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Accumulating evidence has suggested that farnesoid X receptor (FXR) agonists, such as obeticholic acid (OCA) are therapeutically useful for non-alcoholic steatohepatitis (NASH). However, it is still unclear how FXR agonists protect against NASH and which cell type is the main target of FXR agonists. In this study, we examined the effects of OCA on the development of NASH using melanocortin 4 receptor-deficient (MC4R-KO) mice that progressively developed hepatic steatosis and NASH on Western diet (WD). Treatment with OCA effectively prevented chronic inflammation and liver fibrosis in WD-fed MC4R-KO mice with only marginal effect on body weight and hepatic steatosis. Hepatic crown-like structure (hCLS) is a unique histological structure characteristic of NASH, which triggers hepatocyte death-induced interstitial fibrosis. Intriguingly, treatment with OCA markedly reduced hCLS formation even after MC4R-KO mice developed NASH, thereby inhibiting the progression of liver fibrosis. As its mechanism of action, OCA suppressed metabolic stress-induced p53 activation and cell death in hepatocytes. Our findings in this study highlight the role of FXR in hepatocytes in the pathogenesis of NASH. Collectively, this study demonstrates the anti-fibrotic effect of OCA in a murine model of NASH with obesity and insulin resistance, which suggests the clinical implication for human NASH.

Although recent evidence has pointed to the role of organ- and pathogenesis-specific macrophage subsets, it is still unclear which subsets are critically involved in the pathogenesis of nonalcoholic steatohepatitis (NASH). Using melanocortin-4 receptor-deficient (MC4R-KO) mice fed Western diet (WD), which exhibit liver phenotypes similar to those of human NASH, we found a histological structure, termed hepatic crown-like structure (hCLS), in which CD11c+ macrophages surround dead/dying hepatocytes, a prominent feature of NASH. Here, we demonstrate that hCLS-constituting macrophages could be a novel macrophage subset that drives hepatocyte death-triggered liver fibrosis. In an "inducible NASH model," hepatocyte death induces hCLS formation and liver fibrosis sequentially in the short term. In combination with the long-term WD feeding model, we also showed that resident macrophages are a major cellular source of CD11c+ macrophages constituting hCLS, which exhibited gene expression profiles distinct from CD11c- macrophages scattered in the liver. Moreover, depletion of CD11c+ macrophages abolished hCLS formation and fibrogenesis in NASH. Our clinical data suggest the role of CD11c+ macrophages in the disease progression from simple steatosis to NASH. This study sheds light on the role of resident macrophages, in addition to recruited macrophages, in the pathogenesis of NASH.

It has been shown lately that activity of G protein-coupled receptors (GPCRs) is regulated by an array of proteins binding to carboxy (C)-terminus of GPCRs. Proteins of 4.1 family are subsets of subcortical cytoskeletal proteins and are known to stabilize cellular structures and proteins at the plasma membrane. One of the 4.1 family proteins, 4.1G has been shown to interact with the C-terminus of GPCRs and regulate intracellular distribution of the receptors, including parathyroid hormone (PTH)/PTH-related protein receptor (PTHR). PTHR is coupled to trimeric G proteins Gs and Gq, which activate the adenylyl cyclase/cyclic AMP (cAMP) pathway and phospholipase C pathway, respectively. During the course of investigation of the role of 4.1G on adenylyl cyclase/cAMP signaling pathway, we found that 4.1G suppressed forskolin-induced cAMP production in cells. The cAMP accumulation induced by forskolin was decreased in HEK293 cells overexpressing 4.1G or increased in 4.1G-knockdown cells. Furthermore, PTH -(1-34)-stimulated cAMP production was also suppressed in the presence of exogenously expressed 4.1G despite its activity to increase the distribution of PTHR to the cell surface. In cells overexpressing FERM domain-deleted 4.1G, a mutant form of the protein deficient in plasma membrane distribution, neither forskolin-induced nor PTH -(1-34)-stimulated cAMP production was not altered. The suppression of the forskolin-induced cAMP production was observed even in membrane preparations of 4.1G-overexpressing cells. In 4.1G-knockdown HEK293 cells, plasma membrane distribution of adenylyl cyclase 6, one of the major subtypes of the enzyme in the cells, showed a slight decrease, in spite of the increased production of cAMP in those cells when stimulated by forskolin. Also, cytochalasin D treatment did not cause any influence on forskolin-induced cAMP production in HEK293 cells. These data indicate that plasma membrane-associated 4.1G regulates GPCR-mediated Gs signaling by suppressing adenylyl cyclase-mediated cAMP production.

