Direct maxacalcitol injection into hyperplastic parathyroids improves skeletal changes in secondary hyperparathyroidism.

Direct maxacalcitol (OCT) injection into a parathyroid gland (PTG) ameliorates several important etiologic factors of resistance to medical treatments for secondary hyperparathyroidism (s-HPT): the upregulations of vitamin D receptor (VDR) and Ca-sensing receptor (CaSR) in PTGs and the regression of PTG hyperplasia by the induction of apoptosis. In this study, we evaluated the bone histomorphology on the basis of maintaining these effects in advanced s-HPT. Five/six nephrectomized Sprague-Dawley rats were fed a high-phosphorus and low-calcium diet for 8 weeks. These rats were divided into four treatment groups: (1) basic uremic (at the baseline), (2) direct OCT single injection into PTGs (DI-OCT) followed by OCT intravenous administration for 4 weeks (IV-OCT), (3) direct vehicle injection and IV-OCT, and (4) no treatment for an additional 4 weeks. The effects of these treatments on serum intact-parathyroid hormone (PTH) level, PTG weight, VDR and CaSR expression levels in PTGs, and bone histomorphometric parameters were investigated. In the DI-OCT+IV-OCT group, the significant decrease in serum intact-PTH level was maintained by the following IV-OCT. A significant decrease in PTG weight and the upregulations of VDR and CaSR expression levels in PTGs were also observed. Bone histomorphometric analysis showed significant improvements in osteitis fibrosa in both cancellous and cortical bones. However, these findings were not observed in the other groups. These results suggest that osteitis fibrosa caused by advanced s-HPT can be successfully reversed by a control of PTH at an appropriate level through the improvement of PTG hyperplasia as induced by DI-OCT+IV-OCT.

Abnormal uterine bleeding and prognosis of endometrial cancer.

OBJECTIVES: The aim of this study was to analyze the relationship between the period of abnormal uterine bleeding (AUB) and the prognosis of endometrial cancer. METHODS: We reviewed 304 endometrial cancer patients who were diagnosed and treated between 1985 and 1998 in our hospital, and whose history of AUB and clinical parameters were clearly available from their charts. Pathological data and overall survival were compared between groups having different periods of AUB. RESULTS: Duration of AUB had no impact on the prognosis of endometrial cancer. Patients diagnosed with endometrial cancer without AUB showed a significantly better 5-year overall survival rate than the patients diagnosed after the onset of AUB. The distribution of clinical stages and histological grades did not differ depending on AUB status. CONCLUSIONS: The prognosis of endometrial cancer was determined by the histopathological character of the tumor. However, the diagnosis and treatment of endometrial cancer with some suspicious signs other than AUB might improve the prognosis.

Molecular regulation of the oxytocin receptor in peripheral organs.

The oxytocin receptor belongs to the G-protein-coupled seven transmembrane receptor superfamily. Its main physiological role is regulating the contraction of uterine smooth muscle at parturition and the ejection of milk from the lactating breast. Oxytocin receptor expression is observed not only in the myometrium and mammary gland but also in the endometrium, decidua, ovary, testis, epididymis, vas deferens, thymus, heart and kidney, as well as in the brain. The expression profile shows a tissue-specific as well as a stage-specific pattern. The oxytocin receptor gene is a single-copy gene consisting of four exons and three introns, localized at 3p25-3p26.2 in the human chromosome. In transfection studies using a fusion construct containing the promoter region of the oxytocin receptor gene inserted in a reporter plasmid, neither proinflammatory cytokines nor oestrogen directly activate the gene. The nuclear fractions from up-regulated (term myometrium) and down-regulated (non-pregnant myometrium) tIssues show differential patterns of protein binding to the 5'-flanking region, and a human homologue of chicken MafF has been cloned as a term-myometrium-specific oxytocin receptor modulator. The oxytocin receptor gene appears to be highly methylated. Methylation around intron 1 and in intron 3 might contribute to tIssue-specific suppression of the gene. The oxytocin receptor is also regulated by desensitization, whose mechanism appears to involve loss of ligand-binding activity of the protein as well as suppression of the oxytocin receptor mRNA transcription. These findings taken together indicate that the oxytocin receptor is regulated in a very complicated manner, and the transcriptional regulatory elements critical for this regulation should be investigated further.

