Fas ligand-induced caspase-1-dependent accumulation of interleukin-18 in mice with acute graft-versus-host disease.

Acute graft-versus-host disease (aGVHD), the fatal side effects of bone marrow transplantation, was shown to be accompanied by elevation of serum levels of interleukin 18 (IL-18). In this study, the mechanism underlying the accumulation of IL-18 in aGVHD in mice was investigated. Lethally irradiated recipients having transplantation with H-2 disparate donor splenocytes demonstrated aGVHD and contained markedly elevated serum levels of IL-18. In contrast, recipients having transplantation with gld/gld spleen cells, which lack functional Fas ligand (FasL), contained only normal ranges of IL-18, indicating FasL-mediated IL-18 release in aGVHD. The wild-type hosts engrafted with caspase-1-deficient cells revealed marked increases of IL-18 similar to those engrafted with wild-type cells, whereas caspase-1-deficient recipients engrafted with wild-type cells showed only a slight elevation of serum IL-18, indicating that IL-18 elevation is derived from host cells in a caspase-1-dependent manner. These results suggest FasL-mediated caspase-1-dependent IL-18 secretion in aGVHD in mice.

Comparison of the long-term effects of oral estriol with the effects of conjugated estrogen on serum lipid profile in early menopausal women.

OBJECTIVE: We investigated the long-term effects of oral estriol (E(3)) on serum levels of total cholesterol (t-Cho), high-density lipoprotein cholesterol (HDL-Cho), low-density lipoprotein cholesterol (LDL-Cho), and triglycerides in early menopausal women. METHODS: We studied 67 healthy early menopausal women who were treated for 48 months with 2.0 mg of E(3) plus 2.5 mg of medroxyprogesterone acetate daily (E(3) group, n=21), 0.625 mg of conjugated estrogen plus 2.5 mg of medroxyprogesterone acetate daily (CE group, n=19), or 1.0 microg of 1alpha-hydroxyvitamin D(3) daily or 1.8 g of calcium lactate containing 250 mg of elemental calcium daily (control group, n=27). The serum levels of t-Cho, HDL-Cho, LDL-Cho, and triglycerides were evaluated at baseline and every 6 months. RESULTS: After 48 months of treatment, the t-Cho decreased significantly by 4.3+/-2.1% (mean+/-SE) from baseline in the E(3) group, did not change in the CE group (-1.9+/-2.1%), and significantly increased (5.4+/-3.4%) in the control group. The HDL-Cho significantly increased in the CE group (10.7+/-2.4%), but not in the E(3) group (3.8+/-3.3%) or in the control group (-3.6+/-3. 0%). The LDL-Cho significantly decreased in the CE group (-11.4+/-4. 0%), did not change in the E(3) group (-5.2+/-3.6%), and significantly increased in the control group (11.8+/-6.3%). The triglyceride level decreased significantly in the E(3) group (-6. 7+/-4.9%), whereas it significantly increased in the CE group (17. 6+/-11.4%), and did not change in the control group (6.1+/-6.4%). CONCLUSIONS: Oral E(3) prevented a postmenopausal rise in the t-Cho. Oral estriol did not induce the hypertriglyceridemia that was seen after treatment with conjugated estrogen. Oral E(3) may be a useful alternative therapy in women with hypertriglyceridemia and in women who are reluctant to continue conventional hormone replacement therapy because of uterine bleeding.

[Locoregional adoptive immunotherapy using LAK cells and IL-2 against liver metastases from digestive tract cancer].

We investigated the clinical efficacy of locoregional and systemic adoptive immunotherapy (AIT) with or without interleukin-2 (IL-2) against solid metastatic lesions from digestive tract cancer. Eighteen patients, who were treated with more than 10(10) lymphokine-activated killer (LAK) cells, were enrolled in this study. Seven of the 18 patients received hepatic arterial infusion (HAI) of LAK cells with or without IL-2 against metastatic liver tumors (locoregional therapy group). The remaining 11 patients received systemic transfer of LAK cells with IL-2 against metastatic lesions located in organs other than the liver (systemic therapy group). Three of 7 locoregional therapy group patients showed clinically significant tumor regressions that were evaluated as being equivalent to partial response (PR). Two of the 11 systemic therapy group patients showed significant tumor regressions, but this response rate was much lower than that of the locoregional therapy group. The 2 effective cases in the systemic therapy group were esophageal cancer patients. Locoregional AIT with or without IL-2 against liver metastases from digestive tract cancer could be an effective therapeutic modality in some patients who are refractory to conventional therapies (e.g., chemotherapy and radiotherapy). It is necessary to find a new way to augment the anti-tumor effect of this therapy in combination with prior or concomitant chemotherapy.

