Comparative analysis of MCP-1 and TF in elderly patients with acute exacerbations of COPD and its clinical significance.

OBJECTIVE: This study was conducted to investigate the variation of monocyte chemoattrant protein-1 (MCP-1) and tissue factor (TF) in elderly patients with acutely exacerbated chronic obstructive pulmonary disease (AECOPD) and their clinical significance. PATIENTS AND METHODS: Serum specimens were obtained from 49 AECOPD. Patients and 30 health controls, with mean age of 76.1 ± 10.2 and 62.8 ± 6.5 years. Patients in AECOPD group were further grouped into two subgroups, with high or normal procaletonin (PCT) levels. Plasma TE, MCP-1 and PCT were qualified by enzyme-linked immunosorbent assay (ELISA). RESULTS: TF and MCP-1 were found to be higher in AECOPD patients than in health people (p < 0.01), and TF was linearly and positively related to MCP-1. In the subgroups TF was significantly higher in patients with higher PCT than those with normal PCT (p < 0.05). CONCLUSIONS: In AECOPD patients blood cells are activated to hypercoagulation state, particularly when their PCT level is high. Extrinsic pathway activated by TE plays important role in development of the hypercoagulation state. Our results indicate that plasma TF level was positively correlated to the level of MCP-1. This suggests that monitoring of plasma TF and MCP-1 levels in AECOPD patients could be a very useful way to prevent and cure blood hypercoagulability, cardiovascular and cerebrovascular thrombotic diseases.

Tranilast inhibits the proliferation of human coronary smooth muscle cell through the activation of p21waf1.

Restenosis after percutaneous transluminal coronary angioplasty (PTCA) occurs due to vascular smooth muscle cell proliferation and migration. Recently, tranilast, an anti-allergic drug, has been used for the prevention of restenosis after PTCA. To determine the molecular mechanism involved, the effect of tranilast on the proliferation of human coronary smooth muscle cells (SMCs) was investigated. Tranilast arrested the proliferation of human coronary SMCs at the G0/G1 phase of the cell cycle. In association with this inhibitory effect, tranilast increased p21waf1 and p53 tumor suppressor factor, and decreased cyclin-dependent kinase 2 (CDK2) activity. These results suggest that tranilast inhibits the proliferation of human coronary SMCs during restenosis after PTCA via an induction of p21waf1 and p53. Tranilast may thus allow us to prevent restenosis after PTCA by interfering with this mechanism.

Effects of 1-bromopropane, 2-bromopropane, and 1,2-dichloropropane on the estrous cycle and ovulation in F344 rats.

The present study was performed to investigate comparatively the toxic effects of inhalation exposure of 1-bromopropane, 2-bromopropane, and 1,2-dichloropropane on reproductive physiology, particularly on the estrous cycle and spontaneous ovulation in female F344 rats. The rats received inhalation exposure to different halogenated propanes, and were exposed daily for 8 h throughout almost 3 weeks to 0,50,200 and 1000 ppm of 1-bromopropane or 2-bromopropane, or to 0,50,100 and 200 ppm of 1,2-dichloropropane. Throughout the exposure period of 1-bromopropane or 2-bromopropane, the ratio of the number of estrous cycle of 6 days or longer to the number of all cycles in both 1000 ppm groups were about two-fold the ratio in each control group, however, no significant difference was found between the ratios of exposed and control groups. The ratios of such long estrous cycles in groups exposed to 100 or 200 ppm of 1,2-dichloropropane were six- to seven-fold higher than that of the control group. These ratios in exposed rats differed significantly from those of controls. The number of ovulated ova in rats exposed to 1,2-dichloropropane decreased in a dose-dependent manner, and the number of ovulated ova in the 200 ppm group was significantly different from that of control rats. Such significant changes in ovulation were not observed in rats exposed to 1-bromopropane or 2-bromopropane. The absolute and relative weights of the ovaries and uterus in rats exposed to three halogenated propanes were not significantly different from those in each control. Therefore, the present study clarified that: (1) 1,2-dichloropropane prolonged the length of the estrous cycle and inhibited spontaneous ovulation in F344 rats; and (2) the potency of 1,2-dichloropropane to disturb the female reproductive physiology appeared to be greater compared with that of 1-bromopropane and 2-bromopropane under the present conditions of inhalation exposure.

