Modulation of doxorubicin sensitivity by cyclosporine A in hepatocellular carcinoma cells and their doxorubicin-resistant sublines.

BACKGROUND AND AIMS: Cyclosporine A (Cys) and verapamil (Ver) sensitize multidrug-resistant (MDR) cells to various anticancer drugs by interacting with membrane glycoproteins involved in the drug efflux. In the present study, we assessed the effect of Cys on the modulation of doxorubicin (DOR) sensitivity in hepatocellular carcinoma (HCC) cell lines, and their DOR-resistant sublines. METHODS: The sensitivity to DOR and the chemosensitizing effects of Cys were assessed by using two human HCC cell lines, PLC/PRF/5 and Hep-3B, and their DOR-resistant sublines, PLC/DOR and 3B/DOR. The expression of multidrug resistance 1 (MDR1) and multidrug resistance-associated protein (MRP) mRNA in these cells were detected by using a RT-PCR. The HCC cell lines grown in individual wells of 24-well plates were incubated with DOR that were sequentially diluted in culture medium in combination with 5 micromol/L Cys for 24 h. The cell viability in each well was measured by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: The mRNA of MDR1 and that of MRP were readily detectable in the HCC cell lines by RT-PCR. When 5 micromol/L Cys was added to the culture, the 50% inhibiting concentration (IC50) of DOR was reduced from 0.93 +/- 0.29 microg/mL to 0.32 +/- 0.10 microg/mL in PLC/PRF/5, and from 0.25 +/- 0.07 microg/mL to 0.09 +/- 0.04 microg/mL in Hep-3B. Furthermore, in the presence of 5 micromol/L Cys, the IC50 of DOR was reduced from 48.63 +/- 17.04 microg/mL to 0.49 +/- 0.14 microg/mL in PLC/DOR, and from 4.60 +/- 1.22 microg/mL to 0.15 +/- 0.06 microg/mL in 3B/DOR. The amounts of PCR products of MDR1 mRNA in PLC/DOR and 3B/DOR were greater than those in PLC/PRF/5 and Hep-3B, respectively. CONCLUSIONS: In HCC, the amplification of MDR1 mRNA is probably the main mechanism underlying acquired DOR resistance. Cyclosporine is also indicated to be highly active in potentiating the anticancer activity of DOR in HCC cells and their DOR-resistant sublines.

Focal nodular hyperplasia coexistent with hemangioma and multiple cysts of the liver.

We report here a case of hepatic focal nodular hyperplasia (FNH) associated with hepatic hemangioma and multiple hepatic cysts in a 71-year-old man. He was admitted to our hospital because of body weight loss. Ultrasonography detected multiple cysts. and two tumors in the liver one, 3.5 cm and one, 1.6 cm. Color Doppler ultrasonography showed arterial signals within the large tumor. On dynamic computed tomography, the large tumor was a hypodense lesion which was enhanced during the arterial phase and almost isodense during the delayed phase: the small tumor was also a hypodense lesion, and was enhanced during both the arterial and delayed phases. On magnetic resonance imaging using superparamagnetic iron oxides, the large tumor had decreased signal intensity on the T2-weighted image. On hepatic arteriography, the feeding artery of the large tumor showed a spoke-wheel appearance and that of the small tumor showed a cotton-wool appearance. Ultrasonographically guided fine-needle aspiration biopsy of the large tumor revealed hepatocellular hyperplasia. Finally, we diagnosed the two hepatic tumors as FNH and hemangioma. There was no intracranial lesion. The cause of the patient's emaciation was psychogenic anorexia. To our knowledge, this is the first case report that describes the simultaneous occurrence of these three kinds of hepatic lesions. The pathogenesis of FNH still remains unclear, but this association suggests that FNH may arise because of a vascular anomaly.

Increase in CD95 (Fas/APO-1)-positive CD4+ and CD8+ T cells in peripheral blood derived from patients with autoimmune hepatitis or chronic hepatitis C with autoimmune phenomena.

