Analysis of the susceptibility of CD57 T cells to CD3-mediated apoptosis.

After stimulation with anti-CD3 antibody in vitro, CD57(+) T cells showed a greater susceptibility to apoptosis than CD57(-)alphabetaT cell receptor (TCR)(+) T cells (regular alphabeta T cells). The apoptotic fraction of CD57(+) T cells showed an increased production of active caspase-3. An increase in both Fas expression and Fas-ligand (FasL) production was also observed in CD57(+) T cells, whereas the expression of survivin was suppressed in CD57(+) T cells compared to that of regular alphabeta T cells. CD57(+) T cells display a biased expansion of a few Vbeta T cell fractions in individuals, but such Vbeta T cells were not specifically susceptible to CD3-mediated apoptosis. The TCR expression level of CD57(+) T cells was much lower than that of regular T cells and anti-TCR antibody stimulation induced a smaller apoptotic proportion of CD57(+) T cells than did anti-CD3 antibody. Although the CD3epsilon expression levels were similar in both T cell subsets, the CD3zeta level of CD57(+) T cells was significantly higher than that of regular T cells. These results suggest that several apoptotic and anti-apoptotic molecules are involved in the CD3-induced apoptosis of CD57(+) T cells and raise the possibility that the imbalance in expression of the CD3epsilon and CD3zeta chains may also contribute to the susceptibility of CD57(+) T cells to undergo apoptosis.

Interleukin-10 and interferon-gamma levels within the peritoneal cavity of patients with gastric cancer.

BACKGROUND AND OBJECTIVES: Immune status in the peritoneal cavity of patients with gastric cancer remains largely unknown. To clarify the clinical significance of the host immune response within the peritoneal cavity, we examined the levels of interferon-gamma (IFN-gamma), a type 1 cytokine, and interleukin-10 (IL-10), a type 2 cytokine, in peritoneal washings obtained from patients with gastric cancer. METHODS: Both the concentrations of IFN-gamma and of IL-10 in peritoneal washings obtained during surgery from 56 patients with gastric cancer were determined by an enzyme-linked immunosorbent assay. RESULTS: The IFN-gamma level was not correlated with the IL-10 level. The IL-10 level increased in a stage-dependent manner. The high IL-10 level correlated with an unfavorable outcome, whereas there was no relationship between the IFN-gamma level and survival rate. However, among the stage III-IV cancer patients, the high IFN-gamma level correlated with a favorable outcome, while there was no relationship between the IL-10 level and survival rate. CONCLUSION: Although the IL-10 level increases with tumor progression, the outcome of patients with advanced gastric cancer may be affected by the IFN-gamma level, but not by the IL-10 level, in the peritoneal cavity.

Liposome-mediated gene therapy using HSV-TK/ganciclovir under the control of human PSA promoter in prostate cancer cells.

To more accurately determine the tissue-specific expression of the target gene in prostate cancer cells, we introduced the enhancer element (-4,777 to -3,934; PSAR) and the promoter region (PSAP) of the prostate-specific antigen (PSA) gene. Furthermore, to elucidate the advantages of using liposomes as a gene carrier, we screened more than 20 liposome preparations in this study. The 5' upstream region of PSA gene (PSARPSAP) was conjugated to either the chloramphenicol acetyltransferase (CAT) gene or herpes simplex virus thymidine kinase (TK) gene, and the transfection of these plasmids was carried out using cationic liposomes, DMRIE-C (Gibco) or LipoTAXI (Stratagene). The expression of CAT activity was clearly observed when PSARPSAP-CAT plasmid was transfected into PSA-positive LNCaP cells, whereas no CAT activity was detected in PSA-negative DU145 cells or bladder carcinoma T24 cells. The CAT activity increased after the addition of testosterone. We then evaluated the therapeutic effect of the PSARPSAP-TK plasmid in vitro. When PSARPSAP-TK plasmid was transfected and ganciclovir was added to the medium, the growth of LNCaP cells was inhibited, while no growth inhibition was observed in DU145 cells. Furthermore, this inhibitory effect was observable even when the cells were cultured in a medium supplemented with dialyzed fetal bovine serum. These results suggest that the liposome-mediated transfection of PSARPSAP-TK appears to be a potentially effective approach for selecting the optimal treatment for tumor cells producing PSA even with the low androgen levels seen in patients treated by anti-androgen therapy.

