改性的纳米羟基磷灰石/聚乳酸复合材料的亲水性与溶胀性研究

评价用聚乙二醇系列的表面改性剂PEG、F127及PELA改性的纳米羟基磷灰石／聚乳酸复合材料的亲水性和溶胀性，先对纳米羟基磷灰石进行表面改性处理后，再综合用传统的溶液共混、流延法及热压法将改性的和未改性的纳米羟基磷灰石分别与聚乳酸制备成复合薄膜。检测结果表明：改性剂分别被涂敷于纳米羟基磷灰石上，改性处理能够改善纳米颗粒在基材内的分布；改性的纳米羟基磷灰石／聚乳酸复合材料比未改性的对比材料的表面接触角小、表面能大、亲水性好、溶胀度大，达到饱和溶胀度的时间长。改性的纳米羟基磷灰石比未改性的羟基磷灰石改善基体聚乳酸的亲水性和溶胀性效果更显著。     

A novel lysophospholipid- and pH-sensitive receptor, GPR4, in brain endothelial cells regulates monocyte transmigration.

Abundant evidence documents the highly proinflammatory actions of lysophosphatidylcholine (LPC). Further, LPC, found in high amounts in oxidized low-density lipoprotein (LDL), is implicated as an atherogenic factor. In endothelial cells, LPC impairs endothelial barrier function through GPR4, a novel receptor hypothesized to be sensitive to LPC and protons. The authors investigated the stimulation by LPC or low pH of GPR4 in human brain microvascular endothelial cells (HBMECs) and whether the activated GPR4 regulates in vitro monocyte transmigration. The results indicated that HBMECs stimulated by LPC (5 microM), but not low pH, showed a twofold increase in monocyte transmigration. Using retroviruses containing siRNA to GPR4, a > 60% reduction of GPR4 expression resulted in blockade of the LPC-stimulated transmigration. The inhibited response was restored by co-expression with an small interference RNA (siRNA)-resistant, but functional, GPR4 mutant construct. To investigate potential signaling mechanisms, the siRNA-mediated knockdown of GPR4 also prevented LPC-induced RhoA activation. C3 transferase, a Rho inhibitor, prevented approximately approximately 65% of the LPC-stimulated transmigration. LPC also increased MLC phosphorylation by 5 min, which was inhibited by the Rho kinase inhibitor, Y-27632 (10 microM) or ML-7 (myosin light chain kinase (MLCK) inhibitor). The findings indicate that the proinflammatory and atherogenic LPC stimulated endothelial GPR4, which promoted monocyte transmigration through a RhoA-dependent pathway.     

Prominent proliferative response of peripheral blood mononuclear cells to a recombinant non-structural (NS3) protein of hepatitis C virus in patients with chronic hepatitis C.

The proliferative response of peripheral blood mononuclear cells (PBMC) to a recombinant non-structural (NS3) protein of hepatitis C virus (HCV) was studied in 41 patients with chronic hepatitis C. Of them, 28 had chronic persistent hepatitis (CPH) and 13 chronic active hepatitis (CAH). The positive proliferation rate of PBMC to the recombinant NS3 protein, T9Ag, was 66% in the 41 patients (77% in CAH versus 61% in CPH; P > 0.05) when stimulation index (SI) = 4 was set as the cut-off value. However, mean SI of CAH patients was significantly higher than that of CPH patients (8.3 +/- 5.2 versus 5.1 +/- 3.6; P < 0.05). Six other chronic hepatitis patients who were repeatedly negative for anti-HCV antibody but positive for serum HCV RNA also had an SI of > or = 4.0. The frequency of cellular immune response to the T9Ag is among the highest results obtained by using HCV antigens tested so far. Our studies thus indicate that NS3 is an immunologically important region of HCV for T cells. Moreover, the proliferative response to T9Ag may help to establish hepatitis C etiology in chronic hepatitis patients who are seronegative with currently available anti-HCV assays.     

Two cases of common bile duct stone after liver transplantation

Biliary complications after orthotopic liver transplants are a continuing cause of morbidity and mortality. Biliary stones and sludge are less well known complications of hepatic transplantation, although they have long been recognized. Recently we experienced two cases of biliary stones developed after liver transplantation. One 32-year-old male, who frequently admitted due to recurrent cholangitis, was treated with percutaneous transhepatic biliary drainage and choledochojejunostomy with cholecystectomy. The other 58-year-old male, who had stones in commone bile duct, was treated by endoscopic manipulation. They are in good condition without recurrent bile duct stones or its accompanying complications. Although stones and sludge are relatively infrequent after liver transplantation, surgical or interventional radiologic treatments are usually performed for treatment.     

