Progesterone enhances macrophage colony-stimulating factor production in human endometrial stromal cells in vitro.

Increasing evidence suggests that macrophage colony-stimulating factor (M-CSF) is produced in the uterine endometrium and that it plays an important role in the reproductive process. In the present study, using an in vitro decidualization model and human endometrium, we investigated M-CSF messenger RNA (mRNA) expression in human endometrial stromal cells (ESC) by Northern blotting and in situ hybridization. The secreted M-CSF in the culture medium of ESC was measured by enzyme-linked immunosorbent assay. ESC were cultured in the presence of progesterone (P) or estrogen. After a 9-day culture with P, when in vitro decidualization was confirmed by the production of PRL, M-CSF mRNA and protein levels were 3.1 +/- 0.5- and 3.2 +/- 0.8-fold (mean +/- SEM) higher, respectively, than those in cultures without P (P < 0.01). The P-induced increase was dose dependent. On the other hand, estrogen did not increase M-CSF mRNA expression. M-CSF mRNA expression in the first trimester deciduae that expressed PRL mRNA was higher than that in the endometria. By in situ hybridization, ESC as well as epithelial cells were shown to express M-CSF both in vitro and in vivo. These findings indicate that human ESC (decidua cells) express M-CSF mRNA and suggest that they secrete M-CSF in a P-dependent manner during the process of decidualization.

Progesterone-dependent secretion of macrophage colony-stimulating factor by human endometrial stromal cells of nonpregnant uterus in culture.

Recent studies have suggested that macrophages colony-stimulating factor (M-CSF), a hematopoietic glycoprotein essential to the proliferation and differentiation of mononuclear phagocytes and their progenitor cells, is also involved in the reproductive process in mice and humans. In this study, we examined, by enzyme-linked immunosorbent assay, the supernatants of stromal cell-enriched fraction (SF) of human nonpregnant endometrium for the presence of M-CSF during culture with progesterone (P) or estrogen. The bioactivity of M-CSF was assessed in a colony-forming assay of murine bone marrow cells. In addition, the M-CSF level in the culture supernatant of SF further purified by subculture, of epithelial cell-enriched fraction purified from human endometrium, and of peripheral blood lymphocytes, including about 10% monocytes, was examined with or without P, because SF is contaminated by epithelial cells and macrophages, both of which are suggested to secrete M-CSF. During 2-week culture, the level of M-CSF in the supernatants of SF cultured with P was markedly higher than that of control culture and estrogen-treated culture on any day tested, except for the first 2 days. P had a dose-dependent effect on M-CSF production by SF. Estrogen also enhanced M-CSF production by SF, but did not show dose dependency. The SF culture supernatants showed a colony-forming activity that was completely blocked by neutralizing anti-M-CSF antibody. SF subcultured three times, which was confirmed to be of more than 99% purity, secreted M-CSF in a P-dependent manner. M-CSF was also detected in the culture supernatants of epithelial cell-enriched fraction and peripheral blood lymphocytes, but P-dependent M-CSF production was not shown in these cultures. These results suggest that human endometrial stromal cells themselves can secrete bioactive M-CSF in a P-dependent manner in vitro, indicating that the M-CSF reported to be present in human endometrium is secreted in part by stromal cells and may play a role in the regulation of endometrial function under P control.

Interleukin-1 inhibits in vitro decidualization of human endometrial stromal cells.

Interleukin-1 (IL-1), a critical cytokine for the initiation of the immune response to infection or antigenic challenge, is known to also possess a variety of biological functions outside the immune system. We examined whether IL-1 could affect the decidualization of human endometrial stromal cells (ESC), a conspicuous part in the process of implantation, by assessing PRL production and morphological transformation in an in vitro system. Purified human ESC were cultured in the presence of progesterone (P) with or without the addition of IL-1. IL-1 markedly suppressed the induction of PRL production by P in a dose-dependent manner. The morphological decidualization of ESC in response to P was also inhibited by IL-1. This report demonstrates for the first time the possibility that IL-1 blocks decidualization, the functional differentiation of human endometrial stromal cells in response to ovarian steroids.

