Safety evaluation of the use of calcineurin inhibitor to prenatal and postpartum women in Japan from a health administrative database.

BACKGROUND: To investigate the use of calcineurin inhibitors (CNIs) in pregnant Japanese women and to evaluate their safety in infants. METHODS: Data were extracted from the claims database of the Japan Medical Data Center. The prevalence of CNIs was evaluated 180 days before pregnancy onset, during pregnancy, and within180-days post partum. We investigated the characteristics of the infants, including the presence of major malformations and their diagnoses, for 1 year after birth. RESULTS: A total of 91,865 pregnancies in 80,049 women were included. Fifty-three women were prescribed CNIs between 180-day before pregnancy onset and 180-day postpartum; 35 of the 53 women were prescribed the drugs during pregnancy, and 10 of their infants were born preterm. Three were diagnosed with major congenital malformations, such as patent ductus arteriosus. Six preterm infants presented with infant respiratory distress syndrome. CONCLUSIONS: No congenital anomalies were clearly attributable to the use of CNIs during pregnancy.

Deletions of chromosome 13q, mutations in Retinoblastoma 1, and retinoblastoma protein state in human hepatocellular carcinoma.

Twenty-one tumors from 18 patients (two independent tumors were obtained from each of three patients) with hepatocellular carcinomas were examined for loss of heterozygosity (LOH) at loci on chromosome 13q, using 15 polymorphic nucleotide sequences of microsatellites as genetic markers. The results revealed LOH in a common region between the centromeric D13S127 locus (13q12.2-q14.1) and the telomeric D13S137 (13q14.3) locus, including the RB1 locus, in nine of 21 tumors. Immunohistochemical staining of paraffin-embedded tumor sections indicated loss of retinoblastoma (RB) protein expression in all tumors showing LOH except one. Absence of RB protein expression was also observed in three of 12 tumors without LOH. Single-strand conformation polymorphism analysis of polymerase chain reaction products using primers flanking all 27 exons of the RB1 gene, as well as nucleotide sequencing, revealed tumor-specific small deletions of the RB1 coding region in the remaining RB1 allele of two tumors having LOH at the RB1 locus with concomitant loss of RB protein expression. Our results indicate that the loss of a region of chromosome 13q including the RB1 locus significantly (P < 0.006) correlates with loss of RB protein in hepatocellular carcinomas. However, tumor-specific mutations of the RB1 gene were detected in only two of 13 tumors with LOH and/or lack of RB protein expression, indicating that analysis of the RB1 status at the protein level in these tumors may be more sensitive than the actual mutational analysis.

Telomerase expression in respiratory epithelium during the multistage pathogenesis of lung carcinomas.

To investigate the role of telomerase in the multistage pathogenesis of lung cancer, we examined 205 fresh and archival tissue samples obtained from 40 patients, 34 of whom had invasive lung carcinoma, 5 with carcinoma in situ (CIS) without invasion, and 1 without lung carcinoma. We analyzed samples for telomerase enzyme activity using the semiquantitative PCR-based telomeric repeat amplification protocol assay (131 samples) or by a radioactive in situ hybridization method for expression of the RNA component of human telomerase (hTR; 74 samples). A subset of samples was assayed by both methods, and the correlation was excellent (30 of 36; 83%). With the exception of a carcinoid tumor and a necrotic squamous cell carcinoma, all tumor cells were moderate to strongly positive for both hTR and telomerase activity, except for foci of keratinization in squamous cell carcinomas. Telomerase positivity, with weak enzyme activity and/or low hTR expression, was present in basal epithelial cells of large bronchi, both histologically normal (26%) and hyperplastic (71%), and in 23% of peripheral lung samples (in epithelium of small bronchi and bronchioles or lymphoid aggregates). More advanced epithelial changes (metaplasia, dysplasia, and CIS) were associated with telomerase dysregulation. Dysregulation in preneoplasia was manifested in three ways: almost all such lesions expressed hTR, although enzyme activity levels were several-fold lower than in the corresponding invasive tumors; cells throughout these multilayered processes expressed hTR; and intense, focal up-regulation of hTR occurred in CIS foci in the vicinity of invasive cancers. Alveolar cells and areas of atypical adenomatous hyperplasia (possible precursor lesions for peripheral adenocarcinomas) were negative. Our studies demonstrate that dysregulation of telomerase occurs early in the multistage pathogenesis of bronchogenic lung carcinomas and that intense focal localized hTR expression in CIS may indicate imminent invasion.

