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Introduction: Mechanical stress-induced chronic hypertension can initiate the development of cardiac hypertrophy and, ultimately, congestive heart failure. It is widely accepted that various chemokines are expressed in the heart in response to pressure overload. These chemokines might be involved in the cardiac remodeling and pathogenesis of heart failure. In this study, we investigated the roles of CCR5 in pressure overload-induced cardiac hypertrophy and heart failure in mice. Methods: Male C57BL/6J (WT) and CCR5-deficient (CCR5-KO) mice were subjected to transverse aortic constriction (TAC). Aortic constriction was achieved by tying around the transverse thoracic aorta against a 27-gauge needle using a 7–0 silk suture, and then removing the needle. Cardiac hypertrophy, fibrosis, cardiac function by echocardiography and expression of hypertrophy related molecules were examined. Results and discussion: Three weeks after TAC, CCR-5-KO mice exhibited more significant loss of cardiac function with cardiac hypertrophy, compared with WT mice, indicating that CCR5 plays a protective role in TAC-induced cardiac hypertrophy and heart failure. In WT mice, intracardiac Ccr5 expression was increased at 3 day and 1 week TAC, and CCR5 protein was expressed in the cardiac fibroblasts. Mechanical stress-induced inflammation is known to be indispensable for compensatory hypertrophy, however, which was significantly attenuated in CCR5-KO mice, compared with WT mice. Consistently, CCR5-KO mice lacked the initial compensatory hypertrophy. The lack of initial compensatory cardiac hypertrophy might lead CCR5-KO mice to pathological cardiac hypertrophy. Enhanced CCR5 expression in intracardiac fibroblasts might be crucially involved in the compensatory hypertrophy-induced by mechanical stress.

The conditions required for a takeoff in surfing, are discussed, with the waves simulated numerically, considering two types of wave breaking, i.e., a plunging type, and a spilling type. First, a surfer is required to obtain a sufficient value for the horizontal component of paddling speed, not to be overtaken by a wave peak. Second, when the surfer stops paddling, he needs to be floating at a location where the force on him is downward, along the wave front face. On the basis of both conditions, the time variation of the required value for the horizontal component of paddling speed, is evaluated for both the plunging-type, and spilling-type, cases. When the paddling speed is sufficient, the surfable area is larger in the former case, than in the latter, on the offshore side of the wave-breaking point.

Our purpose was to evaluate the metabolism of bile acids in the fetus by analyzing the bile acid composition of meconium of preterm (less than 30 weeks' gestational age) and full-term infants and comparing the results with the bile acid composition of feces of preterm and full-term infants 6 days after delivery.The concentrations of individual bile acids were determined by gas chromatography-mass spectrometry after solvolysis and hydrolysis of bile acid conjugates.In meconium, the main bile acids were chenodeoxycholic and hyocholic acids. The main bile acid of feces from preterm infants at 6 days of age was the same as that of meconium. We also detected large amounts of secondary bile acids, especially deoxycholic acid and ursodeoxycholic acid. The ratio of cholic acid relative to chenodeoxycholic acid in meconium of preterm and full-term infants and in feces of preterm infants was less than 1, 0.36, 0.55, and 0.55, respectively. The percentage of chenodeoxycholic acid relative to total bile acids in meconium of preterm (P0.05) and full-term (P0.01) infants was significantly higher than that in feces of 6-day-old full-term infants.More than half of the main pathway, at least, for bile acid synthesis in preterm infants may be the acidic pathway until the infants reach about 7 days of age.

In this report, we describe a fatal case of pontine hemorrhage related with methamphetamine abuse. A 54-year-old male was found dead in a prone position in his parents’ house, and a medico-legal autopsy was carried out to determine the cause of his death. Externally, although an injection mark-like injury with subcutaneous hemorrhage was observed in the left cubital fossa, the autopsy revealed no severe trauma leading to death. Internally, every organ was moderately congested. The brain weighed 1330 g. Macroscopically, there was no vascular abnormality such as aneurysm or malformation. In the sections of the brain stem, a massive hematoma occupied the central area of the pons. Drug screening test using Triage™ was weakly positive for amphetamines. Moreover, in the blood and urine samples, methamphetamine was quantitatively detected at concentrations of 0.4 and 0.6 mg/l, respectively, by gas chromatography–mass spectrometry. Other drugs and poison were not detected in the blood and urine samples collected at autopsy. Histopathologically, necrotizing angiitis characterized by fibrinoid necrosis of the intima and media was observed with cell infiltration. Thus, the pontine hemorrhage seemingly resulted from methamphetamine-induced angiitis, with an acute elevation of blood pressure after methamphetamine abuse.

