Okayama University Medical School

In order to elucidate the role of erythrocyte catalase in the accumulation of mercury in erythrocytes, labeled erythrocytes and plasma were prepared by exposing normal and acatalasemic mice to radioactive mercury vapor (203Hg0: 6.8mg/m3) for 30 min. Labeled erythrocytes (or plasma) were mixed with unlabeled plasma (or erythrocytes) of normal or acatalasemic mice and incubated at 0 degrees C for 1 h. After incubation, the radioactivity of mercury in the erythrocytes and the plasma was measured by a gammascintillation counter. When labeled erythrocytes were incubated with unlabeled plasma, the ratio of mercury transferred from acatalasemic erythrocytes to normal plasma (11.6%) or to acatalasemic plasma (13.3%) were significantly higher than that from normal erythrocytes to normal plasma (1.8%) or to acatalasemic plasma (2.2%). When labeled normal (or acatalasemic) plasma was incubated with unlabeled normal or acatalasemic erythrocytes, the uptake of mercury by acatalasemic erythrocytes from normal plasma was 2.0%, and 1.2% from acatalasemic plasma, which tended to be lower than that by normal erythrocytes from normal plasma (3.4%) or from acatalasemic plasma (2.2%). The results indicated impaired accumulation of mercury in acatalasemic erythrocytes, suggesting the importance of catalase in taking up mercury in erythrocytes and protecting other organs from toxic effects of metallic mercury.

DeVega's annuloplasty was performed on 41 patients with tricuspid regurgitation (TR) associated with combined valvular disease and results were assessed based on Doppler echocardiographic findings in an attempt to examine the applicability of this surgical technique. TR was quantitatively evaluated via Doppler echocardiography before and after surgery. Clinical symptoms, cardiac function, and surgical results were assessed, and the severity of left ventricular myocardial degeneration was determined using electron microscopy. There were no differences in the following factors between the TR recurrence and TR improvement groups: previous heart surgery, number of involved valves, presence or absence of a giant left atrium, preoperative New York Heart Association (NYHA) functional class, and type of prosthetic valve (Bjork-Shiley vs. St. Jude Medical). We found no differences between these two groups in TR severity and tricuspid annulus diameter measured during surgery. Severity of myocardial degeneration was closely associated with the recurrence of TR. Clinically, most had diminished cardiac function before surgery. DeVega's technique appears to be remarkably effective in patients with well-preserved myocardium because no TR recurrence was detected even in examinations with the most accurate Doppler echocardiography. However, such long-term effectiveness of DeVega's technique cannot be expected in patients with degenerated myocardium.

Gross specimens are valuable sources in morphology education. In this study, we investigated how the fixation of gross specimens may be accelerated. For this purpose, whole organ specimens from freshly killed rabbits: extremities, kidney, heart, liver, stomach and uterus were fixed in a mercaptoethanol-formaldehyde mixture for 3-3.5h under the following conditions: 1, at room temperature; 2, at gradually increasing temperatures up to 45 degrees C; and 3, at a gradually increasing vacuum ranging from 20 kPa to 40 kPa. The results were compared with those of formaldehyde-fixed controls, and the mercaptoethanol-formaldehyde mixture was found to be useful in shortening the fixation time and providing good fixation. Both heat and vacuum enhanced these phenomena.

We determined plasma human atrial natriuretic peptide (hANP) levels in normal pregnancy and pregnancy induced hypertension (PIH). The plasma hANP levels slightly decreased in the first trimester of normal pregnancy and tended to recover as pregnancy advanced, although these changes were slight. However, the plasma hANP level in puerperium was higher than that in the third trimester of normal pregnancy. The plasma hANP level in mild PIH was not significantly higher than that in the third trimester of normal pregnancy. In contrast, the plasma hANP level in three cases of severe PIH was approximately 200% higher than those in the normal third trimester and mild PIH.