Telomerase enzyme activity and RNA expression during the multistage pathogenesis of breast carcinoma.

Telomerase, an RNA-containing enzyme, is associated with cellular immortality and malignancy. We investigated the role of telomerase during the multistage pathogenesis of breast cancer. We used the semiquantitative, PCR-based telomeric repeat amplification protocol assay for enzyme activity (42 specimens from 42 patients) and a radioactive in situ assay for expression of its RNA component (human telomerase RNA; hTR) for the identification of telomerase-positive cells in archival resection samples (n = 67 from 39 patients). Low telomerase activity was detected in 1 (14%) of 7 samples of benign breast disease, in 4 (67%) of 6 fibroadenomas, in 11 (92%) of 12 carcinoma in situ (CIS) lesions, and in 16 (94%) of 17 invasive breast cancers. There was a progressive increase in the mean telomerase levels with progressive increase in severity of histopathological change (P < 0.05). Almost all of 67 resection samples expressed hTR, irrespective of histology. Expression was low to moderate in some samples of normal epithelium and nonproliferative fibrocystic changes. hTR expression was limited to epithelial cells; expression in stromal cells, including those in fibroadenomas, was negative. Increased hTR expression was observed in some foci of apocrine metaplasia and atypical hyperplasia. Increased hTR expression was also observed in all CIS and invasive lesions, although considerable heterogeneity was noted. Focal up-regulation was frequently noted in CIS lesions in the vicinity of invasive tumors. Thus, up-regulation of hTR may be a predictive marker for invasive tumor development.

Aberrations of the tumor suppressor p53 gene and p53 protein in solar keratosis in human skin.

Aberrations of the p53 gene in 27 solar keratoses were examined by the polymerase chain reaction and single-strand conformation polymorphism and DNA sequencing analyses. In a series of Japanese patients, eight of 27 cases (30%) of solar keratosis showed structural abnormalities of the p53 gene. Six of eight aberrations of p53 gene were determined to be single nucleotide substitutions, and five of these were located at a dipyrimidine site. In solar keratosis, noticeable mutations were C to T in three cases, and one each of C to A and T to C nucleotide changes. p53 protein was detected immunohistochemically in the nuclei of six of 27 cases (22%) of solar keratosis. Nuclear staining for p53 protein was only significantly correlated with the presence of missense mutation of p53 gene (p < 0.01). Aberrations of the p53 gene in solar keratosis may be a marker to predict early cancerous lesions.

Expression of telomerase RNA component correlates with the MIB-1 proliferation index in ependymomas.

Although there is general agreement that certain morphologic subtypes of ependymoma are benign, the biologic behavior of other ependymal neoplasms is poorly understood and not clearly related to conventional histopathologic criteria. The absence of universally accepted standards has prompted the search for more objective biologic markers. Telomerase is an RNA-containing enzyme associated with immortality in proliferating stem cells and many tumors. We investigated the proliferative activity of 26 ependymomas as determined by MIB-1 immunolabeling and compared the results with the in situ expression of human telomerase RNA (hTR) and WHO tumor grade. The study included 9 WHO grade I ependymomas (6 subependymomas and 3 myxopapillary ependymomas), 13 WHO grade II ependymomas, and 4 anaplastic (WHO grade III) ependymomas. The proliferation index (PI) and telomerase RNA expression were significantly increased in grade III ependymomas (p < 0.0001 for PI and p = 0.0015 for hTR). In these tumors, the PI and hTR expression were highly correlated (p = 0.0001). Of note, a single case designated grade II showed both increased proliferative activity and the highest hTR expression detected in this series of ependymal neoplasms. Our results suggest that the PI and hTR expression may be important biologic markers, independent of other histopathologic criteria of tumor grade. Future studies examining the correlation of MIB-1 cell kinetics and hTR expression with clinical parameters in selected ependymoma subtypes are needed to determine the prognostic relevance of these markers.

