Development and characterization of Porphyromonas gingivalis-specific rat T-cell clones.

Porphyromonas gingivalis has been implicated as a major pathogen in periodontitis. To determine the role of T cells in the regulation of this disease, a method was developed for the generation and characterization of rat T-cell clones with antigen specificity to P. gingivalis whole cells. The clones studied so far demonstrated a T-helper (Th) phenotype W3/13+, W3/25+, OX8− and OX22−. These T-cell clones proliferated in vitro in response to P. gingivalis, but not to other bacteria (Prevotella intermedia, Actinobacillus actinomycetemcomitans, Wolinella recta, Fusobacterium nucleatum, Streptococcus sanguis). Limiting dilution analysis showed W3/25+, OX8− T cells preferentially respond to P. gingivalis, rather than W3/25−, OX8+ T cells. P. gingivalis-reactive W3/25+ T cells belonged to the OX22− population, suggesting that the OX22− T cells may represent memory cells. All clones tested produced interferon γ, but not interleukin 2. The cloned T-cell F1 significantly enhanced P. gingivalis-specific antibody production (p < 0.03). The availability of these cloned T cells should bring new insight into the mechanism by which T cells regulate oral health and periodontal disease.     

The complex formation of ions with a phospholipid monolayer adsorbed at an aqueous|1,2-dichloroethane interface

The complex formation of phosphatidylcholine adsorbed at a water|1,2-dichloroethane, W|DCE, interface with ions was evaluated based on the desorption behavior of lipids at the interface accompanied by an ion transfer and was confirmed by measuring voltammograms for the ion transfer facilitated by the complex formation with lipids. The desorption behavior of lipids was measured according to the Wilhelmy vertical plate method, the cell of which was improved to achieve polarization of the W|DCE interface. Voltammograms for the ion transfer at the W|DCE interface facilitated by the complex formation with lipids were recorded at a micro W|DCE interface in order to avoid the influence of convection caused by the desorption of lipids at the interface. Lipids adsorbed at the W|DCE interface showed complex formation with alkali metal ions, ${\mathrm{NH}}_4^{+}\text{,}({\mathrm{CH}}_3){\mathrm{NH}}_3^{+}\text{,}({\mathrm{CH}}_3{)}_2{\mathrm{NH}}_2^{+}\mathrm{or}({\mathrm{CH}}_3{)}_3{\mathrm{NH}}^{+}$, but not with $({\mathrm{CH}}_3{)}_4{\mathrm{N}}^{+}$ or anions such as ${\mathrm{SO}}_4^{2-}$, CH₃COO^?, Cl^?, Br^?, ${\mathrm{NO}}_3^{-}$, I^? or ${\mathrm{ClO}}_4^{-}$. Lipids also showed strong complex formation with a basic amino acid such as an arginine cation, which has two amino groups in the molecule. The strength of the complex formation with lipids was ${\mathrm{Arg}}^{+}>{\mathrm{Li}}^{+}\text{,}{\mathrm{Na}}^{+}>{\mathrm{K}}^{+}>{\mathrm{NH}}_4^{+}>{\mathrm{CH}}_3{\mathrm{NH}}_3^{+}>({\mathrm{CH}}_3{)}_2{\mathrm{NH}}_2^{+}>{\mathrm{Cs}}^{+}>({\mathrm{CH}}_3{)}_3{\mathrm{NH}}^{+}$.     

IL-4- and IL-6-producing cells in human periodontal disease tissue

IL-4- and IL-6-producing cells in human periodontal disease tissues were investigated using immunohistochemical and in situ hybridization techniques. Immunohistochemical analysis demonstrated the presence of IL-4-producing cells within the CD45RO⁺ subset and the percentage of IL-4+ cells was significantly higher in periodontal lesions than in gingivitis tissues (p<0.01). The percentage of IL-6-producing memory cells was higher in periodontal lesions compared with gingivitis tissues, although it was not statistically significant (p<0.05). A reverse tendency in IL-4- and IL-6-positive cells was observed in a few individual cases. No IL-4 mRNA could be detected using the in situ hybridization technique. However, high levels of IL-6 mRNA were present in clinically healthy tissues, with a further increase in both epithelium and connective tissues affected by gingivitis, although only the former was significant (p< 0.025). There was a significant decrease in IL-6 mRNA in both the connective tissue (p<0.025) and epithelium (p<0.01) in periodontitis tissues compared with levels in gingivitis tissues. However, the levels of IL-6 mRNA in periodontal tissues were high compared with those of IL-1 mRNA, which was used in this study as a positive control. These results suggest that Th2–type cells may accumulate in periodontal lesions.     

