Construction and characterization of a fimA mutant of Porphyromonas gingivalis.

Although fimbriae of Porphyromonas gingivalis have been implicated as playing a major role in adherence to gingival tissue surfaces, no conclusive genetic evidence has yet been obtained. The fimA gene, the determinant for the major fimbrial subunit protein, was cloned and sequenced (D. P. Dickinson, M. A. Kubiniec, F. Yoshimura, and R. J. Genco, J. Bacteriol. 170:1658-1665, 1988). We undertook to inactivate the fimA gene by a homologous recombination technique and examined the fimA mutant for changes in surface properties, including production of fimbriae, adherence to human gingival fibroblasts and epithelial cells, hemagglutinating activity, and surface hydrophobicity. To inactivate the fimA gene, we disrupted a fimA clone by insertion of a DNA segment containing an erythromycin resistance (Emr) gene. This was then delivered into P. gingivalis ATCC 33277 from an Escherichia coli K-12 strain, SM10 lambda pir, by using a mobilizable suicide vector, pGP704; recombination at the fimA locus led to the isolation of a fimA mutant. Disruption of the fimA locus and disappearance of FimA production were confirmed by Southern hybridization with a fimA-specific DNA probe and Western immunoblotting with a monoclonal antibody against the FimA protein, respectively. The fimA mutant constructed failed to express long (0.5- to 1.0-micron) fimbriae from the bacterial surface and had a diminished adhesive capacity to tissue-cultured human gingival fibroblasts and epithelial cells. Observation of the bacteria adhering to human gingival fibroblasts by scanning electron microscopy revealed that the wild-type strain had dramatic local changes in the appearance of the microvilli at the point of contact with large bacterial clumps, whereas the fimA mutant did not. In contrast, neither the hemagglutinating activity nor the surface hydrophobicity was changed in the fimA mutant. These data thus constitute the first direct genetic evidence demonstrating that the FimA protein of P. gingivalis is essential for the interaction of the organism with human gingival tissue cells through a function(s) encoded by the fimA gene.     

Anti-arthritic properties of FK506 on collagen-induced arthritis in rats

Objective and design:To determine the effect of FK506 (tacrolimus) on paw inflammation, TNF- expression in joint, and bone and cartilage destruction in type II collagen-induced arthritis (CIA) model in rats.Methods:CIA was induced by immunization of female Lewis rats with an emulsion of bovine type II collagen and incomplete Freunds adjuvant. Paw inflammation was assessed by the increase in paw volume. Tumor necrosis factor (TNF) - expression in hind knee joint was assessed by immunohistochemical analysis. Lesions of bone and cartilage were assessed on the basis of histological change in knee joint, radiographic analysis in hind paw, bone mineral density in femora and proteoglycan contents in the cartilage of femoral heads. FK506 at doses of 1, 1.8 and 3.2 mg/kg or its placebo formulation was orally administered to rats for 28 days from the day after immunization (n = 10). Effect of FK506 was compared with that of vehicle (distilled water).Results:FK506 at a dose of 1.8 mg/kg significantly suppressed paw swelling (p < 0.01) and histological change in knee joint (p < 0.05). Tumor necrosis factor (TNF)- was mainly expressed in the region with a marked infiltration of inflammatory cells in the hind knee joint. FK506 (3.2 mg/kg) markedly reduced TNF- expression. FK506 at a dose of 1.8 mg/kg suppressed radiographic changes in hind paw (p < 0.05) and also recovered the decrease in bone mineral density in the femora (p < 0.05). Proteoglycan contents in the cartilage of femoral heads were determined to evaluate the cartilage destruction more quantitatively and found to significantly decrease in CIA rats. FK506 at a dose of 1.8 mg/kg recovered the loss of proteoglycan contents (p < 0.01).Conclusion:These results show that FK506 is effective in suppressing inflammation, TNF- expression in joint, and damage to bone and cartilage in rat CIA, and may be useful in the treatment of rheumatoid arthritis.     

Mechanisms of blood coagulation induced by latex particles and the roles of blood cells

Latex particles with highly negative or positive charges shortened the clotting time of whole blood and platelet-rich plasma and activated platelet factor 3. Platelet-poor plasma was clotted by the particles with a highly negative charge, but not by those with a positive charge, except hydrophobic particles. Blood coagulation by positively-charged particles was attributed to platelet activation. An enhancement of blood coagulation was also observed in the presence of erythrocytes, leucocytes, their cell membranes or negatively charged phospholipids, and phosphatidylserine instead of platelets. Hydrophilic and low-charged particles suppressed blood coagulation.     