Preoperative serum interleukin-18 level as a postoperative prognostic marker in patients with gastric carcinoma.

BACKGROUND: Interleukin-18 (IL-18), a recently described cytokine produced mainly by macrophages, stimulates interferon-gamma (IFN-gamma) production by natural killer cells and T cells. Although it has been reported that serum IL-18 levels are higher in patients with advanced tuberculosis and acute graft-versus-host disease compared with normal controls, the authors found no reports regarding serum IL-18 levels in patients with malignant solid tumors. The purpose of this study was to determine serum IL-18 levels and their clinical significance in patients with gastric carcinoma. METHODS: Peripheral blood samples were obtained from 94 patients with gastric carcinoma who underwent curative surgery and from 50 healthy volunteers. The serum IL-18 level, the IFN-gamma, level, and the Helicobacter pylori (HP) serology status were determined in each sample with an enzyme-linked immunosorbent assay. RESULTS: The mean serum IL-18 level for all patients was significantly higher compared with the mean level in healthy volunteers (P < 0.01). IFN-gamma titers were below the level of detection in all samples tested. When the patients were subdivided into groups, it was found that the serum IL-18 level in patients with Stage II and III disease was significantly higher compared with the level found in healthy volunteers (P < 0.01). The serum IL-18 level decreased after patients underwent surgical resection. However, there was no significant difference in the serum IL-18 level between healthy controls and patients with Stage I or IV disease. Patients with IL-18 levels >or= 310 pg/mL (i.e., equal to or greater than the mean levels +/- 1 standard deviation in the healthy volunteers) experienced a significantly lower survival rate compared with patients who had IL-18 levels < 310 pg/mL after undergoing surgery (P < 0.05) despite a lack of any discernible difference in clinicopathologic factors between the two groups. The serum IL-18 level was identified as an independent postoperative prognostic factor in multivariate survival analysis using a Cox proportional hazards model (hazard ratio, 4.89; P = 0.01). There was no significant correlation between HP serology status and serum IL-18 levels. CONCLUSIONS: The preoperative serum IL-18 level may represent a significant postoperative prognostic determinant in patients with gastric carcinoma. Its function in the host immune system remains to be elucidated.

Activation of the maternally preset program of apoptosis by microinjection of 5-aza-2'-deoxycytidine and 5-methyl-2'-deoxycytidine-5'-triphosphate in Xenopus laevis embryos.

The present study examines the effects on embryogenesis of microinjecting Xenopus laevis fertilized eggs with 5-aza-2'-deoxycytidine (5-Aza-CdR), which induces hypomethylation of DNA, and 5-methyl-2'- deoxycytidine-5'-triphosphate (5-methyl-dCTP), which induces hypermethylation of DNA. Embryos injected with either one of these analogs cleaved normally until the mid-blastula stage, but underwent massive cell dissociation and stopped development at the early gastrula stage. Dissociated cells that appeared here were positive by terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end-labeling and contained fragmented nuclei with condensed chromatin. The DNA from these cells formed a "ladder" on electrophoresis. Furthermore, the induction of cell dissociation by 5-Aza-CdR and 5-methyl-dCTP was postponed by 2-3 h by co-injection of Bcl-2 mRNA and the normal metabolite (CdR and dCTP, respectively). Using a specific antibody against 5-methyl-cytosine, we confirmed that 5-Aza-CdR induces hypomethylation, whereas 5-methyl-dCTP induces hypermethylation in X. laevis embryos before the onset of cell dissociation. Incorporation of radioactive precursors revealed that synthesis of DNA, and also RNA, is inhibited significantly in both 5-Aza-CdR-injected and 5-methyl-dCTP-injected embryos. These results show that 5-Aza-CdR and 5-methyl-dCTP are incorporated into DNA and induce apoptosis, probably through alteration of DNA methylation coupled with inhibition of DNA replication and/or transcription.