BACKGROUND: The expressions of CD95 (Fas/APO-1) and Bcl-2 are determinants of apoptosis in normal lymphocytes, and abnormalities in their expressions might contribute to the induction of autoimmunity. In this study, we examined the expressions of CD95 and Bcl-2 on freshly isolated T and B cells from patients with autoimmune hepatitis (AIH) or chronic hepatitis C associated with autoimmune phenomena (CH-C(AI)). METHODS: The CD95 and Bcl-2 expressions within CD4+ T, CD8+ T, and CD19+ B cell subsets were analysed by two-colour flow cytometry. RESULTS: The surface expression of CD95 was significantly high in both the CD4+ T and CD8+ T cell subsets derived from the patients with AIH and those with CH-C(AI), compared with expression in patients with CH-C and normal subjects. The increase in CD95 expression was associated with the phenotypic conversion of naive CD45RO- to primed CD45RO+ CD4+ T cells. Bcl-2 was detected in the vast majority of peripheral T and B cells. There was no significant difference in the percentage of Bcl-2-positive cells in the CD4+ T cell, CD8+ T cell and CD19+ B cell subsets among the patient groups and normal subjects. CONCLUSIONS: These results indicate that an increase in CD4+ T cells expressing CD45RO and CD95 marks an important subset of AIH and CH-C(AI) patients. These expanded CD95+ CD45RO+ primed T cells most likely reflect a continuous antigen-specific or non-specific activation of T lymphocytes, and/or the persistent presence of activated lymphocytes as a consequence of abnormalities in the peripheral deletion of activated lymphocytes. These persistently activated lymphocytes might play a role in the induction of autoimmunity in AIH and CH-C(AI).

Association of chronic infection of Chlamydia pneumoniae and coronary heart disease in the Japanese.

The association of Chlamydia pneumoniae with atherosclerosis of coronary and carotid arteries and the aorta has been demonstrated by seroepidemiology and by detection of the organism in atheromata. We investigated the frequency of C. pneumoniae seropositivity in patients with acute myocardial infarction (AMI). C. pneumoniae-specific antibodies were measured by the microimmunofluorescence test in 160 AMI patients and 160 control subjects matched for age and gender. The odds ratios (ORs) were 2.2 (95% confidence interval (CI), 1.2 to 3.9) for immunoglobulin (Ig)G and 2.7 (95% CI, 1.7 to 4.3) for IgA. After adjustment for other cardiovascular risk factors of age, gender, hypertension, diabetes, cigarette smoking and serum cholesterol, the ORs were essentially unchanged. This study confirmed that the observations of an association between antibody against C. pneumoniae and coronary heart disease in Western nations is also present in Japan. Our results are comparable to the previous seroepidemiological studies reporting ORs of 2.0 or greater.

[Mechanism and clinical usefulness of S4-S1 interval in heart failure associated with left ventricular inflow pattern].

The interval between S4 and S1 detected by auscultation or phonocardiography is prolonged by exacerbation and shortened by improvement of heart failure. The timing of S4, S1, and the terminal point of the A wave of transmitral inflow velocities on pulsed Doppler echocardiography (At) was studied to elucidate the mechanism of the prolongation of the S4-S1 interval on exacerbation of heart failure. The study population consisted of 30 patients, nine with old myocardial infarction, six with dilated cardiomyopathy, six with hypertensive heart disease, nine with chronic hemodialysis, and 17 normal subjects. The interval from the peak of the A wave by apexcardiography and At to the onset of main vibration of S1 were measured as the S4-S1 interval and At-S1 interval, respectively. The P-Q interval and Q-S1 interval were also measured. Both intervals were compared during exacerbation and improvement of heart failure. Patients with P-Q prolongation were excluded. The S4-S1 interval was 102 +/- 24 msec during exacerbation of heart failure or before hemodialysis, and shortened to 76 +/- 18 msec after improvement of heart failure or after hemodialysis. The At-S1 interval was concordantly shortened from 59 +/- 31 msec to 30 +/- 23 msec (p < 0.001). However, both the P-Q interval and Q-S1 interval were not significantly changed before and after improvement of heart failure. The timing of S4 becomes parallel to that of At earlier during the exacerbation of heart failure. Thus, S4-S1 interval is a convenient and useful index to investigate patients with heart failure.

Genomic organization and mutational analysis of HERG, a gene responsible for familial long QT syndrome.

