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In 1973, rotaviruses A (RVAs) were discovered as major causative agents of acute gastroenteritis in infants and young children worldwide. The infectious RV virion is an icosahedral particle composed of three concentric protein layers surrounding the 11 double-stranded (dsRNA) segments. An in vitro replication system for RVs in permanent cell lines was developed in 1982 and expanded to replication in intestinal organoids in 2015. However, the details of rotavirus (RV) entry into cells and particle maturation mechanisms at the molecular level remain incompletely understood. Slowing down human RVA replication in cell culture on ice allowed morphological visualization of virus particle entry and the assembly of triple-layered particles (virion). Although RVAs are non-enveloped viruses, after virus attachment to the cell membrane, the virus enters the cell by perforating the plasma membrane by a fusion mechanism involving VP5* of the cleaved VP4 protein, as the alternative virus entry route besides the receptor-mediated endocytosis which is generally accepted. After assembling double-layered particles (DLPs) in viroplasm or cytoplasm, they appear to be connected with the endoplasmic reticulum (ER) membrane and become coated with outer capsid proteins (VP4 and VP7) in a coating process. The perforation of the ER membrane is caused by an unknown mechanism following interaction between non-structural protein 4 (NSP4) and the inner capsid protein VP6 of the DLPs. The coating process is closely related to the formation of a hetero-oligomeric complex (NSP4, VP4 and VP7). These lines of evidence suggest the existence of novel mechanisms of RV morphogenesis.

Retaining experienced nurses is an indispensable part of securing the safety and reliability of medical care. In this study, we investigate how the working conditions affect staff nurses' intention to remain in their current employment positions (intention to remain employed). A total of 319 staff nurses working for three private hospitals were given a questionnaire in order to evaluate their working conditions. A stepwise multiple logistic regression analysis selected 5 out of 44 question items as significant independent variables for their intention to remain employed; they were assumed to directly affect their intentions (direct modulators). From the remaining 39 items, seven meaningful factors were extracted via factor analysis. We constructed a path network model so that these factors would indirectly affect the intention to remain employed via any of the five direct modulators (indirect modulators). In this network, the factor, "Rapport with coworkers" had the strongest positive effect on three of the five direct modulators (beta = 0.28-0.55, p < 0.05). According to factor loadings, positive teamwork together with appropriate supervision and recognition from superiors and colleagues seems to characterize this factor, and therefore, may play a key role in retaining staff nurses in their current positions. Two indirect modulators related to "Management by Objectives" (MBO)--"Understanding MBO" and "Clear personal objectives" also played negligible roles in this path model. These findings could be helpful in improving management systems of the nursing sections of hospitals, which could contribute to retaining staff nurses in their current employment positions.

Knee pain is extremely common among the elderly, particularly women. Hence, there is an urgent need for applicable community-based intervention models for halting the progression of knee pain and related disabilities in elderly women. We aimed to assess the efficacy of home-visit physiotherapy as a new intervention model. This non-randomized 5-month-long controlled trial enrolled elderly community-dwelling women (aged 60-83 years) with mild knee pain. The intervention consisted of two home visits by a physiotherapist, with instructions on routinely performing muscle-strengthening exercises at home and implementing simple environmental modifications when necessary. Outcome measures were assessed at baseline and 5 months later. The primary outcomes were measured as the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and a newly devised seiza-style sitting score. People in Japan, especially elderly women, are accustomed to seiza-style sitting that involves kneeling on one's lower legs while resting the buttocks on the heels. The secondary outcomes included quadriceps isometric strength, knee alignment in the frontal and sagittal planes, and passive knee extension range. Forty-two subjects (20 in the intervention group and 22 in the control group) completed the study. At baseline, characteristics and the primary outcomes did not significantly differ between the 2 groups. At 5 months, the primary outcomes improved significantly in the intervention group. Estimated differences in the change from baseline for each outcome between the 2 groups were computed, adjusting for outcome variables imbalanced at baseline. Even after the adjustment, the home-visit physiotherapy regimen provides favorable improvement in the seiza-style sitting score.

Hepcidin, a key iron-regulator secreted from the liver, consists of 25 amino acids (hepcidin-25), blocks iron release from macrophages via internalization and degradation of cellular iron exporter ferroportin, and restrains the use of iron in organs. Hepcidin mRNA and protein are also expressed in the human heart. A short form of hepcidin that lacks 5 amino-acid residues in the N-terminus (hepcidin-20) has been found in human serum, although its physiological role is unknown. Here, we successfully measured the serum levels of hepcidin-25 and hepcidin-20 in 12 patients with acute myocardial infarction (AMI) using surface-enhanced laser desorption ionization time of flight mass spectrometry. Among the selected 10 patients, whose blood samples were taken within 4 hours after a heart attack, all the patients showed elevated serum levels of hepcidin-20 [between 31.7 and 285.1 arbitrary unit (AU); normal level < 9.3 AU], while 8 patients showed high levels of hepcidin-25 (9.3-271.4; normal < 25.5 AU). The hepcidin-20 level was decreased to nearly the normal level on day 7 (range of 2.9 to 12.5 AU) in the 12 patients, whereas the hepcidin-25 level remained high on day 7 in 8 patients. Furthermore, the elevated levels of hepcidin-25 and hepcidin-20 were not correlated with the serum levels of markers for inflammation, interleukin-6 and C-reactive protein, in the patients with AMI. In conclusion, the serum hepcidin-20 is transiently elevated in response to acute cardiac ischemia. Measurement of serum hepcidin-20, rather than hepcidin-25, is helpful for diagnosis of acute myocardial infarction.