Loss of heterozygosity among tumor suppressor genes in invasive and in situ carcinoma of the uterine cervix.

The aim of the present study was to further clarify the histogenesis of cervical carcinoma by investigating loss of heterozygosity (LOH) among a number of tumor suppressor genes in invasive and in situ carcinoma of the cervix. Materials consisted of 16 in situ and 29 invasive carcinomas (16 squamous cell carcinomas, nine adenocarcinomas, and four adenosquamous carcinomas). DNA samples were collected by microdissection from ordinary formalin-fixed, paraffin-embedded tissues, both from the lesions and from normal tissues. LOH was analyzed using eight DNA polymorphic tumor suppressor markers. Of the 16 cases of carcinoma in situ, three cases exhibited LOH at one locus. Of the 29 cases of invasive carcinomas, six cases exhibited LOH at two loci and nine cases exhibited LOH at one locus. Overall, LOH was found more frequently in invasive carcinomas than in in situ carcinomas. LOH was most frequently detected at the PTCH (Drosophila patched gene) locus. There was no significant correlation between LOH at a specific site and either histologic subtype or clinical stage. These results suggest that LOH might already occur in a fraction of preinvasive squamous lesions and that accumulation of LOH may in part play a role in carcinogenesis of the cervix.

Effects of a GnRH analogue on human smooth muscle cells cultured from normal myometrial and from uterine leiomyomal tissues.

Leiomyomas are tumours of uterine smooth muscle tissue that are oestrogen and progesterone dependent. When explants of these tumours were grown in culture, the proliferating tissue formed characteristic ball-like aggregates (BLA), rather than the usual hill and valley (HV) pattern of growth of normal myometrial tissue in culture. Immunocytochemical staining with fluorescein isothiocyanate (FITC)-labelled gonadotrophin-releasing hormone (GnRH) revealed that both myometrial and leiomyomal cells have membrane receptors for this hypothalamic releasing hormone. Furthermore, polymerase chain reactions (PCR) with primer sets that were specific for GnRH receptor mRNA, as well as GnRH mRNA, showed that transcripts for both of these nucleic acids are present in myometrial and leiomyomal tissues. The treatment of cultured explants of leiomyomal tissue with a GnRH analogue (buserelin, HOE766) diminished the formation of BLA, but this synthetic hormone had only a moderate effect on the HV topography of normal myometrial tissue. A colorimetric assay indicated that GnRHa inhibited cell proliferation in leiomyomal tissue in a dose-dependent manner. Western blotting, to detect the expression of G1 phase cell cycle-related gene products, showed that cyclin E and p33cdk2 formation in leiomyomas were inhibited by high concentrations of GnRHa. In conclusion, GnRHa might suppress leiomyomal growth by interfering with the expression of cell cycle factors.

Prognostic significance of bcl-2 expression in leiomyosarcoma of the uterus.

We examined bcl-2 expression as well as p53 expression and mutation in human uterine smooth muscle tumours to determine the influence of bcl-2 expression on prognosis in patients with uterine leiomyosarcomas. bcl-2 protein was expressed in nearly all benign smooth muscle tumours but in only 57% of leiomyosarcomas. Benign smooth muscle tumours were usually negative for p53 protein, but 16 out of 21 (76%) leiomyosarcomas were positive. A p53 gene mutation was detected in nine of the 16 leiomyosarcomas that showed p53-positive staining. A significant positive correlation was observed between p53 mutation and p53 expression, between the number of mitoses and the Ki-67 labelling index, and between clinical stage and p53 mutation. A significant negative correlation was observed between bcl-2 expression and p53 mutation, and between bcl-2 expression and p53 overexpression. Univariate survival analysis revealed that bcl-2 expression, p53 mutation and clinical stage (stage 1 vs stages 2-4) all showed a significant correlation with prognosis. In a multivariate stepwise regression analysis, positive bcl-2 expression and stage 1 disease were the independent predictors of a favourable prognosis. Our results suggest that bcl-2 is frequently expressed in human uterine smooth muscle tumours, and that its expression may correlate with a favourable prognosis in patients with uterine leiomyosarcoma.