High-pressure carbon dioxide in contact with water dissolves to form carbonic acid, causing a decrease in pH. By use of these characteristics of a CO2/H2O biphasic system, chitosan-coated cationic liposomes of l-alpha-dipalmitoylphosphatidylcholine were successfully prepared by an improved supercritical reverse-phase evaporation (ISCRPE) method. Liposome-chitosan complexes carrying a positive charge were prepared in a single-step procedure without the use of acid or organic solvent, including ethanol. The maximum trapping efficiency of liposomes prepared by the ISCRPE method was 17%, with or without the addition of chitosan, compared to only 2% for liposomes prepared by the Bangham method. Furthermore, the liposomal dispersion was stable at room temperature in a sealed tube for over 30 days.

Liposomes of various phospholipids were prepared using an improved supercritical reverse phase evaporation (ISCRPE) method that utilizes supercritical carbon dioxide (scCO(2)) as an alternative to organic solvents. Using this method, in the absence of any organic solvent including ethanol, the maximum trapping efficiency of glucose reached 36% for 20 mM l-alpha-dioleoylphosphatidylcholine (DOPC), compared to less than 10% using the Bangham method. Liposomes prepared by the ISCRPE method were highly stable for one month at room temperature. Freeze fractured TEM observations, osmotic shrinkage measurements, and DSC measurements revealed that the liposomes prepared by the ISCRPE method are unilamellar vesicles with loosely packed phospholipids. Comparison of nitrogen with scCO(2) revealed that the presence of CO(2) is necessary for the formation of liposomes.

Antimitochondrial antibodies (AMA) are the serum hallmark of primary biliary cirrhosis (PBC). However, AMA-positivity can be found in non-PBC sera when lower dilutions are used, thus raising issues about the specificity and sensitivity of the test. AMA reacts primarily with the lipoylated domains of pyruvate dehydrogenase-E2 (PDC-E2) which is highly conserved across species, including bacteria. We studied 77 serum samples, including 24 from patients with anti-PDC-E2-positive PBC and 53 controls (16 with autoimmune hepatitis (AIH), 10 with primary sclerosing cholangitis (PSC), and 27 healthy individuals) for their reactivities at serial dilutions (1:10, 1:20 and 1:40) against Escherichia coli DH5 alpha lysate overexpressing human PDC-E2 using immunoblotting (IB). A murine anti-human PDC-E2 monoclonal antibody (mAB) was used as control. We further studied positive sera using adsorption with a synthetic E. coli peptide sharing similarity with human PDC-E2. Finally, we verified whether a unique buffer for E. coli preparation could reduce non-specific serum reactivity. Results demonstrated that 100% of anti-PDC-E2-positive PBC and up to 38% of control sera at 1:10 dilution recognized E. coli PDC-E2 at IB while dilution tests indicated that the overall potency of PBC reactivity was 100-fold higher compared to controls. In fact, a subgroup (20-38%) of non-PBC sera were positive at low titers but lost the reactivity when absorbed with the synthetic E. coli peptide. Finally, our unique buffer reduced the reactivity of non-PBC sera as measured by ELISA. In conclusion, we demonstrated that weak cross-reactivity with E. coli PDC-E2 occurs in non-PBC sera at lower dilutions and that such reactivity is not due to AMA-positivity. The use of a specific buffer might avoid the risk of false positive AMA determinations when E. coli-expressed recombinant antigens are used.

症例は53歳, 男性. 1981年1月から慢性糸球体腎炎による慢性腎不全にて血液透析を導入された. 1995年4月から透析時に高血圧発作が出現するようになり, 血中ノルアドレナリンは高値を呈し, CTにて左副腎腫瘍が認められ, また131I-MIBGシンチで同部に集積像が認められたことから左副腎褐色細胞腫と診断され, 同年8月1日に当科入院した. また, CTにて後天性腎嚢胞 (ACDK) と右腎中部外側に径約2cmの充実性腫瘍が認められ, 腹部超音波検査, MRIにてACDK合併腎癌と診断した.同年8月31日全身麻酔下, 上腹部孤状切開にて左副腎摘除, 右腎摘除術を同時に施行した. 摘出副腎は病理組織学的に褐色細胞腫であり悪性所見はなかった. 摘出右腎には径2cmの腫瘍が認められ, 腎細胞癌であった. 術後血中ノルアドレナリン値は正常化し, 降圧剤の内服も不要となり術後18日目に退院した. 現在高血圧発作および両腫瘍の再発は認められず, 他院にて週3回の維持透析を施行しつつ経過観察中である.血液透析患者に副腎褐色細胞腫とACDK合併腎癌が同時に発見された症例は, われわれが検索した範囲内では本症例が本邦2例目と思われた.

