Activation of human neutrophil by cytokine-activated endothelial cells.

Cytokine activation of vascular endothelial cells renders the hyperadhesiveness for neutrophils. During the processes of inflammation and atherosclerosis, the production of reactive oxygen species by neutrophils contributes to endothelial cell (EC) damage and injury. However, the precise mechanisms for neutrophil activation by ECs remain unknown. Thus, we investigated what kinds of pathophysiological factors synthesized by inflammatory cytokine-activated ECs potentiated the activity of neutrophil functions. The magnitude of O(2)(-) release from neutrophils, which is one of pivotal neutrophil functions, was measured as an indicator potentiated by activated ECs. Neutrophils release massive amounts of O(2)(-) on coculture with activated ECs. Anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody (Ab) or specific platelet-activating factor (PAF)-receptor antagonist suppressed the O(2)(-) release from neutrophils on coculture with the activated ECs by 50% to 70%. The supernatants from activated ECs also induced O(2)(-) release by neutrophils. This stimulatory effect of activated EC supernatants on O(2)(-) release by neutrophils was abolished by anti-GM-CSF Ab or by PAF-receptor antagonist. As we previously reported, we demonstrated the expression of GM-CSF mRNA by Northern blotting and protein synthesis of GM-CSF by ELISA on tumor necrosis factor as well as interleukin-1-activated ECs. Although phosphorylation of mitogen-activated protein kinases was observed in ECs stimulated by tumor necrosis factor and interleukin-1, treatment of ECs with PD98059 (MEK1 inhibitor) and SB203580 (p38 mitogen-activated protein kinase inhibitor) in the presence of the cytokine failed to attenuate the stimulatory effect of activated ECs on neutrophil activation. We found that activated ECs regulated neutrophil function on coculture. We show here for the first time, to our knowledge, that the collaboration between GM-CSF and PAF synthesized by activated ECs markedly potentiated neutrophil activation.

Anemia-inducing substance from plasma of patients with advanced malignant neoplasms.

Patients with advanced malignant neoplasms develop anemia and immunosuppression. During an attempt to identify the causes, we have found that plasma from such patients makes RBCs more fragile in hypotonic buffer, according to results obtained with a coil planet centrifuge. Plasma from these patients suppresses mitogen-stimulated lymphocyte proliferation. In this study, we identified the substance with these effects as a protein. During two-dimensional gel electrophoresis, two isomers with M(r) 50,000 and slightly different isoelectric points near 6.0 were found. Cell fractionation showed that these proteins were in both the cytosol and the nuclear fraction of cells in neoplasms. Another protein with the same antigenicity and a M(r) 100,000 found in the nuclear fraction of cells in neoplasms.

Elimination of anemia-inducing substance by cyclic plasma perfusion of tumor-bearing rabbits.

We carried out a fundamental study to search for a therapeutic modality that would remove the anemia-inducing substance (AIS) from the plasma of cancer patients because it is thought to be one of the substances responsible for anemia and immunodeficiency in advanced cancer patients. Using AIS isolated from the plasma of patients with advanced ovarian carcinoma, we confirmed that adsorption of AIS to noncoated charcoal was nonspecific and high. Moreover, it was verified that VX2 carcinoma-bearing rabbits are an optimal experimental model for plasma perfusion. The data obtained on day 40 after transplantation (hemoglobin, 9.1+/-2.1 g/dl; osmotic pressure inducing RBC lysis, 137+/-11 mosmol/kg; lymphocyte stimulation index, 8.8+/-8.6; and RBC fragility-inducing activity, 40+/-9 mosmol/kg) proved similar to the hematological findings in patients with cancer cachexia. A 1-h plasma perfusion (3 ml/min) through noncoated charcoal was performed in tumor-bearing rabbits, and it resulted in the restoration of RBC fragility-inducing activity and suppression of lymphocyte blast formation to pretransplantation values. When plasma perfusion was performed every 3 days, RBC fragility-inducing activity, which increased again 3 days after perfusion, was diminished, and RBC osmotic resistance was within the normal range from the fourth perfusion onward. These results showed that cyclic plasma perfusion is effective in sustained removal of RBC fragility-inducing factor from plasma, suggesting that it might have the potential for clinical application.

Impact of erythrocytes on mouse embryonal development in vitro.