Although enhanced expression of IL-1 family proteins, including IL-1α, IL-1β, and IL-1 receptor antagonist (IL-1ra) during wound healing has been observed, the pathophysiological roles of these factors, particularly IL-1ra, still remain elusive. We explored skin wound-healing processes in IL-1ra-deficient mice. Compared to wild-type (WT) mice, IL-1ra-deficient mice exhibited impaired wound healing, as evidenced by attenuated collagen deposition and delayed neovascularization. In contrast, neutrophil recruitment was significantly exaggerated, with the augmented expression of IL-1s, TNF-α, and CXC chemokines, MIP-2 and KC, in IL-1ra-deficient mice compared with WT mice. Because the transcription of these proinflammatory cytokines and CXC chemokines requires the activation of NF-κB, a major target of IL-1- and TNF-α-mediated signal pathway, we examined the activation states of NF-κB. Nuclear translocation of NF-κB p65 was significantly enhanced and prolonged in IL-1ra-deficient mice, compared to that in WT mice. The cross-talk between NF-κB and TGF-β-mediated signals has been proposed based on in vitro observations. Indeed, compared to WT mice, the amounts of total and phosphorylated Smad2 and Smad3 were decreased with a reciprocal increase in the amount of Smad7 in skin wound sites of IL-1ra-deficient mice. Moreover, the gene expression of vascular endothelial growth factor, a target gene of TGF-β1, was decreased in IL-1ra-deficient mice. Thus, the absence of IL-1ra may suppress TGF-β-mediated signaling pathway, which is crucial for collagen deposition and vascular endothelial growth factor-mediated neovascularization in wound healing.

Sepsis is asevere, systemic inflammatory disease caused by various kinds of microbes. In the present study, we immunohistochemically examined tumor necrosis factor (TNF)-α expression in sepsis-induced lung injury, and discuss its availability for the postmortem diagnosis of sepsis. Lung samples were obtained from different lung lobes of nine sepsis and eight control cases with postmortem intervals between 12 and 48 hours. Immunohistochemical analysis using anti-human TNF-α rabbit polyclonal antibodies was carried out. In sepsis and control groups, immunoreactivity for TNF-α was strongly detected in round-shaped mononuclear cells. The intensity of the immunohistochemical staining reaction was homogeneous in all lobes of the lungs examined. Furthermore, a double-color immunofluorescence analysis revealed that macrophages were a main cellular source of TNF-α in the lungs. To semiquantitatively evaluate the expression of TNF-α in the lungs, the ratios of the number of TNF-α-positive macrophages to total number of macrophages were calculated. Morphometrically, in lungs of the sepsis group, the ratio of TNF-α-positive macrophages was significantly higher, compared with the control group. TNF-α expression in the lungs can become a clue for the postmortem diagnosis of pulmonary inflammation, especially, TNF-α-positive ratios of 20% of more might suggest sepsis as the cause of death.

We report a case of fatal water intoxication due to polydipsia. A 69-year-old schizophrenic male was found dead at his room of the hospital in which he had been admitted. Medico-legal autopsy was carried out to determine the cause of his death. The autopsy revealed no severe trauma leading him to the death. Internally, it was noticed that the stomach was vigorously expanded, including fluid contents. Intracardiac blood, being dark-red in color, seemed to be diluted. The both lungs ballooned aqueously, showing apparently edema. However, there was neither macroscopic nor histopathological lesion, being responsible for his death. Postmortem biochemical analyses revealed severe hyponatremia of 92 mEq/ml. In cases with short postmortem interval, serum sodium level almost similarly reflected antemortem level. According to his psychiatric doctor, he had been diagnosed as water intoxication due to polydipsia. Moreover, at 2 h before the discovery of his body, he had been found to drink much running water. It was concluded the cause of his death as fatal water intoxication.

To elucidate the contribution of signal transducer and activator of transcription (STAT) 6 to the pathophysiology of chronic renal injury, STAT6-/- mice were subjected to unilateral ureteral ligation together with wild-type control mice. STAT6-/- kidneys had more apoptotic cells and a greater influx of F4/80-positive cells than wild-type kidneys following ureteral obstruction. There was a much larger alpha-smooth muscle actin-positive area in STAT6-/- kidneys than in wild-type kidneys after ureteral ligation. However, renal fibrosis, as quantified by Masson-Trichrome staining, was not significantly exaggerated in STAT6-/- kidneys compared with wild-type kidneys. The accumulation of collagen I was significantly less in STAT6-/- kidneys than in wild-type kidneys. These observations indicate that the STAT6 signal transduction pathway exerts a protective role on renal cell apoptosis in chronic obstructive uropathy. Our findings also suggest that the STAT6 pathway may have a promotive effect on renal fibrosis by activating collagen synthesis following ureteral obstruction.