Familial long QT syndrome (LQTS) is characterized by prolonged ventricular repolarization. Clinical symptoms include recurrent syncopal attacks, and sudden death may occur as a result of ventricular tachyarrhythmias. Three genes responsible for this syndrome (KVLQT1, HERG, and SCN5A) have been identified so far, and mutations have been reported on the basis of partially characterized genomic organization. To optimize the search for HERG mutations, we have determined the genomic structure of HERG and investigated mutations in LQTS families. Human genomic clones containing the HERG gene were isolated from a human genomic library by using reverse-transcribed polymerase chain reaction (RT-PCR) products from this gene as probes. We determined exon/intron boundaries and flanking intronic sequences by using primers synthesized on the basis of the HERG cDNA sequence available in the DNA database. HERG was shown to consist of 15 exons spanning approximately 19 kb on chromosome 7q35. Subsequently, we synthesized oligonucleotide primers to cover the entire coding region and searched for mutations in 36 Japanese LQTS families. When genomic DNA from each proband was examined by the PCR/single-strand conformation polymorphism technique followed by direct DNA sequencing, five novel mutations were detected. Each mutation was present in affected relatives of the respective proband. This work should increase the efficiency of screening mutations associated with HERG.

[Estimation of left ventricular systolic function based on the electrocardiograms in cases with left bundle branch block].

The electrocardiographic features indicating left ventricular dysfunction were studied in 32 consecutive patients having left bundle branch block including 10 with idiopathic genesis without significant underlying disease, 6 with dilated cardiomyopathy, 8 with old myocardial infarction, and 8 with hypertensive heart disease. The patients were divided into two groups; those with favorable left ventricular systolic function and those with poor left ventricular systolic function evaluated by using non-invasive methods. Electrocardiographic findings were compared between these two groups. Ten patients had favorable and 22 poor left ventricular systolic function. One or more of the following electrocardiographic findings were observed in the poor group, but none in the favorable group: low voltage in the limb leads, prolonged intraventricular conduction (QRS duration wider than 0.17 sec), transitional zone between V5 and V6, depression of the ST-J point by more than 0.2 mV in V6, reverse progression of the R wave in V1-V5, marked left axis deviation (axis beyond: 30 degrees), left atrial overload (positive Morris index), PQ prolongation, and abnormal Q waves in I, aVL, V6. No significant differences in the distribution of these findings was observed in any of the underlying diseases. The clinical background of patients with left bundle branch block who had no significant underlying disease showed favorable left ventricular systolic function except the patients above 80 years of age, who showed poor left ventricular systolic function. In contrast, patients with underlying mild hypertensive heart disease may have a favorable left ventricular systolic function. Thus, left ventricular systolic function in patients with left bundle branch block may be suspected by observing these electrocardiographic findings.

Electrocardiographic features differentiating dilated cardiomyopathy from hypertrophic cardiomyopathy.

To determine the usefulness of electrocardiographic (ECG) features in differentiating between hypertrophic cardiomyopathy with features mimicking dilated cardiomyopathy (D-HCM) and true dilated cardiomyopathy (DCM), we compared ECGs of 52 consecutive patients (11 with D-HCM, 41 with DCM). Left atrial dimension, left ventricular internal dimension, and septal and posterior wall thickness were employed as echocardiographic indexes, while QRS duration, amplitude of RV5 or V6 + SV1, number of abnormal Q waves, P-terminal force in V1, and frontal plane QRS axis were used as ECG parameters. The patients with D-HCM demonstrated a larger number of abnormal Q waves (P < .0001), greater prolongation of QRS duration (P < .0001), and lower amplitude of RV5 or V6 + SV1 (P < .0001). In all cases of D-HCM, atrial overload was observed and abnormal QRS axis in 9 (82%) of the 11 patients. These features were noted in 21 (51%) and 17 (41%), respectively, of the 41 DCM patients (P < .005 and P < .05, respectively). Despite significant differences in the echocardiographic parameters between D-HCM and DCM, excluding left ventricular end-diastolic dimension, ECG abnormalities were more significant between the two groups. The results indicate that ECG features are extremely useful in differentiation between DCM and D-HCM.

Four novel KVLQT1 and four novel HERG mutations in familial long-QT syndrome.

