Regulatory mechanisms of C4b-binding protein (C4BP)alpha and beta expression in rat hepatocytes by lipopolysaccharide and interleukin-6.

BACKGROUND: C4b-binding protein (C4BP), a multimeric protein structurally composed of alpha chains (C4BPalpha) and a beta chain (C4BPbeta), regulates the anticoagulant activity of protein S (PS). Patients with sepsis have increased levels of plasma C4BP, which appears to be induced by interleukin (IL)-6. However, it is not fully understood how lipopolysaccharide (LPS) and IL-6 affect the plasma C4BP antigen level and C4BPalpha and C4BPbeta expression in hepatocytes. OBJECTIVES: To assess the effect of LPS and IL-6 on plasma C4BP, PS-C4BP complex levels, PS activity, and C4BP expression by rat liver in vivo and on C4BP expression by isolated rat hepatocytes in vitro. RESULTS: Plasma C4BP antigen level transiently decreased from 2 to 12 h after LPS (2 mg kg(-1)) injection, and then it abruptly increased up to 24 h after LPS injection. Plasma C4BP antigen level increased until 8 h after IL-6 (10 microg kg(-1)) injection, and then gradually decreased up to 24 h after IL-6 injection. LPS significantly decreased the protein and mRNA expression of both C4BPalpha and C4BPbeta in rat hepatocytes, and this effect was inhibited by NFkappaB and MEK/ERK inhibitors. IL-6 mediated increase in C4BPbeta expression in rat hepatocytes, which leads to increased plasma PS-C4BP complex level and to decreased plasma PS activity, was inhibited by inhibition of STAT-3. CONCLUSION: LPS decreases both C4BPalpha and C4BPbeta expression via the NFkappaB and MEK/ERK pathways, whereas IL-6 specifically increases C4BPbeta expression via the STAT-3 pathway, causing an increase in plasma PS-C4BP complex, and thus decreasing the anticoagulant activity of PS.

Lipopolysaccharide-induced decreased protein S expression in liver cells is mediated by MEK/ERK signaling and NFkappaB activation: involvement of membrane-bound CD14 and toll-like receptor-4.

BACKGROUND: The vitamin K-dependent protein S (PS), mainly synthesized in hepatocytes and endothelial cells, plays a critical role in the anticoagulant activity of plasma. The decreased plasma level of PS in sepsis is associated with thrombotic tendency, but the mechanism is unclear. OBJECTIVES: In the present study, we examined the effect of lipopolysaccharide (LPS) on PS expression in vivo in rat liver, and in vitro in isolated hepatocytes and sinusoidal endothelial cells (SECs) from normal rats. RESULTS: LPS induced a progressive decrease of plasma PS antigen level up to 12 h with a slight recovery at 24 h, and a transient decrease of liver PS mRNA level at 4-8 h with a complete recovery at 24 h. In the in vitro studies, LPS decreased PS antigen and mRNA levels in both hepatocytes and SECs. After LPS treatment, tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interferon-gamma (IFN-gamma) transiently increased in plasma. IL-6 increased the protein expression of PS from hepatocytes, while TNF-alpha decreased it from SECs. LPS increased CD14 in hepatocytes and decreased it in SECs, but did not affect toll-like receptor-4 (TLR-4) expression in both cells. Antirat CD14 and antirat TLR-4 antibodies inhibited LPS-induced NFkappaB activation, and a NFkappaB inhibitor suppressed LPS-induced decreased PS expression in both cells. Furthermore, MEK inhibitor blocked LPS-induced decreased PS expression in both cells. CONCLUSIONS: These findings suggest that LPS-induced decreased PS expression in hepatocytes and SECs is mediated by MEK/ERK signaling and NFkappaB activation and that membrane-bound CD14 and TLR-4 are involved in this mechanism. These findings may explain in part the decreased level of plasma PS and thrombotic tendency in sepsis.

Brain malformations caused by retroviral vector-mediated gene transfer of hippocampal cholinergic neurostimulating peptide precursor protein into the CNS via embryonic mice ventricles.