Topical application of FK506 (tacrolimus) ointment inhibits mite antigen-induced dermatitis by local action in NC/Nga mice

BACKGROUND: FK506 ointment (tacrolimus ointment, protopic) is a new drug therapeutically effective for patients with atopic dermatitis (AD). However, the mechanism of action of FK506 ointment on AD is not fully understood. METHODS: We examined the effect of FK506 ointment on mite antigen-induced dermatitis in NC/Nga mice. Clinical symptoms and ear thickness were recorded, and histopathological studies and in vitro analyses were performed. RESULTS: Topical application of FK506 ointment (0.03-0.3%) suppressed the development of dermatitis. In the lesional skin, both interleukin (IL)-4 and interferon (IFN)-gamma were detected, even though the IL-4+/IFN-gamma- T helper 2 (Th2) population was predominant in the regional lymph nodes (LNs). Topical application of FK506 treatment reduced the elevated level of both IL-4 and IFN-gamma in the skin, but did not decrease the expansion of the Th2 population in the LNs. CONCLUSIONS: Topical application of FK506 ointment suppresses dermatitis by inhibiting the activation of inflammatory cells locally, without systemic immune suppression, in this AD model.     

The predictive ability of six pharmacokinetic models of rocuronium developed using a single bolus: evaluation with bolus and continuous infusion regimen

Purpose

Rocuronium concentration prediction using pharmacokinetic (PK) models would be useful for controlling rocuronium effects because neuromuscular monitoring throughout anesthesia can be difficult. This study assessed whether six different compartmental PK models developed from data obtained after bolus administration only could predict the measured plasma concentration (Cp) values of rocuronium delivered by bolus followed by continuous infusion.

Methods

Rocuronium Cp values from 19 healthy subjects who received a bolus dose followed by continuous infusion in a phase III multicenter trial in Japan were used retrospectively as evaluation datasets. Six different compartmental PK models of rocuronium were used to simulate rocuronium Cp time course values, which were compared with measured Cp values. Prediction error (PE) derivatives of median absolute PE (MDAPE), median PE (MDPE), wobble, divergence absolute PE, and divergence PE were used to assess inaccuracy, bias, intra-individual variability, and time-related trends in APE and PE values.

Results

MDAPE and MDPE values were acceptable only for the Magorian and Kleijn models. The divergence PE value for the Kleijn model was lower than ?10 %/h, indicating unstable prediction over time. The Szenohradszky model had the lowest divergence PE (?2.7 %/h) and wobble (5.4 %) values with negative bias (MDPE = ?25.9 %). These three models were developed using the mixed-effects modeling approach. The Magorian model showed the best PE derivatives among the models assessed.

Conclusions

A PK model developed from data obtained after single-bolus dosing can predict Cp values during bolus and continuous infusion. Thus, a mixed-effects modeling approach may be preferable in extrapolating such data.

     

Effect of Mass Screening for Breast Cancer from the Aspect of Psychosocial Assessment of the Quality of Life

To assess the quality of life (QOL) in patients with breast cancer receiving mass screening, a collaborative matched case-control study was conducted in nine hospitals throughout Japan. A total of 122 patients detected by mass screening (study group) and 226 patients found in out-patient clinics (control group) were assessed psychosocially on the basis of questionnaire information. The incidence of patients with early stage breast cancer was significantly higher in the study group than in the control group ( P <0.05). Chest wall pain was observed in 35.2% of the study group and in 46.5% of the control group ( P <0.05). Although control patients were more optimistic than study group patients, disturbed daily life and anxiety about recurrence were a little more frequent in the former group than in the latter. In particular, shoulder stiffness was frequently seen in the control group ( P <0.05). Early detection and information do not create anxiety in mass screening patients ( P <0.01). We should recommend mass screening to patients to detect early stage breast cancer and provide better QOL.     

Contribution of whole body FDG-PET to the detection of distant metastasis in pancreatic cancer

OBJECTIVE: Accurate baseline staging is necessary to appropriately treat pancreatic cancer. The present study was undertaken to evaluate the clinical contribution of whole body FDG-PET to the detection of distant metastasis in pancreatic cancer. METHODS: A total of consecutive 42 patients with previously untreated pancreatic cancer were examined. Whole body FDG-PET imaging for initial staging was performed with a 3D acquisition and iterative reconstruction on Siemens ECAT HR+ scanner at 1 hour post 185-200 MBq 18F-FDG injection. PET findings were correlated with clinical and radiological data to determine the impact of PET on staging. RESULTS: In 16 patients, there were one or more sites of metastasis based on clinical data. FDG-PET correctly identified the presence of metastasis in 13 of 16 patients and its absence in 23 of the remaining 26 patients. Thus, FDG-PET missed 4 metastatic sites in 4 patients (liver and lung metastasis). FDG-PET correctly identified 8 metastatic sites in 7 patients (peritoneal dissemination and liver, bone and supraclavicular lymph node metastasis), which were missed on CT imaging. Based on whole body FDG-PET, the clinical stage was changed in 5 of 42 patients (11.9%). CONCLUSIONS: These results suggest that FDG-PET and CT appear to have a complementary role in the detection of distant metastasis in patients with pancreatic cancer.