Death-associated protein kinase (DAPK) is a Ca2+/calmodulin-dependent serine/threonine kinase that is thought to mediate apoptosis. We have shown that the kinase domain of DAPK is crucial for the induction of renal tubular cell apoptosis in chronic obstructive uropathy (COU) created by unilateral ureteral ligation. DAPK-mutant mice, generated by deletion of 74 amino acids from the catalytic kinase domain, were used to investigate the role of the DAPK kinase domain in renal fibrosis following COU. Interstitial collagen and alpha-smooth muscle actin (alpha-SMA) expressions in situ were compared between obstructed kidneys in wild-type and mutant mice. As a result, tubulointerstitial fibrosis, as quantified by interstitial collagen expression, was significantly augmented in mutant kidneys compared with wild-type kidneys following COU. Furthermore, deletion of the kinase domain from DAPK significantly increased the appearance of alpha-SMA-positive myofibroblasts in the renal interstitium during COU. Thus, our results suggest that the kinase domain deleted by gene targeting plays a suppressive role for the development of renal fibrosis through inhibition of the tubular epithelial-to-mesenchymal transition in a mouse model of COU.

Summary Background Recently, the novel antimicrobial peptide named dermcidin (DCD) was reported in human eccrine sweat glands. Objectives We investigated the expression of DCD in a variety of cutaneous tumours in order to assess the usefulness of the monoclonal antibody (G-81), which recognizes a fragment of DCD. Patients/methods We studied the immunoreactivity of the G-81 antibody on 197 cutaneous tumours. Results A total of 13 of 26 cutaneous mixed tumours showed substantial immunoreactivity. In contrast all the following cases were completely unreactive: (i) epithelial tumours (seborrhoeic keratosis, squamous cell carcinoma, Bowen's disease, actinic keratosis, genital Paget's disease); (ii) follicular tumours (basal cell carcinoma, trichilemmoma, trichoepithelioma, trichoblastoma, keratoacanthoma, proliferating trichilemmal tumour, pilomatricoma); (iii) melanocytic tumours (malignant melanoma, naevus cell naevus, Spitz naevus, blue naevus); (iv) neural tumours (schwannoma, neurofibroma, Merkel cell neoplasm); (v) mesenchymal tumours (soft fibroma, dermatofibroma, dermatofibrosarcoma protuberans, vascular leiomyoma, leiomyosarcoma, lipoma, juvenile xanthogranuloma, angiomyoma); and (vi) other sweat gland tumours (poroid neoplasms, syringoma, cylindroma, clear cell hidradenoma, spiradenoma, syringoid eccrine carcinoma, mucinous carcinoma, apocrine cystadenoma, syringocystadenoma papilliferum, apocrine adenocarcinoma). Twenty-six cutaneous mixed tumours were considered from histopathological findings to be the apocrine type, but 13 of 26 mixed tumours contained some DCD-immunopositive cells that possibly differentiate into eccrine secretory glands. Conclusions We found the expression of DCD in tubular structures of 50% of cutaneous mixed tumours with apocrine differentiation. These results suggest that a number of cutaneous mixed tumours show both eccrine and apocrine differentiation in the same neoplasm.

Objective: To clarify the clinical features and pathogenesis of intussusception in neonates. Methods: Fourteen neonates were diagnosed with intussusception between June 1974 and January 2001. Patients were divided into two groups according to whether or not signs were present on the first day of life. The clinical features were interrelated with the pathological findings. Results: All six patients in the group whose signs were present on the first day of life also had intestinal atresia or malrotation. Among patients whose signs began less than 24 h after birth, five of eight patients suffered hypoxia. Moreover, it was very difficult to establish the diagnosis of intussusception, particularly in the group of late-onset type intussusception. Conclusions: Hypoxic events may play a crucial aetiologic role in the pathogenesis of late-onset neonatal intussusception.

In the hair of individuals with blood group AB, the level of blood group A glycosphingolipids is much lower than that of blood group B. We hypothesized that in hair, blood group A determinants are converted by alpha-N-acetylgalactosaminidase (alpha-NAGA, E.C.3.2.1.49) to H determinants. To address our hypothesis, the relative amount of ABH glycosphingolipids in hairs and nails of normal subjects, patients with Kanzaki disease, and heterozygous carriers of alpha-NAGA deficiency were analyzed by dot-blotting and enzyme-linked immunosorbent assay. In hair from normal subjects with blood group B, ABH glycosphingolipids consisted of 88% blood group B- and 12% blood group H glycosphingolipids. In blood group A subjects, 14% were group A- and 86% were group H glycosphingolipids. In Kanzaki patients, 81% were blood group A- and 19% were blood group H glycosphingolipids. In 2 alpha-NAGA deficiency carriers, the ABH glycosphingolipids consisted of 67% blood group A- and 33% blood group H glycosphingolipids. These results indicate that blood group A glycosphingolipids are catabolized to H glycosphingolipids by alpha-NAGA, resulting in lower levels of blood group A glycosphingolipids in the hair of normal subjects, and alpha-NAGA deficiency causes accumulation of blood group A glycosphingolipids in the hair of Kanzaki patients. This finding is of clinical relevance because it suggests that hair may be used to diagnose and assess the alpha-NAGA status of individuals.