Capsular Polysaccharide-Fimbrial Protein Conjugate Vaccine Protects against Porphyromonas gingivalis Infection in SCID Mice Reconstituted with Human Peripheral Blood Lymphocytes

The effect of immunization with either a Porphyromonas gingivalis fimbrial protein, a capsular polysaccharide, or a capsular polysaccharide-fimbrial protein conjugate vaccine were compared in hu-PBL-SCID mice. A significantly higher human immunoglobulin G antibody response and the highest degree of in vivo protection against bacterial challenge was observed in the group immunized with the conjugate vaccine. It was concluded that capsular polysaccharide-fimbrial protein conjugate from P. gingivalis could potentially be developed as a vaccine against periodontal infection by P. gingivalis.Porphyromonas gingivalis has been implicated as one of the major periodontal pathogens, and specific humoral and cell-mediated immune reactions to this organism have been demonstrated in periodontal diseases (13, 16). Attempts to induce protection against experimental infection with P. gingivalis by active immunization procedures have been studied by immunization with selected cell wall fractions, outer membrane proteins, and capsular polysaccharides (CPS) of P. gingivalis (8, 14). While most of these approaches afforded significant levels of protection (8, 14), problems such as maintaining functional levels of specific antibodies for extended periods of time (immune memory) (9), the multiple antigenicity of various pathogenic organisms, and the inability to activate T-cell-dependent immune responses (12) remain to be overcome. One strategy may be to develop a conjugation vaccine composed of CPS coupled with an outer membrane protein of P. gingivalis which can function as an immunodominant antigen as well as a carrier protein to activate T-cell-dependent immune responses.An additional area of improvement in vaccine strategies is the development of an adequate animal model system for simulating humanized antibody responses. Conventional animal models have disadvantages, since the animals may be qualitatively different from humans with respect to oral microbial environments and histological components in the development of periodontal lesions, and the nature of animal immune functions differs from that of human immune responses. Also, immunogenetic makeup (i.e., immunoglobulin [Ig] allotypes) and control over Ig class and subclass responses differ in animals and humans. Recently, mice with severe combined immunodeficiency (SCID) were identified (3, 15). The SCID mice lack functional T and B cells due to a mutation affecting the recombinase system that impairs the rearrangement of antigen receptor genes in both T and B cells. As postimmunization levels of IgG subclasses in vaccinees or in hu-PBL-SCID mice were closely associated with human Ig allotypes (5, 10, 11), we reconstituted the SCID mouse phenotype with human peripheral blood lymphocytes (PBL) whose Ig allotypes were positive for the phenotype fnb. As an extension of our previous experiments (5), we evaluated the protective effect of a newly developed polysaccharide-fimbrillin (FIM) protein conjugate vaccine with hu-PBL-SCID mice.Twenty-six SCID mice (C.B.-17-scid; Charles River Japan, Inc., Kanagawa, Japan) initially examined for IgG against P. gingivalis whole cells were reconstituted with 0.5 ml of human PBL (8 × 10⁷/ml) from periodontally healthy donors who were positive for the Ig phenotype fnb (either agfnb or axgfnb). Two weeks after reconstitution with human PBL, the expression of human Ig allotype markers was identified by the hemagglutination inhibition assay described previously (4).CPS of P. gingivalis 53977 was prepared by a modification of the method previously described (14). Briefly, bacterial cells were suspended in water (0.2 to 0.4 g [wet weight]/ml) and extracted with an equal volume of 90% phenol for 20 min at 65 to 68°C. The aqueous phase was obtained by centrifugation at 4,000 × g and dialyzed against distilled water with Spectrapor 1 tubing. The dialyzed solution was brought to 0.15 M sodium chloride, 4 mM MgCl₂, 1 mM CaCl₂, and pH 7.5 with Tris-HCl, treated with RNase A (0.04 mg/ml) and