Androgens induce prolactin production by human endometrial stromal cells in vitro.

Although there is a significant quantity of androgens in the endometrium, the function of these hormones has not been clarified, except for being estrogen precursors. Human endometrial stromal cells (ESC) were cultured in the presence of testosterone (T) and 5 alpha-dihydrotestosterone. Following culture, prolactin (PRL), a biochemical marker of stromal cell differentiation (decidualization) which is produced by ESC, was examined. T induced PRL production in a time- and dose-dependent manner, as reported previously for progesterone (P) stimulation. In addition, 5 alpha-dihydrotestosterone, which cannot be converted to estrogens, similarly induced PRL production. T in combination with P enhanced PRL production in cultured ESC significantly more than either P or T stimulation alone. A specific androgen receptor blocker, flutamide, when added to cultures containing T, inhibited PRL production in a dose-dependent manner, but did not affect the production of PRL induced by P. These results indicate that in vitro PRL production by human ESC is induced not only by P, but also by androgens through specific receptors and further suggest that androgens play an important role in human endometrial differentiation.

Combination effect of adenovirus-mediated pro-apoptotic bax gene transfer with cisplatin or paclitaxel treatment in ovarian cancer cell lines.

To develop a novel therapeutic strategy for ovarian cancer, we constructed a recombinant adenovirus which highly expresses pro-apoptotic Bax protein and examined its therapeutic effect on a series of ovarian cancer cell lines: A2780, A2780/cDDP, OVCAR-3 and SK-OV-3. A recombinant adenovirus carrying the Bax-alpha gene (AxCALNKYbax) induced high expression of the Bax-alpha protein in all the cell lines. The cytotoxic effect of Bax was observed in three ovarian cancer cell lines: the per cent reduction in the number of cells was 40.0% for cisplatin-sensitive A2780, 50.0% for cisplatin-resistant A2780/cDDP, and 64.8% for marginally cisplatin-resistant OVCAR-3. In contrast, it was only 12.3% for cisplatin-resistant SK-OV-3. Cisplatin-resistant A2780/cDDP had a p53 mutation and exhibited attenuated Bax induction after cisplatin treatment, which may explain why supplementation of Bax was effective in this chemoresistant ovarian cancer. Combination with cisplatin or paclitaxel enhanced the cytotoxic effect of Bax induction in all but one cell line including cisplatin-resistant A2780/cDDP. It appears that adenovirus-mediated Bax induction, with or without combination with conventional chemotherapy, useful strategy for the treatment of ovarian cancer.

Depression of proliferative response to fetal lymphocytes of retroplacental blood lymphocytes in human pregnancy.

Retroplacental blood lymphocytes (RPL) in human pregnancy were studied for proliferative response to related fetal lymphocytes and it was compared with that of peripheral blood lymphocytes (PBL) of the same donor. RPL proliferated poorly to related cord blood lymphocytes in 9 of 10 cases when compared with autologous PBL. RPL also showed lower proliferative response to unrelated adult lymphocytes in 7 of 12 cases. Proliferation of RPL was weaker to related cord blood lymphocytes than to allogeneic adult lymphocytes. These results indicate that the response of T cells at feto-maternal interface is impaired, especially to fetal antigens.

Comparison of human cytomegalovirus DNA polymerase activity for ganciclovir-resistant and -sensitive clinical strains.

Previously, we found that a ganciclovir (GCV)-resistant clinical human cytomegalovirus (HCMV) isolate had an amino acid substitution at codon 501 (Leu --> Phe) in the delta-region C of the DNA polymerase gene. DNA polymerases have now been (partially) purified from both the GCV-resistant and sensitive parental strains and the activity of DNA polymerase and 3'-5' exonuclease compared. With respect to DNA polymerase activity, the Michaelis constant (Km) and maximum velocity (Vmax) of the GCV-resistant strain for the DNA template were lower than those of the GCV-sensitive strain. With respect to 3'-5' exonuclease activity, the Km and Vmax of the GCV-resistant strain for the DNA substrate in the presence of ammonium sulfate were lower than those of the GCV-sensitive strain, while being similar in the absence of ammonium sulfate. Although the polymerase activity of the two strains showed almost the same sensitivity for the different polymerase inhibitors, the 3'-5' exonuclease activity of the GCV-resistant strain was more resistant to these inhibitors than that of the GCV-sensitive strain.