Expression of cyclins and cyclin-dependent kinases in smooth muscle tumors of the uterus.

To investigate the cell cycle regulatory mechanisms involved in the growth of smooth muscle tumors, we studied the expression of Ki-67, cyclins E and A, and their catalytic partners, the cyclin-dependent kinases cdk2 and cdc2 by using tissue specimens from benign and malignant smooth muscle tumors. These included 20 cases of usual leiomyoma (UL), 18 of cellular leiomyoma (CL), 8 of bizarre leiomyoma (BL), 8 of uncertain malignant potential tumors (UMP) and 20 of leiomyosarcoma (LMS). The proliferation rate detected by Ki-67 was low in normal myometrium and leiomyomas (UL, CL and BL), but it was markedly increased in LMS. The expression of the cyclins (E and A) and cdks (cdk2 and cdc2) was also low in normal myometrium and leiomyomas. However, the expression of these factors was markedly increased in LMS. In addition, a survival analysis using Log-rank test, revealed that LMSs with positive staining for cyclin A and with diffusely staining for cyclin E were associated with significantly shorter survival. Our results suggest that expression of cyclins and cdks may be involved in the growth control of uterine smooth muscle tumors.

Mast cells in smooth muscle tumors of the uterus.

Mast cells (MCs) have been reported in the myometrium and uterine smooth muscle tumors. We examined the number of MCs in various uterine smooth muscle tumors (including leiomyosarcomas) and assessed whether this feature might be of value in their pathologic diagnosis. The number of MCs in 95 uterine smooth muscle tumors, including 55 ordinary leiomyomas, 17 cellular leiomyomas, 8 bizarre leiomyomas, and 15 leiomyosarcomas, was counted using toluidine blue and immunohistochemical staining. The number of MCs that stained for tryptase was lowest in leiomyosarcoma and next lowest in ordinary leiomyoma; the number in each of these two groups was significantly lower than in the myometrium (p < 0.001). In cellular and bizarre leiomyomas, the number of MCs was significantly higher than in ordinary leiomyoma (p < 0.001 and p < 0.001, respectively) and leiomyosarcoma (p < 0.001 and p < 0.005, respectively). Statistical analysis revealed that counting the number of MCs and using a cut-off value of 16 MCs per high-power-field is useful for the differential diagnosis of leiomyosarcomas from cellular leiomyoma and bizarre leiomyoma, yielding 100% sensitivity and 96% specificity. The number of MCs was significantly lower in leiomyosarcomas at TNM stages III and IV than in those at stages I and II (p < 0.05), but there was no significant correlation between the number of MCs and patient survival. These results suggest that the number of MCs might be useful as part of a multivariate approach to the differential diagnosis of leiomyosarcoma from bizarre or cellular leiomyoma.

Frequent occurrence of loss of heterozygosity among tumor suppressor genes in uterine leiomyosarcoma.

OBJECTIVE: Leiomyosarcoma of the uterus is a rare smooth muscle tumor; it is extremely malignant and the rates of local recurrence and metastasis are high. Since tumor suppressor genes are commonly altered in malignant tumors, it is possible that mutations in such genes are involved in the development of uterine leiomyosarcoma. METHODS: Fifty-five patients (37-70 years of age) diagnosed as having smooth muscle tumors of the uterus were selected. DNA was extracted from four or five 8-microm-thick consecutive tissue sections of each smooth muscle tumor from the paraffin-embedded blocks. Loss of heterozygosity (LOH) was investigated at nine loci within or close to tumor suppressor genes (TP53, RB1, DCC, NM23, WT1, D14S267, P16, DPC4, PTCH). RESULTS: Nineteen of twenty leiomyosarcomas revealed at least one instance of LOH among eight of the nine markers tested (one locus showed no LOH at all). In fact, 11 of the 20 cases exhibited two or more instances of LOH and, of the remaining 9 cases, 4 showed a point mutation of p53 in addition to an alteration in one of the 9 markers, while one exhibited a p53 mutation only. CONCLUSION: An accumulation of genetic alterations among tumor suppressor genes may play a key role in the tumorigenesis and progression of uterine leiomyosarcoma.