Human telomerase RNA expression and MIB-1 (Ki-67) proliferation index distinguish hemangioblastomas from metastatic renal cell carcinomas.

Hemangioblastomas are low-grade, capillary rich neoplasms of the cerebellum and spinal cord that can occur sporadically or in the setting of Von Hippel-Lindau syndrome. The present study analyzed the utility of proliferation potential in differentiating hemangioblastoma from RCC metastatic to the central nervous system using a MIB-1 (Ki-67) labeling index and assessment of expression of the RNA component of telomerase. Immunohistochemical analysis for epithelial membrane antigen (EMA) and MIB-1 was performed on paraffin-embedded sections of 27 hemangioblastomas and 5 RCC metastatic to the central nervous system. All but one hemangioblastoma demonstrated low or negative MIB-1 immunoreactivity, while 4 of 5 RCC metastases had moderate or high labeling indices. Telomerase RNA expression was assessed in 10 hemangioblastomas and in all 5 metastatic RCC by in Situ hybridization. All 10 hemangioblastomas demonstrated a lack of expression of telomerase RNA, while all 5 metastatic RCCs showed moderate to strong expression. Our results suggest that the MIB-1 labeling index is useful in differentiating hemangioblastoma from metastatic RCC and assessment of telomerase expression can also provide novel information on the difference in growth potential of these tumors.

Expression of the RNA component of telomerase in Wilms tumor and nephrogenic rest recapitulates renal embryogenesis.

Telomerase is a ribonucleoprotein enzyme associated with cellular immortality and has been detected in the vast majority of adult tumors. Wilms tumor is a histologically diverse embryonal malignancy of childhood, and the histological features of Wilms tumor and its precursor lesion, the nephrogenic rest, recapitulate the components of normal renal embryogenesis. Both the epithelial and the stromal components of Wilms tumor arise by differentiation of primitive mesodermal blastema. We compared expression of the RNA component of human telomerase (hTR) in normal developing kidneys, Wilms tumors, and nephrogenic rests and correlated expression of hTR with cell proliferation. Using a radioactive in situ hybridization method, we examined archival material from 17 Wilms tumors (including nine with nephrogenic rests), four therapeutically aborted embryos (37 to 56 days), three fetuses on whom autopsies had been performed, and one neonate for expression of hTR. Proliferative index was measured by immunohistochemical staining for MIB1. In the embryonic kidney, Wilms tumors, and nephrogenic rests, the patterns of hTR expression were similar: expression was usually maximal within the immature epithelial elements followed by the poorly differentiated blastema, but was weak or absent in the immature stroma. Mature tubules, glomeruli, and stroma were negative for hTR expression, as were differentiated heterologous elements present in post-therapy Wilms tumors. There was only a partial relationship between proliferative index and hTR expression. In the embryonic kidney, Wilms tumors, and nephrogenic rests, blastema had the highest proliferative index, whereas the indices were significantly lower in the immature epithelium and stroma. The proliferative index in mature and heterologous elements was low or zero. Thus, the pattern of hTR expression in Wilms tumor and its precursor lesion recapitulates embryogenesis precisely and may represent that aspect of the persistent fetal phenotype which predisposes to the development of malignancy.

Effects of long-term administration of sulindac on APC mRNA and apoptosis in colons of rats treated with azoxymethane.