Death-associated protein kinase (DAP kinase) is a Ca2+/calmodulin-dependent serine/threonine kinase implicated as a positive apoptosis mediator. However, little is known about DAP kinase involvement with apoptosis in renal diseases.In order to determine whether DAP kinase has a role in renal cell apoptosis in kidney diseases, we performed an immunohistochemical study using a monoclonal antibody to DAP kinase. Firstly, by examining the cellular distribution of DAP kinase in normal human renal tissues and cultured proximal tubule cells. We then used western blotting and immunohistochemical analysis to examine directly whether DAP kinase protein levels could be modulated in rat kidneys with chronic obstructive uropathy created by unilateral ureteric ligation.Immunohistochemistry of normal human kidney tissues showed that DAP kinase was exclusively localized in the cytoplasm of renal tubule cells. Expression analysis of DAP kinase using cultured cells confirmed DAP kinase mRNA and protein presence in human renal tubule cells. Immunocytochemical analysis directly visualized DAP kinase in the cytoplasm of the renal tubule cells in culture. Finally, DAP kinase was found up-regulated in renal tubule cells of rat kidneys with chronic obstructive uropathy.Our study demonstrates that DAP kinase is localized to renal tubule cells, implying a crucial role for DAP kinase in renal tubular cell apoptosis in progressive renal diseases.

An immunohistochemical study combined with morphometry was carried out to examine the time-dependent expression of vascular endothelial growth factor (VEGF) using 53 human skin wounds with different wound ages (groups I: 0-12 h, II: 1-4 days, III: 7-14 days and IV: 17-21 days). In the human wound specimens aged 4-12 h, neutrophils recruited at the wound showed no positive signals for VEGF. With an increase in wound ages ofor =7 days, granulation tissue and angiogenesis were observed, with the migration of macrophages and fibroblasts of which the cytoplasm expressed VEGF-positive reactions. Morphometrically, the average VEGF-positive ratio was highest in group III, followed by that of group IV. In groups III and IV, 13 out of 26 wound samples had VEGF-positive ratios of more than 50%. However, all of the wound samples in groups I and II showed VEGF-positive ratios of less than 50%. With regard to the practical applicability and forensic validity, these observations suggest that a VEGF-positive ratio of more than 50% possibly indicates a wound age of 7 days or more.

Death-associated protein kinase (DAPK) is a Ca2+/calmodulin-dependent serine/threonine kinase that has been implicated as a positive mediator of apoptosis. However, little is known about the involvement of DAPK in the apoptosis associated with several pathological states, except for cancer. Here, DAPK-mutant mice were used in order to examine the role of DAPK in renal cell apoptosis in chronic obstructive uropathy (COU) created by unilateral ureteral ligation. These mice express mutant DAPK with a deletion of 74 amino acids from the catalytic kinase domain. Obstructed kidneys in wild-type and mutant mice were examined for both DAPK protein levels and renal cell apoptosis during the course of COU. Obstructed kidneys in wild-type and mutant mice showed a marked increase in the DAPK and mutant DAPK protein levels, respectively, at day 14 after ureteric ligation. The obstructed kidneys in DAPK-mutant mice displayed a significant attenuation of tubular cell apoptosis, compared with wild-type mice. In contrast, no significant difference in interstitial cell apoptosis was observed between the obstructed kidneys from wild-type and mutant mice. Thus, these results indicate that the part of the kinase domain deleted by the gene targeting is crucial for the execution of tubular cell apoptosis, but is not essential for interstitial cell apoptosis in a COU model in mice.

Background: We investigated the efficacy of natural interferon (IFN)-α treatment in 34 Japanese children with chronic hepatitis C. Methods: Thirty-four children completed 6 months of therapy with natural IFN-α and were followed for 12 months or longer. We examined the serum hepatitis C virus (HCV) RNA titer and liver histology before, during, and after IFN treatment. Results: At 6 months after the cessation of IFN-α treatment, 16 patients (47%) had normal serum alanine aminotransferase concentration and no detectable serum HCV RNA. There were no major side-effects, excluding some influenza-like symptoms during the IFN-α treatment. Most genotype 2a patients had a complete response (80%). Moreover, patients who had a low HCV RNA titer (

To profile postmortem degradation of mRNA, total RNA was extracted, at given postmortem intervals, from the brain, lung, heart and liver of rats left at 20 degrees C. In electrophoretic analysis, total RNA was most stable in the brain, moderately stable in the lung and heart, and most unstable in the liver. Northern blot analysis of total RNA extracts from the brain and liver of dead rats with a cDNA probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) showed that GAPDH mRNA degraded in a similar fashion to total RNA. Analysis of the postmortem degradation profile of GAPDH mRNA with real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) gave results consistent with those above, indicating that real-time RT-PCR is reliable for estimation of the mRNA level in specimens from dead bodies. Real-time RT-PCR analysis showed that degradation rates of three housekeeping genes, GAPDH, beta-actin and hypoxanthine guanine phosphoribosyltransferase, in the brains of dead rats were similar. The degradation rate of interleukin-1beta (IL-1beta) mRNA induced by intravenous injection of LPS to rats was higher than that of GAPDH mRNA in the lung. In real-time RT-PCR analysis using GAPDH mRNA as an internal standard, the detection level of IL-1beta mRNA decreased in the postmortem interval. However, enhanced expression of IL-1beta was detected for at least 3 days postmortem.