To elucidate the role of reactive oxygen species (ROS) in the development of mouse embryo, effects of superoxide dismutase (SOD), catalase and erythrocytes were studied in vitro. Oocytes were fertilized and 2-cell-cleaved embryos were cultured in the presence or absence of either erythrocytes, SOD or catalase. Under standard culture conditions, the fertilization and cleavage rates were 77.4 and 12.5%, respectively. In the presence of xanthine and xanthine oxidase, those rates decreased to 28.2 and 4.5%, respectively. The hazardous effect of ROS was completely inhibited by erythrocytes. These results suggested that small amounts of erythrocytes might effectively degrade ROS during the development of cultured embryos.

Role of glutathione metabolism and apoptosis in the regression of liver hemopoiesis.

Although apoptosis has been believed to play important roles in ontogenic development of animals, the molecular mechanism that triggers the regression of liver hemopoiesis during perinatal period is not known. Apoptosis is induced by many factors, such as decrease in growth factors and increased oxygen stress. Because hepatic gamma-glutamyltransferase (GGT) changes markedly during the perinatal period of a rodent, metabolism of glutathione (GSH), a naturally occurring major antioxidant, might change significantly in and around liver cells. To know the possible involvement of apoptosis and GSH metabolism in the regression of hemopoiesis, hepatocytes and hemopoietic cells were isolated from fetal rat liver. Biochemical analysis revealed that, during the perinatal period, hepatic GGT levels transiently increased predominantly with hepatocytes, suggesting a marked change in thiol status in and around these cells. Cell culture analysis revealed that hemopoietic cells but not hepatocytes exhibited a marked apoptosis in a thiol-free medium, as judged from DNA fragmentation. The apoptosis of hemopoietic cells was inhibited by various thiols, such as L-cysteine, N-acetyl-L-cysteine (NAC), and GSH. These observations suggested that a marked change in GSH status in and around liver cells might play critical roles in triggering apoptosis of hemopoietic cells, thereby enhancing the regression of liver hemopoiesis.

Survivin, bcl-2 and matrix metalloproteinase-2 enhance progression of clear cell- and serous-type ovarian carcinomas.

Clear cell adenocarcinomas (CA), unlike serous adenocarcinomas (SA) of the ovary, are often at stage I, are resistant to platinum-based drugs and have a poor prognosis. The causes of these differences are unclear. In this study, the differences in progression between CA and SA were examined in terms of apoptosis-related and tumor invasion-related factors. The 16 cases of CA and the 16 cases of SA were reviewed. Excised tissues were classified into primary or metastatic loci, and the expressions of survivin, Bcl-2 and matrix metalloproteinase-2 (MMP-2) in each locus immunohistochemically assayed. Whether the expression of each protein was correlated to prognosis was investigated and additionally the invasion ability of cell strains established from CA and SA were examined using in vitro invasion assay. CA at stage I showed significantly higher survivin expression than SA (p<0.05). In CA, survivin tended to be expressed higher in primary locus than in metastatic locus (p=0.068), however, Bcl-2 was expressed relatively higher in the latter (p=0.087). SA did not have these tendencies. While MMP-2 was expressed significantly higher in SA than in CA (p<0.05), and more so in metastatic locus than in primary locus of SA (p<0.05). Invasion assay showed that the invasion of cells derived from SA was significantly inhibited by tissue inhibitors of metalloproteinase-2, an MMP inhibitor. The disease-free interval was significantly shorter when survivin expression was observed in the nucleus. These results suggest that the expression of apoptosis inhibiting factors and enhanced invasion ability affect progression of CA and SA, respectively.

Angiogenesis in the adipose tissue of tumor-bearing rabbits treated by cyclic plasma perfusion.

The mechanisms of cancer cachexia have not been elucidated. We previously reported that cyclic plasma perfusion using non-coated charcoal was effective in cancer cachexia. In the present study we investigated the angiogenic effect of cyclic plasma perfusion on adipose tissue. Twenty rabbits were divided into two groups, i.e., tumor-bearing rabbits subjected to cyclic plasma perfusion (n=10, PP group), and tumor-bearing rabbits subjected to sham-perfusion (n=10, SP group). The changes in body weight, total body fat, and expression of angiogenic factors were investigated. Loss of body weight and total body fat was significantly suppressed in the PP group. Apoptosis of adipocytes was seen in both groups only in the early stage of tumor bearing. Lipolytic activity in the PP group showed a lower ratio than that in the SP group. In the PP group, increased microvessel density and expression of vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) were seen at 40 days after transplantation. Similar findings were not seen in the SP group. These results suggest that cyclic plasma perfusion not only decreased lipolytic activity but induced angiogenesis in the adipose tissue.

Effect of cyclic plasma perfusion on apoptosis regulatory proteins in primary tumors of VX2-carcinoma-bearing rabbits.