Increased expression of Fas (APO-1, CD95) on CD4+ and CD8+ T lymphocytes during total body irradiation.

Fas/APO-1 (CD95) is a cell surface molecule that can transduce apoptotic signals into cells. We examined the expression of Fas antigen on CD4+ and CD8+ T cells of patients who received total body irradiation (TBI) as a preparative regimen for allogeneic bone marrow transplantation. Numbers of peripheral blood lymphocytes were significantly reduced after TBI. Cytofluorometric analysis revealed a significantly higher expression of Fas on CD4+ and CD8+ T cells after TBI. Serum soluble Fas concentrations were significantly elevated after TBI. Changes in the Fas system were therefore accompanied by TBI-induced lymphocytopenia, suggesting that Fas plays a role in irradiation-induced apoptosis in vivo.

Comparison of the long-term effects of oral estriol with the effects of conjugated estrogen, 1-alpha-hydroxyvitamin D3 and calcium lactate on vertebral bone loss in early menopausal women.

We investigated the long-term effects of oral estriol (E3) on bone mineral density (BMD) at the lumbar spine and biochemical indices of bone turnover in early menopausal women. We studied 64 healthy early menopausal women who were treated for 24 months with 2.0 mg E3 plus 2.5 mg medroxyprogesterone acetate daily (E3 group, n = 15), 0.625 mg of conjugated estrogen plus 2.5 mg medroxyprogesterone acetate daily (CE group, n = 19), 1.0 microgram 1-alpha-hydroxyvitamin D3 daily (D3 group, n = 13), or 1.8 g calcium lactate containing 250 mg of elemental calcium daily (Ca group, n = 17). The BMD at the third lumbar vertebra was determined by quantitative computed tomography, and serum levels of osteocalcin (OC) and total alkaline phosphatase (Alp), as well as urinary ratios of calcium-to-creatinine (Ca/Cr) and hydroxyproline-to-creatine (Hyp/Cr), were evaluated at baseline and every 6 months. After 24 months of treatment, the BMD decreased significantly by 12 +/- 4.5% (mean +/- S.E.) in the D3 group and 14 +/- 2.5% in the Ca group, but not in the E3 group (-4.1 +/- 4.8% from baseline) and in the CE group (-0.9 +/- 3.2% from baseline). The serum levels of Alp and OC decreased or remained unchanged in the E3 and CE groups, but increased in the D3 and Ca groups. The urinary Ca/Cr was decreased in the E3 and CE groups, but not in the D3 and Ca groups. The urinary Hyp/Cr decreased in the CE group, was unchanged in the E3 and D3 groups, and increased in the Ca group. Uterine bleeding occurred less frequently in the E3 than in the CE group (2.4 +/- 4.2 versus 13.1 +/- 14.8 days/person per year, P < 0.001). The bone-preserving effect of 2.0 mg of oral E3 was comparable to that of 0.625 mg of conjugated estrogen and was superior to that of 1.0 microgram 1-alpha-hydroxyvitamin D3 and 1.8 g Ca. Our findings suggest that a reduction in bone turnover in the E3 group may have contributed to the preservation of bone.

[Occurrence of numerical aberrations of chromosomes in metachronous liver metastases of colorectal cancers].

To investigate the change of numerical aberrations of chromosome 17 or 18 during progression of Colorectal carcinoma, we applied FISH, using chromosome specific probes, to 3 colorectal primary carcinomas and a total of 4 metachronous metastatic lesions in the liver. We also investigated the relationship between the DNA content of whole nucleus and the number of chromosome 17 by multiparametric analysis using autostage cytofluorometry. When compared with the results for primary lesions, in 4 metastatic lesions, the population of tetrasomy 17 cells increased in 2 lesions, and that of monosomy 18 cells increased in 1 lesion. The nuclear DNA contents histograms for disomy 17 and aneusomy 17 cells analyzed individually for 3 primary and 4 metastatic lesions, showed almost no appreciable difference of peak DNA values. These results indicate that the numerical aberrations of chromosome 17 and 18 accumulate during the progression of colorectal carcinomas, and that the subpopulation of aneusomy 17 consist mainly of those cells that involve the numerical aberrations in a few chromosomes.

[Clonal analysis of hepatocellular carcinoma].

