Ischemic preconditioning upregulates vascular endothelial growth factor mRNA expression and neovascularization via nuclear translocation of protein kinase C epsilon in the rat ischemic myocardium.

Ischemic preconditioning (IP) exerts cardioprotection through protein kinase C (PKC) activation, whereas myocardial ischemia enhances vascular endothelial growth factor (VEGF) mRNA expression. However, the IP effect or the involvement of PKC on the VEGF expression is unknown in myocardial infarction. We investigated whether IP enhances VEGF gene expression and angiogenesis through PKC activation in the in vivo myocardial infarction model. Sprague-Dawley rats were assigned into the following 3 groups: the sham group; the IP group, which underwent 3 cycles of 3 minutes of ischemia and 5 minutes of reperfusion (IP procedure); and the non-IP group. The latter 2 groups were subsequently subjected to left anterior descending coronary artery occlusion. To examine the involvement of PKC, the PKC inhibitor chelerythrine (5 mg/kg) or bisindolylmaleimide (1 mg/kg) was injected intravenously before the IP procedures. PKCepsilon was translocated to the nucleus after 10 minutes of ischemia after the IP procedure but was not translocated in the non-IP and the sham groups. VEGF mRNA expression 3 hours after infarction was significantly higher in the IP group than in the non-IP and the sham groups. Capillary density in the infarction was significantly higher, whereas the infarct size was smaller in the IP group than in the non-IP group at 3 days of infarction. Chelerythrine but not bisindolylmaleimide blocked all of the IP effects on the nuclear translocation of PKCepsilon, enhancement of VEGF mRNA expression and angiogenesis, and infarct size limitation. These results show that IP may enhance VEGF gene expression and angiogenesis through nuclear translocation of PKCepsilon in the infarcted myocardium.

Isolation and characterization of a TATA-less promoter for the human RAG-1 gene.

Human recombination activating gene-1 (RAG-1) genomic DNA clones containing the first exon coding for the 5' untranslated region and the second exon coding for the remaining 5' untranslated region, coding region, and 3' untranslated region were cloned. Primer extension analysis and RNase protection analysis demonstrated the multiple RAG-1 transcription start sites, clustered in a 31 nucleotide (nt) region. Sequence analysis showed that the RAG-1 promoter lacked a TATA box as well as an initiator sequence. Transient expression assays using a luciferase reporter gene with truncated promoter fragments and substitution mutants, showed that the 5' promoter region containing the CCAAT box between -110 and -86, is indispensable for its basal promoter activity in RAG-1 expressing Nalm 6 cell line. Comparative transient expression assays in various cell lines revealed that the 854 nt upstream promoter region was active, not only in RAG-1 expressing cell lines but also in RAG-1 non-expressing cell lines. These data indicate that the 854 nt upstream region of RAG-1 gene confer basal promoter activity, and that the tissue- and stage-specific expression of RAG-1 is controlled by elements present outside of the promoter region and/or differential chromatin structure(s) of the individual cells.

[Complete resection for giant thymic carcinoma after simultaneous combination chemotherapy].

A 19-year-old man visited our hospital complaining of dyspnea. Chest X-ray and computed tomography (CT) showed a huge mass in the right anterior mediastinum. We diagnosed this as invasive thymoma by microscopic examination of specimens obtained by echo-guided needle biopsy. The patient underwent 6 courses chemotherapy [1st course : carboplatin (CBDCA) + doxorubicin hydrochloride (DXR) + vincristine sulfate (VCR) + cyclophosphamide (CPA), 2nd, 3rd-6th course : cisplatin (CDDP) + ADM + VCR + CPA]. At achievement of partial response (the reduction rate of the tumor size : 91.4%), the tumor was completely resected. The pathological examination of the resected specimens yielded a diagnosis of large cell carcinoma. Preoperative chemotherapy with ADOC regimen may be effective in advanced thymic carcinoma.

Increased urinary levels of adrenomedullin in patients with cystitis.