[Concurrent chemoradiation therapy with nedaplatin for high-risk cervical cancer--clinical investigation of adverse events].

A clinical investigation of adverse events was conducted to confirm the safety of concurrent chemotherapy using nedaplatin (cisplatin derivative) and radiotherapy in the high-risk carcinoma of the uterine cervix. Seven patients who were treated with radical radiotherapy and 5 patients who were treated with adjunctive radiotherapy after radical hysterectomy and pelvic lymphadenectomy were eligible for the study. Nedaplatin was given intravenously at 70 mg/m2 on day 1 and day 29, and a total of 24 courses of nedaplatin administration were observed. None of the planned radiotherapy was postponed or discontinued due to side effects. Major adverse effects observed were gastrointestinal effects such as anorexia (66.7%), nausea and vomiting (33.3%) and diarrhea (66.7%). Grade 3 (in the 2nd course) and Grade 4 (in the 1st course) diarrhea was observed in one patient, which was easily relieved by antidiarrheal. Hematologic side effects were also major, including leukopenia (62.5%), neutropenia (75.0%), anemia (75.0%), and thrombocytopenia (33.3%). Hematologic effects were generally moderate; no Grade 4 (severe) effects were observed. Although these hematologic effects were lasting longer compared with radiation therapy alone, there were no significant differences in the seriousness of these side effects. Concurrent chemoradiation therapy with nedaplatin 70 mg/m2 every 4 weeks was safe and adverse effects were self-limited or resolved with medical management. Dose escalation in the phase III clinical study may be considered.

beta(2)-adrenergic agonists and pelvic floor exercises for female stress incontinence.

OBJECTIVE: We compared beta(2)-adrenergic agonist therapy with clenbuterol (DT) and physiological therapy (PT) in a randomized study to establish the first line therapy for stress incontinence (SI). METHOD: The clinical efficacy of DT (group A), PT (group B), and a combination of DT and PT (group C) was investigated in 61 patients with SI by means of a 12-week randomized controlled study. The frequency and volume of SI and the patients' own impressions were used as the basis for the assessment of efficacy. RESULTS: The SI improvement rates in groups A, B, and C were 76.9, 52.6, and 89. 5%, respectively (P=0.0361). A significant therapeutic effect on the frequency of SI was observed in group B and group C at 2 weeks after the start of treatment (both P<0.05), and in all groups at 6 weeks (all P<0.01). The efficacy rates based on the patients' own impressions in groups A, B, and C were 84.6, 31.6, and 68.4%, respectively (P=0.0064). CONCLUSION: The beta(2)-adrenergic agonist appeared to be clinically useful as a drug of choice for SI.

Cytokine production in chorioamnionitis.

Lymphohematopoietic cytokines play a significant role in many biological mechanisms including a number of reproductive processes such as ovulation, implantation, placentation, cervical dilation and parturition. Recent experiments have suggested that cytokines play a crucial role in the mechanisms of preterm labor and delivery, which are the leading causes of perinatal morbidity and mortality. Growing evidence suggests that infection is deeply concerned in the pathogenesis of preterm labor and delivery. Chorioamnionitis, a subset of intrauterine infection, has been identified in 20-33% of women with preterm delivery, and the inflammatory and related cytokines, interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-8 (IL-8), showed substantial increases in the amniotic fluid at women with intrauterine infection. Although the precise mechanism for chorioamnionitis-driven preterm labor mediated via cytokines is still unknown, both IL-1 and TNF-alpha along with IL-6 enhance prostaglandin production by human amnion cells, chorionic cells and decidual cells. Analysis of the regulatory sequences in the 5' upstream regions of receptor gene for human oxytocin, a potent uterotonic agent, suggests a close relationship between preterm labor and inflammatory cytokines through induction at the oxytocin receptor. Prompt identification of the patients with intra-amniotic infection may be useful in clinical practice. At present, the measurement of IL-8 in maternal serum or the measurement of IL-6 in cervical secretion may be helpful as a non-invasive screening for chorioamnionitis.