Oseltamivir has been used for treatment of influenza A and B infections, but recent reports documented that it was less active against the latter. We compared the effectiveness of oseltamivir in children between laboratory confirmed influenza A and B over 4 influenza seasons from 2001 to 2005 in a pediatric clinic in Japan. Among 1,848 patients screened, 299 influenza A and 209 influenza B patients were administered oseltamivir (treated groups), and 28 influenza A and 66 influenza B patients were assigned as non-treated groups. The duration of fever, defined as period when patients had the maximum temperature higher than 37.5 degrees C in three-time measurements in a day after the clinic visit, was evaluated among the four groups. In uni-variate analysis, the duration of fever was shorter for treated group than non-treated for influenza A (1.8 +/- 0.9 days vs 2.6 +/- 1.3 days, p0.01), but it was not significant for influenza B (2.4 +/- 1.3 days vs 2.8 +/- 1.2 days, p = 0.9). The fever duration was longer in treated influenza B than A patients (p0.01). Multi-variate analysis indicated younger age (6 years old) and higher body temperature at the clinic visit prolonged the duration of fever. Adjusted average duration of fever indicated that oseltamivir was effective for both types, but more effective on influenza A, and the benefit increased for younger children. Our data provide evidence that oseltamivir is beneficial for influenza infections, but the effectiveness is differed by type and age.

In recent years, a dramatic increase of amantadine-resistant influenza A has occurred globally, but limited data have been available on the clinical course of patients developed amantadine-resistant viruses. We compared fever reduction between patients who developed resistance or remained sensitive in a pediatric clinic in Niigata, Japan, from 2000 to 2006. A total of 2,802 clinical samples were collected from patients who visited the pediatric outpatient clinic with influenza like illness during the seven influenza epidemic seasons. Patients were divided into 4 groups and analyzed for the fever reduction after amantadine treatment: emerged amantadine-resistant (n = 15); amantadine-sensitive (n = 35); patients administered no antiviral drugs (n = 42); and oseltamivir-treated patients (n = 320), which served as references. All 4 groups showed alleviation of fever up to day 3. The amantadine-resistant group had a significant recurrence of fever on day 4 and/or 5, and as a consequence, the course of illness was prolonged. Considering the pattern of fever, recurrent and persistent patterns were found significantly at higher rates in children with emerged resistant virus compared to other groups, and the age tended to be younger in amantadine-resistant compared to amantadine-sensitive group (3.9 +/- 3.0 vs 6.7 +/- 4.1 years old, n.s.). Therefore, we concluded that younger children were prone to develop amantadine-resistance after treatment and showed a significant recurrence of fever on day 4 and/or 5, and the course of illness was consequently prolonged.

An off-season community influenza outbreak with high prevalence of amantadine-resistant influenza A/H3N2 occurred during September-October 2005 in Nagasaki Prefecture, Japan, prior to standard influenza circulation. A total of 48 patients with influenza-like-illness (ILI) visited a clinic during the outbreak and 27 (69.2%) of 39 ILI patients were positive for influenza A with rapid antigen testing (Quick Vue Rapid SP Influ). Nine patients were not tested because their symptoms were compatible for influenza without examination. Nasopharyngeal swabs were obtained from 4 of 27 rapid test positive patients, and influenza H3N2 strain was isolated from one out of four. The 4 nasopharyngeal samples were positive for influenza A M2 gene in polymerase chain reaction, and sequencing results all showed identical mutation at position 31, serine to asparagine (S31N) in the gene, conferring amantadine resistance. The phylogenetic tree analysis demonstrated that the hemagglutinin (HA) gene sequences of the 4 samples formed a distinct cluster (named clade N) from recent circulating H3N2 strains, characterized by dual mutations at position 193, serine to phenylalanine (S193F), and at position 225, asparatic acid to asparagine (D225N). Our findings suggested that an off-season community influenza outbreak in Nagasaki was caused by a distinct clade in H3N2 (named clade N), which possessed characteristics of amantadine resistance.

Use of high-density barium (= or > 180 w/v%) has been widely spread in community-based mass screening for gastric cancer in Japan. However, the impact on outcomes of the screening has not been fully evaluated. Then, we investigated the advantage of high-density barium use by comparing the outcomes between a period with high-density barium and that with low-density barium (< 180 w/v%) each for 3 consecutive years between 1991 and 2002 at 5 medical examination organizations in Niigata Prefecture, Japan. Multivariate logistic regression analysis adjusted for age, sex and medical organization revealed that the introduction of high-density barium decreases the positive finding rate in initial mass screening x-ray examinations by 12% (odds ratio, 0.88; 95% confidence interval, 0.86 - 0.90) and increase the gastric cancer detection rate by diagnostic examination by 15% (odds ratio, 1.15; 95% confidence interval, 1.01-1.31). We conclude that introduction of high-density barium has improved the efficiency of mass screening for gastric cancer by decreasing unnecessary diagnostic examinations and reducing the total personal and public health costs. Thus, nationwide use of high-density barium is recommended for mass gastric cancer screening in Japan.

Kawasaki disease (KD) is a systemic vasculitis and occurs among Japanese children at a high incidence. Serum bilirubin and heme oxygenase-1 (HO-1) expression are known to play a significant role in the protection of vascular endothelial cells. Japanese have unique polymorphic distribution patterns of (TA)7 or G71R of the bilirubin UDP-glucuronosyltransferase (B-UGT) gene and of (GT)n repeats of the HO-1 gene. We investigated the relationship of KD susceptibility with these polymorphisms. There were no significant differences in the distribution of allele frequencies and genotypes of these polymorphisms between KD patients and controls. These polymorphisms are not associated with KD susceptibility.