Deep vein thrombosis (DVT) is a major cause of pulmonary thromboembolism, a leading cause of death in individuals with DVT. And DVT is multifactorial and often results from a combination of risk factors such as genetic conditions, obesity, drugs, pregnancy, aging, trauma, and malignancy. We demonstrated the pathophysiological roles of cytokines such as IFN-g and TNF-α in the processes of thrombus resolution using genetically engineered mice. In this study, we examined the pathophysiological roles of IL-6 in the resolution of DVT by the use of Il6 KO mice. Upon the ligation of the inferior vena cava (IVC) of WT mice, venous thrombi formed and grew progressively until 5 days, and the thrombus weight decreased after that. Concomitantly, the gene expression of Il6 was detected in the thrombi. When Il6 KO mice were treated in the same manner, thrombus size was much larger than WT mice. The intrathrombotic IL-6 was produced by intrathrombotic infiltrated macrophages on double color immunofluorescence staining of thrombi. And the number of intrathrombotic IL-6-positive cells changed in proportion to the number of macrophages. Moreover, the blood flow of the IVC was more recovered in WT than in Il6 KO mice. And intrathrombotic Mmp2 , Mmp9 , and Plau mRNA expression was significantly reduced in Il6 KO mice than WT mice. Supportingly, the administration of anti-IL-6 antibody delayed the thrombus resolution in WT mice. Furthermore, IL-6 treatment enhanced gene expression of Plau , Mmp2 , and Mmp9 in WT-derived macrophages. These IL-6 effects were significantly suppressed by Stat3 inhibitor. Collectively, IL-6 can have a beneficial role in the thrombus resolution by upreglation of MMP-2, MMP-9 and alpha expression. IL-6 can be a good molecular target for the DVT treatment.

A method to obtain surfable waves by amplifying ship generated waves with both wave concentration structures and a reef is discussed to build surf points in calm areas of nearshore zones. The appropriate wave height for ordinary surfers is defined according to a questionnaire to local surfers in Kagoshima, Japan. The most effective shapes of the structures are determined on the basis of amplification factors of wave height through numerical calculation. We performed hydraulic experiments, as well as numerical computations, of ship generated waves amplified using the structures. Consequently, it is feasible to build a surf point in Kagoshima Bay by amplifying waves generated by the ferries through the proposed method with the structures which consist of the wave concentration structures and the reef.

To investigate the role of placental transport of bile acids in fetal bile acid metabolism, such as with regard to synthesis of the unusual bile acids (1 beta- and 6 alpha-hydroxylated and unsaturated bile acids), we measured the concentrations of bile acids in umbilical cord blood, amniotic fluid, maternal serum and maternal urine at delivery by means of gas chromatography-mass spectrometry. Serum and urine from healthy nonpregnant women were used as controls. We detected large amounts of unusual bile acids, especially hyocholic acid and 3 beta-hydroxy-delta 5 bile acids, in amniotic fluid and umbilical cord blood. The concentration of total bile acids in maternal serum was less than that of control serum and umbilical cord blood, and the concentration of total bile acids in maternal urine was higher than that of control urine and amniotic fluid. In conclusion, the fetus synthesized large amounts of unusual bile acids, and these compounds were transported from fetus to mother by placental transfer. We suggest that pregnant women may excrete large amounts of bile acids into the urine to control serum concentration of bile acids in fetus.

Of the primary dementing disorders that cause frontotemporal dementia, the best-known is Pick disease. We report on a 44-year-old woman with progressive frontal lobe dementia and spastic paraplegia. Examination revealed increased serum levels of cholestanol with abnormal cholesterol metabolism and a heterozygous mutation of the sterol 27-hydroxylase gene (CYP27). Biochemical findings were compatible with cerebrotendinous xanthomatosis (CTX); however, the clinical manifestations were very dissimilar. To our knowledge, a symptomatic carrier of this mutation among CTX patients has not been reported. We speculate that the present patient has a previously undescribed neurodegenerative disease related to abnormal cholesterol metabolism with this heterozygous mutation.

A microplate enzyme immunoassay (EIA) was developed for the measurement of glycine- and taurine-conjugated 7alpha-hydroxy-3-oxo-4-cholenoic acids (CDCA-delta4-3-one) in human urine. The antiserum was prepared by immunizing rabbits with N-(7alpha-hydroxy-3-oxo-4-cholen-24-oyl)-3-aminopropionic acid--bovine serum albumin conjugate. A colorimetric EIA was established using horseradish peroxidase-labeled antigen having a shorter bridge length than that of the immunogen, and 3, 3', 5, 5'-tetramethylbenzidine /hydrogen peroxide for the measurement of the enzyme activity. The reactivities of the antiserum for glycine and taurine conjugates of CDCA-delta4-3-one was almost the same. The specificity of the antiserum was investigated by determining the cross-reactivities of various bile acids and related compounds. An appropriate dose-response curve for conjugated CDCA-delta4-3-one was obtained in the range of 0.05-10 ng/well. This method was used for direct analysis of conjugated CDCA-delta4-3-one in urine of healthy infants and patients with liver diseases.