The stereocontrolled total synthesis of (−)-myriocin 1 is reported. Optically active epoxide 9 was converted from symmetrical cyclohexadiene 8, utilizing an enzymatic kinetic resolution. The three sequential stereogenic centers of 1 were constructed by a regioselective epoxide-opening reaction and a Hofmann rearrangement. Elongation of the side chain was efficiently accomplished by the Julia–Kocienski reaction.

Purpose: We report our experiences treating 5 patients who had filarial chyluria using an endoscopic approach.Materials and Methods: Two men and 3 women 47 to 83 years old with chyluria were treated with endoscopic coagulation using guide tube methods. Intrarenal pelvic instillation of silver nitrate was not effective in 4 patients and catheterization was impossible in 1.Results: The responsible lesion was successfully coagulated in all 5 patients. Because the lesion was in the ruptured portion of the caliceal fornix, we thought that chyluria had arisen in the fragile portion of the fornix (fistulization). After endoscopic treatment there was no recurrence in any patient.Conclusions: Endoscopy is effective and minimally invasive therapy for filarial chyluria.

We reviewed the findings of adrenocortical scintigraphy with 131I-6-beta-iodomethyl-19-norcholesterol (NCL-6-131I) of 39 patients to clarify its role in the evaluation of unilateral adrenal or juxtaadrenal masses incidentally discovered by CT, ultrasonography or plain radiography. Twenty-seven benign adrenal masses showed various scintigraphic findings (hot nodule: 12 silent adenomas, warm nodule: one solid mass, normal appearance: one cyst and 2 solid masses, diffuse decrease: each one; solid mass, myelolipoma, ganglioneuroma and calcified adrenal and partial or complete defect: each one; solid mass, myelolipoma and ganglioneuroma and 2 cysts and 2 pheochromocytomas); while a partial or complete defect was shown in a nonfunctioning carcinoma and 3 metastases and a complete defect or inhomogeneous uptake without opposite adrenal visualization was shown in 2 patients with cortisol-producing carcinoma. Therefore a hot nodule and an inhomogeneous uptake or complete defect with nonvisualization of the opposite adrenal are specific to a benign tumor and a cortisol-producing carcinoma, respectively. The impaired tumor uptake of NCL-6-131I is a nonspecific finding. The scintigraphic findings of juxtaadrenal masses were normal in 4 and deviated adrenals in 2. Thus adrenocortical scintigraphy can identify silent adenomas and cortisol-producing carcinomas among the adrenal masses and may help to differentiate juxtaadrenal from adrenal masses.

A 63-year-old man visited our hospital with complaints of the chest pain and loss of appetite. A computed tomography of chest showed wall thickening in the lower portion of the esophagus and carinal and para-aorta lymph node swelling. Upper gastrointestinal endoscopy revealed an irregular ulcerated lesion in the middle portion of the esophagus, which was pathologically diagnosed as small cell carcinoma. A computed tomography of the abdomen showed multiple liver metastases and para-aortic, cardiac, and common hepatic arterial lymph node swelling. One course of combined chemotherapy with CPT-11 and CDDP, then 3 courses of chemotherapy with CPT-11 showed clinical complete remission.

Background: We determined the long-term result of our percutaneous antegrade balloon dilation technique performed for adult patients with ureterointestinal anastomotic stricture between 1992 and 1997. Methods: Balloon dilation was performed on 13 ureterointestinal anastomotic strictures in 10 patients. After a nephrostomy was performed, a guide wire was introduced into the intestinal loop through the stenotic portion under direct observation using a ureterorenoscope. Dilation was performed using the Olbert balloon dilator (30-Fr) inserted along a guide wire into the stenotic portion. A 20-Fr or 22-Fr multihole catheter was left for approximately 6 weeks. No major complications were encountered during or after these procedures. After removal of the indwelling catheters, the progress of each patient was followed for at least 14 months. Results: Additional dilation was necessary in three of 10 patients for the recurrent stricture. The balloon dilation was ineffective in two patients with a long stenosis of the ureter or a previous history of radiation therapy for uterine cancer. Eight of 10 patients showed satisfactory outcomes during the mean follow-up period of 47.1 months. Conclusions: Based on these results, we believe that the balloon dilation could be the first line of treatment for strictures of uro-digestive anastomosis, except for some patients with a long stenosis or a previous history of intrapelvic radiation.