Microangiopathic hemolytic anemia and thrombocytopenia have been reported in patients with primary pulmonary hypertension, but not in patients with congenital heart disease even if accompanied with pulmonary hypertension. We present a 7-year-old boy with atrial septal defect and pulmonary hypertension who developed microangiopathic hemolysis and thrombocytopenia. Microangiopathic hemolytic anemia and thrombocytopenia should be remarked as a complication in patients with congenital heart disease.

Ebstein's anomaly is a rare congenital cardiac anomaly showing significant clinical manifestations with a high mortality rate in the neonatal period. The prognosis of the patient is essentially determined by the severity in morphological changes, however, high pulmonary vascular resistance in the neonatal period may aggravate tricuspid regurgitation and lead to functional pulmonary atresia. We describe a critically ill neonate with morphologically mild Ebstein's anomaly who was successfully managed with intensive care including isoproterenol administration for functional pulmonary atresia. Isoproterenol is a potent pulmonary vasodilator with inotropic and chronotropic effects, and seemed to decrease the pulmonary vascular resistance allowing increased antegrade blood flow to the pulmonary artery and improved cardiac output. If tachycardia is not present, isoproterenol administration is recommended in critically ill neonates with anatomically mild Ebstein's anomaly and no associated cardiac defects.

An anonymous unlinked HIV antibody test was conducted on 1632 Chlamydia trachomatis (C. trachomatis) antibody positive women from 10 institutes of 7 prefectures in Japan. All the sera were negative for both HIV-1 and HIV-2 antibodies. The result may support the suggestion that HIV prevalence is low among general population in Japan. Such a test as this study will be useful not only for developing a reliable HIV surveillance system but also for the study of sexual behavior of general population, since C. trachomatis infection is sensitive to reflect sexual contact.

SAKAMOTO, M., YAZAKI, N., KATSUSHIMA, N., MIZUTA, K., SUZUKI, H. and NUMAZAKI, Y. Longitudinal Investigation of Epidemiologic Feature of Adenovirus Infections in Acute Respiratory Illnesses among Children in Yamagata, Japan (1986-1991). Tohoku J. Exp. Med., 1995, 175 (3), 185-193 - Using human embryonic fibroblast (HEF) and HEp-2 cell cultures, adenoviruses were isolated from 989 (3.7%) out of 26, 793 pediatic patients with ARI in Yamagata, Japan from January, 1986 to December, 1991. All isolates were identified as types 1 (Ad1)-6 and no other serotypes were identified. Epidemiologic feature was different depending on the subgenus group. Ad1, 2, 5 and 6 (group C) were endemic and the infections occurred frequently in the summer season. Ad3 (group B) was epidemic in the autumn to winter season, although the virus was isolated every month in non-epidemic season. No seasonal distribution of Ad4 (group E) could be determined because the number of patients was limited. Neutralizing antibody positive ratio for group C were more than 40% at 1-2 years of age and almost 100% by 10 years of age but those for Ad3 (group B) were 40% by 10 years of age. The neutralizing antibodies for Ad4 (group E) or Ad7 (group B) became negative by 10 years of age. With group C infections, most cases were infants and young children less than 2 years of age, but Ad3 infections were older children with the peak at 4 and 5 years of age.

Thirty-two children with bone marrow transplantation (BMT) received intravenous injections of gammaglobulin (IVIG) with a high titer of neutralizing (NT) antibody against human cytomegalovirus (HCMV) (200 mg/kg/week) from 1 week before to 4 months after transplantation. NT antibody titers before BMT and the highest levels in serial determinations conducted after BMT were compared for each patient. They were classified into three groups according to the antibody response: primary HCMV infection as group I, endogenous reactivation or external reinfection as group II, and indeterminable cases as group III. Two (6.3%) out of 32 patients examined had BMT-associated primary HCMV infections, but did not show any clinical symptoms. Significant changes in clinical parameters were also lacking in all the other 30 patients, independent of whether they shed viruses into the urine, or demonstrated on antibody boost. It was concluded from the group variation that the antibody response was indeed due to the engraftment of BMT, rather than to a direct effect of treatment with IVIG. Our results further indicate that passive immunization with HCMV antibody does not prevent infection, but confers some protection against symptomatic disease.

The large number of child deaths in developing countries is associated with delays in care-seeking by families, but the community-based efforts of the Integrated Management of Childhood Illnesses developed by WHO/UNICEF has remained ineffectual. To improve caregivers' ability to recognise potentially life-threatening symptoms of major childhood illnesses, we provided education about the importance of danger signs and immediate care-seeking practices through a community-based intervention of 'the Growth Monitoring Programme Plus (GMP+)' in low-income areas of Lusaka, Zambia. Using repeated cross-sectional data from interviews, we compared attendance and non-attendance groups to assess the impacts of intervention on mothers' care-seeking. Of 1717 and 1546 attendance mothers in the baseline and the final survey, 1097 and 1035, respectively, sought care from a health centre after perceiving the danger signs. The proportion of mothers with immediate response increased from 35.7% (392/1097) to 51.5% (533/1035) (p < 0.01). In the final survey, the attendance mothers became more likely to respond immediately to the danger signs than the non-attendance mothers (adjusted odds ratio: 2.140, 95% confidence interval: [1.408-3.252]), and the higher educational level the attendance mothers had, the more likely they were to respond immediately to the danger signs (primary level: 2.067 [1.050-4.068], secondary level and above: 2.174 [1.098-4.306]). In conclusion, GMP+ with danger sign education can improve mothers' care-seeking for severely sick children. Therefore, GMP+ has the potential to reduce child death in developing countries, i.e., contribute to the Millennium Development Goal 4 aiming at reducing child mortality by two-thirds by 2015.