Objective. To characterize the clinical and histologic features of chronic hepatitis C virus (HCV) infection after blood transfusion in Japanese children. Study Design. We studied 231 children with a history of blood product transfusion. Patients were divided into two groups: 116 patients with a history of malignant disease (group 1), 115 patients who had undergone open heart surgery (group 2). We examined changes in serum alanine aminotransferase (ALT) activity and HCV markers, and patients' clinical course. Moreover, in 38 patients in whom the time of HCV infection could be defined, we examined liver histology. Results. The proportions of patients in each group who were anti-HCV-positive were 35 out of 116 (30%) and 20 out of 115 (17%), respectively. Of the anti-HCV-positive patients, the proportions of HCV RNA-positive patients in each group were 30 out of 35 (86%) and 12 out of 20 (60%), respectively. Levels of ALT activity in patients with HCV infection varied widely for several years after blood transfusion; thereafter ALT activity fell to Conclusions. Sixty percent to 80% of children with HCV infection in this study developed chronic hepatitis C. However, examination of liver histology findings in children with chronic hepatitis C showed only mild changes.

The administration of ABO blood group active glycoproteins via the rear footpads of BALB/c mice with subsequent fusion of popliteal lymph node lymphocytes produced hybridomas differentially secreting antibodies to the peptide moiety of antigens. On the other hand, conventional intraperitoneal immunization of the same antigens and intraperitoneal boost followed by splenic lymphocyte fusion produced hybridomas differentially secreting antibodies to the carbohydrate moiety of antigens. Similar results were obtained in the production of monoclonal antibodies to erythrocyte membranes. Almost all hybridomas obtained by rear foodpad immunization secreted antibodies to the peptide moiety of erythrocyte membrane proteins, and those obtained by intraperitoneal immunization secreted antibodies to both peptide and carbohydrate moieties of erythrocyte membrane components. These results suggest the possibility of differential production of monoclonal antibodies to the carbohydrate moiety and the peptide moiety of some glycoproteins by different immunization routes.

A 3-oxo-delta4-steroid 5beta-reductase (5beta-reductase) deficiency is difficult to diagnose because severe liver damage can result in a similar pattern of metabolite excretion. We investigated the usefulness of immunoblot analysis for diagnosis of 5beta-reductase deficiency and quantitatively analysed urinary bile acids by gas chromatography-mass spectrometry in a 5-month-old Japanese boy with severe neonatal cholestasis associated with hypertyrosinaemia. A liver sample was examined by immunoblot analysis using monoclonal antibodies against 5beta-reductase. Urinary 3-oxo-delta4 bile acids accounted for 88.3% of total bile acids, 5alpha-bile acids for 0.9%, and primary bile acids for 9.1%. Immunoblot analysis of the liver tissue showed an indistinct band of 5beta-reductase.These findings suggest that this patient had a secondary 5beta-reductase deficiency due to severe liver damage, even though 3-oxo-delta4 bile acids constituted more than 70% of total urinary bile acids. However, the patient may possibly have had an inherited 5beta-reductase deficiency.

Background: Ursodeoxycholic acid absorption in the proximal intestine may be impaired in patients with inflammatory bowel disease. Methods: We examined the intestinal absorption of ursodeoxycholic acid by the oral ursodeoxycholic acid tolerance test in 19 children and adolescents with inflammatory bowel disease at various stages, including 8 patients with unoperated Crohn's disease, 3 patients with ileal-resected Crohn's disease, 8 with ulcerative colitis, and 8 healthy control subjects. Results: Ursodeoxycholic acid malabsorption was present in all patients with unoperated Crohn's disease in the first diagnosed active stage, in 3 of 5 patients in a relapsing active stage, and in 2 of 8 patients in remission. Ursodeoxycholic acid absorption was significantly lower in patients in the first diagnosed active stage than in the healthy controls (p < 0.01) or in patients in remission (p < 0.01). There was no significant difference between healthy controls and the patients in a relapsing active stage or in remission. Ursodeoxycholic acid absorption was abnormal during the first postoperative month in patients with ileal-resected Crohn's disease, but normalized over time. Malabsorption of ursodeoxycholic acid was not observed in any patients with ulcerative colitis. Conclusions: These findings suggest that absorption of ursodeoxycholic acid in the proximal intestine is impaired in patients with Crohn's disease and that the oral ursodeoxycholic acid tolerance test is a convenient and useful means of evaluating the absorption of bile acid in the proximal intestine in pediatric patients with ileal or ileocolic Crohn's disease.