In this study, we examined urinary levels of adrenomedullin (AM) in 18 healthy volunteers and 18 patients with cystitis. We also compared urinary levels of AM in 11 patients with cystitis before and after antibiotic treatment. Urinary AM concentrations were measured by a radioimmunoassay specific for human AM. Urinary AM levels in patients with cystitis were significantly elevated compared with those of healthy volunteers and correlated positively with the number of urine leukocytes. By antibiotic treatment, urinary AM levels significantly decreased as compared with before the treatment. By RNA blot analysis of AM transcript, we detected significant levels of AM mRNA in canine urinary bladder and ureter. Intravenous administration of lipopolysaccharide elevated the AM mRNA level in the urinary bladder. These data suggest that infection and inflammation stimulate AM production in the urinary tract, which results in increased urinary AM levels in patients with cystitis. Based on these results, it is deduced that AM participates in the pathophysiology of cystitis, and its urinary level could be used as an index of the degree of cystitis.

Pituitary response to luteinizing hormone-releasing hormone in women with variant luteinizing hormone.

OBJECTIVE: To assess the LH response of the pituitary gland to GnRH stimulation in healthy women with a mutant beta-subunit (Trp8 to Arg8 and Ile15 to Thr15). DESIGN: Clinical study. PATIENTS: We studied 40 healthy non-pregnant Japanese women of known zygosity for the LH beta-subunit gene (3 homozygotes for the mutant gene, 17 heterozygotes, and 20 homozygotes for the wild type). All women had normal ovulatory cycles. MEASUREMENTS: Serum LH status was determined by comparing LH immunoassays results using a monoclonal antibody recognizing only wild-type LH with those from a polyclonal antibody assay recognizing both variant and wild-type LH. The ratio of monoclonal to polyclonal immunoassay results determined the serum LH status. LH secretion in response to a GnRH stimulation test was measured. RESULTS: All women with the wild-type LH showed a normal response of LH to GnRH according to both assays. Over the time course of the response, the ratios in women with wild-type LH showed no remarkable changes. The response curves in women heterozygous for the mutant peaked 15-30min after GnRH injection; their response patterns included a statistically significant decrease in the rates of response at 15min after injection. CONCLUSIONS: There are the differences in circulatory kinetics between the two LH forms and in regulation of the two types of LHbeta genes. The maximal response of the variant LH to pituitary stimulation with GnRH appears to be greater than that of wild-type LH.

Diagnostic difficulty in polycystic ovary syndrome due to an LH-beta-subunit variant.

We initially failed to confirm a case of polycystic ovary syndrome (PCOS) because underestimation of LH concentrations due to a variant form of this hormone resulted in a misleadingly low LH/FSH ratio. A 26-year-old woman presented to our hospital with infertility. Given the presence of bilateral polycystic ovaries, oligomenorrhea and hirsutism. PCOS was suspected, but a normal LH/FSH ratio as measured by RIA led to diagnostic problems. When we remeasured LH and FSH using a chemical luminescence enzyme immunoassay (CLEIA), the ratio of the LH concentration measured by RIA to that measured by CLEIA was 0.29, and the ratio of LH to FSH measured by CLEIA was 3.3 compared with 0.81 measured by RIA. We then diagnosed PCOS. The point mutations Trp8 to Arg8 and Ile15 to Thr15 in the LH subunit were detected in the corresponding gene. The patient's LH status represented variant and wild-type LH equally. She was therefore diagnosed as heterozygous for the mutant LH-beta. Histologic assessment of ovarian tissue after laparoscopic biopsy was compatible with a polycystic ovary.

Increased prevalence of luteinizing hormone beta-subunit variant in patients with premature ovarian failure.

