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The effect of tumor necrosis factor (TNF) and interferon (IFN)-gamma on antibody-dependent cellular cytotoxicity (ADCC) in patients with ulcerative colitis (UC) was investigated. ADCC activity was measured by the 51Cr release assay, using peripheral blood mononuclear cells of healthy subjects as effector cells and RPMI 4788 cells derived from human colon cancer as target cells. ADCC activity under sera from healty subjects remained low whether or not the effector cells were pretreated with TNF (100 U/ml, 16h). Under sera from UC patients, ADCC activity of 13.9%, compared to 9.6% when pretreatment was deleted. The effect of IFN pretreatment (100 U/ml, 16h) was also examined under sera from UC patients; in that experiment activity rose to 26.8%, in comparison to a 10.7% when IFN-gamma pretreatment was deleted. Finally, when the effector cells were pretreated with both TNF and IFN-gamma (100 U/ml of each, 16h) the ADCC activity under sera from UC patients was higher than when either TNF or IFN-gamma were used alone. These results suggest that TNF and IFN-gamma, by increasing ADCC activity in UC lesions, are involved in cell injury in the colonic epithelium. IFN-gamma appears to increase ADCC activity by increasing the number of high affinity monocyte Fc gamma RI receptors, while TNF increases ADCC activity by a different mechanism. more...

Patients with chronic idiopathic neutropenia (CIN) usually show chronic inflammation in their bone marrow stromal cells, but the etiologies of this disorder are still unknown. We suspected viral infection of the bone marrow stromal cells and used polymerase chain reaction (PCR) analysis to test for cytomegalovirus (CMV) infection in 4 patients with CIN and in 6 healthy volunteers. Peripheral blood mononuclear cells did not yield signals in any of the patients or healthy volunteers, but PCR analysis for CMV DNA in bone marrow stromal cells showed positive signals in 2 of the patients with CIN. CMV DNA was not detected in the other 2 patients or in the healthy volunteers. The 2 patients who were positive for stromal CMV received 500 mg/day ganciclovir for 14 days. After this treatment, the PCR band showing the presence of stromal CMV disappeared, and the neutrophil numbers of the 2 patients who received this viral eradication therapy normalized 6 months after treatment. In conclusion, the etiology in some cases of CIN is speculated to be infection by CMV in bone marrow stromal cells, and treatment with ganciclovir may be effective in treating such patients. more...

Sera from 14 normal control subjects, 30 patients with alcoholic liver diseases (fatty liver, n = 8; hepatitis, n = 13; liver cirrhosis, n = 9), 7 controls with chronic hepatitis B, and 8 controls with chronic hepatitis C were masured for their concentrations of antibodies against HepG2 membrane protein by a binding assay utilizing 125I-labeled protein A. When the cut-off level was set as the mean value plus 2 SD of normal control subjects, the incidence of positivity was 75%, 69.2%, and 77.8% in patients with alcoholic fatty liver, alcoholic hepatitis, and alcoholic cirrhosis, respectively. Both the mean serum antibody values and the positive incidence were significantly higher in patients with alcoholic liver diseases than in either the normal controls or in the control patients with chronic hepatitis. Sodium dodecylsulfate polyacrylamide gel electrophoresis of 125I-labeled HepG2 membrane protein precipitated with IgG from patients with alcoholic liver diseases revealed an immunoreactive band at a molecular weight of 78,000 daltons (gp78). The antibody activity remained after immunoabsorption by human liver-specific lipoprotein (LSP) but decreased when HepG2 cells were pre-treated with trypsin or neuraminidase. Consequently, gp78 appears to be a glycoprotein distinct from LSP, and is specifically recognized by IgG from patients with alcoholic liver diseases. This assay may provide a new system to measure autoantibody to hepatocytes in alcoholic liver diseases. more...