Although the mechanism of cancer cachexia is unknown, in previous studies we reported that cyclic plasma perfusion through non-coated charcoal was effective in preventing cancer cachexia. In the present study we investigated the effect of cyclic plasma perfusion on VX2 carcinoma. Sixteen tumor-bearing rabbits were divided into two groups: i) a group subjected to cyclic plasma perfusion (n=8, group PP) and ii) a group subjected to sham-perfusion (n=8, group SP), and changes in body weight, tumor size, and morphological findings were investigated. Body weight loss and tumor growth were significantly suppressed in group PP. The tumor cells in group PP showed apoptosis and a high Bax/Bcl-2 ratio, and the survival rate was significantly higher than in group SP. These results suggest that cyclic plasma perfusion is effective not only in preventing cancer cachexia but in suppressing tumor growth and improving outcome.

Expression of apoptosis regulatory proteins in advanced cancer of the uterine cervix after cyclic balloon-occluded arterial infusion chemotherapy.

We have already reported satisfactory therapeutic results of cisplatin-based cyclic balloon-occluded arterial infusion chemotherapy (BOAI) because it enabled advanced cervical cancer of the uterus (cervical cancer) to be treated by simple total hysterectomy (STH). In the present study, we investigated the expression of apoptosis regulatory proteins in advanced cervical cancer treated by cyclic BOAI. The results showed that the proportion of Bax-positive cells was 75.2 +/- 5.6% after the first BOAI in the cases in which STH became possible (group C), and significantly lower, 11.6 +/- 11.7% (p=0.0001), in the cases in which STH remained impossible (group I). The proportion of Bax-positive cells was significantly higher in group C than in group I throughout the treatment period, but there was no significant difference in Bcl-2 expression between the two groups. The survival rate by the Kaplan-Meier method was significantly higher in group C than in group I. These results suggest that the antitumor effect of cyclic BOAI is closely associated with apoptotic cell death, which may in part be influenced by the expression of Bax.

Association of the shrinkage of uterine leiomyoma treated with GnRH agonist and deletion of long arm of chromosome 7.

Specific chromosomal abnormalities, such as del(7q), t(12;14), 12 trisomy, or the rearrangement of 6p, are seen in approximately 30% of uterine leiomyomas despite their benign status. We investigated the association between the shrinkage of uterine leiomyomas treated with a GnRH agonist and the interstitial deletion of chromosome 7q, which is one of the most common chromosomal abnormalities in uterine leiomyomas. This study covered 29 women with uterine leiomyomas who were treated with a GnRH agonist before surgery. The volume of the largest myoma nodule was measured by means of MRI before and at 12 weeks after the beginning of GnRH agonist treatment, and the percentage of the reduction in volume was calculated. Genomic DNA was extracted from leiomyoma tissue and peripheral blood, and amplified by PCR using fluorescently-tagged oligonucleotide primers of twelve microsatellite loci on chromosome 7. The PCR products were analyzed for loss of heterozygosity (LOH) using an automated fluorescent DNA sequencer. Of the 29 informative tumors, five (17%) showed LOH with deletion of the common region, D7S491. The mean percentages of the reduction in volume of the largest myomas with LOH or without LOH were 32+/-13 and 18+/-58%, respectively (not significant). One tumor showing interstitial deletion of both alleles (homo-deletion) reduced in volume by 19%. Another tumor showing an extraband increased in volume by 13%. Although tumor specific chromosomal deletion suggested the existence of tumor suppressor genes in this region, there was no significant association between the shrinkage of uterine leiomyomas treated with a GnRH agonist and the interstitial deletion of chromosome 7q.

Combined analysis of p53 and RB pathways in epithelial ovarian cancer.

Disruptions of the p16-CDK4/cyclin D1-pRb pathway (RB pathway) and the p14ARF-MDM2-p53 pathway (p53 pathway) are important mechanisms in the development of human malignancies. In this study, we investigated RB and p53 pathways in 46 epithelial ovarian cancers (EOCs). In the RB pathway, 16 (34.8%) of 46 cases had p16 gene alterations or loss of expression. The deletion of the p16 gene was a rare event. In 7 cases, we observed methylation in the 5'CpG island in the promoter region of the p16 gene. Abnormal expressions of pRb and CDK4/cyclin D1 were 10.9% and 30.4%, respectively. In the p53 pathway, 10 (21.7%) of 46 cases had p14ARF gene alterations or abnormal expression. In 4 cases, methylation in the 5'CpG island in the promoter region of the p14ARF gene was present. MDM2 overexpression was a rare event. Thirty-six (78.3%) of 46 patients had p53 gene alterations or expression. In our studied cases, p14ARF abnormalities were independent of p16 abnormalities. Abnormal RB and p53 pathways were present in 60.9% and 80.4% of cases, respectively. In conclusion, disruptions of p53 and RB pathways are frequent events and the inverse correlations were present between the abnormality of p16 and p14ARF in EOCs.

