large numbers of subgingival plaque samples.

Aggregatibacter actinomycetemcomitans↗

An interleukin-1 inhibitor in gingival crevicular fluid of patients with chronic inflammatory periodontal disease.

The gingival crevicular fluid of a patient(s) with marginal periodontal disease contained an activity inhibitory to interleukin-1 (IL-1). The inhibitory activity could be detected after the depletion of IL-1 alpha by the use of a specific antibody (anti-human recombinant IL-1 alpha monoclonal antibody)-conjugated Sepharose column. The inhibitory activity was not due to a toxic effect on the thymocytes since IL-1 alpha-depleted gingival crevicular fluid did not affect the incorporation of [3H]thymidine in either the presence or absence of concanavalin A. The inhibitory activity was exerted against both IL-1 alpha and IL-1 beta. The inhibitory factor did not have any effect on IL-2-induced proliferation of concanavalin A-activated spleen cells. The inhibitor was heat labile. Gel filtration on a Superose 12 column revealed the IL-1 inhibitor to have two major peaks, one in the molecular size range of 12 to 14 kDa and the other below a molecular size of 10 kDa.

Animals↗

Partial characterization of an interleukin-1-like factor in human gingival crevicular fluid from patients with chronic inflammatory periodontal disease.

A significant level of interleukin-1-(IL-1)-like activity was detected in gingival crevicular fluid obtained from sites in patients with chronic inflammatory periodontal disease, confirming a previous report of IL-1-like activity detected in human gingival crevicular fluid from patients with chronic inflammatory periodontitis (J. A. Charon, T. A. Luger, S. E. Mergenhagen, and J. J. Oppenheim, Infect. Immun. 38:1190-1195, 1982). In the present study, we sought to investigate whether this IL-1-like activity belonged to IL-1 alpha or IL-1 beta and to characterize some of the biochemical properties of this factor. Polyclonal antibodies against recombinant human IL-1 alpha or IL-1 beta (rIL-1 alpha or rIL-1 beta) have been used for serological comparison of the IL-1-like factor. IL-1-like activity was completely neutralized by anti-human rIL-1 alpha antiserum, but not by anti-human rIL-1 beta antiserum. On gel filtration with a high-pressure liquid chromatographic Superose 12 column, IL-1-like activity was separated into two peaks, one with a molecular weight of about 43,000 and the other with a molecular weight of less than 17,000. The majority of the IL-1-like factor with a low molecular weight in human gingival crevicular fluid migrated at a molecular weight of about 17,000 under the reducing conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specificity of the IL-1-like factor was further confirmed by an immunochemical method (Western blotting [immunoblotting]) by using anti-human rIL-1 alpha monoclonal antibodies. On isoelectric chromatography with a high-pressure liquid chromatographic Mono P column, the pI of this IL-1-like factor was between pH 4.9 and 5.2. These results suggest that the IL-1-like factor in human gingival crevicular fluid from diseased sites in patients with chronic inflammatory periodontitis consists predominantly of IL-1 alpha.

Adult↗

Synthetic oligonucleotide probes complementary to the HLA-B variable region hybridize with Klebsiella pneumoniae.

Three oligonucleotide probes complementary to base sequences of the HLA-B variable region were used to probe clinical and foodborne isolates of Klebsiella spp. by DNA colony hybridization. One oligonucleotide (RR-3) corresponding to amino acid residues 66-74 of the HLA-B27.1 sequence and one corresponding to residues 66-74 of the HLA-B7 sequence (RR-5) hybridized with K. pneumoniae under conditions of high stringency. A genomic library was constructed using K. pneumoniae K43 chromosomal DNA, and a 1 kb PstI restriction fragment was found to contain the sequence associated with specific binding of the oligonucleotide probes. After purification and radiolabeling, the cloned fragment hybridized with 96.2% of the K. pneumoniae isolates by DNA colony hybridization. These results confirm the presence of sequence similarities between bacterial DNA and the MHC Class I variable region.

Antigens, Bacterial↗

Isolation of T cell line capable of protecting mice against collagen-induced arthritis.