A method has been developed for the determination of 3-oxo-delta4- and 3-oxo-delta4,6-bile acids and related bile acids in biological fluids of infants by gas chromatography-mass spectrometry (GC-MS) of the methyl ester-dimethylethylsilyl ether-methoxime derivatives. The 7alpha-hydroxylated 3-oxo-delta4-bile acids were partially dehydrated to give the 3-oxo-delta4,6-bile acids by trimethylsilyl or dimethylethylsilyl derivatization and other pretreatments under acidic or alkaline conditions for GC-MS analysis. To prevent dehydration, the 3-oxo-delta4-bile acids were derivatized to the oximes by treatment with O-methylhydroxylamine prior to pretreatments such as solid-phase extraction, enzymatic solvolysis and hydrolysis of the conjugates, and silylation with dimethylethylsilylimidazole. Calibration curves for the bile acids were linear over a range of 5-250 ng and the detection limit was 100 pg for each 3-oxo-delta4-bile acid. Recoveries of the bile acids and their glycine and taurine conjugates from bile acid-free urine and serum ranged from 94.2 to 105.9% of their added amounts. The bile acids in urine and serum of four patients with severe cholestatic liver disease were measured by the analytical method, and the 3-oxo-delta4-bile acids were determined to be the major bile acids (59-68%) in the urines associated with 3-oxo-delta4-steroid 5beta-reductase deficiency or dysfunction.

The key to improving the heat load of vacuum plasma sprayed tungsten coatings on low activation ferritic steel maintained at low temperatures is elimination of stratified low-density layers with many large pores, in which thermal cracks propagate preferentially. The low-density layers are formed owing to the deposition of large solidified tungsten particles, which remain mainly at the periphery of the spray stream. In this study, by shading this periphery, partially homogeneous tungsten coatings without large pores were successfully obtained. The coatings are expected to show good heat load, which is feasible for nuclear fusion applications.

Direct enzymatic assay of urinary sulfated bile acids is a sensitive, rapid, minimally invasive, and convenient method of detecting cholestasis in young infants. It may replace measurement of serum direct bilirubin for selective screening for biliary atresia and neonatal hepatitis syndrome at 1 month of age.

Increased expression of a brain/embryo-type myosin heavy chain isoform (MIIB2) in mesangial proliferative glomerulonephritis . Proliferation of mesangial cells is frequently found in glomerulonephritis, such as IgA glomerulonephritis. Recent reports suggest that a brain/embryo-type myosin isoform (MIIB2) is involved in cell proliferation. We have studied the expression of MIIB2 in renal biopsy samples from patients with various renal diseases and in the renal tissues from the rat model of mesangial proliferative glomerulonephritis induced with anti-Thy 1.1 antibody. Immunohistochemical analysis of the biopsy samples using an anti-brain-type myosin heavy chain-specific monoclonal antibody (HBM1) indicated that 92% of the samples from patients with IgA glomerulonephritis contained a significant population of mesangial cells that reacted with the antibody. Most of the samples from patients with other types of proliferative glomerular diseases also contained HBM1-reactive mesangial cells, while none of the samples from patients with non-proliferative glomerular diseases contained a significant population of HBM1-reactive mesangial cells. The expression of a brain/embryo-type myosin heavy chain isoform (MIIB2) in the mesangial cells began at five days after injection of anti-Thy 1.1 antibody and peaked at the tenth day. On the other hand, the expression of the proliferating cell nuclear antigen in the mesangial cells was induced at two days after injection of anti-Thy 1.1 antibody and was maximal at the fourth day. These results indicate that the expression of the MIIB2 isoform by mesangial cells is accelerated in proliferative glomerulonephritis and suggest that the myosin isoform is involved in the phenotypic transformations of the glomerular tissues rather than in the cell proliferation.

We present two male siblings with intrahepatic cholestasis and prolonged indirect hyperbilirubinaemia. Their familial intrahepatic cholestasis syndrome was characterized by Coombs’negative haemolytic anaemia, without giant cell transformation of hepatocytes and high concentrations of serum γ-glutamyl transpeptidase and cholesterol. By gas chromatography-mass spectrometry, we detected large amounts of 1β-hydroxylated bile acids, especially lβ,3α,7α,12α-tetrahydroxy-5β-cholan-24-oic acid (25.5-67.9% of total urine bile acids) in the urine during phenobarbital therapy. However, the amount of urinary 1β-hydroxylated bile acids gradually decreased as the disease progressed. At the end-stage, we detected large amounts of 7α,12α-dihydroxy-3-oxochol-4-en-24-oic acid (19.6% of total urine bile acids). The ratio of 7α,12α-dihydroxy-3-oxochol-4-en-24-oic acid to cholic acid in the urine was 0.8. We conclude that in infants with end-stage liver failure, the microsomal hydroxylation of bile acids is impaired and the excretion of Δ4-3-oxo bile acids is increased.Familial intrahepatic cholestasis, Coombs’negative haemolytic anaemia, 1β-hydroxylated bile acids, unsaturated ketonic bile acids