Neonatal thrombocytopenia induced by maternal anti-HLA antibodies: a potential side effect of allogenic leukocyte immunization for unexplained recurrent aborters.

Allogenic leukocyte immunization is one of several treatments tried for unexplained recurrent aborters, and is reported to have few maternal and neonatal side effects after the immunotherapy having been reported to date. In the present study, we report a rare case of neonatal thrombocytopenia (41000 cells/microl) observed in a female infant delivered by an unexplained habitual aborter. The mother was immunized with her husband's leukocytes once before pregnancy and twice at the 5th and 6th week of her successful pregnancy. Serological studies using mixed passive hemagglutination assays (MPHA) showed that maternal serum did not contain any antibodies which were reactive to 11 platelet-specific antigens, or to granulocyte antigens extracted from 9 persons. Lymphocyte cytotoxicity tests, however, showed that maternal serum but not infant serum had anti-HLA antibodies against both paternal and infant lymphocytes. Moreover, the maternal serum was found to have anti-HLA IgGs against platelet antigens extracted from the father and the infant. It is highly likely that this case of neonatal thrombocytopenia was caused by transplacental perfusion of maternal anti-HLA antibodies whose production was induced or enhanced by the allogenic leukocytes immunizations.

A simplified method for measuring fetal hemoglobin.

Despite confirmation of the placental location by ultrasound examination, bloody tap during diagnostic and therapeutic amniocentesis may occur. If the blood originates from the fetus, such unfavorable complications as fetomaternal transfusion or maternal sensitization may result. Thus, it is necessary to determine immediatley the origin of the blood by the presence of fetal hemoglobin (HbF) in the specimen. To meet an urgent demand for a simpler and faster method for measuring HbF within a few minutes at bedside without requiring complicated procedures or instrumentation, a method has been developed which uses the alkali denaturation technic.

Correlation between shrinkage of uterine leiomyoma treated with buserelin acetate and histopathologic findings of biopsy specimen before treatment.

OBJECTIVE: To analyze histopathologic features related to myoma volume reduction after treatment with a GnRH agonist, buserelin acetate, in needle biopsy specimens obtained before treatment. DESIGN: Prospective clinical study. SETTING: University teaching hospital. PATIENT(S): Twenty women with normal menstrual cycles and symptomatic uterine myomas. INTERVENTION(S): Buserelin acetate was administered intranasally (900 micrograms/d) for at least 16 weeks; transcervical needle biopsy of the uterine myoma was done before this treatment. MAIN OUTCOME MEASURE(S): The relation between histopathologic features (degree of cellularity, hyaline change, and collagen content) and the percent decrease in the volume of the largest myoma at 16 weeks measured by magnetic resonance imaging. RESULT(S): The correlation between the degree of hyaline change and the percent decrease in the myoma volume was significant, as was that with the proportion of collagenous tissue in the specimens. CONCLUSION(S): We predicted myoma volume reduction after treatment with a GnRH agonist by histologic analysis of the uterine leiomyoma before treatment.

Correlation between the growth of uterine leiomyomata and estrogen and progesterone receptor content in needle biopsy specimens.

OBJECTIVE: To examine the relationship between estrogen receptor (ER) and progesterone receptor (PR) content in needle biopsy specimens and the growth of uterine leiomyomata after biopsy. DESIGN: Prospective clinical study. SETTING: University teaching hospital. PATIENT(S): Thirty-one women with uterine leiomyomata and a normal menstrual cycle. INTERVENTION(S): Transcervical needle biopsy of uterine leiomyomata. MAIN OUTCOME MEASURE(S): The relationships between histologic features (smooth muscle content, immunohistochemical expression of ER and PR) and the percent increase over a 12-month observation period in the volume of the largest myoma nodule measured by magnetic resonance imaging were analyzed. RESULT(S): Both the density and intensity of immunohistochemical staining of PRs in uterine leiomyoma tissue showed significant positive correlation with leiomyoma growth. CONCLUSION(S): The growth of uterine leiomyomata can be determined by histologic and immunohistochemical analysis of needle biopsy specimens from uterine leiomyomata.