DNase I (0.01 mg/ml) (Sigma, St. Louis, Mo.) for 2 h at 37°C, treated with proteinase K (0.04 mg/ml) for 1 h at 60°C, dialyzed against dH₂O, and lyophilized. The lyophilized extract was dissolved in 0.05 M Tris-HCl buffer (pH 9.5) containing 0.3% deoxycholate and 0.001 M trisodium EDTA, applied to a column of Sephacryl S-400 HR (1.0 by 47 cm) (Pharmacia, Piscataway, N.J.), and eluted with the deoxycholate-containing buffer. Fractions were assessed for lipopolysaccharide and CPS by double immunodiffusion in agarose, for LPS contamination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and for protein contamination. Fractions containing only CPS were pooled, sodium chloride was added to 0.15 M, and CPS was precipitated with 4 volumes of 95% ethanol. The precipitates were isolated by centrifugation, dissolved, dialyzed, and lyophilized.Fimbriae of P. gingivalis 381 were purified as follows (17). Briefly, cells were harvested by centrifugation and suspended in 20 mM Tris-HCl (pH 7.4)–0.15 M NaCl–10 mM MgCl₂ by repeated pipetting. The suspension was agitated by magnetic stirrer for 30 min, and the supernatant was obtained after centrifugation at 8,000 × g for 20 min. Ammonium sulfate was added to 40% saturation, precipitated proteins were collected by centrifugation, and the precipitate was dissolved in 20 mM Tris-HCl (pH 8.0) and dialyzed against 20 mM Tris-HCl (pH 8.0). The dialysate was clarified by centrifugation at 8,000 × g for 20 min and applied to a column of DEAE-Sepharose CL-6B (1.5 by 16 cm) (Pharmacia) equilibrated with the above-described buffer. The column was washed with 20 mM Tris-HCl, pH 8.0, and eluted with a linear gradient of 0 to 0.3 M NaCl. The 43,000-molecular-weight protein (43K protein) band was not detected in fractions eluted after 0.17 M NaCl. Fractions containing the 43K protein were concentrated by ammonium sulfate precipitation and dialyzed against 3 mM Tris-HCl (pH 8.0) or 3 mM sodium bicarbonate (pH 8.0).The carboxylate group of the P. gingivalis CPS was conjugated to free amino residues of either cationic bovine serum albumin (CPS-BSA) or 43-kDa P. gingivalis fimbrillin (CPS-FIM) via a 1-ethyl-3-(dimethylaminopropyl)-carbodiimidide (EDC) intermediate. A total of 1.5 mg of CPS was dissolved in 0.5 ml of EDC conjugation buffer (Pierce, Rockford, Ill.), and 2 mg of BSA (SuperCarrier; Pierce) or 2 mg of fimbrial protein dissolved in 0.2 ml of water was mixed and added to the EDC (10 mg in 1 ml of water) and reacted for 2 h at room temperature. The mixture was separated from carbodiimidide by gel filtration on a Sephacryl S-300 column (1.0 by 10 cm) with 0.9 M sodium chloride and 0.083 M sodium phosphate, pH 7.2. Fractions were screened by immunodiffusion in agarose gel with rabbit antisera to CPS, fimbrial protein, or BSA.Only mice with no detectable amount of murine IgG against P. gingivalis were included in the study. Two weeks following PBL reconstitution, mice were examined for the expression of human Ig allotypes. Three of 26 mice were found to be leaky; a total of 23 mice were used in the experiment. Group I (n = 6) was immunized with FIM, group II (n = 6) was immunized with the CPS-BSA vaccine, group III (n = 6) was immunized with the CPS-FIM conjugate vaccine, and group IV (control, n = 5) was immunized with BSA. Each immunization procedure consisted of two intraperitoneal injections (at 2-week intervals) with 0.2 ml of immunogen adjuvant (Imject Alum; Pierce) mixture. The final amount of immunogen was 10 μg. Two weeks after the final immunization, mice were challenged by two dorsal subcutaneous injections (0.1 ml each) of whole P. gingivalis 53977 cells (1 × 10¹¹/ml) and evaluated for protective effects for 3 weeks based on the following criteria: general appearance, cachexia, weight loss, size and nature of localized abscess formation, development and size of secondary lesion, and death. Preimmune, postimmune (2 weeks following final immunization), and postinfection (3 weeks following infection) total IgG and IgG subclass antibody titers were determined by enzyme-linked immunosorbent assay (ELISA) with an alkaline phosphatase assay system. Microtiter plates (96 well) were coated with 0.1 