Indocyanine green (ICG) was injected into rat liver nodules induced by 2-acetylaminofluorene (2-AAF) via portal vein. The relationship between ICG staining and cell atypism of liver nodules was examined by means of histology and DNA flow cytometry. After 2-AAF administration, many small nodules appeared on the liver surface. All hyperplastic nodules were ICG stained until 10 weeks after the administration, but some nodules were not stained after 14 weeks. ICG-stained nodules histologically consisted of benign tissues and borderline lesions, and many of them showed "diploidy" in DNA cytometry. ICG-unstained nodules consisted of hepatocellular carcinoma (HCCs) and borderline lesions, and many of them showed "aneuploidy". In this way, it has been suggested that HCC could derive from hyperplastic nodules and that they might lose an ability to take up ICG in the process of hepatocarcinogenesis. Immunohistochemical staining for glutathione-S-transferase alpha (GST-alpha), a carrier protein of ICG in hepatocytes, was well correlated with ICG staining in the nodules, suggesting that the loss of ICG uptake in HCC was partly due to the decrease of GST-alpha. Moreover, the appearance of ICG unstained and aneuploid nodules was significantly inhibited in rats which were fed on diet containing Syosaiko-to after the administration of 2-AAF. Chemopreventive effect of Syo-saiko-to on hepatocarcinogenesis was identified.

A new model of status epilepticus (SE), which was induced by intermittent electrical stimulation (20 Hz for 20 sec every min for 180 min) of the deep prepyriform cortex, has been developed in the conscious rat. SE was induced in 9 of 16 rats in the drug-free group. The number of stimulation trains required to induce SE in this status subgroup was 125.6 +/- 12.7 (mean +/- SEM) and the mean duration of self-sustained seizure activity (SSSA) occurring after cessation of the stimulation session was 295.4 +/- 111.4 min. Some animals showed secondary generalized seizures. Significant cell loss was observed in the hippocampal CA3 pyramidal cell layer ipsilateral to the stimulation site and bilateral CA1 areas in the status subgroup compared with the group subjected to sham operation. In addition, there was a significant negative correlation between the duration of SSSA subsequent to the stimulation session and the total number of intact pyramidal neurons observed in the bilateral CA1 and ipsilateral CA3 subfields of the status subgroup. There were significant differences between the status and non-status subgroups with respect to the number of afterdischarges (ADs) and the total AD duration during the stimulation session. Pretreatment with phenobarbital (30 mg/kg) prevented the development of SE and hippocampal cell loss completely. Pretreatment with MK-801, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist (0.25 or 1 mg/kg), also prevented hippocampal cell loss, although it did not block SE generation completely, which suggests dissociation of the mechanisms underlying the development of SE and hippocampal damage. These results indicate that prolonged SSSA actually causes hippocampal damage and it is critically dependent upon NMDA receptor participation.

The proliferative activity of various parts of normal and malignant endometrium was evaluated using an immunohistochemical approach and flow cytometry (FCM). The two monoclonal antibodies, Ki-67 and anti-DNA polymerase alpha antibody (anti-poly alpha antibody) were used to detect the proliferative activity of cells, and the percentage of the Ki-67 and anti-poly alpha positive cells were measured. Proliferative indices (PI; percentage of S and G2M phase) and DNA ploidy were measured by FCM. Normal endometrial specimens from 29 patients with benign diseases were used and three different parts (fundus, middle, and low part of the uterus) were examined. In the proliferative phase of normal endometrium, there was no significant difference in the proliferative activity in the three parts. In 20 patients with endometrial carcinomas with myometrial invasion, tissues were taken from the myometrial invasive site and the central part of the tumor tissue. In the cases of endometrial carcinoma, the myometrial invasive site had a higher proliferative activity than central part of the tissue. The proliferative activity measured by the immunohistochemistry was correlated with the histological grade of malignancy, but it was not consistent with PI by FCM. This suggests that the proliferative activity measured by the immunohistochemistry is independent of flow cytometric PI.

Radiological findings on the fate of grafted Kiel bone implants for the treatment of bone tumors were evaluated in 25 lesions. The mean follow-up period was 14.8 years, ranging from 5 to 21.8 years. We classified the radiological findings into 4 grades; Excellent (4 lesions), Good (14 lesions), Fair (2 lesions), and Poor (5 lesions). All cases of the Poor grade were polyostotic fibrous dysplasia. The younger the patient at the time of the operation, the more rapidly Kiel bone grafts tended to be incorporated. The grafted bone can become enmeshed in the structure of the recipient bed (Good or Excellent grades) within 10 years in most cases, except in polyostotic fibrous dysplasia.