PURPOSE: Non-steroidal anti-inflammatory drugs, including sulindac, have been shown to exhibit anti-colon cancer activity; however, the detailed mechanisms concerning continuous long-term administration are still unclear. Therefore, we examined the anti-colon carcinogenesis effects of sulindac after prolonged administration. METHODS: Administration of AOM, a colon-specific carcinogen, induced colonic preneoplastic lesions, which can progress to carcinomas about 40-50 weeks after AOM administration. We studied the effects of sulindac on the incidence of preneoplastic lesions, proliferative activity of colonic cells (AgNORs), tumor suppressor adenomatous polyposis coli (APC) gene expression, and apoptosis using AOM-treated rat colon mucosa at 4 weeks and 40 weeks (early and late stage of colon carcinogenesis, respectively). RESULTS: Sulindac suppressed the development of preneoplastic lesions induced by AOM at 4 weeks and 40 weeks by about 50% ( P<0.01); the proliferative activity of colonic cells increased by AOM was suppressed almost completely. Furthermore, APC expression was significantly increased by sulindac at both the early and late stages ( P<0.01). However, apoptosis was clearly increased at the early stage ( P<0.01), but not at the late stage. CONCLUSIONS: APC overexpression induced by sulindac can suppress colon carcinogenesis at both the early and late stages, but apoptosis might work as one of anti-cancer mechanisms at the early stage of colon carcinogenesis.

Telomerase activity and in situ telomerase RNA expression in malignant and non-malignant lymph nodes.

AIMS/BACKGROUND: Telomerase, an enzyme associated with cellular immortality, is expressed by most malignant tumours, but is inactive in normal somatic cells except for male germ cells and proliferating stem cells. Thus, the measurement of telomerase activity in tissue samples may provide useful diagnostic and prognostic information. The aim of this study was to determine whether telomerase expression is useful for the detection of occult malignant cells in lymph nodes. METHODS: Telomerase activity was compared with histological findings in 123 surgically removed lymph nodes submitted for routine or frozen section diagnosis. Telomerase activity was measured using a modified, semi-quantitative PCR-based telomeric repeat amplification protocol (TRAP). The assay was adapted for single 5 microns OCT embedded cryostat sections. In either fresh tissues or cryostat sections, normalised activity was linear when compared with protein concentration. Furthermore, using an in situ hybridisation method, the human telomerase RNA (hTR) component was measured in a subset of negative and positive nodes. RESULTS: Most (96%) of the 97 histologically negative nodes expressed low levels of activity (mean value of positive samples = 3.0 units/microgram protein) which may be derived from activated lymphocytes that express telomerase activity. All 26 malignant nodes (17 metastases, nine lymphomas) expressed telomerase (mean value = 17.8 units/microgram protein). The rank order levels between the two groups differed significantly (p = 0.0002). In situ results showed clearly that the hTR was expressed relatively highly in metastatic cancer cells and at lower levels in germinal centres of secondary follicles. CONCLUSIONS: Although expression of telomerase by activated lymphocytes may limit its usefulness, measurement of enzyme activity combined with detection of hTR using in situ hybridisation may assist in the histopathological diagnosis of lymph nodes.

Telomerase activity in benign and malignant thyroid diseases.

BACKGROUND: Telomerase, an enzyme associated with cellular immortality, is expressed by most malignant cells and is inactive in most normal somatic cells, with the excitation of proliferative stem cells, male germ cells, and activated lymphocytes. The measurement of telomerase activity in clinically obtained tissue samples may provide useful information as both a diagnostic and prognostic marker. In this study, we sought to determine whether telomerase activity might prove helpful in the assessment of benign and malignant thyroid tumors. METHODS: A modified, semiquantitative polymerase chain reaction-based telomeric repeat amplification protocol assay was used for detection of telomerase activity in 59 samples obtained at thyroidectomy, including 15 thyroid cancers, 22 benign thyroid diseases, and 22 adjacent normal thyroid tissues. RESULTS: Four of 13 differentiated thyroid carcinomas (30%) and 2 of 2 medullary carcinomas (100%) expressed telomerase activity. Unexpectedly, we also detected activity in 3 of 22 (14%) adjacent normal thyroid tissues and 6 of 22 (28%) benign thyroid diseases. Pathologic review of the telomerase-positive benign specimens revealed that many contained extensive lymphoid infiltrates with germinal centers (six of nine, 67%), as did two of four telomerase-positive papillary carcinomas. CONCLUSIONS: In contradistinction to other epithelial carcinomas, telomerase does not appear to be frequently reactivated in differentiated thyroid carcinomas.