It has been shown that some types of tumour cells produce activated transforming growth factor beta-1 (TGF-beta 1). However, the mechanism for the activation of TGF-beta 1 derived from tumour cells has not been fully elucidated. The present study was undertaken to characterise an activator of latent TGF-beta 1 secreted from a human gastric cancer cell line, KATO-III. Western blot analyses using antibodies for TGF-beta 1, latency associated peptide (LAP) and latent TGF-beta 1-binding protein (LTBP) revealed that, in the cell lysate of KATO-III, TGF-beta 1 protein was expressed as a small latent complex of TGF-beta 1 and LAP. This was also confirmed by a gel chromatographic analysis of the cell lysate obtained from KATO-III. A 2.5 kb transcript of TGF-beta 1 mRNA was detected in KATO-III cells by Northern blot analysis. A gel chromatographic analysis of the conditioned medium from KATO-III cells revealed, in addition to the active form of TGF-beta 1, a factor which activated latent TGF-beta 1 from NRK-49F cells at fractions near a molecular size of 65,000. This factor was inactivated by heat (100 degrees C), acidification, trypsin and serine protease inhibitors. TGF-beta 1 activity in KATO-III cell lysate was not detected in the untreated state, but potent TGF-beta 1 activity was detected after acid treatment. These results suggest that KATO-III releases not only a latent TGF-beta 1 complex but also a type of serine protease, different from plasmin, plasminogen activator, cathepsin D, endoglycosidase F or sialidase, which activates the latent TGF-beta 1 complex as effectively as acid treatment. Images Figure 1 more...

We characterized tumor-infiltrating lymphocytes (TIL) from ascites of patients with ovarian or pancreatic cancer in which the human tumor necrosis factor (TNF) gene was successfully transduced with retrovirus vector. The TNF-gene-transduced TIL (TNF-TIL) from these patients showed a higher level of TNF production and higher cytotoxic activity against K562 and Daudi cells than did neomycin-phosphotransferase-gene-transduced TIL (neo-TIL). Of these TIL preparations, only that from pancreatic cancer was further characterized since it was collected in a relatively large amount. In spite of the fact that the autologous tumor cells showed resistance to soluble TNF, the TNF-TIL clearly demonstrated enhanced cytotoxicity against them as compared with neo-TIL. The enhanced cytotoxicity was ascribed to autocrine effects of secreted TNF on TIL, which included augmentation of adhesion molecule (CD2 and CD11a) and interleukin-2 receptor expression, and elevation of production of interferon gamma, lymphotoxin and granulocyte/macrophage-colony-stimulating factor and its paracrine effect on target cells to facilitate them to be more susceptible to TIL. more...

Recently, we produced a monoclonal antibody, 5D4, against the PRAD1/cyclin D1 product and suggested positive nuclear staining to be associated with mantle cell lymphoma (MCL). Now we have further characterized the specificity of this antibody and studied the relation of immunohistochemical detection to PRAD1/cyclin D1 mRNA expression and DNA rearrangement. Immunofluorescence and immunoblotting studies demonstrated the 5D4 antibody to be crossreactive with cyclin D2, but not cyclin D3. On immunostaining, 15 of 19 MCL cases (79%) presented the nuclear staining pattern and PRAD1/cyclin D1 mRNA expression was detected by Northern blot analysis in 12 of 15 MCL cases studied (80%): all cases with the mRNA expression showed the nuclear staining pattern. Southern blot analysis with 11q13 BCL-1 probes detected DNA rearrangements in 8 of 19 MCL cases (42%), all 8 exhibiting PRAD1/cyclin D1 mRNA expression. In 21 lymphoma cases of types other than MCL, neither the mRNA expression nor the nuclear staining were observed, although cytoplasmic staining was often apparent. These results indicated that positive nuclear staining of lymphoma cells by 5D4 antibody reflects PRAD1/cyclin D1 mRNA expression, and showed that this monoclonal antibody has diagnostic value for differentiating MCL from other types of lymphomas. more...