BACKGROUND: Familial long-QT syndrome (LQTS) is characterized by prolonged ventricular repolarization. Clinical symptoms include recurrent syncopal attacks, and sudden death may occur due to ventricular tachyarrhythmias. Three genes responsible for this syndrome (KVLQT1, HERG, and SCN5A) have been identified so far. We investigated mutations of these genes in LQTS families. METHODS AND RESULTS: Thirty-two Japanese families with LQTS were brought together for screening for mutations. Genomic DNA from each proband was examined by the polymerase chain reaction-single-strand conformation polymorphism technique followed by direct DNA sequencing. In four of the families, comprising 16 patients, mutations were identified in KVLQT1; five other families (9 patients) segregated mutant alleles of HERG. All 25 of these patients carried the specific mutations present in their respective families, and none of 80 normal individuals carried these alleles. Mutations were confirmed by endonuclease digestion or hybridization of mutant allele-specific oligonucleotides. No mutation in SCN5A was found in any family. CONCLUSIONS: We identified nine different mutations among 32 families with LQTS. Eight of these were novel and account for 25% of all types of mutations reported to date. Such a variety of mutations makes it difficult to screen high-risk groups using simple methods such as endonuclease digestion or mutant allele-specific amplification.

Clinical significance of abnormal postexercise systolic blood pressure response in patients with hypertrophic cardiomyopathy.

The clinical significance of abnormal postexercise systolic blood pressure (SBP) response in patients with hypertrophic cardiomyopathy (HCM) was assessed by treadmill exercise testing in 30 normal subjects and 96 patients with HCM. SBP recovery ratios were derived by dividing the SBP at 1 and 3 min after exercise by the peak exercise SBP. The upper normal limit of the SBP ratio was defined by two standard deviations (SD) from the mean in 30 normal subjects. A postexercise SBP ratio that exceeded the upper normal limits of the SBP ratio was considered to be an abnormal SBP response in patients with HCM. Twenty-seven (28%) of 96 patients with HCM showed an abnormal SBP response. Compared with the normal SBP response group (69 cases) among patients with HCM, the abnormal SBP response group had lower SBP response during exercise (22 +/- 25 vs 62 +/- 26 mmHg : mean +/- 1 SD, p < 0.05), more prolonged QRS width (110 +/- 21 vs 92 +/- 20 msec, p < 0.05), higher incidences of ventricular tachycardia (12 vs 7 cases, p < 0.001), and sudden death (7 vs 0 cases, p < 0.0001). The defect area of Tl-201 myocardial scintigraphy was more extensive in the abnormal SBP response group (2.9 +/- 1.7 segments) than in the normal SBP response group (1.1 +/- 1.3 segments, p < 0.05). During atrial pacing (120/min), pulmonary artery wedge pressure was slightly elevated from 10 +/- 2 (at rest) to 14 +/- 3 mmHg (during pacing) (p<0.001), cardiac index showed no significant changes, and time constant T was shortened from 58 +/- 13 to 48 +/- 10 msec (p < 0.001) in the normal SBP response group, but in the abnormal SBP response group pulmonary artery wedge pressure was highly elevated from 12 +/- 5 to 20 +/- 3 mmHg (p < 0.0001), cardiac index was decreased from 2.5 +/- 0.7 to 2.1 +/- 0.6 l/min/m2 (p < 0.05), and the time constant T had no significant changes. These observations suggest that patients with HCM and abnormal postexercise SBP response have an abnormal cardiac response during exercise and extensive myocardial damage.

[Prognostic importance of pseudonormalized left ventricular inflow pattern especially for sudden cardiac death].