Hippocampal cholinergic neurostimulating peptide precursor protein (HCNP-pp) is a unique multifunctional protein, being not only the precursor of HCNP, which promotes the phenotype development of septo-hippocampal cholinergic neurons, but also the binding protein of phosphatidylethanolamine, ATP, Raf-1 kinase (known as "Raf-1 kinase inhibitory factor" in peripheral organs), and serine protease. We obtained a high-titer retroviral vector harboring HCNP-pp cDNA by the use of a modified packaging cell line and centrifugation, and by injecting it into embryonic mouse ventricles, we investigated the function of its gene product within the central nervous system (CNS). We found that efficient transduction into hippocampal pyramidal neurons can be achieved by injecting the vector into embryonic brain ventricles on embryonic day 14 (E14). Three days after receiving the intraventricular injection of the high-titer HCNP-pp retrovirus vector on E14, the tissues around the ventricles showed an overexpression of HCNP-pp. This was accompanied by a reduced amount of activated MEK and Erk (as analyzed by histochemical and Western blot methods), suggesting that HCNP-pp also regulates the MAP-kinase cascade within the CNS. Surprisingly, mouse brains that received the HCNP-pp retroviral vector showed massive malformation of the hippocampus and cerebellum when examined 30 days after birth. This shows that strictly regulated HCNP-pp gene expression is necessary for the normal development of the mouse brain, and that the moderate overexpression achieved by retroviral vector-mediated gene transfer is sufficient to cause severe abnormality of entire brain structures.

External validity of a randomised clinical trial of temporomandibular disorders: analysis of the patients who refused to participate in research.

PURPOSE: To assess the external validity of a randomised clinical trial (RCT) of a painful condition. METHOD: Consecutive patients with painful temporomandibular disorders (TMDs) were invited to participate in a clinical trial. Patients who refused to participate were compared to those who agreed to participate in this study with respect to degree of symptoms at time of presentation. RESULTS: The patients who refused to participate had more pain, and their condition interfered more with their daily life than those who accepted the invitation to participate. CONCLUSION: Selection bias in RCTs of painful conditions can skew the results, and external validity should be analysed before the results are generalised.

Classification of surgical difficulty in extracting impacted third molars.

Few studies have attempted to analyse preoperative factors that complicate the surgical removal of impacted mandibular third molars. We studied this problem in two steps. We found that difficulty in extraction is associated with depth (depth is deep occlusal level: level C), ramus relationship/space available (ramus relationship/space available is no space: class 3), width of root (the width of the middle root is thicker than that of the neck and the roots do not separate, incomplete roots excluded: bulbous), or a combination of these factors. The index was tested in 20 patients whose extractions were difficult, and 24 in whom they were not. The new index has an odds ratio (relative risk) of 62.3 (95% confidence interval, 9.3-415.9), a sensitivity of 0.85 and a specificity of 0.92. We consider that the new index is superior to the conventional Pederson's index.

Adjustment of prognostic effects in prevalent case-control studies on genotype.

Since genotypes are unchangeable, adjustment of prognostic effects in prevalent case-control studies may produce an unbiased estimate of odds ratio (OR) for disease occurrence. In this paper, the prognostic effects on OR is demonstrated, then three approaches to examine and/or adjust the OR are presented. The demonstration shows that the prognostic effects are larger in diseases with poor prognosis than in those with better prognosis. Genotypes increasing disease risk and fatality rate are underestimated, while those increasing the risk and improving prognosis are overestimated. The simplest approach to examine the OR derived from prevalent case-control studies is to conduct stratified analysis according to the interval between diagnosis and study enrollment. When the stratified analysis finds no substantial difference in the estimate, the OR reflects mainly the relative risk for disease occurrence. The proportion of genotype among putative cases at diagnosis can be estimated from prevalent cases by a logistic model, producing the OR adjusted for the interval from diagnosis. An incomplete-data case-control design is also applicable to adjust the prognostic effects. An actual prevalent case-control study on breast cancer is used to demonstrate the three approaches. They are useful to compensate the disadvantage of prevalent case-control studies.

Randomized clinical trial of primary treatment for temporomandibular joint disk displacement without reduction and without osseous changes: a combination of NSAIDs and mouth-opening exercise versus no treatment.

OBJECTIVE: The effectiveness of nonsteroidal anti-inflammatory drugs (NSAIDs) and physical therapy for disk displacement without reduction is unknown. This study compared this treatment method with nontreatment controls. METHODS: Sixty patients with painful disk displacement without reduction and without osseous changes were randomly divided in 2 groups, consisting of NSAID and physical therapy and a nontreated control group. Both groups were observed at 2 weeks and, for those patients who did not show any improvement, again at 4 weeks. RESULTS: There was 60% improvement in the treatment group compared with 33% in the control group during the entire 4 weeks of the study. The number needed to treat for benefit was 3.75, with a 95% CI 2.103 to 65.935. CONCLUSIONS: A combination of NSAID and physical therapy for 4 weeks is effective as a primary treatment of patients with disk displacement without reduction and without osseous changes.