To establish the most proper method of in situ hybridization in detection of HCV-RNA in the liver, various detailed procedures were examined using frozen as well as paraffin-embedded sections of tissue derived from patients. In frozen sections of the liver from hepatitis C patients obtained at autopsy or surgery, HCV-RNA was detectable by in situ hybridization using thymine-thymine dimerized oligonucleotide DNA probes when the sections were treated with ethanol-acetic acid at first, then 0.2 N hydrochloric acid, proteinase K (0.02 u/ml) and DNase. When the paraffin-embedded liver sections were used, more intense proteinase K treatment (0.2-2 u/ml) was required to expose viral RNA and even after that, the positive HCV-RNA signals were less than those in frozen sections, because the cytoplasmic RNA in the routine paraffin-embedded sections was preserved unevenly and less than in frozen sections. These findings indicate that in situ hybridization of HCV-RNA is useful for diagnosing HCV infection and should be a potent tool for monitoring the state of virus activities during therapy. However, the liver biopsy method should be modified so that RNA is retained properly to utilize biopsies more effectively for the routine diagnosis of HCV infection.

We detected an antibody to HCV envelope protein (E1) in sera of patients with HCV-related chronic liver diseases (20 patients with chronic hepatitis and 5 patients with liver cirrhosis) by Western blotting using the fusion protein of E1 envelope protein and beta-galactosidase as an antigen. The antibody to HCV E1 (anti-HCV E1) was detected in 8 (42%) of 19 patients positive for HCV-RNA (16 were positive and 3 were negative for antibody to C100-3) and in 1 (17%) of 6 patients negative for HCV-RNA but positive for antibody to C100-3. HCV-RNA was detected in 8 (89%) of 9 anti-HCV E1 positive sera. The value of alanine aminotransferase was significantly higher in patients positive for anti-HCV E1 than in patients negative for the antibody. Although an antibody to the envelope protein of HCV is suspected to be one of the candidates of virus-neutralizing antibodies, our results suggest this hypothesis appears to be unlikely.

Trans-sternal bilateral thoracotomy was performed to resect the right upper lobe and the left S1 + 2 + S3, and to complete lymphadenectomy in a 35-year-old female case of lung cancer in whom multiple lesions were suspected. Trans-sternal bilateral thoracotomy was considered to be useful for one-stage surgery in patients in whom bilateral lung cancer is suspected or confirmed, because it provides a sufficient surgical field enabling the resection of lung and lymph nodes. This may be the first case report of trans-sternal bilateral thoracotomy to treat multiple primary lung cancer.

Cytotoxic anti-thyroid microsomal autoantibodies are highly prevalent in sera of patients with Graves' disease, but in Graves' disease thyroid tissues rarely show destructive changes. We postulated that this might be due to membrane-associated complement regulatory proteins which protect target cells from injury by complement activation. We, therefore, investigated the expression of membrane attack complex inhibitory factor (MACIF) and decay accelerating factor (DAF) in the thyroid tissues from patients with Graves' disease, Hashimoto's thyroiditis, thyroid adenocarcinoma and normal human thyroid tissues. We found a high level of expression of MACIF and DAF in Graves' thyroid tissues. Using the membrane immunofluorescence and cell-ELISA techniques, we also investigated the factors which enhanced the MACIF and DAF expression in cultured thyroid cells. Thyroid stimulating hormone, phorbol 12, 13-dibutyrate and thyroid stimulating autoantibody enhanced the MACIF and DAF expression. These findings suggest that the membrane complement regulatory proteins increase in response to the thyroid stimulating factors such as thyroid stimulating autoantibody in Graves' disease and that this increase then protects the cells from damage due to complement activation by thyroid autoantibodies.