Introduction Cardiac hypertrophy is an adaptive response of the heart to prolonged increases in hemodynamic workload. This compensatory process is initially beneficial in normalizing wall stress, and sustained left ventricular hypertrophy significantly increases the risk of developing heart failure. Although various molecules have been shown to be involved in the compensatory process, its molecular mechanism still remains unclear. We investigated roles of IFN-γ in the process of compensatory cardiac hypertrophy induced by pressure overload. Methods Male Balb/c (WT) and IFN-γ-deficient (IFN-γ-KO) mice were subjected to transverse aortic constriction (TAC).Aortic constriction was achieved by tying around the transverse thoracic aorta against a 28-gauge needle using a 7–0 silk suture, and then removing the needle. As the control, sham operation was also performed without constricting the aorta. Cardiac hypertrophy, survival rate and hypertrophy related signaling pathways were examined. Results Intracardiac gene expression for IFN-γ was rapidly increased in TAC mice. Although TAC induced rapid increase of heart weight/body weight ratio (HW/BW ratio) in WT mice even at 3 days after TAC, most mice survived for 3 weeks after TAC. On the other hand, IFN-γ-KO mice showed no significant increase of HW/BW ratio at 3 days after TAC, and about 60% of them died within 6 days after TAC. Western blotting analysis showed that AKT phosphorylation in the left ventricles of WT mice was significantly increased at 3 days after TAC, compared with sham operated mice. The downstream signal molecules, GSK-3 β and GATA4, were also highly phosphorylated in the left ventricles of WT mice, compared with sham operated mice. However, the activation of PI3K/AKT signals were significantly attenuated in the left ventricles of IFN-γ-KO mice, indicating that IFN-γ plays a crucial role in the hypertrophic response through PI3K/AKT signaling. Conclusion Our results demonstrate blunted hypertrophy with reduced survival rate and attenuated PIK3/AKT signaling following pressure overload in IFN-γ-KO mice, and suggest that IFN-γ plays a crucial role in the compensatory hypertrophic response.

Introduction Bone marrow-derived cells are known to play important roles in repair/regeneration of injured tissues, but their roles in pathological fibrosis are less clear. Here, we report a crucial role for CX3C chemokine receptor 1 (CX3CR1) in the recruitment of lung fibrocytes. Methods An intratracheal injection of bleomycin into wild-type mice caused a massive infiltration of leukocyte subsets, such as neutrophils, T lymphocytes, and macrophages, followed by the development of diffuse pulmonary fibrosis with accumulation of fibrocytes characterized by CD45+/collagen type I+ cells. Results Intrapulmonary CX3CR1 and its ligand CX3CL1 expression were enhanced significantly and remained at elevated levels until pulmonary fibrosis developed. Compared with wild-type mice, collagen deposition was attenuated in CX3CR1−/− mice with reduced number of lung fibrocytes although bleomycin increased leukocyte infiltration to a similar extent in wild-type and CX3CR1−/− mice, implying that less intrapulmonary recruitment of fibrocytes directly correlated with decreased collagen deposition in CX3CR1 deficiency. Furthermore, bone marrow transplantation from CX3CR1−/− to wild-type mice, but not that from wild-type to CX3CR1−/− mice, recapitulated the phenotypes in CX3CR1−/−- mice. Conclusion: These results demonstrated that CX3CL1-CX3CR1 interaction was involved in bleomycin-induced recruitment of bone marrow-derived fibrocytes and subsequent development of pulmonary fibrosis.

Fetal bile acids (1β-hydroxylated, 6α-hydroxylated and unsaturated bile acids), especially 1β,3α,7α, 12α-tetrahydroxy-5β-cholan-24-oic acid (CA-1β-ol), have been detected in urine and feces early in life. To investigate whether a fetal pathway of bile acid synthesis exists in infancy, we measured the concentrations of bile acids in the urine, meconium and feces from normal newborns and infants by means of gas chromatography—mass spectrometry. The mean ratio of total bile acids to creatinine in urine increased between birth and 7 days and then gradually decreased; however, the concentration of total bile acids in urine remained significantly higher than that in adult urine until 3 mo of age. The main urinary bile acid was CA-1β-ol, and substantial amounts of fetal bile acids were detected in urine until 3 mo of age. The ratio of cholic acid to chenodeoxycholic acid was abnormally low in meconium (mean, 0.44; range, 0.19 to 0.74), and hyocholic acid constituted 19.3% of total bile acids. The mean total bile acid content of feces decreased between birth and 7 days of age and thereafter increased. The mean percentage of fetal bile acids in feces decreased after birth, but substantial amounts were present in feces until 1 mo of age. Our data support the notion that a primitive pathway for the synthesis of fetal bile acids—such as CA-1β-ol, hyocholic acid and 3β-hydroxy-5-cholen-24-oic acid—may be responsible for excretion mechanisms of cholestatic bile acids during immaturity of hepatic excretory function in infants from birth until 3 mo of age. (Hepatology 1994;20:819–824).