Immunohistological analysis of memory T lymphocytes and activated B lymphocytes in tissues with periodontal disease

Memory T-cells and activated B-cells were identified in cryostat sections of adult periodontitis (AP) lesions and categorized in terms of frequency and distribution. Nineteen periodontitis biopsies were obtained at the time of periodontal surgery to remove residual periodontal pockets following the completion of initial preparation. Gingival tissues exhibited various degree of inflammation (Gl of 0-2) but probing depths of < 4 mm and < 5 mm loss of attachment. As a control, 5 gingivitis specimens (Gl of 1, probing depth and loss of attachment of ≤ 3 mm) were obtained from premolar and third molar sites requiring extraction for either orthodontic treatment or pericoronitis. Serial cryostat sections (6 μm in thickness) were prepared from each biopsy, on which a double staining avidin-biotin immunoperoxidase and avidin-biotin alkaline phosphatase technique was used to identify CD4+, CD45RO+ memory T-cells and activated CD19+ B-cells expressing CD23 or CD25. In periodontitis lesions the mean percentage of CD4+ cells expressing CD45RO was consistently high (65.9% in the crevicular (C) one-third (1/3), 61.2% in the middle (M) 1/3 and 62.5% in the oral (O) 1/3). This contrasts with the low mean percentage of CD4+, CD45RA+ naive T-cells (17.1% in the C 1/3, 14.8% in the M 1/3 and 12.4% in the O 1/3). In gingivitis specimens, the incidence of CD4+, CD45RO+ was 81.9% in the C 1/3, 81.1% in the M 1/3 and 89.0% in the O 1/3. This was higher than that of periodontitis biopsies. With CD4+, CD45RA+ the incidence was 10.0% in the C 1/3, 8.0% in the M 1/3, and 6.6% in the O 1/3 and the relationship to the periodontitis biopsies was reversed. However, the percentage of CD23+ and CD25+, CD19+ B-cells which were identified in 13 out of 19 samples from periodontitis varied significantly (0-100% for CD23, 0-36.2% for CD25) in spite of similar clinical status. The frequency of B-cells and activated B-cells in the gingivitis was much lower than that of periodontitis. These results indicate that both T-cells and B-cells were in active stage in periodontitis lesions. Differences of immunohistological features between gingivitis and periodontitis may be attributable to the heterogeneity of profiles of cytokine production by CD4+, CD45RO+“memory’ cells.     

Stress distribution in implant-supported prostheses using different connection systems and cantilever lengths: digital photoelasticity

Photoelastic analysis was used to evaluate the biomechanical behaviour of implant-supported, double-screwed crowns with different connection systems and cantilever lengths. Three models were made in PL-2 photoelastic resin and divided into six groups, on the basis of the implant connection system (external hexagon [EH] or Morse taper [MT]), type of abutment (Mini Pilar [Neodent, Curitiba, Paraná, Brazil] or “UCLA”) and number of crowns in the cantilever (one or two). The implant-prosthesis unit was placed in a circular polariscope. Occlusal surfaces of the crowns were subjected to 100-N loads in the axial and oblique (45°) directions in a universal testing machine (EMIC). Generated stresses were recorded and analysed qualitatively in a graphics program (Adobe Photoshop). Under axial loading, all of the groups had similar numbers of fringes, which were increased when the crowns were subjected to oblique loading. The highest number of fringes was found during oblique loading in the EH?+?Mini Pilar group. In conclusion, although the type of implant connection system did not have a direct influence on the stress distribution for axial loading, the cantilever length did have a direct influence on stress distribution. Models with two crowns in the cantilever showed more stress, with a greater concentration of force on the cervical part of the implant.     

Pre- and post-serial metagenomic analysis of gut microbiota as a prognostic factor in patients undergoing haematopoietic stem cell transplantation

The human gut harbours diverse microorganisms, and gut dysbiosis has recently attracted attention because of its possible involvement in various diseases. In particular, the lack of diversity in the gut microbiota has been associated with complications of haematopoietic stem cell transplantation (HSCT), such as infections, acute graft-versus-host disease and relapse of primary disease, which lead to a poor prognosis. However, few studies have serially examined the composition of the intestinal microbiota after HSCT. In this study, we demonstrated, using next-generation sequencing of the bacterial 16S ribosomal RNA gene, combined with uniFrac distance analysis, that the intestinal microbiota of patients undergoing allogeneic HSCT substantially differed from that of healthy controls and recipients of autologous transplants. Faecal samples were obtained daily throughout the clinical course, before and after transplantation. Notably, the proportions of Bifidobacterium and genera categorized as butyrate-producing bacteria were significantly lower in patients with allogeneic HSCT than in healthy controls. Furthermore, among allogeneic transplant recipients, a subgroup with a preserved microbiota composition showed a benign course, whereas patients with a skewed microbiota showed a high frequency of complications and mortality after transplantation. Thus, we conclude that the stability of intestinal microbiota is critically involved in outcomes of HSCT.     