The mechanism of a defective IFN-gamma response to bacterial toxins in an atopic dermatitis model, NC/Nga mice, and the therapeutic effect of IFN-gamma, IL-12, or IL-18 on dermatitis.

NC/Nga (NC) mice raised under conventional conditions (Conv. NC mice) spontaneously develop dermatitis similar to human atopic dermatitis, whereas NC mice raised under the specific pathogen-free conditions do not develop dermatitis. In the present study, we show that the representative Th1 cytokine, IFN-gamma levels in the sera of NC mice, injected with either staphylococcal enterotoxin B or endotoxin (LPS), to be severalfold lower than those of normal mice. The low IFN-gamma response to staphylococcal enterotoxin B was correlated to the lack of regular Vbeta8(+) T cells and Vbeta8(+) NK T cells, and the low IFN-gamma response to LPS was correlated to an impaired IL-18 production of macrophages. The CD3-stimulated IL-4 production from liver and spleen T cells from Conv. NC mice in vitro was greatly augmented. The serum IL-4 levels of untreated Conv. NC mice also were higher than those of normal mice and specific pathogen-free NC mice. Treatment of Conv. NC mice either with IFN-gamma, IL-12, or IL-18 twice a week from 4 wk of age substantially inhibited the elevation of the serum IgE levels, serum IL-4 levels, and dermatitis, and IL-12 or IL-18 treatment also reduced the in vitro IL-4 production from CD3-stimulated liver T cells. The systemic deficiency in the Th1 response to bacterial stimulation thus leads to a Th2-dominant state and may induce an abnormal cellular immune response in the skin accompanied with an overproduction of IgE and a susceptibility to dermatitis in NC mice.

Effects of para-nonylphenol on 92 kDa gelatinase secretion by human peripheral lymphocytes and U937 cells in vitro.

Much attention has focused on environmental estrogenic chemicals such as para-nonylphenol which disrupt various tissues via the estrogen receptor. We studied effects of para-nonylphenol on gelatinase secretion by human lymphocytes in vitro. para-Nonylphenol (0.05-50 microM) dose dependently suppressed 92 kDa gelatinase secretion. The suppressive effect of 25 and 50 microM para-nonylphenol was completely blocked by tamoxifen. We also studied the effects of para-nonylphenol (0.05-50 microM) on 92 kDa gelatinase secretion by human leukemia U937 cells. para-Nonylphenol suppressed 92 kDa gelatinase secretion in a dose-dependent manner. The suppressive effect of 50 microM para-nonylphenol was completely blocked by tamoxifen. Estradiol did not significantly suppress 92 kDa gelatinase secretion. Our results suggest that para-nonylphenol suppressed 92 kDa gelatinase secretion via the estrogen receptor, however, para-nonylphenol interacts with the estrogen receptor in a manner distinct from estradiol. As this assay system is simple and rapid, it may prove useful to evaluate toxic effects of para-nonylphenol on human blood cells.

Adsorption of anti-annexin V using dextran sulfate bound cellulose beads.

Anti-annexin V (Anx V) antibodies are detected in SLE patients and patients with habitual fetal loss or preeclampsia. Several case reports have indicated that recurrent abortion based on antiphospholipid syndrome (APS) could be successfully treated with immunoadsorption by using dextran sulfate (DS) columns. The purpose of this study is to clarify whether or not anti-Anx V is also adsorbed by DS-bound cellulose beads. Sera from anti-Anx V-positive patients were mixed with DS-bound cellulose beads in vitro, and the titers of anti-Anx V were measured both before and after incubation. The anti-Anx V titers significantly decreased after incubation. The Anx V also bound to bovine serum albumin-conjugated DS immobilized on microtiter plates. The results of the present study lend support to the basic rationale for immunoadsorption therapy using DS columns in the treatment of habitual abortion closely associated with anti-Anx V antibodies.

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) activates the maternal program of apoptosis shortly after MBT in Xenopus embryos.