KOBAYASHI, K., SUZUKI, H., UENO, Y., NAGATOMI, R., KANNO, A., OTSUKI, M. and TOYOTA, T. Anti-Idiotypic Antibodies Directed against Anti-HBs among the Patients with Chronic Hepatitis B. Tohoku J. Exp. Med., 1990, 16 (4), 261-271 -Anti-idiotypic antibodies (anti-Id) against anti-HBs were found in the sera of patients with chronic hepatitis type B. Anti-idiotypic antibodies were detected by an enzyme-linked immunosorbent assay using horseradish peroxidase conjugated mouse monoclonal anti-HBs. Ten of 72 HBsAg positive sera contained anti-Id (13.9%). The prevalence of anti-Id did not appear to correlate with HBeAg/anti-HBe system. However, HB virus specific DNA polymerase activity was significantly higher in anti-Id positive sera. In the sera obtained from the patients treated with predonisolone before, anti-Id positive rate was higher than that in the patients without a history of predonisolone therapy. These results suggest that anti-Id may be related to the immunoregulatory mechanism of HB virus replication.

The present study investigated smoking prevalence and attitudes toward smoking in student and staff nurses, and activities to promote tobacco intervention by staff nurses. A total of 743 student nurses in 2 colleges and one university and 490 staff nurses in the university hospital in Niigata, Japan participated in the study and filled in self-administered anonymous questionnaires focused on smoking habits, attitudes toward smoking, and activities to promote tobacco intervention. The smoking prevalence among student nurses was 6% and increased with the year of study, whereas that of among staff nurses was 16% and increased by employment up to 3-5 years. Almost half of the student nurse smokers started the habit before admission to higher education facilities, and more than half of staff smokers also started smoking in their school years, in high school or college. Nicotine dependence, as defined by over 11 cigarettes per day and the first cigarette within 30 min after getting up, was high in smokers who started smoking before admission to college or university, but not in those who took up the habit thereafter. Thirty-six percent of student nurses and 25% of staff nurses agreed with the statement that "nurses should not smoke." Only 12% of staff nurses were involved in anti-tobacco interventions routinely, partly due to lack of broad knowledge and the necessary skills. Greater efforts are needed as a high priority to educate student and staff nurses systematically and comprehensively about the range of tobacco interventions available in Japan.

SUZUKI, H., KITAOKA, S., KONNO, T. and SATO, T. The Localization and Function of the Neutralizing Protein, VP4, in Human Rotavirus. Tohoku J. Exp. Med., 1991, 163 (1), 73-75 - The binding sites of a monoclonal antibody (mAb) to human rotavirus VP4 (K-1532) on the virion surface were examined with electron microscopy and the Markham photorotation method. The mAb, which had both hemagglutination inhibiting and neutralizing activities, appeared to bind to the outer margin of double-shelled particles of human rotavirus, especially to the parts overlaying the tubelike channels, which seemed to be the “cover lid” of the tubelike channels that extruded radially from the core region. This binding point may be a putative fusion site of the virus (Mackow et al. 1988) as well as a viral nucleus component ejection gate (Suzuki et al. 1986). Rotavirus particles treated with the mAb were shown to be capable of binding to MA104 cells by ultra-thin section examination. These observations suggest that neutralization via VP4 is related to virus uncoating but not to attachment of rotavirus.

Sixty of 128 (46.9%) residents of a nursing home were immunized with two doses of the trivalent split influenza vaccine. They developed 7.4-11.5-fold antibody increases, with a 69-82% protection rate, presenting good immune response rates to the influenza vaccine. Two outbreaks of influenza A (H3N2) occurred. There were no significant antigenic differences among the vaccine strain and the strains isolated from both outbreaks in haemagglutination-inhibition tests, suggesting that the second might have been a reoccurrence. There were no residents who were infected in both outbreaks. The vaccine efficacy against clinical illness in the first outbreak of typical influenza-like-illness (ILI) was 51% (relative risk: 0.49), and the febrile period was reduced significantly by vaccination. In the second outbreak, however, in which all patients had atypical ILI with a high fever but not respiratory symptoms, vaccine efficacy was not apparent for unknown reason.

2450 sera from students and 5215 sera from pregnant women were examined for the presence of Chlamydia trachomatis (CT) antibody. CT antibody positive rates were less than 5% with the students and 24.5% with the pregnant women suggesting the latter is significantly higher than former. The results provided a base for discussing possibility of using CT infection as a reliable method for studying sexual behavior.

A histopathologic study was carried out on pulmonary vessels of lung tissues from 5 children with primary pulmonary hypertension (PPH), in whom diagnosis was made on the basis of cardiac catheterization. Histological sections of lung were obtained in 4 patients by open lung biopsy and in one patient by closed needle necropsy soon after death. In all cases the vascular diseases were classified as grade 6 of the Heath-Edwards. Morphologic studies of small pulmonary arteries revealed medial hypertrophy (41.0%), intimal cellular proliferation (2.5%), intimal fibroelastic proliferation (36.9%) and medial destructive changes (19.6%) such as dilatation lesions, plexiform lesions, arteritis, or fibrinoid necrosis. Index of pulmonary vascular disease (IPVD) was 1.6 to 3.4 (mean 2.5). The advancement of these pulmonary changes sufficiently explains the extremely poor prognosis of children with PPH.