OBJECTIVE: To evaluate the significance of an LH variant with a mutant beta-subunit (Trp8 to Arg8 and Ile15 to Thr15) in gynecologic disease, including infertility. DESIGN: Clinical study. SETTING: Department of Obstetrics and Gynecology, Shimane Medical University Hospital, Izumo, Japan. PATIENT(S): Two hundred forty-five Japanese women with endocrine disorders and/or gynecologic disease and 153 healthy, nonpregnant, fertile Japanese women. INTERVENTION(S): A blood sample was collected. MAIN OUTCOME MEASURE(S): The ratio of LH values from the SPAC-S and Immulyze assays (LH ratio: SPAC-S LH/Immulyze LH) was used to determine variant (< or =0.5) or wild-type (>0.5) LH status according to a demonstrated relation between the ratio and the sequence of the LH beta-subunit gene. RESULT(S): The LH ratio was lower (0.80+/-0.31) in the 245 patients than in the controls (1.00+/-0.38), and the variant was more frequent in the patients (18.4%) than in the controls (8.5%). We found no difference in the frequency of the variant between infertile and fertile patients. The prevalence of infertility did not differ between patients with variant LH and patients with normal LH. Ovulatory disorders, hyperprolactinemia, premature ovarian failure, menstrual disorders, and luteal insufficiency were significantly more frequent in patients with the variant. CONCLUSION(S): Variant LH may contribute to female reproductive disorders, including infertility and premature ovarian failure.

Estradiol-17beta measurement in women receiving conjugated estrogens. Dissociation between two commercial methods.

To investigate problems associated with measurement of estradiol-17beta (E2) in hormone replacement therapy (HRT), two commercial immunometric methods (Coat-A-Count E2 and Immulyze E2) were used to assay E2 concentrations and the two results were expressed as E2 ratios. Samples were obtained from 97 Japanese women receiving HRT and 168 unmedicated women. The largest differences between methods (P < 0.001) occurred in patients receiving oral conjugated estrogen (CE), while the best concordance was found in unmedicated women; like these controls, patients receiving oral estriol or transdermal E2 showed no significant difference between methods. Defining an E2 ratio > or = 2.0 as an abnormal discordance, the mean E2 ratio and the frequency of abnormal discordance in the CE group were 2.15 +/- 1.18 and 43.6%, respectively. No abnormal discordance for E2 occurred in other groups. In serial serum samples from the control group, no significant difference was seen between the mean E2 ratio at first measurement and those at a subsequent measurement. Similarly, no significant difference in the ratio was seen when two serial samples from CE patients were compared. However, E2 ratios after prescription of CE were significantly higher than before treatment in all patients. In conclusion, although measurement of E2 is important in patients receiving HRT, validity of the test methods must be carefully weighed for patients receiving CE.

Vascular endothelial growth factor mRNA synthesis by peripheral blood mononuclear cells in patients with acute myocardial infarction.

We studied vascular endothelial growth factor (VEGF) mRNA synthesis by peripheral blood mononuclear cells (PBMCs) in 30 patients with acute myocardial infarction (AMI) and 20 healthy individuals. PBMCs were isolated from all patients on days 3 and 14 after the onset of aMI, and from all of control individuals. To prepare samples containing identical amounts of GAPDH cDNA, competitive PCR was performed by co-amplifying serial dilutions of GAPDH mutant templates. Next, to measure VEGF cDNA quantitatively in the samples containing identical amounts of GAPDH, we also used competitive PCR by co-amplifying mutant templates of VEGF. The serum VEGF concentrations on day 14 in patients with aMI were measured by an ELISA method. Higher levels of VEGF mRNA in PBMCs were present on day 14 than either on day 3 or in the control group. Serum VEGF concentrations correlated with the VEGF mRNA levels of PBMCs on day 14. Peak serum CK levels correlated well with VEGF mRNA levels of PBMCs on day 14. The present findings suggest that PBMCs may be one of the candidates responsible for elevated circulatory VEGF protein following aMI. In addition, VEGF mRNA may be overexpressed in PBMCs in response to cardiac muscle damage.

Efficacy and safety of oral estriol for managing postmenopausal symptoms.