A case of postpartum hemolytic uremic syndrome with severe elevations of liver enzymes.

An unusual case of multifocused postpartum thrombotic microangiopathy was encountered in a woman with pregnancy-induced hypertension, which presented clinical features different from those generally seen in postpartum hemolytic uremic syndrome or thrombotic thrombocytopenic purpura. These changes included acute disseminated intravascular coagulation, severe elevations of liver enzymes, severe renal dysfunction, and hemolytic anemia. The patient was treated successfully with heparin, gabexate mesilate, furosemide, fresh whole blood, and large doses of corticosteroid with antibiotics, and she recovered completely without sequelae. The hypothetical pathogenesis of the complex symptoms of this catastrophic clinical condition is proposed.

[Conditions for low-intensity voluntary wheel running in rats and its chronic effects on health indexes].

Studies were undertaken to evaluate the fundamental conditions for a low-intensity voluntary wheel running model in rats and its chronic effects on health indexes. Male Fischer rats (SPF) 5 weeks of age were housed in individual sedentary conditions or in individual wheel-cage units which allowed free access to voluntary wheel running for 8 months. Voluntary running averaged 640 +/- 198 m/day, reached a peak (965m) at the 2nd month and waned over time, reaching a plateau after the 6th month (about 400-500m). Exercising rats consumed more food (+23%), but exhibited decreased body weight gains (-9%), suggesting a remarkable lowering of fat. A lowering effect on resting blood pressure (-5%) was also recognized. In addition, preventive effects on oxygen toxicity and effective bactericidal activity of neutrophils and pulmonary alveolar macrophages (PAM) were suggested. Although the amount of exercise in this study was the smallest of the other preceding ones conducted with a voluntary wheel running model, many potential health benefits were recognized. Such health promoting and protective effects by low-intensity voluntary exercise and the harmfulness of forced exercise in rats have been reported in researches on cancer, lowering fat and hypertension. Therefore it is important to set up conditions for low-intensity voluntary running. It was also demonstrated by this study that strictly controlled environmental conditions, such as room temperature and humidity, a 12-hr light-dark cycle and prevention of infection and psychological stress to rats, as well as using male rats, which are more inactive, were important factors to establish this model.

Increased cyclooxygenase-2 expression is correlated with suppressed antitumor immunity in cervical adenocarcinomas.

Cyclooxygenase-2 (COX-2) inhibition suppressed the growth of various tumors. The augmentation of antitumor immunity by increasing cytotoxic lymphocytes may be an important mechanism for COX-2 inhibition. Among cervical cancers, adenocarcinomas present more aggressive behavior and overexpressed COX-2. The expression of COX-2 and the CD8+ lymphocyte infiltrations were evaluated in this study by immunohistochemistry. We studied COX-2 expression and CD8+ lymphocyte infiltration in 55 women with cervical adenocarcinomas. COX-2 expression and tumor stromal CD8+ lymphocytes were evaluated by semiquantified methods. Tumor intraepithelial lymphocytes were counted under microscopic field of x200. Correlations between these data and other clinicopathologic features were investigated. Thirty-seven out of 55 (67.3%) cervical adenocarcinomas significantly expressed COX-2. Patients who died within 5 years showed higher percentage of COX-2 expression than survivors (100% vs 58.1%, P < 0.05). Victims also showed lesser intraepithelial CD8+ lymphocyte counts than survived patients (3.4 vs 26.4, P < 0.05). COX-2 expression and tumor intraepithelial lymphocyte count were reversely correlated with each other (correlation index: -0.38, P < 0.01). Up-regulated COX-2 expression and lesser tumor intraepithelial CD8+ lymphocyte count are poor prognostic indicators for cervical adenocarcinoma patients. COX-2 may play an important role in the suppression of host antitumor immunity in cervical adenocarcinomas.