Infrequent occurrence of high-risk human papillomavirus and of p53 mutation in minimal deviation adenocarcinoma of the cervix.

To assess the occurrence of high-risk human papillomavirus (HPV) infection and p53 alterations in minimal deviation adenocarcinoma (MDA) of the uterine cervix, paraffin sections were used to investigate the presence of HPVs 16 and 18 and p53 expression and mutation in six cases of MDA. By polymerase chain reaction, only one case was positive for HPV 16 and none was positive for HPV 18. By in situ polymerase chain reaction in the case positive for HPV 16, HPV 16 was detected in a low-grade squamous intraepithelial lesion overlying the MDA but not in the MDA itself. All of the MDAs were negative or only focally positive for p53 by immunohistochemistry. Our polymerase chain reaction DNA sequencing study failed to detect p53 mutation in exons 5 to 8 in those cases focally positive for p53 expression. These results suggest that MDA may be associated only occasionally with the high-risk HPVs or with p53 gene alterations.

Distribution and heterogeneity of mast cells in the human uterus.

Mast cells (MCs) are widely distributed in most human tissues. Those cells that contain only tryptase are designated as T-MCs, while those that also contain chymase are referred to as TC-MCs. This study uses immunohistochemical staining for tryptase and chymase to assess the distribution and heterogeneity of these two types of MCs in the human uterus. The greatest number of MCs was found in the inner (i.e. luminal) half of the myometrium, with this area containing approximately equal proportions of T-MCs and TC-MCs. There were fewer MCs in the outer half of the myometrium and the cervix, but the proportion of TC-MCs in both of these areas was substantially higher. In contrast, the endometrium contained significantly fewer MCs, but proportionally more T-MCs. There was no change in the number of MCs between the proliferative and secretory phases of the menstrual cycle; however, there was a significantly lower number in all areas after menopause. Most of the MCs were observed in close association with uterine smooth muscle cells, as well as in the vicinity of fibroblasts and collagen, and it appears they may play an important role in the reconstruction of uterine tissues during the menstrual cycle.

Genetic alterations in microsatellite marker sites among tumor suppressor genes in endometriosis.

Four endometriotic lesions were examined for the presence of genetic alterations in microsatellite marker sites among eight tumor suppressor genes. For this, a microdissection method was used on paraffin sections. Only one instance of loss of heterozygosity was detected at the PTCH locus. Heterozygosity was retained (indicating the absence of both loss of heterozygosity and microsatellite instability) at the other seven tumor suppressor gene loci in all the cases. Among the tumor suppressor genes examined, genetic defects in these microsatellite regions are certainly not ubiquitous in endometriosis and may be uncommon.

Phosalone applied to cotton (Gossypium hirsutum L.): its deposition and persistence on foliages, soils and final residues in seeds.

Phosalone, O,O-diethyl-S-(6-chloro-1,3-benzoxazol-2(3H)-onyl)methyl phosphorodithioate, was field-applied by ground equipment to cotton (Gossypium hirsutum L.) at the rates of 1050 and 2100 g a.i./ha, respectively, to determine its dissipation on leaves and soils and the residues in seeds at harvest. The insecticide concentrations on cotton leaves and soils were measured periodically for 14 days following its application. It was found that the half lives of the insecticide on cotton leaves at the dosages of 1050 and 2100 g a.i./ha were 6.8 and 6.3 days, respectively. And the half lives on soils for the 2 dosages were 7 and 5.8 days, respectively. The residues remaining in soils at harvest time were 0.072 and 0.121 mg/kg 14 days post-application and the residues in cotton seeds were relatively low (less than 0.02-0.12 mg/kg).

In-vitro model of uterine leiomyomas: formation of ball-like aggregates.