A T cell line specific to human type II collagen (CII) was selected and propagated from DBA/1J mice immunized with human CII. The line cells were not reactive to type I or type III collagen of human origin, but they were cross-reactive to bovine, rat, and rabbit CII and they recognized both native and heat-denatured human CII. The cells were reactive to an N-terminal three-quarters fragment of human CII, produced by tadpole collagenase digestion of human CII, but not to a C-terminal one-quarter fragment of human CII. The cells showed Thy-1+, Lyt-1+, Lyt-2-, and L3T4+ phenotypes characteristic of T helper cells or delayed-type hypersensitive cells, determined by the immunofluorescence method. To clarify the role of T cells in the pathogenesis of collagen-induced arthritis, we inoculated this cell line into DBA/1J mice and found that they developed clinical arthritis, albeit at a low incidence. The cells attenuated by x-ray were capable of inducing resistance to the subsequent induction of collagen-induced arthritis of DBA/1J mice. The sera from mice protected by inoculation of the cell line exhibited anti-idiotypic antibody response against conventional and monoclonal anti-CII antibodies. Anti-T cell receptor response may be involved in the mechanism for the protective effect of the cell line against autoimmune murine arthritis.

Animals↗

An experimental mouse model of Yersinia-induced reactive arthritis.

An experimental animal model of Yersinia-induced arthritis was successfully developed in mice using Yersinia enterocolitica WA. In order for the Yersinia to induce arthritis the strain had to be cured of plasmid. The arthritis-susceptible mouse strains were DBA/2 and BDF1. C57B1/6, Balb/c, CBA and DBA/1 were arthritis-resistant. Both the dose of bacteria and the route of administration were critical.

Animals↗

Muramyl dipeptide induces acute joint inflammation in the mouse.

Acute joint inflammation was produced in BALB/c mice by a single intravenous injection of synthetic muramyl dipeptide (MDP), its stereoisomers and 6-O-acyl derivatives of MDP. Four adjuvant-active, but not five adjuvant-inactive MDP analogs induced acute swelling and erythema of the ankles and wrists which were detected around 6-10 hr, reached the maximum severity by 18-24 hr and subsided by days 3 to 4 after injection. Introduction of the stearoyl group, but not the alpha-branched long chain fatty acid group into the C-9 hydroxyl group of MDP enhanced and prolonged the joint lesions compared with MDP.

Acetylmuramyl-Alanyl-Isoglutamine↗

Acute joint inflammation in mice after systemic injection of the cell wall, its peptidoglycan, and chemically defined peptidoglycan subunits from various bacteria.

The systemic injection of an aqueous suspension of cell wall or its peptidoglycan (PG)-rich sonicate derived from group A streptococcus and Lactobacillus casei induced acute joint lesions in BALB/c, DBA/1J, (BALB/c X DBA/1J)F1, and C3H/He mouse strains, but not in C57BL/6, DBA/2, and AKR strains. Cell walls and their enzymatically degraded PG fragments from other bacteria as well as the synthetic disaccharide dipeptide and Lactobacillus plantarum cell wall-derived disaccharide tripeptide produced similar acute inflammation in susceptible BALB/c mice. Acute swelling and erythema of the ankles and wrists were observed as early as 3 h, reached maximum severity by day 2, and generally subsided by days 4 to 6 after injection. Histological studies showed synovial proliferation, marked infiltration of many mononuclear cells and a few polymorphonuclear leukocytes in the soft tissues, and extensive deposition of fibrinous exudate in the joint space. Antibody response was detectable against the PG fraction. However, anti-PG antibody does not seem to be responsible for the pathogenesis of this disease. On the other hand, experiments on decomplementation by cobra venom factor suggest that complement components are involved in the early phase of this arthritic model.

Animals↗

Characterization of monoclonal antibody specific for human type II collagen: possible implication in collagen-induced arthritis.

We obtained monoclonal antibodies specific for human type II collagen and characterized them using human collagen type I, II, III and V and tropocollagen A (3/4) (TCA) and tropocollagen B (1/4) (TCB) fragments of type II collagen which were obtained by digestion with tadpole collagenase. These antibodies were of the IgG2a class and specific for the conformational determinant of TCA fragment of type II collagen. When injected intravenously into DBA/1J mice, one of the monoclonal antibodies induced arthritis, which was characterized by early onset, mildness in severity and preferential localization mainly in the peripheral joints of the lower extremities. These results suggest that, at least, one of the arthritogenic determinants of type II collagen for collagen-induced arthritis of mice exists in the three quarter region from the N-terminus of type II collagen.

Animals↗

Specificity of anti-type II collagen antibody response in rats.

Specificity of the rat antibody against type II collagen was investigated using rat, bovine and human type II collagen preparations. PVG/c rats showed the highest antibody level in assays with autologous rat type II collagen than with heterologous (bovine or human) type II collagen even when immunized with heterologous one. On the other hand, the rat exhibited significantly low antibody response when they were immunized with autologous type II collagen. The results indicate that rats develop strong antibody response against self type II collagen when immunized with heterologous collagen and this unique specificity of anti-type II collagen antibody response suggests that autoimmunity is involved in the pathogenesis of collagen-induced arthritis in rats.

Animals↗