Expression of RANTES by IL-1 beta and TNF-alpha stimulated nasal polyp fibroblasts.

OBJECTIVE: Accumulation of eosinophils (Eo) is one of the most characteristic feature of nasal polyps. However, the question remains why eosinophils accumulate into the nasal polyp tissue. RANTES (regulated upon activation, normal T cell expressed and presumably secreted) is a recently described chemokine that is said to play a role in the recruitment of eosinophils into inflammatory tissue sites. Fibroblasts are a rich source of cytokines and inflammatory mediators. The objective of this study was to demonstrated the expression of the chemokine RANTES in nasal polyp fibroblasts after stimulation with proinflammatory cytokines like TNF-alpha and IL-1 beta. METHODS: Fibroblast lines were established from human nasal polyp biopsy tissues taken from patients with chronic sinusitis who had no other associated diseases. Cultured nasal polyp fibroblasts were stimulated with TNF-alpha or IL-1 beta at various doses (0.1, 1.0, 1 ng/ml) or for various times (l, 6, 12, 24, 48, 72 h). To detect the RANTES gene expression, RT-PCR was performed. The resulting supernatants were assayed with ELISA for the level of RANTES. RESULTS: We demonstrated that TNF-alpha and IL-1 beta induced the gene expression and protein production of RANTES in nasal polyp fibroblasts. This responsiveness to TNF-alpha and IL-1 beta was time and dose-dependent. CONCLUSION: These findings suggest that nasal polypfibroblasts may also play an important role in the recruitment of Eo through the production of RANTES.

Phase II study of irinotecan and cisplatin as first-line chemotherapy in advanced or recurrent cervical cancer.

Irinotecan (CPT-11) and cisplatin are singly active against cervical cancer. We evaluated the efficacy and toxicity of CPT-11 plus cisplatin as first-line chemotherapy in patients with advanced or recurrent cervical cancer. Twenty-nine chemotherapy-naive patients with advanced or recurrent cervical cancer were treated with CPT-11 (60 mg/m(2)) on days 1, 8, and 15 by intravenous infusion over 90 min, followed by cisplatin (60 mg/m(2) i.v.) on day 1 over 90 min. The patients' median age was 57 years (range 35-75). Nineteen patients (66%) had advanced primary disease. Six patients with recurrent disease (21%) had been treated with prior radiotherapy. The remaining 4 patients (14%) had residual or recurrent disease after radical surgery. The histologic diagnoses were squamous cell carcinoma in 25 patients (87%), adenocarcinoma in 3, and adenosquamous cell carcinoma in 1. All eligible patients were included in the toxicity and response analysis based on the intent to treat. Two patients (7%) achieved a complete response and 15 (52%) a partial response (overall response rate: 59%, 95% confidence interval; 41-74%). Stable disease was recorded in 6 patients (21%) and progressive disease in 3 patients (10%). In 3 patients, image-guided evaluation of response was judged to be unfeasible at the time of independent extramural review (10%). The median time to response was 32 days (range 16-62 days). The median survival was 27. 7+ months (range, 6.4-52.8+ months). Two dose-limiting side effects were observed: grade 3 (28%) or 4 (45%) neutropenia and grade 3 (7%) or 4 (7%) diarrhea. Other severe toxicities included anemia (45%), thrombocytopenia (3%), nausea/vomiting (31%), and alopecia (7%). The combination of CPT-11 with cisplatin is an active regimen for treatment of advanced or recurrent cervical cancer albeit with a significant degree of myelosuppression.

Distinct expression of RANTES and GM-CSF by lipopolysaccharide in human nasal fibroblasts but not in other airway fibroblasts.