To clarify the biological characteristics of uterine leiomyomas, cells explanted and cultured from uterine leiomyomas and from normal myometrial tissue were observed by time-lapse cinemicrography and phase-contrast microscopy. The histological characteristics were evaluated by electron microscopy and immunofluorescence microscopy, and these observations revealed significant differences. By time-lapse cinemicrography, the cells cultured from leiomyomas and myometrium differed in their behaviour. Cells from the myometrium started to grow in parallel with the cell's major axis and formed topographically uniform hills and valleys by day 21 of culture. In contrast, the cells from leiomyomas started to grow irregularly, as if having no contact inhibition, and formed ball-like aggregates of cells by day 21 of culture. The aggregates resembled the nodules of leiomyoma in vivo. Ultrastructurally, cells from both leiomyomas and myometrium had typical features of smooth muscle. Immunofluorescently, a different distribution of alpha-smooth muscle actin-positive filaments and different staining of cellular fibronectin and N-cadherin between the cells from leiomyomas and myometrium were observed, which may contribute in part of the different behaviour of the cells. Given that the explant cell culture system resembles the features of uterine leiomyomas in vivo, this suggests that it can be used as an in-vitro model.

Expression of steroid receptors, Ki-67, and p53 in uterine leiomyosarcomas.

The expression of estrogen receptor (ER), progesterone receptor (PR), tumor suppressor oncogene p53, and Ki-67 was compared in uterine smooth muscle tumors, including leiomyosarcoma (LMS), tumor of uncertain malignant potential (UMP), cellular leiomyoma (CL), bizarre leiomyoma (BL), and usual leiomyoma (UL). ER and PR were expressed in all ULs. PR was expressed in UL irrespective of the phase of the menstrual cycle; this staining was also observed in CL, UMP, and BL, although BL showed variable staining for ER. Compared to these tumors, the expression of both ER and PR was markedly reduced in LMS. The results of ER and PR transcripts by reverse transcription-polymerase chain reaction were compatible with those of immunohistochemistry. The number of Ki-67 positive cells in LMS was significantly higher than in UMP, BL, CL, and UL. p53 immunoreactivity was seen in 10 of 14 LMSs, and missense mutation in the p53 gene was found in 4 of 10 LMSs. These results suggest that abnormal expression of ovarian steroid receptors, p53, and Ki-67 is frequently associated with LMS of the uterus.

Immunohistochemical analysis of cell cycle regulatory gene products in normal trophoblast and placental site trophoblastic tumor.

Intermediate trophoblast (IT) rarely gives rise to a placental site trophoblastic tumor (PSTT) To examine the different growth mechanisms present in normal and neoplastic IT, the expression of cell cycle regulatory molecules was compared at normal implantation sites and in PSTTs. Normal implantation sites in early gestation (19 patients) and PSTTs (6 patients) were immunohistochemically studied using antibodies against cytokeratin, human chorionic gonadotropin, and human placental lactogen to identify IT, and antibodies against Ki-67, cyclins (A, B, D1, and E), cyclin-dependent kinases (cdks), and p53 to investigate the proliferative activity of the trophoblast. Marked proliferative activity was observed in the trophoblast of the cell columns. Normal IT exhibited a very low labeling index for Ki-67, with negative expression for cdks and cyclins, except for cyclins B and E. The tumor cells of PSTT exhibited a high labeling index for Ki-67 with positive expression for all the cyclins and cdks examined. Expression of p53 was identified in tumor cells of PSTTs and the distribution of p53-positive cells correlated topographically with that of the cyclin A-positive cells. The transformed IT of PSTT has high proliferative activity with an abnormal expression of cell cycle regulatory molecules, which is not observed in normal IT.

Demonstration of focal p53 expression without genetic alterations in endometriotic lesions.

Their monoclonal origin (as indicated by recent investigations) indicates the neoplastic nature of most endometriotic lesions. p53, a representative tumor suppressor, regulates cell proliferation, and genetic alterations in p53 are involved in carcinogenesis in a wide variety of human cancers. The aim of this study was to examine endometriotic lesions for p53 expression and genetic alterations in p53. An immunohistochemical study revealed that 20% (13/64) of endometriotic lesions showed focal p53 expression in the epithelial cells. Using serial paraffin sections, we employed a microdissection method to extract DNA from the endometriotic tissues that showed p53 expression. No mutations were found in exons 5-8 in p53 by cleavase fragment length polymorphism scanning and polymerase chain reaction-DNA sequencing. Moreover, neither loss of heterozygosity nor microsatellite instability was detected at the microsatellite marker sites of p53. These results suggest that the focal p53 expression recognized in the endometriotic epithelia may be due to overproduction of wild-type p53 protein.