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) mRNA in 1- and 2-cell stage Xenopus embryos induces cell autonomous dissociation at the late blastula stage and developmental arrest at the early gastrula stage. The induction of cell dissociation took place "punctually" at the late blastula stage in the SAMDC-overexpressing cells, irrespective of the stage of the microinjection of SAMDC mRNA. When we examined the cells undergoing the dissociation, we found that they were TUNEL-positive and contained fragmented nuclei with condensed chromatin and fragmented DNA. Furthermore, by injecting Xenopus Bcl-2 mRNA together with SAMDC mRNA, we showed that SAMDC-overexpressing embryos are rescued completely by Bcl-2 and becometadpoles. These results indicatethat cell dissociation induced by SAMDC overexpression is due to apoptotic cell death. Since the level of S-adenosylmethionine (SAM) is greatly reduced in SAMDC-overexpressing embryos and this induces inhibition of protein synthesis accompanied by the inhibition of DNA and RNA syntheses, we conclude that deficiency in SAM induced by SAMDC overexpression activates the maternal program of apoptosis in Xenopus embryos at the late blastula stage, but not before. We propose that this mechanism serves as a surveillance mechanism to check and eliminate cells physiologically damaged during the cleavage stage.

Expression of carbonic anhydrase I or II and correlation to clinical aspects of colorectal cancer.

BACKGROUND/AIMS: This study was undertaken to elucidate the correlation between the expression of carbonic anhydrase I or II and the characteristic features of colorectal cancer. METHODOLOGY: The carbonic anhydrase I or II expressions of 74 colorectal cancer patients were analyzed by Western blotting. The relative intensity of cancer to the paired normal mucosa was calculated, and then compared with the clinicopathological parameters. Furthermore, a multivariate analysis for synchronous distant metastasis was undertaken. RESULTS: The expression of carbonic anhydrase I in colon cancer or carbonic anhydrase II in rectal cancer with Duke's D was found to be significantly lower than that with Duke's B or C, respectively. Similarly, carbonic anhydrase I in colon cancer or carbonic anhydrase II in rectal cancer with moderate-severe budding was found to be significantly lower than that with none-mild budding, respectively. Based on the findings of a logistic regression analysis, carbonic anhydrase I was adopted for colon cancer (P = 0.057) and carbonic anhydrase II for rectal cancer (P = 0.008) regarding synchronous distant metastasis. CONCLUSIONS: The expressions of carbonic anhydrase I and II correlated with biological aggressiveness of colorectal cancer and synchronous distant metastasis, especially carbonic anhydrase I for colon cancer and carbonic anhydrase II for rectal cancer.

Inhibition of glucocorticoid-induced apoptosis by the expression of antisense gene of mitochondrial ATPase subunit 6(1).

To isolate the apoptosis-linked genes involved in the cell death of thymocytes induced by glucocorticoids, we developed a functional cloning assay. Murine CD4(+)CD8(+) thymic cell line 2-257-20 cells were transfected with cDNA expression libraries obtained from a dexamethasone-resistant cell line. The transfected cells were selected in the presence of dexamethasone, and the plasmids which episomally expanded were then extracted from the surviving cells. One of the rescued cDNAs was found to be an antisense cDNA fragment identical to the mouse mitochondrial ATPase 6 gene. In the stable transfectants with the ATPase 6 antisense gene, the induction of apoptosis by dexamethasone was significantly delayed. Furthermore, the ATP synthesis in these transfectants was also reduced to some extent. ATPase 6 is a subunit of F(o)F(1) ATPase and our results support that ATP synthesis from the mitochondria is necessary for the induction of apoptosis induced by glucocorticoids.

Maternal program of apoptosis activated shortly after midblastula transition by overexpression of S-adenosylmethionine decarboxylase in Xenopus early embryos.