Employing in vitro and in vivo models of catheter-associated infection, biofilm formation of Pseudomonas aeruginosa in artificial urine and bactericidal activity of several classes alone or in combination with a macrolide antibiotic were investigated. In the course of continuous urine flow, bacteria adherent to a Teflon catheter formed microcolonies at 6 h and a biofilm at 24 h. Following 24 h of urine flow, the thickness and the density of the biofilm increased to about 20 microm and 10(8) cfu/mm3, respectively. Of the antibiotics examined, the fluoroquinolones showed the most potent bactericidal activity against the P. aeruginosa biofilms. However, those antibiotics were not always potentiated by the combination with a macrolide antibiotic.

Objectives. To compare bactericidal activities of antimicrobial agents against Pseudomonas aeruginosa biofilms by employing an in vitro model of catheter-associated infection because such infections are refractory to antimicrobial treatment. Methods. Bactericidal activities of piperacillin (PIPC), ceftazidime (CAZ), panipenem (PAPM), amikacin (AMK), ciprofloxacin (CPFX), and levofloxacin (LVFX) were examined against a P. aeruginosa biofilm generated on a Teflon catheter in artificial urine. The colony-forming activities of biofilm bacteria were determined for 48 hours during the treatment with each drug at concentrations of 1 up to 128 times the minimal bactericidal concentration (MBC), and time-kill curves were constructed by plotting the viable cell counts against time. Results. Although CAZ was more bactericidal to the biofilm bacteria than PIPC, the biofilm bacteria still remained on the catheter during CAZ treatment at a concentration 128 times the MBC for 48 hours. Biofilm bacteria were completely eradicated within 48 hours by treatment with PAPM and AMK at a concentration 64 and 128 times the MBC, respectively. Both CPFX and LVFX eradicated biofilm bacteria completely by 24 hours at a concentration 32 times the MBC. Conclusions. These results indicate that fluoroquinolones have the most potent bactericidal activity against the P. aeruginosa biofilm generated in urine.

Using the checkerboard titration method as well as the time-kill curve technique, we investigated the activities of beta-lactams, fluoroquinolones, amikacin, and fosfomycin alone and in combination against Pseudomonas aeruginosa isolated from patients with complicated urinary tract infections. In the checkerboard titration studies, none of 21 combinations demonstrated antagonism against 26 strains tested, and the mean fractional inhibitory concentration (FIC) indices for these combinations ranged between 0.4694 and 0.9828. Corresponding to the respective FIC indices, the bactericidal activity determined in combinations of meropenem with ciprofloxacin or amikacin and ceftazidime with ciprofloxacin at sub-minimum inhibitory concentrations (MICs) produced a great reduction in bacterial counts (/=2 log10 CFU/ml) within 6 h of administration against most of the strains, including strains resistant to one or both drugs, and these synergistic effects were confirmed morphologically by scanning electron microscopy. In time-lag combinations, the first administration of ciprofloxacin or amikacin supplemented by meropenem with 1-h lag diminished bactericidal activity, in comparison with the simultaneous administration of the drugs. These results suggest that simultaneous combinations of beta-lactams with fluoroquinolones or amikacin may be useful alternatives for the treatment of serious infections due to P. aeruginosa.

A 46-year-old woman presented with a hypogastric mass. The preoperative diagnosis was a malignant ovarian tumor involving multiple organs, including the urinary bladder. Surgical exploration was performed with wide resection of the right ovary and uterus, including the affected ileum, sigmoid colon, and omentum. An intraoperative histopathological examination of the paravesical tissue revealed abdominal actinomycosis. Consequently, bladder resection was not done. The cause of abdominal actinomycosis in this patient was probably due to implantation of an intrauterine device 3 years previously.

Background: The successful treatment of bacterial prostatitis depends on an effective antimicrobial concentration in prostatic tissue against the infecting organism. In this study we compared the diffusion of 5 types of antimicrobials into the prostate. Methods: The concentrations of piperacillin, cefotiam, minocycline, amikacin, and ofloxacin were determined in prostatic fluid, prostatic tissue, and serum 2.5 to 3 hours after a single administration from 55 patients with benign prostatic hypertrophy. Results: Although amikacin showed the highest mean concentration both in prostatic tissueand prostatic fluid, the prostatic tissue/serum ratio was significantly higher (P < 0.01) for ofloxacin (1.49 ± 0.80) and minocycline(0.94 ± 0.39)compared with those for amikacin (0.49 ± 0.2l)and piperacillin (0.21 ± 0.15). Also, the prostatic fluid/serum ratio was lower than the prostatic tissue/serum ratio for each drug, however, the prostatic fluid/serum ratio of ofloxacin was significantly higher than that of other antimicrobials tested (P < 0.01). Conclusion: These results support earlier studies demonstrating that fluoroquinolones are a useful class of antimicrobials for the treatment of chronic bacterial prostatitis. They also suggest that in view of the pharmacokinetic properties and antimicrobial activities, amikacin and minocycline may be alternate antimicrobial options for selected patients with bacterial infections of the prostate.