Metachronous primary hyperparathyroidism due to a parathyroid adenoma and a subsequent carcinoma: report of a case.

An extremely rare case of metachronous primary hyperparathyroidism (PHP) due to a parathyroid adenoma and subsequent carcinoma with local lymphatic spread is presented herein. A 55-year-old woman was operated on for a parathyroid adenoma in the right inferior gland. Thirteen years after the first operation, she was again hospitalized for hypercalcemia and the presence of a hard mass in the right anterior neck region. Exploratory surgery and a histological examination of the resected tumor provided evidence of a parathyroid neoplasm in the right superior gland but the malignancy was equivocal. Postoperatively, her serum parathyroid hormone (PTH) level remained at 1.5-fold the upper limit of the normal range, and hypercalcemia again gradually developed. The results of higher positive rates by Ki-67 immunohistochemical staining and an aneuploid pattern by a flow cytometric analysis of the second neoplasm were consistent with a histological diagnosis of carcinoma, and she therefore underwent further surgery. A radical neck dissection revealed two lymph node metastases which were both successfully removed. The postoperative serum PTH and calcium levels then returned to within the normal ranges. These findings indicate the usefulness of Ki-67 immunohistochemical staining and a flow cytometric analysis for differentiating malignant lesions from benign parathyroid lesions, and the importance of surgically treating cases limited to local regions without distant metastasis.

[Investigation for smoking cessation support on Internet. Internet survey of health and smoking awareness of users].

PURPOSE: This paper is intended as investigation of level of awareness of "health and smoking" of Internet users as a foundation to guide no smoking efforts on the Internet. METHODS AND SUBJECTS: A questionnaire was posted on a homepage only from September 1 to 30, 1999 only. RESULTS: There were 1687 valid responses. Respondents using a cellular phone with the Internet totaled 148. In reference to smoking, 47% of women and 54% of men replied when encountering "smoking by a stranger" that they were "a little troubled." As for "knowledge of medical treatment to stop smoking," over 70% of the respondents replied that they "only know it by name." In regards to responsibility of "who is wrong," respondents that answered "the country that is not regulated" were broken down into about 16% smokers and over 30% non-smokers. Personal computer users that are smokers accounted for 295 people, or 37.5%, and of the total respondents using a cellular phone with the Internet, 49 or 60.5% are smokers. Clearly these results are significant.

Serum antioxidant micronutrients and the risk of oral leukoplakia among Japanese.

A population-based case-control study was designed for the investigation of any association between serum micronutrient levels and oral leukoplakia. Out of a total of 9536 subjects over the age of 40 years who participated in the oral mucosal screening programme in Tokoname city, 48 cases detected with oral leukoplakia (38 male:10 female) were recruited. For each case, four controls matched by age and sex were selected from the same cohort. We examined the fasting serum levels of retinol, alpha-tocopherol, zeaxanthin and lutein, cryptoxanthin, lycopene and carotenoids (alpha-carotene and beta-carotene) by high-performance liquid chromatography. Among males with leukoplakia mean serum lycopene and beta-carotene levels (0.175+/-0.202, 0.357+/-0.295 micromol/l) were significantly lower than those of controls (0.257+/-0.252, 0.555+/-0.408 micromol/l) (P<0.05, P<0.005). Logistic regression analysis with leukoplakia as the dependent variable showed that high serum levels of beta-carotene were related to low risk of oral leukoplakia (odds ratio 0.160, 95% C.I.: 0.029-0.866, P<0.05). There were no significant differences in any of the serum nutrients estimated in female subjects. Our results suggest for the first time that high serum levels of beta-carotene may provide protection against oral precancer for the Japanese male.

[A case of malignant fibrous histiocytoma of the chest wall].

A case of malignant fibrous histiocytoma of the chest wall. A 56-year old man was admitted to the hospital with a complaint of showing abnormal shadow on chest X-ray. Chest X-ray and chest CT scan showed a calcified tumor of the chest wall. Histological diagnosis could not be made prior to the operation. Wide resection of the right chest wall containing the tumor and partial resection of the right lung were done. Malignant fibrous histiocytoma (MFH) showing a striform-pleomorfic pattern was recognized histologically with resected specimen. MFH commonly arises in the soft tissues of the extremities, but rarely in those of the chest. This paper also presents a review of 38 reported cases of MFH originated from the chest wall.

[A case of sarcoid meningoencephalitis with an isolated supratentorial lesion].