When we studied polyamine metabolism in Xenopus embryos, we cloned the cDNA for Xenopus S-adenosylmethionine decarboxylase (SAMDC), which converts SAM (S-adenosylmethionine), the methyl donor, into decarboxylated SAM (dcSAM), the aminopropyl donor, and microinjected its in vitro transcribed mRNA into Xenopus fertilized eggs. We found here that the mRNA injection induces a SAM deficient state in early embryos due to over-function of the overexpressed SAMDC, which in turn induces inhibition of protein synthesis. Such embryos developed quite normally until blastula stage, but stopped development at the early gastrula stage, due to induction of massive cell dissociation and cell autolysis, irrespective of the dosage and stage of the mRNA injection. We found that the dissociated cells were TUNEL-positive, contained fragmented nuclei with ladder-forming DNA, and furthermore, rescued completely by coinjection of Bcl-2 mRNA. Thus, overexpression of SAMDC in Xenopus embryos appeared to switch on apoptotic program, probably via inhibition of protein synthesis. Here, we briefly review our results together with those reported from other laboratories. After discussing the general importance of this newly discovered apoptotic program, we propose that the maternal program of apoptosis serves as a surveillance mechanism to eliminate metabolically severely-damaged cells and functions as a 'fail-safe' mechanism for normal development in Xenopus embryos.

The monoclonal antibody TER-119 recognizes a molecule associated with glycophorin A and specifically marks the late stages of murine erythroid lineage.

The antigen specificity of a rat monoclonal antibody TER-119 was investigated. In adult mice, TER-119 reacted with mature erythrocytes, 20-25% of bone marrow cells and 2-3% of spleen cells but not with thymocytes nor lymph node cells. In fetal haematopoietic tissues, 30-40% of d 10 yolk sac cells, 80-90% of d 14 fetal liver cells and 40-50% of newborn liver cells were reactive with TER-119. TER-119+ cells in adult bone marrow expressed significant levels of CD45 but not myeloid (Mac-1, Gr-1) or B-cell (B220) markers. Morphological examination and haematopoietic colony-forming assays for isolated TER-119+ cells revealed that TER-119 reacts with erythroid cells at differentiation stages from early proerythroblast to mature erythrocyte, but not with cells showing typical erythroid blast-forming unit (BFU-E) and erythroid colony-forming unit (CFU-E) activities. Erythroleukaemia cell lines do not express the TER-119 antigen even after stimulation with dimethylsulphoxide. TER-119 immunoprecipitated protein bands with molecular masses of 110 kDa, 60 kDa, 52 kDa and 32 kDa from erythrocyte membrane, whereas only a 52-kDa band was detected by TER-119 in Western blot analysis. Further molecular and cellular analyses indicated that the TER-119 antigen is a molecule associated with cell-surface glycophorin A but not with glycophorin A itself.

Mouse CD8+ CD122+ T cells with intermediate TCR increasing with age provide a source of early IFN-gamma production.

Although CD8+ IL-2Rbeta (CD122)+ T cells with intermediate TCR reportedly develop extrathymically, their functions still remain largely unknown. In the present study, we characterized the function of CD8+ CD122+ T cells with intermediate TCR of C57BL/6 mice. The proportion of CD8+ CD122+ T cells in splenocytes gradually increased with age, whereas CD8+ IL-2Rbeta-negative or -low (CD122-) T cells conversely decreased. The IFN-gamma production from splenocytes stimulated with immobilized anti-CD3 Ab in vitro increased with age, whereas the IL-4 production decreased. When sorted CD8+ CD122+ T cells were stimulated in vitro by the anti-CD3 Ab, they promptly produced a much larger amount of IFN-gamma than did CD8+ CD122- T cells or CD4+ T cells, whereas only CD4+ T cells produced IL-4. The depletion of CD8+ CD122+ T cells from whole splenocytes greatly decreased the CD3-stimulated IFN-gamma production and increased the IL-4 production, whereas the addition of sorted CD8+ CD122+ T cells to CD8+ CD122+ T cell-depleted splenocytes restored the IFN-gamma production and partially decreased IL-4 production. It is of interest that CD8+ CD122+ T cells stimulated CD4+ T cells to produce IFN-gamma. The CD3-stimulated IFN-gamma production from each T cell subset was augmented by macrophages. Furthermore, CD3-stimulated CD8+ CD122+ T cells produced an even greater amount of IFN-gamma than did liver NK1.1+ T cells and also showed antitumor cytotoxicity. These results show that CD8+ CD122+ T cells may thus be an important source of early IFN-gamma production and are suggested to be involved in the immunological changes with aging.