Eosinophil infiltration of tissue is a hallmark of nasal polyposis and asthma in both atopic and nonatopic patients. Structural cells like airway fibroblasts are a rich source of cytokines and inflammatory mediators. In order to verify whether airway fibroblasts play a role in eosinophilic infiltration, we investigated the release of eosinophil chemotactic and activating factors from airway fibroblasts when stimulated with nonallergenic exogenous agents such as endotoxin (lipopolysaccharide; LPS). Using a number of primary human airway tissue-derived fibroblast lines, we demonstrated that LPS could induce the gene expression and production of RANTES (regulated and normal T cell expressed and presumably secreted) and granulocyte/macrophage colony-stimulating factor (GM-CSF) only in nasal but not in pharyngeal, tracheal, bronchial, and lung fibroblasts. This selective responsiveness of nasal fibroblasts to LPS was time and dose dependent. These findings suggest that nasal fibroblasts may play an important role in the recruitment and activation of eosinophils into nasal polyps through the release of RANTES and GM-CSF.

Human placental cells show enhanced production of interleukin (IL)-8 in response to lipopolysaccharide (LPS), IL-1 and tumour necrosis factor (TNF)-alpha, but not to IL-6.

Interleukin-8 (IL-8) is a chemotactic and activating factor for neutrophils which play important roles in host defence mechanisms. The human placenta constitutively produces IL-8 during pregnancy and enhances its production in chorioamnionitis. The present study was designed to investigate in vitro the regulatory mechanism for IL-8 production in the placentas in normal and inflammatory states. Placental cells produced IL-8 in a dose-dependent fashion when stimulated with lipopolysaccharide (LPS). The purified trophoblasts showed significantly higher IL-8 production than untreated placental cells. The expression of IL-8 gene in the trophoblasts in the third trimester was observed by reverse transcription-polymerase chain reaction (RT-PCR). The placental cells also release IL-8 in a dose-dependent manner, in response to r-(recombinant) IL-1alpha and tumour necrosis factor (TNF)-alpha, but not rIL-6. Moreover, LPS-activated placental cells spontaneously produced a much larger amount of IL-8 and showed increased responses to rIL-1alpha and TNF-alpha. It may, therefore, be proposed that placental cells with multiple endocrine functions exert immunological functions by constitutive production of IL-1 and TNF-alpha, which stimulate placental IL-8 release. This cytokine cascade in the placenta may be augmented by LPS in chorioamnionitis, thereby potentiating the feto-maternal defence mechanisms against infection.

Dynamics of immunoglobulins at the feto-maternal interface.

Transplacental transport of maternal immunoglobulin G (IgG) to the developing fetus is extremely important in the protection of the newborn from infection. Although the exact mechanisms of the selective and active transfer of IgG across the placental barrier are not fully understood, receptors for the Fc part of IgG (FcgammaRs) in the placenta are believed to play a key role. Several known Fc receptors, FcgammaRI, FcgammaRII, FcgammaRIII and FcRn (neonatal FcR), demonstrate heterogeneous expression patterns in placenta. Immunohistochemical analysis shows the expression of FcgammaRI on Hofbauer cells in stromal tissue, FcbetaRII on Hofbauer cells and fetal blood endothelium, FcgammaRIII on Hofbauer cells and trophoblasts, and FcRn on syncytiotrophoblasts and endothelial cells. Recent studies provide evidence for important associations among these receptors and transcytosis of IgG, as well as scavenger mechanisms for clearing immune complexes in the placenta during pregnancy.

Secretory leukocyte protease inhibitor (SLPI) concentrations in cervical mucus of women with normal menstrual cycle.