ml of antigen (10 μg/ml) diluted in 0.01 M phosphate buffer (pH 7.2). After overnight incubation at 4°C, the plates were washed three times with phosphate-buffered saline (PBS) containing 0.1% Tween 20. A total of 0.05 ml of mouse serum samples diluted in PBS containing 0.1% Tween 20 was added to each well and incubated for 2 h at room temperature. The plate was washed three times with PBS containing 0.1% Tween 20, and then 0.1 ml of four mouse anti-human IgG subclasses (affinity-purified monoclonal antibody, γ-chain-specific, IgG1; 8c/6-39, IgG2; HP-6014, IgG3; HP-6050, IgG4; HP-6025; Sigma) diluted in PBS containing 0.1% Tween 20 were added to each well and incubated for 2 h at room temperature. After being washed three times with PBS containing 0.1% Tween 20, 0.05 ml of goat anti-mouse IgG (heavy- and light-chain specific, affinity purified, alkaline phosphatase conjugated; Calbiochem, Basel, Switzerland) diluted in PBS containing 0.1% Tween 20 were added to each well and incubated overnight at room temperature. After the plates were washed, 0.1 ml of nitrophenyl phosphate (1 mg/ml in diethanolamine buffer, pH 9.8) was added to each well and incubated for 60 min, and 0.1 ml of 3 N NaOH was added to stop the color reaction. For total IgG antibody measurements, goat anti-human IgG (affinity purified, γ-chain specific, alkaline phosphatase conjugated; Calbiochem) was used. Optical densities were plotted as a function of serum dilution factor, regression analysis was performed, and reciprocals of the serum dilution factors at the x axis intersection of an optical density of 0.2 were expressed in ELISA units for each sample. For the comparison of antibody levels between groups or intervals, Student’s t test was done.Except for those mice which were found to be leaky (n = 3), all mice (n = 23) expressed human Ig allotypes, either axgfnbt or agfnbt, according to the donors’ allotypes, confirming our previous observation (5). IgG and IgG subclass titers are summarized in Table ​Table1.1. Both postimmune and postinfection IgG levels against whole cells increased significantly compared to baseline values in all groups. IgG levels to whole cells in group III were significantly higher than those of groups I, II, or IV throughout the experimental period. In groups I and III, IgG4 subclass antibody titers were higher at the postinfection phase than at the postimmunization phase. Our previous studies have shown that early-onset periodontitis patients, whose haplotype fnb frequency was significantly higher than that of the race- and age-matched control group, had significantly higher IgG2 and IgG4 levels to P. gingivalis (4, 6). It was reasoned that the conversion of IgG1-restricted responses to IgG4-restricted responses with prolonged proteineous antigenic stimulation might be also responsible for the higher IgG4 subclass levels (1). IgG2 subclass responses were elevated to the polysaccharide antigen, while IgG1 responses were elevated to the fimbrial antigen, similar to previous results following bacterial infection or vaccination (2, 7, 10, 11). While all mice immunized with the conjugate vaccine survived the high-dose bacterial challenge (2 × 10¹⁰ cells of P. gingivalis 53977), one-third of the mice in the other two experimental groups and all of the mice in the control group died. The magnitude of the humoral antibody response was highest and the in vivo protective effect was greatest with minimal weight loss in group III (Tables ​(Tables11 and ​and2).2). This observation implies that through the use of a sophisticated conjugational vaccine incorporating two kinds of immunodominant antigens (i.e., outer membrane fimbrial protein and CPS), protection against P. gingivalis infection can be enhanced. Moreover, the SCID mice reconstituted with PBL from donors of the same IG allotypes provided a genetics-based simulation model for investigating humanized antibody responses to various vaccine formulas. By establishing T-cell hybridomas from hu-PBL-SCID mice, we are attempting to identify antigenic epitopes for T-cell clonal activation and to identify heavy- and light-chain variable gene usage of the antibodies from the conjugate vaccine.