Selective inhibition of hepatoma cells using diphtheria toxin A under the control of the promoter/enhancer region of the human alpha-fetoprotein gene.

We constructed a plasmid containing human alpha-fetoprotein (AFP) promoter/enhancer to direct the cell type-specific expression of diphtheria toxin fragment A (DTA), designated as pAF-DTA, to AFP-producing hepatocellular carcinoma cells. The transfection was carried out with cationic liposomes (DMRIE-C) and the expression of the DTA gene was confirmed by a northern blot analysis. When pAF-DTA was transfected, the growth of AFP-positive HuH-7 cells was inhibited, whereas growth inhibition was not observed in AFP-negative MKN45 cells. In this experiment, the secretion of AFP was similarly suppressed, but the secretion of carcinoembryonic antigen from MKN45 was not altered. pAF-DTA could also exert its growth inhibitory effect on PLC, a cell line with a low level of AFP. However, no inhibitory effect of pAF-DTA was observed on the proliferation of primary hepatocyte cells. Furthermore, transfection experiments in which HuH-7 and splenic stromal cells were co-cultured revealed the growth inhibition by pAF-DTA to be selective in HuH-7 cells. Finally, the growth of HuH-7 transplanted on BALB/c nu/nu mice was inhibited by the direct injection of pAF-DTA/liposome complex into a tumor mass. These results suggest that use of pAF-DTA may be potentially useful as a novel approach for the selective treatment of tumor cells producing AFP even at low levels, without affecting other types of cells.

Identification of the promoter region of human placental 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene.

The placenta-type isozyme of human 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (HP2K) is expressed in several tissues such as placenta, brain, testis, liver, kidney, skeletal muscle, and primary blood mononuclear cells. To better understand the regulation of HP2K gene expression, we isolated and characterized its genomic DNA, which includes the promoter region. The results of oligo-capping analysis indicate that the transcription start point (tsp) is an adenine residue 329 bp upstream of the translational start codon. DNA sequence analysis of this gene shows that the promoter region that contains the TATA box sequence and the 5'-UTR is different from the other known PFK-2/F2, 6BPase genes. In addition, its 5'-flanking and 5'-UTR both have G + C-rich sequences containing Sp1 binding sites. To identify the promoter/enhancer region of HP2K gene, we performed transfection analyses of human choriocarcinoma BeWo cells with HP2K promoter-luciferase constructs. These experiments identified a promoter region 164 bp upstream from the tsp and an enhancer region between -1265 and -1329 on the 5'-flanking sequences. We also showed that Sp1 sites were not essential for HP2K transcription. Following transfection, stimulation experiments with serum, progesterone and phorbol 12-myristate 13-acetate showed that only the construct with the enhancer containing putative early growth response-1 binding motif was responsive to serum. We propose that the transcription of HP2K is strictly controlled by tissue-specific factors even though its genomic DNA contains several transcriptional elements.

Activation of monocytes and endothelial cells depends on the severity of surgical stress.

Surgical injury not only induces a systemic endocrine-metabolic response but also influences the function of the leukocytes and endothelial cells leading to various systemic responses. These responses appear to depend on the severity of surgical stress, which differs according to the surgical procedures. In this study, we investigated the response of monocytes and endothelial cells, and the development of systemic inflammatory response syndrome (SIRS) in relation to the severity of surgical stress. The postoperative clinical course was evaluated between patients undergoing an esophagectomy (ER group) and a distal gastrectomy (DG group). The tumor necrosis factor alpha (TNF-alpha) production of monocytes, the serum interleukin 6 (IL-6) levels, the CD11b expression on either monocytes or granulocytes, and the intercellular adhesion molecule-1 (ICAM-1) expression on human umbilical vein endothelial cells (HUVECs) stimulated with culture supernatants of monocytes were compared between the 2 groups. The development of SIRS was observed in all patients in the ER group, whereas no patients demonstrated SIRS in the DG group. The serum IL-6 levels, TNF-alpha production of monocytes, and CD11b intensity on monocytes or granulocytes in the ER group were higher than those in the DG group. In the ER group, the ICAM-1 intensity on HUVECs with monocytes immediately after operation significantly increased compared with before the operation. In conclusion, both the CD11b expression on monocytes and the TNF-alpha production of monocytes are considered to reflect the degree of surgical stress, and the activation of endothelial cells stimulated with these activated leukocytes may therefore lead to both tissue and organ injury.