Secretory leukocyte protease inhibitor (SLPI) is a potent inhibitor of human leukocyte elastase. SLPI transcripts in the cervical tissue were detected during the menstrual cycle by reverse transcription-polymerase chain reaction (RT-PCR). Western blot analysis revealed that the intensity of SLPI protein in cervical tissue in the ovulatory phase was stronger than in other phases. Immunohistochemistry using an anti-SLPI polyclonal antibody revealed positive staining in the epithelial cells of the endocervix. Western blot analysis also revealed that SLPI protein was present in the cervical mucus. Again the intensity of SLPI protein in the ovulatory phase was stronger than that in the follicular phase. The SLPI concentrations and SLPI/elastase ratios in the cervical mucus of women in the ovulatory phase were significantly higher than in the follicular and luteal phases. The SLPI and elastase concentrations in the cervical mucus were positively correlated. No significant difference was found in the SLPI serum concentrations of women during the menstrual cycle. These results suggest that production of SLPI from cervical epithelial cells during the ovulatory phase may be important for protection from the effects of elastase.

Endometrial development was improved by transdermal estradiol in patients treated with clomiphene citrate.

The objective of this study was to examine the effect of transdermal estrogen therapy on the endometrial thickness and serum hormone levels in anovulatory patients treated with clomiphene citrate (CC). There was a significant difference in endometrial thickness between the CC + transdermal estrogen group and the CC only group from day -2 to day +2. Serum estradiol (E2) levels in the CC + transdermal estrogen group were significantly higher than those in the CC only group on day -2 and day 0. Our results support that addition of transdermal E2 to the treatment protocol of the women treated with CC elicited a favorable response of the endometrium.

Suicide gene therapy for human uterine adenocarcinoma cells using herpes simplex virus thymidine kinase.

In gene therapy, the herpes simplex virus thymidine kinase (HSV-tk) gene is widely used as a suicide agent. Tumor cells expressing HSV-tk are sensitive to nucleoside analogs such as ganciclovir (GCV). An advantage of this system is the bystander killing effect whereby HSV-tk-positive cells exposed to GCV are lethal to surrounding HSV-tk-negative cells. We transfected the HSV-tk gene into a human cervical adenocarcinoma cell line, BU25TK-, and a human endometrial adenocarcinoma cell line, HHUA, by the Lipofectine method. The sensitivity of HSV-tk-positive cells to GCV and bystander killing effect on HSV-tk-negative cells were examined in vitro. HSV-tk-positive cells were sensitive to GCV at concentrations of 1 to 100 microg/ml in a dose- and time-dependent manner. The growth of HSV-tk-negative cells was inhibited when the population of cultured cells contained more than about 3% HSV-tk-positive cells. Moreover, for BU25TK- cells, HSV-tk-positive cells were injected into SCID mice subcutaneously and the effects of GCV therapy and bystander killing at a daily concentration of 25 mg/kg for 14 days were examined. HSV-tk-positive tumors transduced into SCID mice almost disappeared upon GCV treatment. Furthermore, tumor reduction was observed when mixtures of HSV-tk-negative cells containing more than 20% HSV-tk-positive cells were injected into SCID mice. In conclusion, the HSV-tk/GCV system might be applied to both cervical and endometrial adenocarcinoma.

Eotaxin synthesis by nasal polyp fibroblasts.

Nasal polyps is a chronic inflammatory disease of the upper airway characterized by structural abnormalities including stromal fibrosis. Fibroblasts are a rich source of cytokines and inflammatory mediators and are thought to play an important role in the development of fibrosis. In addition, there is considerable evidence for the participation of eosinophils in the pathophysiology of nasal polyps. Although increased numbers of eosinophils are present in nasal polyps, the mechanisms responsible for their selective accumulation are not completely clear. Eotaxin is a chemokine that promotes the selective recruitment of eosinophils. Thus, it may be an important molecule for the recruitment of eosinophils in nasal polyps. The purpose of this study was to investigate whether nasal polyp fibroblasts synthesize eotaxin after stimulation with lipopolysaccharide, IL-1beta or TNF-alpha. Using primary nasal polyp tissue-derived fibroblast lines, we demonstrated that LPS, IL-1beta and TNF-alpha induced the gene expression and protein production of eotaxin in nasal polyp fibroblasts. This responsiveness to LPS, IL-1beta and TNF-alpha was time- and dose-dependent. These findings support the hypothesis that fibroblasts could play an important role in the recruitment of eosinophils in nasal polyps through the production of eotaxin.

cAMP stimulates the bystander effect in suicide gene therapy of human choriocarcinoma.