To elucidate the role of tumor necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta) in chronic hepatitis type C (CH(C], TNF alpha and IL-1 beta released from peripheral blood mononuclear cells were measured in 6 CH(C) patients before and during interferon therapy. During the therapy, TNF-alpha levels were significantly high in two patients whose serum ALT levels turned normal, while IL-1 beta levels did not show significant change. These results suggest that TNF-alpha takes part in improvement of CH(C).

In order to investigate the reliability of ultrasonography in the diagnosis of fatty liver, retrospective evaluation was made of abdominal echograms in 45 patients with various diffuse liver diseases who underwent liver biopsy within 2 weeks after the ultrasound study. Instead of the well-recognized but subjective diagnostic criterion, i.e. the bright liver pattern, liver-kidney contrast is proposed as a new criterion. The liver-kidney contrast is based on the brightness of the liver in comparison to the renal parenchyma where fatty change seldom occurs. Combination of this liver-kidney contrast with two other well-known ultrasonographical findings of fatty liver, vascular blurring and deep attenuation, enables us to grade fatty change semiquantitatively. When fatty change of over 30% in the hepatic lobule was adopted as the definition of fatty liver, the satisfaction of both liver-kidney contrast and vascular blurring presented an ultrasound diagnostic criterion for fatty liver, with sensitivity of 83%, specificity of 100%, and an accuracy of 96%.

Levels of serum IgG antibodies to somatic antigens of Haemophilus influenzae and Pseudomonas aeruginosa were determined in 127 healthy people, 37 patients with chronic bronchitis and 20 patients with bronchiectasis by enzyme-linked immunosorbent assay. The logarithmic mean titers of antibodies to Haemophilus influenzae and Pseudomonas aeruginosa were significantly higher in the patients with chronic bronchitis and bronchiectasis than in healthy people. The invasion rate of Haemophilus influenzae was identical in healthy people, chronic bronchitis and bronchiectasis, while that of Pseudomonas aeruginosa was significantly lower in healthy people than in chronic bronchitis and bronchiectasis patients. These results indicate that patients with chronic bronchitis and bronchiectasis may often have recurrence and are easily infected because of lowered defense mechanisms in the respiratory tract.

Smooth muscle cells growing in the primary culture derived from outgrowths of the intimal-medial explants of both rat and human arteries were used. The 72-hr sequential glucose uptake by the cells of both species in culture dishes was enhanced only slightly with time by the addition of insulin to culture medium, and this enhancement was statistically not significant. The glucose conversions to CO2 and lipids by the rat and human cells dissociated for tracer study were not affected significantly during the 2-hr incubation by the insulin addition in vitro. The smooth muscle cells of both species cultured for a week in medium enriched with insulin and then dissociated revealed the significantly increased glucose conversion to lipids, while the increase in the glucose coversion to CO2 was not significant in these cells. Thus, the smooth muscle cells of both rat and human seem to show significant metabolic response to chronic, but not acute, exposure to insulin. Therefore, it is likely that the persistent change in the insulin level may lead to abnormal metabolic state in the artery.

Using a quantitative blood endotoxin assay utilizing chromogenic substrate coupled with perchloric acid pretreatment (PCA-LCT), we showed the presence of endotoxin-inactivating factor (EIF) in human plasma in vitro. EIF activity inactivated added endotoxin to about 10(-4) of the initial level within 20 min, followed by a stable phase where the residual endotoxin became resistant to EIF and was not further inactivated. The residual endotoxin may represent the endotoxin in patient plasma which is also EIF resistant. We postulate that endotoxin, upon entering the blood, is rapidly inactivated by chemical modification of its active site, lipid A, through EIF. Subsequently, inactivated endotoxin, mainly consisting of polysaccharide, is gradually removed from circulation by endocytosis in the reticuloendothelial system.

KOBAYASHI, Y., TAZAWA, Y., NAKAGAWA, M., SUZUKI, H., KONNO, T. and YAMAMOTO, T.Y. Ultrastructural Changes in Skeletal Muscle of a Patient with Familial Intrahepatic Cholestasis Associated with Vitamin E Deficiency. Tohoku J. exp. Med., 1984, 142 (3), 337-346 - The skeletal muscle from a patient with familial intrahepatic cholestasis associated with vitamin E deficiency was studied by electron microscopy. The muscle fibers showed a variety of pathologic features including degenerative, necrotic and regenerative changes. Granule-like inclusions found in our biopsy specimens were similar in structure to those observed in vitamin E deficient animals. These inclusions were noted not only in the skeletal muscle fibers but also in endothelial cells of the intramuscular capillaries, Schwann cells and perineural cells. Disruption and disappearance of the plasma membrane, separation, disruption and pleats formation of the external lamina, and multilayered external laminae were observed in muscle fibers most frequently. The nerves among muscle fibers also showed degenerative features. These severely degenerative alterations of the muscle fiber have not so far been reported in vitamin E deficient patients. We discuss the process of cell damages caused by vitamin E deficiency.