We investigated the cell clonality of 12 cases of female solitary hepatocellular carcinoma (HCC) that were associated with hepatitis virus infection. The clonal origin of HCC could be assessed by the method based on restriction fragment length polymorphism (RFLP) of X-chromosome-linked androgen receptor gene (AR) and phosphoglycerate kinase (PGK) gene, taking advantage of random inactivation of one of two X-chromosomes by methylation in females. We extracted DNA samples from both fresh and paraffin-embedded specimens of the same lesion as a source of DNA sample for polymerase chain reaction (PCR). Consequently, it was possible to use methylation-sensitive restriction enzymes and PCR to study differential methylation patterns among alleles of these genes for both DNA samples. The RFLPs of AR gene and PGK gene were found in eight of 12 cases and five of 12 cases, respectively. There were two cases which had no RFLPs in either AR gene or PGK gene. All cases of HCC which had RFLP in either AR gene or PGK gene demonstrated monoclonal origin of the tumor regardless of their histologic patterns.

Evaluation of latex agglutination test for alpha-fetoprotein in diagnosing rupture of fetal membranes.

To assess the utility of a latex agglutination test for alpha-fetoprotein in vaginal fluid for diagnosing rupture of the fetal membranes, we tested 91 pregnant women, including 32 with an established rupture, 19 with no evidence of rupture and 40 with a suspected rupture. Results were compared with those of the pH indicator and the fern tests. The latex agglutination test had better sensitivity, specificity, or both, than the pH indicator and the fern tests. We conclude that the latex agglutination test is more accurate than either the pH indicator or the fern test for diagnosing premature rupture of fetal membranes.

[Image analysis on isolated colorectal tumor cells].

Image analysis of the nuclear morphology was performed on propidium iodide-stained isolated cells from 9 adenomas and 9 cancers of the colorectum. We analyzed DNA content, seven nuclear geometric features and four shape factors of tumor cells using an image cytometry system that has been developed in our laboratory. Nuclear breadth and the degree of contour irregularity of cancer cells were found to be significantly different from those of adenoma cells. The coefficients of variation (CV) of some nuclear features and the degree of contour irregularity were increased in cancer cells, while CV of the degree of circularity was increased in adenoma cells. Moreover, nuclear in G2 phase were found to be larger and more round than those in G1 phase. These results suggest that cell-cycle-related analysis of nuclear features would be a valid means to assess the nuclear morphology of colorectal tumors.

[A case report of spontaneous rupture of bilateral lumbar artery in a patient with von Recklinghausen disease].

Vascular complications in von Recklinghausen disease are rare, especially spontaneous rupture of a major artery. A 50-year-old man with previously diagnosed neurofibromatosis was admitted because of spontaneous rt-sided lumbago, paresthesia in rt-lower limb and severe anemia. Angiographic studies, CT and MRI revealed pseudo-aneurysm of bilateral lumbar arteries, which ruptured into the psoas muscle. Both arteries were ligated and hematoma was removed. The pathogenesis of the spontaneous rupture is discussed.

[Progress in microscopic image analysis and the trend of image cytometry].

Image cytometry has recently developed prominently based on the digital imaging of cell morphology brought about by great advances in microcomputer hardware and software, electronics etc. This method is characterized by the quantitation of both the amount of intracellular bioactive materials and cell morphology, and is expected to further advance many cell analysis techniques involving automated cytology, chromosome image analysis, automated histopathology, viable cell analysis, 3D image construction, etc. The present use is still minimal, and advances in both hardware and software for cyto-histologic recognition are essentially necessary. Perhaps by the end of this century, more sophisticated and faster machines for automated cyto-histologic analysis will appear, replacing many microscopic techniques of presently subjective observation in the biomedical field.

[Analysis of cell proliferation kinetics and the effects of cisplatin on the cell cycle of human gastric cancer cells by autostage cytofluorometry].

Analysis of both cell proliferation kinetics and effects of cis-diamminedichloroplatinum (CDDP) on cell cycle in human gastric cancer cell line (HGC-Y2) by measuring the contents of nuclear DNA, RNA and the Ki-67 antigen using autostage cytofluorometry system was described. In HGC-Y2 cells, RNA content increased during the cell cycle and reached to the maximum at G2/M phase. The results of pulse treatment with CDDP on these cells demonstrated a prolongation of S phase and G2 arrest with increasing of RNA content of these cells. We classified the cells by intranuclear distribution pattern of Ki-67 antigen and thus could identified the cells at G0 and M phases from these classification. The content of Ki-67 antigen was moderate grade at G1 phase and it decreased in the early S phase, then increased gradually during S phase and at the late S phase. It increased rapidly, reaching to the maximum at G2/M phase. After CDDP treatment, the content of Ki-67 antigen increased in the cells in prolonged S phase and in the cells arrested at G2 phase. It was also found that the syntheses of both Ki-67 antigen and RNA were not inhibited by CDDP. These results suggest that the method using autostage cytofluorometry system was useful for the research, on the mechanism of cancer therapy because of making possible to analyze precisely the cell cycle and the influence of anticancer drugs.