In gene therapy, tumor cells expressing herpes simplex virus thymidine kinase (HSV-tk) are sensitive to ganciclovir (GCV) and HSV-tk positive cells exposed to GCV are lethal to adjacent HSV-tk negative cells. This phenomenon has been called the bystander effect, and the gap junction is thought to mediate it. In this study, sensitivity to GCV and bystander effect in a human choriocarcinoma cell line, BeWo, transfected with HSV-tk were investigated. Furthermore, the effect of 8-bromo-cAMP on bystander effect and connexin40 gene transcription were examined. HSV-tk positive cells were sensitive to GCV at the concentration of 10 micrograms/ml in a time-dependent manner. The growth of HSV-tk negative cells was inhibited when the population of cultured cells contained more than 10% HSV-tk positive cells and 8-bromo-cAMP enhanced bystander effect. 8-bromo- cAMP increased connexin40 mRNA expression and gap junctional intercellular communication.

Expression and immunolocalization of the oxytocin receptor in human lactating and non-lactating mammary glands.

The milk ejection reflex is mediated by the release of pituitary oxytocin and its interaction with specific receptors within the mammary gland. Although up-regulation of the oxytocin receptor during lactation has been shown for the rat mammary gland by ligand binding assay, investigation of the receptor expression in human breast at the molecular level has not yet been carried out in detail. Here we report the expression and immunolocalization of the oxytocin receptor in the human breast. It appears that the expression level of the receptor-specific mRNA is not significantly elevated during lactation and the protein remains at a relatively low level. However, this lack of increase may be only a dilution effect because of the high level of milk protein expression. Immunohistochemistry and immunoelectron microscopy using three anti-oxytocin receptor antibodies raised against different epitopes of the receptor indicated the presence of receptor immunoreactivity only to a very limited extent in the myoepithelial cells; more specific expression appeared to occur in the ductal/glandular epithelium in both the non-lactating as well as lactating breast. This finding was also confirmed in a New World monkey, the common marmoset (Callithrix jacchus). These results suggest that, at least for human and marmoset, in addition to--or even instead of--myoid cells, the ductal/glandular epithelium is also a target for oxytocin action, not only during lactation but also in the non-lactating breast. Thus, there may be other physiological effects of oxytocin besides direct myoid cell contraction in the breast.

Secretory leukocyte protease inhibitor (SLP) concentrations in seminal plasma: SLPI restores sperm motility reduced by elastase.

In this study, we quantified secretory leukocyte protease inhibitor (SLPI) and elastase in ejaculates from normal donors and infertile patients with or without leukospermia and investigated the effect of SLPI on sperm motility reduced by elastase. Western blot analysis revealed that SLPI protein was detected in the seminal plasma. The SLPI titre in the seminal plasma with leukospermia was lower than that in the seminal plasma without leukospermia and in the seminal plasma of fertile donors. The elastase concentration in the seminal plasma with leukospermia was significantly higher than that in the seminal plasma without leukospermia. A significant correlation between SLPI and elastase concentrations in the seminal plasma (r = 0.36, P< 0.01) was observed. There was a positive correlation between SLPI titre in the seminal plasma and sperm motility (r = 0.51, P < 0.001). SLPI recovered the sperm motility reduced by elastase in a dose-dependent manner. Our results suggest that SLPI is a potential substance to treat infertile patients with leukospermia.

Effect of sexual intercourse on fetal fibronectin concentration in cervicovaginal secretions.

An investigation was performed evaluating persistence of fetal fibronectin in cervicovaginal secretions after sexual intercourse. Fetal fibronectin samples were positive at 12 to 24 hours, 24 to 48 hours, and 48 to 72 hours after coitus in 22.2%, 11.8%, and 5.6% of samples, respectively. A history of vaginal intercourse should be obtained before sampling for fetal fibronectin to eliminate this potential source of false-positive fibronectin test.