Left ventricular mass data were obtained from biplane cineangiocardiograms in 37 normal infants and children. Left ventricular mass calculated by using thickness of anterior wall (left ventricular mass-anterior) and posterior wall (left ventricular mass-posterior) of the free left ventricular wall were studied in relation to body surface area and left ventricular end-diastolic volume. Both of left ventricular mass-anterior and mass-posterior were expressed as a function of body surface area with exponential relationship; left ventricular mass-anterior = 68.5 (body surface area) g (r = 0.98, p less than 0.01), left ventricular mass-posterior = 84.6 (body surface area) g (r = 0.97, p less than 0.01). Left ventricular mass-anterior/left ventricular end-diastolic volume averaged 0.73 +/- 0.01 g/ml, and left ventricular mass-posterior/left ventricular end-diastolic volume 0.89 +/- 0.02 g/ml, respectively. Thus, left ventricular mass can be predicted from body surface area by using the exponential equations. In addition, normal values for left ventricular mass-posterior must be discriminated from those for left ventricular mass-anterior.

TSUCHIYA, S., YAMAGUCHI, Y., KOBAYASHI, Y., MINEGISHI, M., IMAIZUMI, M., SUZUKI, H., KONNO, T. and TADA, K. An Epstein-Barr Virus-Negative B Lymphoma Cell Line (THP-2) from a Burkitt's Lymphoma of a Japanese Patient. Tohoku J. exp. Med., 1983, 140 (4), 429-434 - An Esptein-Barr virus-negative lymphoma cell line, THP-2, was established from a Burkitt's lymphoma taken from a Japanese patient. THP-2 cells grew in single cell suspension with a doubling time of 24hr; the cells carried surface-bound μ-λ immunoglobulins and formed rosettes with IgG antibody-coated ox erythrocytes. THP-2 cells expressed B1, common acute lymphoblastic leukemia (ALL) (J5) and Ia-like antigens of their surface as defined by monoclonal antibodies. Epstein-Barr virus-associated nuclear antigen (EBNA) was not detected. The cell line THP-2 has been maintained for over 5 years. Among the markers examined, only common ALL antigen has been present on the cell surface of THP-2 for these 5 years.

SATOH, T., NARISAWA, K., TAZAWA, Y., SUZUKI, H., HAYASAKA, K., TADA, K. and KAWAKAMI, T. Dietary Therapy in a Girl with Propionic Acidemia: Supplement with Leucine Resulted in Catch Up Growth. Tohoku J. exp. Med., 1983, 139 (4), 411-415 - A 15-month-old girl with propionic acidemia presented a failure to thrive and muscular hypotonia, who had been treated with diets low in leucine, valine, isoleucine, threonine and methionine. Supplementation with leucine gave her a catch up growth and brought about an improvement in muscular hypotonia in parallel with restoration of plasma leucine which had been below normal range during the treatment with the above diets. Oral loading test of leucine produced no ketoacidosis. These findings indicate that the restriction of leucine is not necessary in the dietary management of propionic acidemia.

Serum gonadotropin response patterns to LH-RH infusions were investigated in women with and without actinomycin D treatment. LH and FSH levels increased until 60 min in the non-ovarectomized premenopausal women and until 120 min in ovarectomized women after infusion in both groups. But, thereafter the levels in women untreated with actinomycin D were much higher than in women treated with actinomycin D. These results support the view that the LH-RH-stimulated -synthesis of gonadotropin involves increased RNA synthesis.

TOYOTA, T., SUZUKI, H., SUGAWARA, H., UMEZU, M., FUJIYA, H., SUZUKI, S. and GOTO, Y. Remission of Insulin Resistant Diabetes in Two Patients with Anti- Insulin Receptor Antibodies. Tohoku J. exp. Med., 1982, 138 (2), 187-197 Since Kahn et al. reported in 1976 insulin resistant diabetes due to anti-insulin receptor antibodies, an unusual form of diabetes mellitus (type B) has been found in many countries. We had two diabetic patients with anti-insulin receptor antibodies, associated with either acanthosis nigricans or systemic lupus erythematosus. Remission occurred 15 months after the onset of insulin resistant diabetes. One patient unfortunately died of acute pneumonia and the other has been followed up. The anti-insulin receptor antibodies were measured according to the method of Omori and Hirata by using the pellet of human placental membrane. The anti-insulin receptor antibodies in both cases diminished as remission occurred. Reverse hemolytic plaque assay (PFG) detected immunoglobulin-producing cells. In Case 2, the plaque forming cells were twenty times as many as the normal value. Immunosuppressive therapy with cyclophosphamide reduced the immunoglobulin secreting cells as remission occurred. The patients with insulin resistance (type B) should be treated with enough insulin inspite of the presence of insulin resistance. Besides, cyclophosphamide, 6-mercaptopurine and prednisolone should be used with caution. Plasma exchange is a treatment to be tried. It is important to note that spontaneous remission may occur more than half a year after the onset of insulin resistant diabetes.

YOSHIDA, T., SUZUKI, H., HATTORI, Y, and NODA, K. Hormonal Changes around the Parturition in Rats. Tohoku J. exp. Med., 1981, 135 (1), 87-91-Concentrations of serum oxytocin, cortisol, progesterone and estradiol in pregnant rats were assayed by radioimmunoassay methods. The levels of these hormones increased in pregnancy. Around the parturition, oxytocin or estradiol did not show any change, cortisol increased, and progesterone decreased prominently. The trigger of initiation of labor was discussed.