Detecting circulating cancer cells using reverse transcriptase-polymerase chain reaction for cytokeratin mRNA in peripheral blood from patients with gastric cancer.

BACKGROUND: Cytokeratins (CKs) 19 and 20 have been used as targets for detecting cancer cells. We attempted to detect circulating cancer cells in patients with gastric cancer using a high-sensitivity reverse transcriptase-polymerase chain reaction (RT-PCR) assay for CK transcripts. METHODS: RT-PCR for CK 19 and CK 20 was performed on peripheral blood samples obtained from 52 patients with gastric cancer, from 24 of whom blood samples were collected on three occasions. Fourteen healthy volunteers served as controls. RESULTS: CK 19 and CK 20 were positive in five (9.6%) of 52 patients with gastric cancer. Of these five, four were classified into stage IV and the other stage I, according to the TNM Classification. In gastric cancer patients, three (12.5%) were positive in the 24 cases examined three times and two (7.1%) were positive in 28 cases examined only once. Among the stage IV cancer, positive cases for CK showed significantly lower survival rates than those negative for CK. Between CK 19 and CK 20 in the 24 cases examined three times, CK 19 was found to be more sensitive in detecting cancer cells. CK 20 was detected in one (7.0%) of 14 healthy volunteers, whereas CK 19 was not detected. CONCLUSIONS: We conclude that repeated blood sampling may be desirable to detect circulating cancer cells in peripheral blood, even in patients with advanced gastric cancer; CK 19 may be superior to CK 20 in detecting these cells. The clinical significance of detecting occult cancer in peripheral blood remains to be determined.

[Indication and effectiveness of endoscopic percutaneous gastrostomy as a route of parenteral alimentation for the home care patient].

We are managing 8 home care patients who have a gastrostomy made using an endoscopic percutaneous technique as a route of parenteral alimentation. Based on our experience, the preconditions for an endoscopic percutaneous gastrostomy as a route of parenteral alimentation are 1. normal gastrointestinal function, 2. difficulty in swallowing, 3. possibility that the caregiver can manage the gastrostomy. When we performed an endoscopic percutaneous gastrostomy as a route of parenteral alimentation for 8 home care patients, we obtained the several advantages mentioned below. 1. Swallowing pneumonia was prevented. 2. Adequate amount of alimental liquid could be infused. 3. Patient could take a bath or shower with the gastrostomy, and good QOL was realized. 4. The home care patient with the gastrostomy could have a satisfactorily long life.

Induction of cytotoxicity by chlorogenic acid in human oral tumor cell lines.

Millimolar concentrations of chlorogenic acid (CGA) showed higher cytotoxic activity against human oral squamous cell carcinoma (HSC-2) and salivary gland tumor (HSG) cell lines, as compared with that against human gingival fibroblast (HGF). The cytotoxic activity of CGA was significantly reduced by catalase or CoCl2, but not affected by FeCl3 or CuCl2. ESR spectroscopy showed that higher (millimolar) concentrations of CGA produced radicals under alkaline conditions, acting as a prooxidant, whereas lower concentrations of CGA scavenged superoxide and hydroxyl radical. CGA produced large DNA fragments (as identified by slightly faster migrating band of DNA on agarose gel electrophoresis) and nuclear condensation (as demonstrated by Hoechst (No. 33258) staining) in tumor cell lines. Activation of caspase was demonstrated by staining with M30 monoclonal antibody, which reacts with degradation products of cytokeratin 18. Contact with CGA for at least 6 h was necessary for irreversible cytotoxicity induction. Pretreatment of the cells with caspase 3 inhibitor partially inhibited the cytotoxic action of CGA. These date suggest that CGA induces cytotoxicity in oral tumor cell lines, possibly by hydrogen peroxide-mediated oxidation mechanism.