The prognostic value of left ventricular inflow velocities by the pulsed Doppler method was studied in 32 patients with congestive heart failure including 18 with old myocardial infarction, 9 with dilated cardiomyopathy, and 5 with hypertensive heart disease, who initially revealed pseudonormalized left ventricular inflow pattern. Pulsed Doppler echocardiography, apexcardiography, and phonocardiography were performed at 3- to 9-month intervals. The prognosis was evaluated for two groups of patients with persistent or transient pseudonormalized inflow patterns. Survival rates at 6 months and 2 years in the total patient population were 78% and 47%, respectively. Twenty-one patients had the pseudonormalized left ventricular inflow pattern (persistent group), while the other 11 patients had a changed pattern (transient group). The survival rate at 2 years was 37% for the persistent group, and 82% for the transient group. Significant differences in patient profiles and the initial data between the two patient groups were the presence of cardiogenic shock (10/21 in persistent group vs 1/11 in transient group; p < 0.001) and the value of left ventricular end-diastolic wall stress (159 +/- 62 g/cm2 in persistent group vs 135 +/- 42 g/cm2 in transient group; p < 0.05). There were no significant differences in NYHA class, the values of left ventricular end-diastolic pressure, left ventricular ejection fraction. A/E ratio, or deceleration half time of left ventricular inflow velocities. Left ventricular end-diastolic wall stress in patients with persistent pseudonormalized left ventricular inflow pattern was significantly increased, and may be related to decreased preload reserve. Atrial fibrillation and atrioventricular dissociation were recorded prior to the development of ventricular fibrillation in two patients with sudden cardiac death. Abrupt loss of atrial contribution as well as ventricular arrhythmias may be a trigger of sudden death. Evaluation and follow-up of the pseudonormalized left ventricular inflow pattern is a sensitive indicator for the management of patients with congestive heart failure.

[Early diagnosis and management of acute pulmonary embolism: clinical evaluation those of 225 cases].

To contribute for making early diagnosis and treatment of acute pulmonary embolism (APE), we investigated on clinical pictures of 225 patients with APE. Common underlying factors were heart disease, prolonged bed rest, post-surgical state, thrombophlebitis, malignant tumor and post-catheterization state in this order. Dyspnea, chest pain, tachycardia and shock were frequently seen as initial symptoms and signs. Blood screening showed leukocytosis, hypoxemia, hypocapnia and elevated serum LDH. Electrocardiographic findings highly demonstrated were ST.T abnormalities, such as T inversion with ST elevation in V1-3, ST depression in V4-6 and sinus tachycardia. Chest X-rays showed diminished pulmonary vascular marking and pulmonary artery dilation. Right ventricular dilatation were frequently seen on 2-dimensional echocardiograms. Pulmonary artery pressure were elevated up to 49/20 (30) mmHg. Twenty-five percent of the patients died, and the recurrence was seen in 4%. Thus, as soon as APE is suspected by above clinical findings, definitive diagnosis should be obtained by the lung perfusion scan and pulmonary arteriography, then oxygen and thrombolytic agents should be given immediately to prevent the fatal outcome.

[Mechanism of discrepancy between mean pulmonary capillary wedge pressure and left ventricular end-diastolic pressure].

It is well known that discrepancies between mean pulmonary capillary wedge pressure (man-PCWP) and left ventricular end-diastolic pressure (LVEDP) exist in the pathological heart with sinus rhythm. We discussed the mechanism of these discrepancies in the aspect of increased LV end-diastolic stiffness. Fifty-two patients observed in this study included 23 with old myocardial infarction (OMI), 4 with hypertrophic cardiomyopathy and 9 with hypertensive heart disease (LVH group), 6 with dilated cardiomyopathy (DCM), and 10 normal subjects (N). All 52 patients had sinus rhythm. Those with significant mitral and aortic regurgitation were excluded. End-diastolic LV stiffness was evaluated by the ratio of increases in LV pressure and volume during atrial systole (delta P/delta V), as proved by cardiac catheterization and cine-angiography. Discrepancies between m-PCWP and LVEDP were 5.9 +/- 4.3 mmHg in OMI group, 4.5 +/- 4.6 mmHg in LVH group, 5.8 +/- 4.5 mmHg in DCM, and 1.6 +/- 1.8 mmHg in N group. These discrepancies correlated well with delta P/delta V (r = 0.74). More significant discrepancies were observed in patients with so-called pseudo-normalized left ventricular inflow velocities proved by pulsed Doppler echocardiography, and in patients with marked concentric LV hypertrophy with increased delta P/delta V. In clinical observation, symptoms of heart failure may be determined by m-PCWP rather than LVEDP. We concluded that discrepancies between m-PCWP and LVEDP were caused by the booster pump function of the left atrium against increased LV end-diastolic stiffness. By the use of apexcardiogram and echocardiogram including the pulsed Doppler method, it was possible to predict these discrepancies non-invasively.