In order to know more about the cytoskeletal structure around the bile canaliculi of hepatocytes, we used the following procedure. Liver specimens were passed through an 18-, a 21- and then a 23-gauge needle, by which they turned into cylindrical or oval blocks of 0.4 mm in diameter. Because of their small size, the cytosolic ground substances of every hepatocyte were washed out clearly after subsequent treatment with the solution containing 0.5% Triton X-100 and 0.5 mg/ml saponin for 15 min. This short-term extraction preserved both the cytoskeleton and the plasma membrane of the hypatocyte quite well, and we could observe the crossbridge filaments connecting between a core bundle of microfilaments and plasma membrane of the microvilli of the bile canaliculi.

Hepatitis associated antigen (HB Ag) was measured using a single radial immunodiffusion (SRID) method or an immunoadherent hemag-glutination (IAHA) method in patients under various clinical conditions including acute hepatitis, chronic hepatitis, liver cirrhosis, hepatoma, various malignant tumors and other diseases as well as in normal subjects. HB Ag was found to be frequently positive in patients with hepatoma and with malignant tumor with liver metastasis by the immunoadherent hemagglutination method. The significance of this finding is discussed.

The mechanical properties of the longitudinally cut preparations of the guinea-pig hepatic duct were studied. About 42% of the preparations (18 of 43 preparations) showed spontaneous phasic contractions which were unaffected by either tetrodotoxin, guanethidine or atropine. About 60% of them exhibited phasic contractions which appeared at irregular frequencies in the same tissue and varied in size. This sort of contractions usually accompanied small contractions. In about 20%, contractions occurred constantly in frequency (about 6-14 every 10 min) and in size. The remaining 20% showed twitch-like contractions which occurred sporadically. These contractions were classified into two types depending upon the susceptibility to the Ca2+ entry blocker, D-600. D-600 (1-100 microM) strongly suppressed both regularly occurring contractions and twitch-like ones. On the other hand, the drug was ineffective on irregularly occurring contractions. The Ca2+ sources underlying these contractions were discussed.

With a quantitative blood endotoxin assay using a chromogenic substrate with a perchloric acid pretreatment (PCA-LCT), endotoxemia in various liver diseases was studied. With PCA-LCT, recovery of added endotoxin in human plasma was nearly 90%, as evidenced by an intra- and inter-assay coefficients of variation of 5.7% and 11%, respectively. Because the recovery of endotoxin was not affected in severely icteric plasmas, PCA-LCT proved to be applicable to patients with liver diseases where various degree of jaundice exist. In none of the plasmas from patients with chronic hepatitis, acute hepatitis without hepatic failure or liver cirrhosis without ascites did the endotoxin level exceed the normal range of less than 5 pg/ml. With the presence of ascites, however, endotoxemia became detectable, but at low levels and not in all cases. At the stage of hepatic failure complicated with renal failure or disseminated intravascular coagulation, endotoxemia was more frequent and endotoxin concentration greater. It is uncertain, at present, whether endotoxemia itself is deteriorating factor in hepatic failure or is merely concomitant phenomenon resulting from Kupffer cell failure.

KITAOKA, S., SUZUKI, H., NUMAZAKI, Y., KONNO, T. and ISHIDA, N. The Effect of Trypsin on the Growth and Infectivity of Human Rotavirus. Tohoku J. exp. Med., 1986, 149 (4), 437-447- The effect of trypsin on the infectivity of human rotavirus (HRV) was examined using HRV strains that are cultivable only when treated with trypsin. Their infectivity in cell culture systems was enhanced in relation to enzyme concentration. The primary effect of trypsin appeared to be on the virus. Trypsin untreated HRV virions were capable of being adsorbed on the cell (MA104) but not of passing into it without trypsin. For multiple cycles of replication the presence of trypsin in the maintenance medium was required.

KOBAYASHI, Y., SUZUKI, H., IINUMA, K., TADA, K. and YAMAMOTO T.Y. Endothelial Alterations of Skeletal Muscle Capillaries in Childhood Myopathies. Tohoku J. exp. Med., 1983, 140, (4) 381-389 - Intramuscular blood capillaries in thirteen biopsied specimens obtained from eleven patients with various childhood myopathies were investigated ultrastructurally. The patients were classified into 4 different groups according to the percent distribution of various stages of endothelial alteration; normal endothelial type (E0 Type), slightly abnormal type (E1 Type), moderately abnormal type (E2 Type) and markedly abnormal type (E3 Type). Endothelial types in Duchenne dystrophy progressed from E0 to E1 and further to E2 Type, which kept pace with muscle degeneration. The endothelial types in Werdnig-Hoffmann disease and central core disease were E0 and those in multicore disease and Fukuyama type congenital muscular dystrophy were E1. Two cases of mitochondrial myopathy showed E3 Type, which was characterized by markedly swollen endothelial cells and narrowed lumens. The endothelium in these two cases showed more marked changes than in other childhood myopathies.

In order to examine the clinical features of severe acute exacerbation in chronic hepatitis B virus (HBV) infection, 297 hepatitis B surface antigen (HBsAg) carriers were followed for 35 +/- 22 months (mean +/- S.D.) in Tohoku University Hospital from 1976 to 1987. Of these, 10 experienced severe acute exacerbation with hepatic decompensation. All of these patients had intense subjective symptoms related to the hepatitis. They were all icteric and 8 had ascites. Three developed to fulminant hepatic failure, eventually died. Histology after the exacerbation showed severe hepatic damage such as massive hepatic necrosis and bridging hepatic necrosis in a half of them. Six cases were suspected to result from spontaneous reactivation and 2 from drug-induced reactivation of chronic HBV infection, and the other 2 from superinfection with non-A, non-B hepatitis agent (s). These results suggest that the reactivation of chronic HBV infection is an important factor of severe acute exacerbations in chronic HBV infection in Japan.