Is there any effect of volcanic eruptions of Mount Sakurajima on human lungs?--histopathological investigation and measurement of intrapulmonary particulate deposits amounts.

In order to investigate effects of volcanic eruptions of Mt. Sakurajima on human lungs, an amount of intrapulmonary particulate deposits (IPD) and histopathological changes were evaluated, using autopsied lungs of deceased residents of Kagoshima (the area affected most by the volcanic eruptions of Mt. Sakurajima, n = 66) in comparison with those of Saitama (n = 73). The amount of IPD was measured by macroscopic image analysis and by alkaline extraction. Correlation was seen in the IPD values obtained by these two methods (Spearman's correlation coefficient: 0.525). The IPD value measured through chemical digestion, which increased with age, was lower in the residents of Kagoshima (17.0 +/- 6.3 mg/g) than in those of Saitama (26.8 +/- 10.6 mg/g). This difference of IPD value between Kagoshima and Saitama residents was also observed in male, female, smoker and nonsmoker subgroups. The incidence of pulmonary emphysema was not different between Kagoshima and Saitama cases. No silicotic nodules were observed in any cases. But, the incidence of bronchial goblet cell hyperplasia and squamous metaplasia in Kagoshima was higher than that in Saitama in male cases and smoker cases. In Kagoshima cases, the incidence of squamous metaplasia was significantly higher in smokers than in nonsmokers. The synergistic influence of volcanic eruptions and cigarette smoking on human airways was suggested.

Effects of acidic phospholipids, nucleotides, and heparin on the activity of lipase from rat liver lysosomes.

Purification and characterization of endogenous lipid factors that stimulate rat liver lysosomal lipase has led to the identification of cardiolipin, phosphatidylserine, and phosphatidic acid as stimulators of this activity. Bovine heart cardiolipin (half-maximal stimulation at 1.5 x 10(-4) m) and bovine brain phosphatidylserine (half-maximal stimulation at 9.5 x 10(-4) m) were the most potent of the phospholipids from other sources tested. The major rate-enhancing effect of phosphatidylserine is expressed as a 35-fold increase in the apparent V(max) of the enzyme. The effect is produced by acid phospholipids specifically, since in no case was there greater than a twofold stimulation by synthetic detergents, zwitterionic phospholipids, taurocholic acid, or gum acacia. The observed degree of stimulation depends upon the detergent used to disperse tripalmitin substrate and the relative concentrations of factor and detergent in reaction mixtures. The concentration of phosphatidylserine to produce half-maximal stimulation is directly dependent upon the Triton X-100 concentration, but the effects of this detergent on cardiolipin stimulation are more complex. Enzyme activity is inhibited 50% by 1 mm nucleoside triphosphate and 2.5 mm ADP, 80% by 1 mm PP(i), 100% by 20 U/ml heparin and 0.25 mg/ml chondroitin sulfate, and 80% by 10 mm sulfate ion. Inhibition is partially prevented by phosphatidylserine.

Serum-free medium for murine and human lymphoid and hybridoma cell lines.

We established a serum-free medium of low protein content (125 micrograms/ml) TYI 100, consisting of three hormones and five growth factors for the growth of lymphoid and hybridoma cell lines. In TYI 100 medium, mouse and human hybridomas grew equally well as in RPMI 1640 supplemented with 10% fetal bovine serum (10% FBS) without adaptation to the serum-free medium. TYI 100 medium allowed several passages of mouse hybridoma lines and the total cell number was more than in 10% FBS. TYI 100 medium also supported growth of myelomas and anchorage dependent cell lines, Bowes and CHO, well. TYI 100 medium is composed of inexpensive supplements and is therefor applicable to large scale culture.

A novel UDP-sugar, UDP-3-ketoglucosamine or UDP-4-ketoglucosamine, from bovine heart muscle reduces metmyoglobin with NAD(P)H.