OBJECTIVE: to assess the therapeutic efficacy and safety of oral estriol for the treatment of climacteric symptoms in postmenopausal women. METHODS: 68 postmenopausal women with climacteric symptoms received oral estriol, 2 mg/day, daily for 12 months. We evaluated the degree of climacteric complaints with estriol therapy; serum levels of gonadotropins, estradiol (E2) and lipids; biochemical markers of bone metabolism; blood pressure; and side effects both at baseline and during treatment. Climacteric symptoms were assessed according to the menopausal index (MI), a version of the Kupperman index that had been modified for Japanese women. RESULTS: oral estriol therapy significantly reduced total MI scores. The greatest relief was noted for hot flushes, night sweats, and insomnia. Estriol treatment significantly lowered serum follicle stimulating hormone (FSH) and luteinizing hormone (LH) concentrations but did not affect any of the other parameters (lipids, bone, liver and blood pressure) during the study period. Slightly vaginal bleeding occurred in 14.3% of those who underwent natural menopausal women. Histologic evaluation of the endometrium and ultrasound assessment of the breasts following 12 months of estriol treatment found normal results in all women. CONCLUSION: Estriol is a safe and effective alternative for relieving climacteric symptoms in postmenopausal Japanese women.

Association of an insertion polymorphism of angiotensin-converting enzyme gene with the activity of lupus nephritis.

BACKGROUND: Lupus nephritis is a common manifestation of systemic lupus erythematosus (SLE). The pathogenesis of lupus nephritis has not been fully understood; however, immunological abnormalities have been considered in the development and activity of lupus nephritis. As angiotensin-converting enzyme (ACE) is implicated in various immunological phenomena, we investigated the correlation between insertion (I)/ deletion (D) polymorphism of the ACE gene and the activity of lupus nephritis. PATIENTS AND METHODS: Eighty-four patients with SLE and 100 healthy subjects were enrolled in this study. Following the extraction of genomic DNA from the peripheral blood, the ACE genotype was determined by the polymerase chain reaction. The patients were classified by the histological findings according to the WHO classification. In addition, the activity index and chronicity index were used to assess the severity of renal involvement. RESULTS: Individuals with II genotype showed a significantly increased activity of lupus nephritis. The allelic frequency was I/D = 0.84/0.16 in patients with WHO class IV renal lesions, and I/D = 0.36/0.64 in those with WHO class I lesions and 0.61/0.39 in patients with WHO class I or WHO class II. The difference in the allelic frequency between patients with WHO class IV and those with WHO class I or WHO class I + WHO class II was statistically significant (p = 0.00016 or p = 0.027, respectively). Moreover, lupus nephritis patients with II genotype showed significantly higher activity index than those with DD genotype (p = 0.0009). CONCLUSION: These results suggest that the insertion polymorphism of the ACE gene may correlate with the activity of lupus nephritis.

Enlarged focal nodular hyperplasia of the liver under the influence of oral contraceptives.

Two cases of focal nodular hyperplasia of the liver in mature women treated with the oral contraceptive are described. Radiological investigations in one case revealed the typical findings of focal nodular hyperplasia with computed tomography and magnetic resonance imaging demonstrating central scar structures while spoke-wheel appearance was evident on arteriography, in the other case however findings were atypical. Routine investigations including liver function tests and alpha-fetoprotein levels were normal while hepatitis B surface antigen and hepatitis C virus antibody were negative. The lesions of these two cases enlarged significantly during the follow-up and they were therefore surgically resected. Pathological features of both resected specimens, such as hepatocellular hyperplasia, bile duct proliferation and vascular abnormalities, were compatible with focal nodular hyperplasia. It has been suggested that tumor growth may be augmented by sex hormone stimulation and therefore estrogen and progesterone receptor expressions in the resected tumors were determined by immunocytochemistry. Interestingly, stainings for both receptors were negative. In case 2, the tumor was enlarging although oral contraceptive use had been discontinued for the past 7 years. These results suggest that there is no direct relationship of focal nodular hyperplasia with oral contraceptives. The role of sex hormones in focal nodular hyperplasia of the liver merits further study.

Esophagogastrostomy reconstruction after limited proximal gastrectomy.