Bronchial asthma was classified by the pathophysiology and by the mechanism of onset of the disease. Forty asthmatics who had serum IgE levels lower than 200 IU/ml were evaluated by two classification methods. 1. In asthma classified by a score based on clinical findings and examinations, the characteristics of the findings and examination results were compared among three asthma types, i.e., Ia. simple broncho-constriction type, Ib. bronchoconstriction+hypersecretion type, and II. bronchiolar obstruction type. Type Ib patients, in addition to manifesting hypersecretion, had a significantly higher proportion of eosinophils in the bronchoalveolar lavage (BAL) fluid compared to other asthma types. Significantly decreased values for ventilatory parameters and an increased proportion of BAL neutrophils were found in type II compared with other asthma types. 2. In a new classification by mechanism of onset, asthma was classified into three types according to the degree of participation of IgE-mediated reactions associated with specific IgE antibodies and serum levels of total IgE: asthma induced by definite IgE-mediated reaction (atopic asthma), possible IgE-mediated reactions (asthma), and asthma induced by non-IgE-mediated reaction (asthma syndrome).

We measured hepatitis C virus antibody titers in 13 patients with chronic hepatitis C to determine whether titration of hepatitis C virus antibody was useful or not, to predict and evaluate the efficacy of interferon (IFN) treatment. During administration of IFN, hepatitis C virus titers declined in all patients. Antibody titers performed before treatment as well as just at the end of treatment did not correlate with change of the alanine aminotransferase levels during administration of IFN. Antibody titers declined continuously after treatment in 5 patients with normal alanine amino-transferase levels for over 6 months after discontinuation of IFN. Antibody titers rose again in 6 patients whose alanine aminotransferase levels fluctuated after treatment. An exceptional pattern of change occurred in 2 patients whose antibody titers declined continuously although their alanine aminotransferase levels fluctuated after treatment. Repeated titration of hepatitis C virus antibody appears to be useful for evaluating the long-term efficacy of IFN treatment.

To test the hypothesis that the endothelium-derived relaxing factor (EDRF) contributes to coronary vasodilation induced by myocardial ischemia, we examined the effect of NG-nitro-L-arginine (a potent and selective inhibitor of EDRF release) on the coronary reactive hyperemic response in the open-chest dogs. Intracoronary infusion of NG-nitro-L-arginine at a coronary plasma concentration of 5 x 10(-5) M had no effect on hemodynamics and myocardial oxygen metabolism, but attenuated repayment of the flow debt by an average of 20.4% and 20.0% following coronary occlusion for 10 sec and 20 sec, respectively. Concomitant infusion of NG-nitro-L-arginine at the same concentration and 8-phenyltheophylline (a potent adenosine receptor blocker) at a coronary plasma concentration of 10(-5) M further attenuated flow debt repayment following 10 sec and 20 sec of coronary occlusion by 47.7 and 59.4%, respectively. These results indicate that EDRF plays a significant role in the coronary reactive hyperemic response and may cause vasodilation independently of adenosine-mediated vasodilation following coronary occlusion.

The present study was undertaken to determine whether a biventricular bypass system operated in an independent variable rate (VR) mode can maintain the entire circulation. Two pusher-plate pumps which incorporated the Hall effect position sensors were used to bypass the right and left ventricles in 10 sheep under fibrillation. The flow distributions of the pump output to the carotid and renal arteries were investigated every 6 h using ultrasonic blood flow meters for 24 h in 5 animals, and the controllability of the VR mode was evaluated in 5 long-term experiments. The carotid artery flow ratio to the pump output decreased significantly from 4.7 +/- 0.8% before the bypass to 2.7 +/- 0.9% after 24 h. However, the renal artery flow ratio did not change throughout the experiments. In the long-term experiments, the animals were kept alive from 3 to 48 days (mean 15.6 days). The mean pump output had been maintained at more than 90 ml/min/kg for the first 7 days. After the surgery, the pump driving conditions were not readjusted in any experiment. The results indicate that the biventricular bypass system operated in the independent VR mode automatically maintains the entire circulation at a satisfactory level.