Ultrastructural comparison of uterine leiomyoma cells from the same myoma nodule before and after gonadotropin-releasing hormone agonist treatment.

OBJECTIVE: To compare ultrastructural features of leiomyoma cells from the same uterine myoma nodule before and after GnRH agonist (GnRH-a) treatment and to examine the relation between these ultrastructural changes and the extent of myoma volume reduction with GnRH-a treatment. DESIGN: Prospective clinical study. SETTING: University teaching hospital. PATIENT(S): Twenty women with uterine leiomyomas who were scheduled for surgery. INTERVENTION(S): Transcervical needle biopsy of uterine myoma, s.c. leuprolide acetate injection (3.75 mg) at least three times every 4 weeks before surgery, and hysterectomy or myomectomy. MAIN OUTCOME MEASURE(S): The changes in ultrastructural features of uterine leiomyoma cells and the percentage decrease in the volume of the largest myoma at 12 weeks of GnRH-a treatment measured by magnetic resonance imaging. RESULT(S): A decrease in myofilaments, mitochondrial swelling, and emergence of the lysosomal body were observed in relation to GnRH-a treatment. Furthermore, a positive correlation was observed between the decrease in myofilaments and myoma shrinkage. CONCLUSION(S): Cellular atrophy due to a decrease in myofilaments plays a major role in the reversible myoma shrinkage resulting from GnRH-a treatment.

Retrospective study of the prognostic factors of remission induction for single-agent chemotherapy with cisplatin in advanced epithelial ovarian cancer.

A retrospective study to explore the prognostic factor was conducted in 39 patients with advanced epithelial ovarian cancer, FIGO stage III-IV, who underwent single-agent adjuvant chemotherapy with cisplatin following primary debulking surgery. The survival rate following the adjuvant chemotherapy was 50.9% after 3 years and 44. 7% after 5 years, with a median survival time of 40.4 months. The actual dose intensity of the cisplatin ranged from 14.38 to 46.30 mg/m2/week, and the total dose/m2 was 143.79 to 645.83 mg/m2. Under these therapeutic conditions, the actual dose intensity was found to correlate with survival time (p<0.05), but there was no correlation between the total dose/m2 and survival time.

Angiogenesis in ovarian clear cell carcinoma and its relation to endometriosis.

Twenty-five patients with ovarian clear cell carcinoma (CC-Ca) were enrolled in this study, and tumor cell expression of vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) was investigated, and correlated with the microvessel count (MVC) and the impact of complication endometriosis. Expression of VEGF was strongly positive in 16 cases, weakly positive in 6 cases, and negative in 3 cases. Expression of PD-ECGF was strongly positive in 11 cases, weakly positive in 6 cases, and negative in 8 cases. VEGF expression and the MVC were significantly correlated (p<0.01), and there were no correlations among complication by endometriosis, expression of VEGF, expression of PD-ECGF, and MVC.

Bleomycin, actinomycin-D, and cisplatin treatment of ovarian germ-cell malignancies contributes to reducing adverse drug reactions.

Sixteen ovarian germ-cell malignancy (OGCM) patients between 1983 and 1999 were randomly divided into the BEP group (n=6; bleomycin, etoposide and cisplatin) and the BAP group (n=10; bleomycin, actinomycin-D and cisplatin). The patients were evaluated for adverse drug reactions (ADRs) based on severity-grading of the National Cancer Institute Common Toxicity Criteria. The ADRs in the BAP group were milder than in the BEP group, as seen in regard to alopecia (p=0.0126), low hemoglobin (p=0.0147), and decreased neutrophil count (p=0.0197). The five-year survival rate was 87.5% in the BAP group and 83.3% in the BEP group, and the difference was not significant (p=0.5954). BAP therapy is likely to be more beneficial for OGCM patients than BEP therapy, because ADRs are reduced with no difference in outcome.

An intranodal morcellation technique employing loop electrosurgical excision procedure for large prolapsed pedunculated myomas.

For minimally invasive resection of large prolapsed pedunculated submucous myomas, improved techniques need to be established. An intranodal loop electrosurgical excision procedure (LEEP) was specifically developed for the morcellation of myoma tissue during vaginal myomectomy. Vaginal myomectomy was performed on a 28-year old woman with a large myoma occupying the small pelvis. A prolapsed pedunculated leiomyoma of the uterus 13 cm in maximal diameter was resected by intranodal morcellation of the myoma tissue employing LEEP. Intranodal morcellation technique utilizing LEEP is an effective method for the minimally invasive resection of large prolapsed pedunculated myomas.