Cyclooxygenase-2 expression in hepatocellular carcinoma.

BACKGROUND/AIMS: Cyclooxygenase-2 (Cox-2) is an isoform of cyclooxygenase, which is the key enzyme converting arachidonic acids to prostaglandins. It has been reported that Cox-2 is overexpressed in colon cancer, and that inhibition of this enzyme activity reduces colon cancer development in humans and animals. However, the significance of Cox-2 in human liver cancer is still unclear. To clarify significance of Cox-2 in liver cancer, we immunohistochemically examined expression of this enzyme in cancerous and non-cancerous tissues of hepatocellular carcinoma (HCC). METHODOLOGY: Twenty-nine patients with HCC were examined; 10 patients had well differentiated HCC, 10 had moderately differentiated HCC, and 9 had poorly differentiated HCC. RESULTS: Twenty-eight of 29 (97%) patients with HCC exhibited a positive staining. More intense staining of Cox-2 in cancer tissue rather than non-cancerous tissue was observed in 7 of 10 (70%) patients with well differentiated HCC, in 3 of 10 (30%) with moderately differentiated HCC, and in 3 of 9 (33%) with poorly differentiated HCC, respectively. Rate of higher expression of Cox-2 in cancer tissue rather than in non-cancerous tissue of well differentiated HCC was increased, compared to that of moderately and poorly differentiated HCC (7/10 vs. 6/19, p < 0.05). CONCLUSIONS: The results of the present study showed that Cox-2 is related to HCC whose histology is well differentiated.

Intestinal absorption of cytidine diphosphate choline and its changes in the digestive tract.

Intestinal absorption of cytidine diphosphate choline (CDP-choline), its structural changes in the digestive tract, and hepatic uptake have been investigated in rats using 14C-labeled (14CH3 attached to N of choline) and 3H-labeled (at C5 of pyrimidine) compounds. The results indicate that: 1)CDP-choline is relatively stable in the stomach, but is quickly degraded into cytidine and choline in the intestine; 2) The hepatic uptakes of 14C and 3H reach the maximum in two to three hours after oral administration; 3) Whereas the amount of 14C remaining in the gut is inversely related to the hepatic uptake, no similar correlation is seen with 3H-labeled CDP-choline, and 4) Extrahepatic uptake of 14C and 3H is very small. The possibility of phosphorylation in the mucosa of choline and cytidine has been discussed, based on the differences in individual broken-down products in the intestinal lumen and mucosa.

[Accuracy of participation rate data for health examination research].

Each local government conducts health examinations based on the Health and Medical Law for the Aged. However, since some residents are able to take health examinations at their own work places, for example, and the local government are allowed to exclude such people from taking the law-mandated health examination, it is difficult to obtain an accurate picture of the examination rate in each area. We investigated the actual participation status for health examination services of all of the 6,080 persons 20 years and over in age in Sakuragawa-mura, Ibaraki Prefecture. A comparative investigation was made on 3,655 non-bedridden/non-hospitalized persons of 40 years and over to ascertain the reliability of responses to questions about participation in lung cancer and gastric cancer examination services given by the village. The rate of valid responses was extremely low in those who had not participated in health examinations (male 54%, female 55% for the lung cancer, and male 53%, female 56% for the gastric cancer). These discrepancies are assumed to be the result of confusing the current health examinations with: (1) the health examination given in the previous year, (2) other kinds of health examinations, or (3) the health examination given in the work place or the like. A comparative investigation through logistic regression analysis, between the responses to the questions in this investigation and the actual health examination participation records, for persons who had not yet taken either lung cancer examinations or gastric cancer examinations (908 males and 938 females for the former, and 1,038 males and 1,187 females for the latter). Results showed that the influence of (1) and (2) were more or less detected in every kind of cancer examination, and the influence of (1) on the gastric cancer examination was particularly clear. No definite result was obtained about (3), because the actual record of the health examination service at the work place, etc. was unavailable. The results of this study suggests the necessity of a careful examination of methods when conducting a comprehensive service investigation for the health examinations.