3-Ketoglucose and similar ketosugars have been identified in microorganisms only and little is known about their functions. UDP-sugars are widely found as an intermediate in sugar metabolism in living organisms. Yet what role UDP-sugars play, or whether they play a direct role in metabolism, is still unknown. UDP-sugars were isolated and purified from bovine heart muscle, and a UDP-sugar fraction capable of NAD(P)H-dependent catalytic reduction of metmyoglobin was detected. Subsequent identification revealed that the active UDP-sugar was UDP-3- or UDP-4-ketoglucosamine. These compounds were purified from bovine cardiac muscle by ultrafiltration, anion-exchange column chromatography and reverse-phase chromatography. They were further characterized by determination of their chemical reducing activity, by comparison with synthetic UDP-3- or UDP-4-ketoglucosamine standards using high-performance liquid chromatography, by estimation of molecular mass using fast atom bombardment mass spectrometry, and by Fourier transform infrared microspectroscopy and electron probe microanalysis. The results suggest that UDP-3- or UDP-4-ketoglucosamine reduces metmyoglobin in bovine cardiac muscle. It is important that the reducing activity displayed by this ketosugar is not the effect of UDP-3- or UDP-4-ketoglucosamine alone but depends on NAD(P)H. In other words, this action of UDP-3- or UDP-4-ketoglucosamine is catalytic.

[A light and electron microscopic study of papillary hidradenoma of the vulva].

Papillary hidradenoma is a rare benign tumor, which occurs mostly on the labia major or in the perianal region. Two cases were studied by light and electron microscopy. Light microscopically, these lesions were seen to consist of papillary adenomatous structures, whose lumina were lined by two to three layered cuboidal epithelial cells. Nuclear atypia and mitotic figures of these epithelial cells were rare. Nipple-like projections of their eosinophilic cytoplasm toward the lumina, resembling those of "apocrine" secretion, were frequently observed. Electron microscopy revealed that the epithelial cells lining the lumina had many secretory granules, lysosome-like structures with lipid, and large atypical mitochondria in their cytoplasm. The apical portion of the cytoplasm, which contained numerous secretory granules, often protruded toward the lumina. These ultrastructural features were considered to be similar to those of apocrine gland secretory cells. Moreover, in the basal layer of epithelium, spindle-shaped cells with features of typical myoepithelial cells were identified. These light and electron microscopic findings suggest that papillary hidradenoma of the vulva is one of the tumors of skin appendages with direction of differentiation toward the apocrine glands.

Suppression of natural killer cell activity by sera from patients with endometriosis.

OBJECTIVE: We determined the effect of sera from patients who have endometriosis on natural killer cell activity. STUDY DESIGN: The natural killer cell activity of lymphocytes from healthy volunteers was examined after incubation with sera from patients who had endometriosis or from controls, with K562 cells used as targets. RESULTS: Lymphocytes treated with sera from patients who had endometriosis expressed significantly lower levels of cytotoxicity compared with lymphocytes treated with control sera. This suppression of cytotoxicity was dose dependent, and the degree of suppression was proportional to the incubation time of the effector cells with the sera. Decreased cytotoxicity after serum treatment was also observed with sera from patients who had been treated with danazol. CONCLUSIONS: These findings show that humoral factors that can inhibit natural killer cell activity in vitro are present in the peripheral blood of patients who have endometriosis; moreover, they suggest that the suppressed natural killer cell activity may allow the development of endometrial cells at ectopic sites.

Hepatocellular carcinoma with sarcomatous proliferation showing an unusual and wide-spread metastasis.

A hepatoma comprised of carcinomatous and sarcomatous proliferation has been considered as collision or carcinoma with sarcomatous proliferation. In this report, an autopsy case of primary hepatocellular carcinoma showing almost equal amount of carcinomatous and sarcomatous proliferations is presented. Serial sections from the tumor showed the frequent transition of the figure from carcinoma to sarcoma, justifying the diagnosis of hepatocellular carcinoma with sarcomatous proliferation. When compared with the usual hepatocellular carcinoma, metastasis with sarcomatous figure was more extensive and both ventricles and atriums of the heart, skin, and intestinal tract were involved. A sarcomatous proliferation in hepatocellular carcinoma and manner of metastasis are discussed with a review of the pertinent literature.