Echocardiographic studies were performed on 110 normal infants and children ranging in age from 2 days to 15 years. The following parameters of the left ventricle were measured: left ventricular end-diastolic and end-systolic diameters (LVEDd and LVESd), shortening fraction (LVSF), end-diastolic volume (LVEDV), ejection fraction (LVEF), posterior wall thickness in end-diastole (LVPWED) and end-systole (LVPWES), mass (LVM), LVEDd/LVPWED, LVESd/LVPWES and mean velocity of circumferential fiber shortening (LV mean Vcf). Each parameter was compared with body surface area (BSA). LVEDd, LVESd, LVEDV, LVPWED, LVPWES and LVM were expressed as a function of body surface area with exponential equations: LVEDd = 41.4 (BSA)0.49 mm, LVESd = 28.1 (BSA)0.49 mm, LVEDV = 74.1 (BSA)1.48 ml, LVPWED = 4.4 (BSA)0.45 mm, LVPWES = 9.2 (BSA)0.44 mm and LVM = 46.4 (BSA)1.48 g. LVSF, LVEDd/LVPWED, LVESd/LVPWES and LV mean Vcf were independent of body surface area: LVSF averaged 32 +/- 1 (mean +/- standard error of the mean)%, LVEF 68 +/- 1%, LVEDd/LVPWED 10.0 +/- 0.2, LVESd/LVPWES 3.1 +/- 0.1 and LV mean Vcf 1.08 +/- 0.02 circ/sec. Thus, normal values of these parameters can be applied to estimate them in pediatric patients with heart diseases.

Serum FSH and LH in women with luteal phase defect were measured. Their serum FSH and LH were lower than those in normal women and higher than those in women with amenorrhea. Administration of clomiphene citrate to the women with luteal phase defect were effective in 2/3 of the cases. These results are consistent with the hypothesis that a relative deficiency of FSH during the follicular phase results in diminished follicular development and subsequent inadequate corpus luteum maintenance.

A new method was devised for the estimation of denatured plasma protein after long-term artificial perfusion. The method was based on the behavior of denatured protein, namely, decreased solubility and increased susceptibility to enzymatic hydrolysis. By this method, the extents of denaturation of plasma protein were compared after 20 hours perfusion using bubble, disc or membrane type oxygenators. Membrane type oxygenater was superior to non-membrane oxygenators in inducing less denaturation of plasma protein.

In order to correct severe metabolic acidosis, peritoneal dialysis was attempted in 2 cases of cyanotic babies. The condition was markedly improved in both of them by peritoneal dialysis with buffer solution, though they died finally of cardiac failure. It could be concluded from our experiences that peritoneal dialysis was effective in the correction of severe metabolic acidosis in newborn babies with marked cyanosis.

Six cell lines transformed in vitro by adenovirus type 12 were characterized by karyologically heterogenous population, but the mode consisted of diploid cells. This diploid stem seems stable for a long time in vitro culture.

YOSHIDA, T., HATTORI, Y., SUZUKI, H. and NODA, K. Response of FSH, Prolactin and HCG to Continuous LHRH Infusion in Pregnant Women. Tohoku J. exp. Med., 1981, 135 (3), 255-257-Serum FSH, prolactin and HCG response patterns to 4-hr infusion of LHRH were investigated in six women in the first trimester of pregnancy. FSH did not show response until 60min after beginning of the infusion and then it increased gradually. Serum prolactin and HCG levels were almost constant during the LHRH infusion.

Electron microscopy of biopsy specimens from the small intestines in infants and children with various gastrointestinal disturbances revealed that the intercellular space of the mucosal epithelium was often filled with electron dense materials. Here apposed cell membranes were arranged almost parallel with each other. The dense materials also filled the space just subjacent to the epithelium to form a thick, dense layer in which the basal lamina was completely buried. The gap junctions were especially well developed in the epithelia filled with these materials. Long-period fasting of patients seemed to facilitate the occurrence of the dense materials and the formation of gap junctions.

AMAKASU, H., SUZUKI, H., MIYAKAWA, T., OOTSUKI, M, and GOTO, Y. A Enzymatic and Fluorimetric Determination of Total Bile Acid Sulfates in Human Urine. Tohoku J. exp. Med., 1986, 149 (4), 343-350- A simple, precise and sensitive method for separation and determination of total bile acid sulfates in human urine is described. The sulfate fraction of urinary bile acids was separated with lipophilic anion exchange gel, piperidinohydroxypropyl Sephadex LH-20 after sample clean-up with Sep-Pak C18 cartridge. The obtained sulfate fraction was submitted to solvolysis with a small volume of dimethoxypropane-HCl solution and subjected to enzymatic-fluorimetrical assay using 3α-hydroxysteroid dehydrogenase and resazurin. In this method, no influence of existing salts in the reaction mixture on fluorescence intensity was observed and solvolysis reaction was almost complete. Overall recoveries of glycine- and taurine-conjugated bile acid 3-sulfates from normal urine ranged from 90.5 to 93.7% and those of unconjugates from 48.7 to 78.0%. The sensitivity of the described method enabled to estimate total bile acid sulfates with 0.5ml of normal urine and precision tests showed the satisfactory accurasy. The level of total urinary bile acid sulfates was estimated on some patients with hepatobiliary diseases and healthy subjects.