[The examination of DNA strand breaks induced by peplomycin-using non-radioactive in situ nick translation method].

In situ nick translation (ISNT) is a method to detect DNA single strand break (nick) at each cellular level. In this study, peplomycin (PEP)-induced DNA strand breaks and its repair were examined using non-radioactive ISNT method. Human fibroblasts were cultured with various concentrations of PEP for different durations, and then DNA strand breaks were evaluated by ISNT method. The signal intensity of DNA strand breaks of the PEP-treated fibroblasts was increased in a dose dependent manner. At the fixed concentration of PEP, it reached to a maximum level after 10 min. of culture and remained unchanged for at least 24 hours. In addition, DNA strand breaks induced by PEP was rapidly repaired in 10 min. after washing. Thus, the non-radioactive ISNT is thought to be a quick, sensitive and specific method not only to detect DNA strand breaks but also to observe its repair.

Inhibition of adsorption of oral streptococci to saliva treated hydroxyapatite by chitin derivatives.

This study estimated the effects of five chitin derivatives low molecular chitosan (LMCS), ethyleneglycol chitin (PEGT), carboxymethyl chitin (PCMT), sulphated chitosan (PSSS), and phosphorylated chitin (PPPT) on the adsorption of three oral streptococci to saliva-treated hydroxyapatite (S-HA). The adsorption was evaluated by measuring the optical density of the bacterial cell suspensions released from saliva-treated hydroxyapatite by 0.5 N HCl. The adsorption of test strains to S-HA progressively decreased in proportion to each additional volume of PEGT, PPPT, or PSSS. PPPT and PSSS quite effectively inhibited the adsorption of S. mutans onto S-HA, but were less effective against S. sanguis and S. mitis. PPPT, PSSS, and PCMT all markedly promoted the desorption of S. mutans cells pre-adsorbed onto S-HA. Pretreatment of S-HA with PPPT, PSSS, or PCMT significantly decreased the subsequent adsorption of S. mutans and S. mitis. Pretreatment of these cells with PEGT also decreased their adsorption to S-HA. These findings suggest that these chitin derivatives may change the ionic natures of the S-HA and the bacterial cell surface, resulting in a less favorable interaction.

[Cellular kinetics and the nuclear ploidy pattern in relation to the gross pathological classification and location of advanced colorectal cancers as analyzed by DNA-RNA cytofluorometry].

Correlated studies on the cellular kinetics in association with the nuclear ploidy pattern and the pathological morphology of advanced colorectal cancers have been carried out using DNA-RNA cytofluorometry with an AO stain. The results showed that advanced colorectal cancers could be separated into the 3 distinct groups that follow: group I, involving a diploid cell population; group II, a polyploid cell population; and, group II', an aneuploid cell population. The group I type was located in the entire colorectal region, where as groups II and II', were in the sigmoid and rectum. Three groups were mostly Borrmann type 3 cancers, and the gross pathological classification was not found to be related to the ploidy pattern.

Villous tumor of the stomach associated with adenocarcinomas--a histochemical study of mucosubstances.

A case is described in which both a moderately elevated villous adenoma associated with adenocarcinoma at the posterior wall and an independent gastric carcinoma at the anterior wall were present. These two lesions were surrounded by the mucosa of incomplete intestinal metaplasia. Consecutive sections of the villous adenoma associated focally with carcinoma and the independent cancer were stained by the periodic acid-Schiff (PAS), pH 2.5 alcian blue (A1-B1), high iron diamine pH 2.5 alcian blue (HID-AB) and carcinoembryonic antigen peroxidase-antiperoxidase (CEA-PAP) methods. Villous adenoma was weakly positive in the PAS stain but negative in A1-B1 and HID stains, showing that it did not produce mucin. On the other hand, the intestinal metaplasia and cancerous lesions were positive in PAS, A1-B1 and HID stains, indicating production of the intestinal type of mucin. The villous adenoma accompanied by malignant changes was positive by the CEA-PAP method. This result shows the biological property of villous adenoma that they can easily change into malignancy. These three lesions in our case are considered to have originated independently from the primordial cells and to have developed the differences in mucin production in the process of cell development.