BACKGROUND/AIMS: Recent advances in diagnostic techniques have led to the detection of an increasing number of early gastric cancers in the upper third of the stomach. The objective of this study was to determine the most appropriate surgical treatment for these cancers. METHODOLOGY: The clinicopathologic characteristics of 35 patients with early gastric cancer in the upper third of the stomach who underwent three different types of gastrectomies were reviewed retrospectively from hospital records between January 1992 and August 1999. RESULTS: Patients undergoing limited proximal gastrectomy with esophagogastrostomy reconstruction had shorter operation times and less blood loss than those for patients undergoing total gastrectomy or proximal gastrectomy with jejunal interposition. No lymph node metastasis was identified in any of these patients. Heartburn due to reflux esophagitis was seen in a few patients of each group, but they were successfully treated by antacids. The extreme reduction in food intake volume was more frequently experienced in patients with total gastrectomy than those with both proximal gastrectomies. When mortality due to other disease was excluded, all patients survived without recurrence. CONCLUSIONS: A limited proximal gastrectomy with esophagogastrostomy reconstruction decreased surgical risk and realized preservation of maximal function.

Live birth after conservative surgery for severe adenomyosis following magnetic resonance imaging and gonadotropin-releasing hormone agonist therapy.

This is a report of a live birth after conservative surgery for severe adenomyosis following diagnosis by MRI and therapy with GnRH-a. A 33-year-old gravida 1 para 1 woman with a 5-year history of secondary infertility received a gonadotropin-releasing hormone agonist (GnRH-a), leuprolide acetate, for 16 weeks to control symptoms of severe adenomyosis and to treat infertility. However, severe dysmenorrhea recurred after the discontinuation of therapy. Because an elevated serum level of CA-125 and MRI findings suggested that she was experiencing a relapse of adenomyosis, GnRH-a therapy was re-instituted. After 24 weeks of the second therapy, her uterus decreased to normal size and an MRI revealed a localized low-signal-intensity myometrial mass with well-defined borders. We easily resected the localized lesion of adenomyosis using the same technique used to treat uterine leiomyoma. The patient became pregnant after 12 weeks of additional danazol therapy. A healthy male infant was delivered at term by cesarean section.

A case of a pregnant woman with luteal insufficiency and a mutation in the beta-subunit of luteinizing hormone.

We report a case of a pregnant woman with luteal insufficiency and infertility associated with a variant luteinizing hormone (LH) beta-subunit and normal follicle-stimulating hormone (FSH) concentration. A 29-year-old woman presented to our hospital with infertility. Given the presence of low mid-luteal serum progesterone concentration and out-of-phase endometrial biopsy, luteal insufficiency was suspected. Several forms of treatment (clomiphene citrate therapy and human menopausal gonadotrophin therapy) were administered, but did not improve the patient's luteal insufficiency. However, administration of hCG during the luteal phase could distinctly improve the luteal insufficiency. She became pregnant after luteal support with progesterone. Sequence analysis of the patient's LH beta-subunit gene indicated heterozygosity for the point mutations Trp8 to Arg8 and Ile15 to Thr15 in the coding sequence. LH hypersecretion resembling that seen in polycystic ovary syndrome was observed. Serum concentrations of variant LH showed a more rapid increase in response to gonadotrophin-releasing hormone and reached a higher apparent value than did those of normal LH. Therefore, abnormal bioactivity and mistimed secretion of variant LH at mid-cycle may have a deleterious effect on the completion of oocyte maturation, ovulation, and subsequent corpus luteum function.

A case of postmenopausal endometriosis unrelated to neoplasm.

A 55-year-old woman was referred to our hospital for evaluation of a lower abdominal mass. Menopause had occurred at age 52 years. Ultrasonography and magnetic resonance imaging showed a partially solid right ovarian tumor, an appearance suggestive of malignancy. A transabdominal simple hysterectomy and bilateral salpingo-oophorectomy were performed. Pathologic examination revealed an endometrial cyst of the right ovary, a uterine leiomyoma, and adenomyosis.