OLETF rats develop hyperglycemia, hyperinsulinemia and mild obesity, which is characteristic of human non-insulin-dependent diabetes mellitus (NIDDM). We cloned and sequenced the cholecystokinin type-A receptor (CCKAR) gene in the rats. Comparing the DNA sequences of the OLETF CCKAR gene and LETO CCKAR gene, normal gene, we found a deletion in the OLETF gene, 6847 bases in length, which was flanked by two 3-base-pair direct repeats (5'-TGT-3') at positions -2407/-2405 and 4441/4443, numbered according to the LETO gene sequence, one of which was lost. The promoter region, the first and second exons were missing in the mutant. The region upstream and downstream of the deletion, including exons 3, 4 and 5, was conserved between the two strains, and did not contain any base changes. We found that the gene mapped to chromosome 14 in rats. OLETF rats are the naturally occurring knockout animals with the homozygously disrupted CCKAR gene.

Background: Although endotoxin elicits a variety of pathophysiological activities in hosts with gram-negative bacterial infections, the clinical significance of plasma endotoxin levels have not been clearly defined in the patients with urinary tract infections. The purpose of the study was to measure plasma endotoxin concentrations in relation to the types of urinary tract infections and to examine their correlation with inflammatory parameters. Methods: Using chromogenic Limulus-amebocyte lysate assay, plasma endotoxin concentrations were measured in a total of 63 patients with various types of nonspecific urinary tract infections. Results: The mean plasma endotoxin concentrations in patients with sterile pyuria, chronic complicated cystitis, acute uncomplicated pyelonephritis or acute exacerbation of chronic complicated pyelonephritis, chronic complicated pyelonephritis, and acute bacterial prostatitis or epididymitis were significantly higher than those in healthy individuals and in patients with acute uncomplicated cystitis. The correlation coefficients between plasma endotoxin concentrations and body temperatures, white blood cell counts in peripheral blood, erythrocyte sedimentation rates, and C-reactive proteins were 0.678 (P 0.01), 0.503 (P 0.01), 0.416 (P 0.01), and 0.330 (P 0.01), respectively. Conclusions: Patients with urinary tract infections may respond to endotoxin locally and generally depending on the sites of infection involved. However, endotoxin levels do not always correlate to clinical findings or inflammatory parameters.

Experimental acute bacterial prostatitis in rats was induced by four different routes of bacterial inoculation. The most simple and reproducible method of producing bacterial prostatitis was to instil the bacterial suspension into the prostatic urethra after the administration of an appropriate antibiotic to prevent associated pyelonephritis.

A newly established human osteosarcoma cell line, HS-Os-1, from an osteoblastic tumor arising in the left humerus of an 11-year-old girl was morphologically characterized in vitro and in vivo. HS-Os-1 cells in a monolayer have been maintained for more than 2 years since the initial cultivation, and were round or polygonal in shape with marked pleomorphism. Their cytoplasm was strongly positive for specific markers of osteoblasts, such as alkaline phosphatase and osteocalcin. Tumors induced in nude mice by HS-Os-1 cell inoculation at passage 12 or 23 revealed typical histological features of osteoblastic osteosarcoma, similar to those observed in the original tumor, producing prominent osteoid matrix with calcification. Ultrastructurally, HS-Os-1 cells in vitro and tumor cells in vivo showed similar well-developed, markedly dilated rough endoplasmic reticulum, polysomes and microfilaments in their cytoplasm. Additionally, many collagen fibers associated with deposition of electron-dense material were detected in the stroma featuring osteoid matrix. Thus, the HS-Os-1 cell line was shown to exhibit its osteoblastic nature in vitro and in vivo, and therefore might become an extremely useful tool for various pathomorphological investigations on human osteosarcomas.

The 1:1 complex formation between dioxygen and the meso -tetraphenylporphinatocobalt(II)pyridine complex in toluene was followed spectrophotometrically using a high-pressure cell. The values of the equilibrium constant K p obtained are 0.0278, 0.0513, 0.0840, 0.157 and 0.278 atm −1 at −30, −36, −42, −48 and −54dgC, respectively. The thermodynamic data are Δ H θ = −10.1±0.6 kcal mol −1 and gD S θ = −49±3 eu at a standard pressure of 1 atm.