A case of biliary cystadenocarcinoma that occurred in a 45-year-old woman is reported. Ultrasonography and computed tomography clearly revealed papillary projections in the cyst of the liver. Percutaneous transhepatic cystography showed connection between the cyst and the common bile duct. The tumor was surgically resected and proved to be a mucinous papillary adenocarcinoma arising from a biliary cystadenoma. The patient is doing well 4 years after surgery. Interestingly, this is the first reported case of a biliary cystadenocarcinoma in the liver with markedly diminished excretion of indocyanine green.

The percentages and absolute numbers of gamma delta T cells per CD3 positive cells (T cells) in four different compartments, namely peripheral blood, synovial fluid, synovial membrane and lungs from patients with rheumatoid arthritis (RA) and in peripheral blood from healthy controls were studied by two color flow-cytometric analysis. The percentages (mean +/- SEM = 6.3 +/- 0.8%, n = 22) and absolute numbers (70 +/- 11/microliters, n = 22) of gamma delta T cells in peripheral blood from RA patients were not different from those of 22 age-matched healthy controls (7.5 +/- 0.9%, 81 +/- 17/microliters, respectively). The gamma delta T cells in peripheral blood from 50 RA patients were, however, significantly decreased in negative correlation with the value of CRP as a marker for inflammation, although they had no correlation with the titer of rheumatoid factor as an autoantibody. The percentages of gamma delta T cells in synovial fluid from 10 patients (3.3 +/- 0.5%, n = 10) or in synovial membrane from 5 patients (4.2 +/- 1.9%, n = 5) and in bronchoalveolar lavage fluid from 6 patients (3.6 +/- 0.8%, n = 6) were not different from those in peripheral blood from the same patients. Thus, gamma delta T cells are not the dominant infiltrating T cell subset in the inflammatory sites of RA patients.

To investigate the role of Kupffer cells in complement activation, we used a rat model of acute hepatic injury induced by D-Galactosamine (GalN) and lipopolysaccharide (LPS). In in vivo study, minimal histological changes were observed after i.p. GalN (200 mg/kg) single administration. Complement hemolytic activity (CH 50) decreased to 70% of its initial value 2-3 h after i.p. LPS (1.5 mg/kg) single administration. Massive hepatic necrosis was induced by simultaneous administration of GalN and LPS. After 2-3 h, CH 50 decreased to 70% of its initial value, and deposition of C3 fluorescence (C3) was observed in Kupffer cells. After 4 h, GPT was greatly increased (1286 +/- 240 IU/l), CH 50 was further reduced, and C3 was observed on hepatocyte membranes and in the cytosol. In in vitro study, we used hepatocyte cultures and co-cultures of hepatocytes and Kupffer cells to investigate the participation of GalN, LPS, complement, and Kupffer cells in hepatic cell necrosis. We found no increase of LDH (% leakage) when LPS and complement were added to the medium (22.7 +/- 5.7%). A moderate increase was observed with the addition of GalN (33.2 +/- 2.6%). A remarkable increase was observed only with the addition of GalN, LPS, and complement to the co-culture (50.0 +/- 8.8%). These results suggest that Kupffer cells activated by LPS are very important in promoting acute hepatic injury by complement.

The influences of ventricular pacing at a rate of 70 beats/min (bpm) on the systemic and coronary hemodynamics, myocardial metabolism, and cardiac work efficiency were evaluated in five patients with bradycardia. The results were compared to those obtained in six normal subjects at rest. In order to elucidate the effects of a relatively high rate of ventricular pacing, cardiovascular and metabolic variables were also obtained at 120 bpm in the normal subjects. It was observed that the patients eventually benefited from ventricular pacing at a rate of 70 bpm and improved in systemic hemodynamics. Although coronary hemodynamics and myocardial metabolism were accelerated, the cardiac work efficiency was not improved. A pacing rate of 120 bpm in the normal subjects did not appear to accelerate systemic hemodynamics, but adverse accelerations of coronary hemodynamics and myocardial metabolism were observed, and the cardiac work efficiency was remarkably reduced as a result. Our observations indicated that the coronary reserve capacity was very important for ventricular pacing, and suggested that an undue increment of the pacing rate not only might be meaningless but also might induce ischemic angina. Therefore, we should be cautious in using a rate-responsive pacing mode, particularly in determination of the upper limit of pacing rates, although many benefits with this pacing mode have recently been advocated.