Unfavorable effect of knee immobilization on Achilles tendon healing in rabbits

This study was undertaken to assess the effect of knee immobilization on the treatment of Achilles tendon rupture. After their Achilles tendons were severed, rabbits were divided into 2 groups. In Group A, only the ankle joint was immobilized. In Group B, both the knee and ankle joints were immobilized. At 4 weeks after surgery, both the ultimate tensile force and stiffness of the severed tendons were significantly greater in Group A than in Group B. In Group A, dense collagen fibers were seen in the repaired tendons, and the bundles of collagen fibers were parallel to one another along the axis of the tendons. In contrast, in Group B, dilated veins and capillaries were seen in the repaired tendons, and the proliferation of connective tissue containing collagen fibers was severely reduced around these veins and capillaries and was in general irregular and uneven. These results suggest that knee immobilization retards the healing of a ruptured Achilles tendon without suture, due to congestion and tension deprivation produced by keeping the tendon static.     

Magnitude and orientation of Zernike terms in patients with keratoconus

PURPOSE: To measure the magnitude and orientation of the Zernike terms in keratoconic eyes, with and without rigid gas-permeable (RGP) contact lenses. METHODS: A total of 76 eyes with keratoconus, 58 eyes with keratoconus suspect, and 105 normal eyes were studied. To determine the effect of RGP lenses, 19 eyes with keratoconus, 9 eyes with keratoconus suspect, and 17 normal eyes, with and without an RGP lenses, were compared. Ocular higher-order aberrations (HOAs) were measured with a wavefront sensor for a 4-mm-diameter pupil, and the magnitudes, axes of trefoil, and coma were calculated by vector analysis. RESULTS: Zernike vector analysis showed prominent vertical coma with an inferior slow pattern, with mean axes of 82.5 degrees or 91.0 degrees in the patients with keratoconus or keratoconus suspect, respectively. The mean axes of trefoil in patients with keratoconus (93.8 degrees ) and keratoconus suspect (100.6 degrees ) differed from that in normal subjects (35.4 degrees ), indicating that keratoconus has a reverse trefoil pattern from that of normal eyes. Although the total HOAs were significantly (keratoconus and keratoconus suspect, P < 0.001 and P = 0.012, respectively) reduced with an RGP lens, the patterns of the axes of coma and trefoil were reversed with the lens. CONCLUSIONS: In addition to the larger amount of trefoil, coma, tetrafoil, and secondary astigmatism, keratoconic eyes tend to have a reverse coma pattern and reverse trefoil aberrations compared with normal eyes. Although RGP lenses correct the irregular astigmatism, smaller comet-like retinal images in the opposite direction remain due to residual vertical coma.     

Analysis of telomerase activity and RNA expression in a patient with acute promyelocytic leukemia treated with all-trans retinoic acid

In this study, we show that all-trans retinoic acid (ATRA) treatment leads to a rapid decrease in telomerase activity, which was associated with the reduction in myeloblasts and occurs before the appearance of myelocytes, in a patient with acute promyelocytic leukemia (APL). Microarray analysis by ATRA treatment for 48 hr in peripheral blood mononuclear cells (in vivo) and in cultured bone marrow mononuclear cells (in vitro) from a patient with APL revealed upregulation of CD11b, CD11c, CCAAT enhancer binding protein epsilon, Rb1, Mad, and tumor necrosis factor-related genes; and downregulation of hTERT, c-Myc, WT1, bcl-2, and eukaryotic translation elongation factor 1alpha2. The results might offer the potential to define the molecular mechanism underlying ATRA-induced granulocytic differentiation in patients with APL, and provide clues to identify novel molecular therapeutic targets.     

De-coupling of blood flow and metabolism in the rat brain induced by glutamate

Objective  Glutamate plays an essential role in neuronal cell death in many neurological disorders. In this study, we examined both glucose metabolism and cerebral blood flow in the same rat following infusion of glutamate or ibotenic acid using the dual-tracer technique. The effects of MK-801, an NMDA receptor antagonist, and NBQX, an AMPA-kainate receptor antagonist, on the changes in the glucose metabolism and cerebral blood flow induced by glutamate were also examined. Methods  The rats were microinjected with glutamate (1 μmol/μl, 2 μl) or ibotenic acid (10 μg/μl, 1 μl) into the right striatum, and dual-tracer autoradiograms of [¹⁸F]FDG and [¹⁴C]IMP were obtained. MK-801 and NBQX were injected intravenously about 45 and 30 min, respectively, after the infusion of glutamate. Results  De-coupling of blood flow and metabolism was noted in the glutamate-infused hemisphere (as assessed by no alteration of [¹⁸F]FDG uptake and significant decrease of [¹⁴C]IMP uptake). Pretreatments with MK-801, NBQX, or combined use of MK-801 and NBQX did not affect the de-coupling of the blood flow and metabolism induced by glutamate. A histochemical study revealed that about 20% neuronal cell death had occurred in the striatum at 105 min after the infusion of glutamate. In addition, a significant increase of the [¹⁸F]FDG uptake and decrease of [¹⁴C]IMP uptake were also seen in the rat brain infused with ibotenic acid. Conclusion  These results indicate that glutamate and ibotenic acid caused a significant de-coupling of blood flow and glucose metabolism in the intact rat brain during the early phase of neurodegeneration. It is necessary to evaluate the relation between metabotropic glutamate receptors and de-coupling of blood flow and metabolism.