Application of wavelet analysis to the plethysmogram for the evaluation of mental stress.

The present study proposes a new stress evaluation technique using the photoplethysmogram (PTG). Heart rate variability (HRV) is often used to evaluate mental stress. HRV can be measured from an electrocardiogram (ECG). The frequency variability of HRV and mental stress response are related. PTG also indicates changes in emotional response. PTG can easily be measured without body surface electrodes. This method is less invasive than ECG measurement. We attempt herein to evaluate mental stress by wavelet analysis of the PTG. PTG was measured in the resting and mental stress states, and wavelet transformed PTGs were compared. In nine out of ten subjects, the wavelet result for PTG revealed a decrease in the frequency band.

Demethylation of a repetitive DNA sequence in human cancers.

To detect DNA alterations in unknown regions in human cancers, we have performed restriction landmark genomic scanning (RLGS) analysis of DNA isolated from cancer and normal cells. One spot with a highly intensified signal was detected in DNA from all six malignant melanoma cell lines, two of five colon cancer cell lines and one of six pancreatic cancer cell lines analyzed. In DNA from normal cells, two placentas and seven cultured lymphocytes, the signal of this spot was not intense. The DNA fragment corresponding to the spot was cloned. By nucleotide sequence analysis, the DNA fragment was revealed to be a part of a repeating unit of a 13 kbp nucleotide sequence of which 200 copies were located in chromosome 8q21. Southern blotting analysis using the cloned fragment as a probe demonstrated that the intensified signal for the DNA fragment observed in cancer cells was due to demethylation in the recognition sequence of the NotI restriction enzyme. The results suggest that marked demethylation in the repeating units might be associated with the genesis or progression of some types of cancers.

Absence of activating mutations of the RAF1 protooncogene in human lung cancer.

Recently, the RAF protein has been demonstrated to be a direct effector of RAS protein in a RAS-mediated signal transduction pathway. Activations of the RAF1 gene by small mutations, such as point mutations in the kinase domain and a tetrapeptide insertion into conserved region 2, have been suggested from analyses of chemically induced lung cancers in mice and by site-directed mutagenesis. We investigated the presence of small mutations of the RAF1 gene in human lung carcinomas, especially in those not carrying the mutated RAS gene, expecting that aberrations of the RAF1 gene might play a role complementary to RAS gene mutations in tumorigenesis. Single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction products of DNA samples from 140 patients revealed no tumor specific mutations of the RAF1 gene in any of these specimens. This result suggests that mutations of the RAF1 gene are not involved in tumorigenesis in human lung.

Significance of rises in urinary bicarbonate contents and pH related with increased atmospheric carbon dioxide in Tokyo.

Atmospheric carbon dioxide concentration was measured at several locations in Tokyo, for two weeks, in December, 1995 and 1996, and was found to be increased up to 550 ppm, while it was shown by us to be 450 ppm in December, 1994. These results demonstrate that atmospheric carbon dioxide is steadily increasing at faster rates in Tokyo than we expect, though it has been considered that the atmospheric carbon dioxide is still as much as 350 ppm. Bicarbonate concentration and pH of urine of 13 medical students in Tokyo were also measured for the same period in December of 1995 and 1996, and were found to be significantly increased compared with the values that were reported in the past. Furthermore, urinary bicarbonate and pH were extensively increased, when 4 and 5 students made 3-hour car trip in two different cars with all windows closed, where carbon dioxide was increased up to about 5000 ppm within 1 hour. These results support our previous hypothesis that the increase of atmospheric carbon dioxide may be reflected by the increase of urinary bicarbonate and pH. Our results also suggest that the environmental situation is being seriously aggravated in Tokyo, year by year, in terms of atmospheric carbon dioxide.