A rare case of sarcoid meningoencephalitis with no systemic lesion is reported here. A 58-year old man was admitted experiencing dull headache and speech disturbance. He had never received a diagnosis of systemic sarcoidosis. On admission, neurological examination revealed dysarthria, a defect of the right-side visual field and accelerated right Achilles tendon reflex. A T2-weighted MRI showed a high-intensity signal in the white matter of the left parieto-occipital lobe surrounded by severe brain edema with a mass effect. The meninges around the lesion were enhanced by gadolinium, but no enhancement was observed in the basal portion. Angiotensin-converting enzyme (ACE) activities of cerebrospinal fluid (CSF) and serum were within normal range. The level of interleukin-6 in the CSF was slightly elevated. Chest X ray films and chest CT revealed no abnormal lesions. Whole body gallium scanning showed a hot region only in the intracranial lesion. A brain biopsy was performed. Histological examination revealed typical granuloma of sarcoidosis accompanied by microvasculitis and epithelioid cell granuloma without caseous necrosis. Oral administration of prednisolone improved all symptoms and MRI findings. These observations suggest that release of cytokines from macrophages and epithelioid cells, as well as disruption of the blood-brain barrier due to microvasculitis, are involved in the mechanism responsible for producing lesions of sarcoid meningoencephalitis.

Controlled release from solid dispersion composed of poly(ethylene oxide)-Carbopol interpolymer complex with various cross-linking degrees of Carbopol.

Solid dispersion composed of the poly(ethylene oxide) (PEO)-Carbopol((R)) (CP) interpolymer complex containing phenacetin (PHE) was prepared by using six grades of CP having various cross-linking degrees. We attempted to control the medicine release from the PEO-CP solid dispersion by varying the CP grade. The powder X-ray diffraction pattern and differential scanning calorimetry curves suggested that PHE existed in the amorphous state, and PEO in the crystalline state disappeared in the solid dispersions. The release profile of PHE varied depending on the CP grade. A small release rate was observed at CP910 and CP971P that are cross-linked at low and middle degrees, respectively. The Fourier transform-infrared (FT-IR) spectra showed that the amount of the PEO-CP complex formed by hydrogen bonding changed depending on the CP grade. With the cross-linked CP, a good correlation was observed between the hydrogen bonding percent and the percent released of the PHE after 60 min (D(60 min)), indicating that PHE release was controlled by the amount of PEO-CP complex formation in the solid dispersion. These results show that it is feasible to control the medicine release from PEO-CP solid dispersion by varying the CP grade.

[Consent to enrollment in randomized clinical trials].

Multi-facility joint randomized clinical trials for cancer treatment are the most common method of current clinical study. However, the difference in the rate of participation in multi-facility randomized clinical trials may damage the resulting general validity. Therefore, we studied whether the rate of participation in randomized clinical trials is different between university hospitals and other general hospitals using a questionnaire (with anonymity preserved). There were 744 subjects from university hospitals and 339 from general hospitals participating in the study. The results showed that 10.9 percent of those from university hospitals were willing to participate, against 28.1 percent who were not. Of those from general hospitals (public hospitals in this study) 10.6 percent answered that they would participate and 27.4 percent that they would not. Little difference was found in the rate of participation in randomized clinical trials between university hospitals and other general hospitals. The focus is thus on doctors to solve the possible differences between the facilities in further multi-facility joint studies.

[Clinical comparison of diffuse malignant mesothelioma of the pleura and pseudomesotheliomatous carcinoma of the lung for each case].

Because we experienced each 1 operative case of diffuse malignant mesothelioma of the pleura and pseudomesotheliomatous carcinoma of the lung discovered with pleural effusion, clinical comparison investigated both. The first case was suspected diffuse malignant mesothelioma of the pleura before operation, and we performed pleuropneumonectomy. But the pathologic diagnosis was adenocarcinoma of the lung, what is so called pseudomesotheliomatous carcinoma. The second case had inhaled asbestos and his pleural effusion revealed high concentrations of hyaluronic acid. Thoracoscopic biopsy showed malignant mesothelioma, and we performed pleuropneumonectomy. The pathologic final diagnosis was diffuse malignant mesothelioma of the pleura. In clinical differential diagnosis of diffuse malignant mesothelioma of the pleura and pseudomesotheliomatous carcinoma of the lung, history of inhalation of asbestos and concentrations of hyaluronic acid in pleural effusion are helpful. And thoracoscopic biopsy is necessary in established diagnosis.

Joint Photographic Experts Group compression of intraoral radiographs for image transmission on the World Wide Web.