[Thoracoscopic repair of diaphragmatic thinning for pleuroperitoneal communication; report of a case].

A 53-year-old man presented with massive right hydrothorax just after introduction of continuous ambulatory peritoneal dialysis (CAPD). Because the glucose concentration of pleural fluid was markedly high compared with that of serum, we diagnosed pleuroperitoneal communication. Thoracoscopic surgery was performed and thinning of the diaphragm was found. We sutured the diaphragm to repair the thin portion and performed pleurodesis with 50% glucose solution. He restarted CAPD 1 month post-operatively and continued at home without pleural effusion. Eight months post-operatively, he experienced dyspnea again and chest X-ray showed right hydrothorax. Although the cause of recurrent hydrothorax is unknown, it may be that not only surgical repair but also more intense pleurodesis is needed.

[Role of the spleen in OK-432 immunotherapy and characterization of effector cells].

The mechanism of anti-tumor effect of OK-432 was examined in C3H/He mice transplanted with methylcholanthrene induced fibrosarcomas. Tumor growth was significantly reduced in mice treated with OK-432 daily for 7 days after tumor inoculation. Tumor growth was significantly reduced in Sham-operated mice treated with OK-432 when compared to untreated controls. On the other hand, splenectomized mice failed to respond to OK-432 immunotherapy, suggesting the spleen is an essential organ in host responsiveness to this immunostimulant. Further dissection of the mechanism of OK-432 immunotherapy was achieved by Winn assay with spleen cells taken from mice inoculated with MCA-F (1 X 10(5) cells) and OK-432 for 7 days. Spleen cells from tumor-bearing mice which had been treated with OK-432 further reduced the tumor growth compared with spleen cells from tumor-bearer and such cytotoxic activity was reduced by the spleen cells with treatment of anti-Thy 1. 2 serum plus complement or by 700 rads irradiation before Winn assay, suggesting that OK-432 generated cytotoxic T cell population in vivo.

[Effect of anabolic steroid on immune response].

Using lymphocyte, monocyte and eosinophil counts of the peripheral blood, PHA-blastoid transformation, immunoglobulin and beta 2-microglobulin, the influence of anabolic steroid on the immune reactivity of the host was dissected by administration of Deca-Durabolin ( nandrolone decanoate) to both tumor-bearing host and tumor-free host after operation for alimentary tract. The number of peripheral lymphocytes and monocytes, the PHA-blastoid transformation of peripheral lymphocytes and the IgG level were increased, and the beta 2-microglobulin level showed the tendency of decrease after the administration of Deca-Durabolin.

[Specific active immunotherapy of murine methylcholanthrene-induced sarcoma using a partially purified soluble tumor specific transplantation antigens].

Immunotherapeutic effects of soluble tumor specific transplantation antigens (TSTA) were studied, Crude 3M KC1 extract of C3H/He methylcholanthrene-induced sarcoma (F tumor) was purified by preparative isoelectric focusing (pIEF). The immunoprotective fraction (Fr. 15, pI 5.7-6.0) possessed about 50 fold greater activity than the crude 3M KC1 extract. Three weekly injections of optimal immunoprotective dose of Fr. 15 retarded the outgrowth of F cells and decreased the outgrowth and tumor incidence of rechallenged inoculation in mice completely resected established 1 cm tumor. Weekly injections of Fr. 15 resulted in decreased local recurrences. This therapeutic effect was immunologically specific. Fr. 15 alone did not show the therapeutic effects on the established tumor, but combined treatment with cyclophosphamide gave the increased survivals. A metastatic variant cell line (F-4) metastasized to the lung spontaneously after the resection of primary subcutaneous tumor. Therapeutic injections of Fr. 15 decreased the rates of spontaneous pulmonary metastasis of F-4. Since cross-immunoprotection tests revealed that F and F-4 share the common TSTA, the therapeutic effect of Fr. 15 upon pulmonary metastases might be due to improved host's specific immunity.