TABLE 1

Baseline, postimmunization, and postinfection IgG and IgG subclass levels for each mouse group (ELISA unit ± standard deviation)

Thyroglobulin synthesis of oxyphilic cells in various types of neoplastic and autoimmune thyroid diseases.

To determine the content of thyroglobulin in oxyphilic cells of the thyroid, which have been considered as non-thyroglobulin producing cells, the degree of stainability of the various oxyphilic cells for thyroglobulin was compared with that of non-oxyphilic follicular cells in either same or different lesion. A total of 13 oxyphilic lesions, including three follicular adenomas containing oxyphilic cell nodules, four pure oxyphilic cell adenomas, and six Hashimoto's thyroiditis were compared with 16 of non-oxyphilic lesions such as, seven follicular adenomas, four chronic lymphocytic thyroiditis, and five Graves' disease. Many oxyphilic cells stained positively for thyroglobulin regardless of their morphologic variation, but less intensely than the usual follicular cells in follicular adenomas, chronic lymphocytic thyroiditis, and Graves' disease. The stainability of oxyphilic cells for thyroglogulin did not show any significant correlation with morphologic features, whereas in follicular adenomas, the non-oxyphilic follicular cells forming microfollicles stained less strongly for thyroglobulin than the same cells lining large mature follicles in a reproducible way. With above findings, we concluded that oxyphilic cells maintain the functional activity in terms of thyroglobulin synthesis, although the content of the thyroglobulin is less than that of non-oxyphilic colloid forming follicular cells.     

Experimental transmission of Bartonella henselae by the cat flea.

Bartonella henselae is an emerging bacterial pathogen, causing cat scratch disease and bacillary angiomatosis. Cats bacteremic with B. henselae constitute a large reservoir from which humans become infected. Prevention of human infection depends on elucidation of the natural history and means of feline infection. We studied 47 cattery cats in a private home for 12 months to determine the longitudinal prevalence of B. henselae bacteremia, the prevalence of B. henselae in the fleas infesting these cats, and whether B. henselae is transmitted experimentally to cats via fleas. Vector-mediated transmission of B.henselae isolates was evaluated by removing fleas from the naturally bacteremic, flea-infested cattery cats and transferring these fleas to specific-pathogen-free (SPF) kittens housed in a controlled, arthropod-free University Animal Facility. B. henselae bacteremia was detected in 89% of the 47 naturally infected cattery cats. A total of 132 fleas were removed from cats whose blood was simultaneously cultured during different seasons and were tested individually for the presence of B. henselae DNA by PCR. B. henselae DNA was detected in 34% of 132 fleas, with seasonal variation, but without an association between the presence or the level of bacteremia in the corresponding cat. Cat fleas removed from bacteremic cattery cats transmitted B. henselae to five SPF kittens in two separate experiments; however, control SPF kittens housed with highly bacteremic kittens in the absence of fleas did not become infected. These data demonstrate that the cat flea readily transmits B. henselae to cats. Control of feline infestation with this arthropod vector may provide an important strategy for the prevention of infection of both humans and cats.     

The nationwide epidemiological study of mental disorders in korea

The lifetime prevalences of DSM-III mental disorders using Korean version of DIS-III are presented. They were studied in 5,100 adults (aged 18 to 65) in household selected by two stage cluster sampling. Comparisons were made between regions, sex and age groups. International comparison with Epidemiologic Catchment Area program was also made.     

Expression of rck/p54, a DEAD-box RNA helicase, in gametogenesis and early embryogenesis of mice.

rck/p54 is a DEAD-box RNA helicase protein with ATP-dependent RNA-unwinding activity. Its ortholog is required for sexual reproduction in yeast and for oocyte survival and sperm fertility in Caenorhabditis elegans. In the current study, we investigated the expression of rck/p54 in mouse gametogenesis and early embryogenesis. Western blot analysis revealed that rck/p54 was highly expressed in both the ovary and testis. In the ovary, maturing oocytes strongly expressed rck/p54 in their cytoplasm. In contrast, in the testis, spermatogonia and primary spermatocytes highly expressed rck/p54 in their cytoplasm, but its expression decreased in the spermatids. Interestingly, rck/p54 was concentrated in the heads of spermatozoa; and then its expression gradually decreased as these cells matured along the epididymal duct. After fertilization, rck/p54 protein and its mRNA remained present in the pronucleus phase; and then their expression levels slightly but definitely decreased in morulae and blastocytes. The injection of a CMV-based rck/p54 expression vector into the pronuclei of fertilized eggs caused a delay in early embryogenesis. In generating RCK transgenic mice, the birth rate of the mice was significantly lower than those of other gene transgenic mice. These findings indicate that rck/p54 may play an important role in gametogenesis and early embryogenesis in mammals.