OBJECTIVE: The purpose of this study was to evaluate the subjective quality of Joint Photographic Experts Group (JPEG) compressed images of intraoral radiographs with file sizes of 30 kilobytes or less, which can be transmitted quickly on the World Wide Web. STUDY DESIGN: Conventional intraoral radiographs were digitized at sampling rates of 100, 200, 300, 400, and 600 dots per inch through use of a flatbed scanner and saved in JPEG format in 11 compression degrees. Fifty-five combinations of sampling rate and compression degree were evaluated by means of a visual analog scale. Sampling rate and compression degree combinations whose quality was inferior to that of an average image were excluded. The quality of the remaining combinations was subsequently evaluated through assessment of 8 anatomical features in each image. RESULTS: Forty of the 55 combinations provided a file size less than 30 kilobytes. Thirty combinations obtained VAS scores of 0 or higher on the standardized VAS. As a result, 16 combinations of sampling and compression conditions were selected for the second part of the study. Only one combination of sampling rate and compression degree was found to provide sufficient image quality for all 8 anatomical features. CONCLUSIONS: Under the file size limit of the study design, the full-sized compressed image of an intraoral radiograph did not always provide sufficient quality. This problem will be reduced by improvements in telecommunications infrastructure, which will permit faster transfer of files of larger size.

Immunohistochemical characteristics of estrogen receptor alpha positive cells in glandular epithelium of the rat seminal vesicle.

Epithelial cells of the rat seminal vesicle stained positively for nuclear estrogen receptor alpha (ER alpha). We studied these cells using immunohistochemical means. We demonstrated in a previous study that some glandular epithelial cells of the seminal vesicles of immature castrated rats treated with estrogen for 1-2 weeks had multilayer features. The present study shows that these glandular epithelial cells are nuclear ER and basal cell-specific cytokeratin (34betaE12) positive. These findings suggested characteristics of basal cells. Moreover, we demonstrated that these cells express transforming growth factor beta1 (TGFbeta1) as a result of castration and estrogen treatment. Our findings indicate that glandular epithelial cells with multilayer features, which stained positively for nuclear ER alpha have basal cell features and may play an important role in the expression of TGFbeta1 through an epithelial-stromal interaction.

Emulsion type new vehicle for soft gelatin capsule available for preclinical and clinical trials: effects of PEG 6000 and PVP K30 on physicochemical stability of new vehicle.

To prevent temperature-dependent gel-sol transformation of an o/w emulsion type new vehicle system for a soft gelatin capsule, which may be available for both preclinical and clinical trials, the basic new vehicle formulation (PEG 400:purified water:medium chain triglyceride:polyoxyethylene (20) cetylether = 77:10:10:3) was modified by partially (1, 2 or 3%) replacing PEG 400 with PEG 6000 or PVP K30. When 2 or 3% of PEG 400 was replaced with PEG 6000, temperature-dependent gel-sol transformation was prevented at temperatures below 40 degrees C, and the vehicle appeared to be stable during 8 weeks of storage at 4 to 40 degrees C; the particle size distribution remained unchanged. When 1% of PEG 400 was replaced with PEG 6000, gel-sol transformation was not prevented, though phase separation was not observed at sol state, and the particle size distribution was shifted to be in a larger particle size range after 2 weeks of storage. When PEG 400 was partially (1, 2 or 3%) replaced with PVP K30, temperature-dependent gel-sol transformation was not prevented and, after 2 weeks of storage at 40 degrees C, the particle size distributions of the vehicles were shifted to be in a larger particle size range and the vehicles were separated into two layers. These results suggested that a small amount of PEG 6000 plays an important role in preventing temperature-dependent gel-sol transformation of our developed vehicle system.

Control of medicine release from solid dispersion composed of the poly(ethylene oxide)-carboxyvinylpolymer interpolymer complex by varying molecular weight of poly(ethylene oxide).

Solid dispersion composed of the poly(ethylene oxide) (PEO)-carboxyvinylpolymer (CP) interpolymer complex containing phenacetin (PHE) was prepared by using nine grades of PEO having different molecular weights from 2000 to 4500000. We attempted to control the medicine release from the PEO-CP solid dispersion by varying the molecular weight of PEO. The physicochemical properties of the solid dispersion were analyzed by powder X-ray diffractometry and thermal analysis. The interaction between PEO and CP was analyzed by IR spectroscopy. Transmittance of the polymer solution was measured to study the complexation between PEO and CP. The release profile of PHE varied depending on the molecular weight of PEO. The minimum release rate was observed at the PEO molecular weight of 35000. It was found that the amount of the PEO-CP complex formation by hydrogen bonding changed depending on the molecular weight of PEO. These results indicate that it is feasible to control the medicine release from the PEO-CP solid dispersion by varying the molecular weight of PEO.