Tumor necrosis factor (TNF) and various chemotherapeutic drugs show synergistic antitumor effects in vitro and in vivo, though the mechanism is not clear. Based on our previous finding that endogenous TNF (enTNF) acts as an intracellular resistance factor against exogenous TNF by scavenging oxygen free radicals (OFR) with induced manganous superoxide dismutase (MnSOD), we examined the suppression of these resistance factors by chemotherapeutic drugs and the resulting increase in TNF cytotoxicity. Pretreatment of HeLa cells, which produce an appreciable amount of enTNF and show apparent TNF resistance, with TNF followed by adriamycin (ADM) resulted in an additive effect, whereas pretreatment with ADM followed by TNF resulted in a synergistic effect. After treatment of HeLa cells with ADM, the expression of enTNF was remarkably suppressed and MnSOD activity was decreased by one-half. These results indicate that suppression of the intracellular resistance factors, i.e., enTNF and MnSOD, by ADM plays an important role in the mechanism of the synergistic antitumor effect of TNF in combination with ADM. more...

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) and erythropoietin (rhEPO) were used to treat patients with aplastic anemia (AA). In terms of effects on erythrocyte recovery, the combined use of rhG-CSF and rhEPO showed a favorable response in 6 of 14 (42.9%) patients with moderate AA following 10 weeks treatment and in 3 of 14 (21.4%) patients thereafter. However, the response was poor in patients with severe AA (3/13). A favorable response in severe AA was observed in 1 of 13 (7.7%) patients following 10 weeks treatment and in 2 of 13 (15.4%) patients thereafter. The overall effect on erythrocytes was observed in 44.4% patients. A dose of 400 micrograms/m2 G-CSF was sufficient to cause an increase in neutrophil count and 100 IU/kg rhEPO appeared to be sufficient to cause an increase in erythrocyte count. In 6 of 27 (22.2%) patients, a trilineage response was observed. Interestingly, a delayed and long-lasting effect was obtained in 5 of 27 (18.5%) patients. These results suggest that rhG-CSF can synergize with rhEPO in erythrocyte response, especially in patients with moderate AA. more...

Various novel recombinant human tumor necrosis factor-alpha (TNF) mutants were prepared using protein engineering techniques, and their cytotoxic activity was compared with that of the intact form of TNF (intact TNF). Mutant 471 (a TNF mutant molecule with the deletion of 7 amino acids at the amino-terminal and the substitution of Pro8Ser9Asp10 by ArgLysArg) had a 6-fold higher cytotoxic activity against murine L929 cells. The mutant TNF had an increased ability to bind to TNF receptor on murine L929 fibroblasts cells. A cross-linking study revealed that mutant 471 had an increased ability to form an active trimer. Mutant 471 also showed higher cytotoxic activity against human KYM myosarcoma cells and human MIA PaCa-2 pancreatic carcinoma cells. The possible cachectin activity of the mutant was almost the same as that of intact TNF. These results suggest that mutant 471 might be a more promising candidate as an anticancer agent than intact TNF. more...

Death by bloodletting among patients with factitious anemia has never been reported to our knowledge. We report the first known case. A 25-year-old woman with severe iron deficiency anemia confessed her habit of bloodletting at her first visit to our hospital, in March 1998. We prescribed oral iron and referred her to a psychiatrist. The diagnosis was borderline personality disorder. The psychiatrist began counseling the patient and prescribed a major tranquilizer. The patient's method of bloodletting was to insert an 18-gauge needle without syringe into her vein after inducing congestion in her arm. This method was considered to involve risk of death, because once the patient fell into a faint caused by blood loss, the bloodletting could not be stopped. Although we attempted to persuade the patient to stop bloodletting by this method, she died after self-bloodletting in September 1999. It is not known whether the death was intentional suicide or an accident. more...

The effect of ethanol on the expression of asialoglycoprotein receptor protein and its mRNA was studied in a human hepatoma cell line, HepG2. The number of asialoglycoprotein receptors on the cell surface was decreased to 60% of the control level, without a loss in affinity, by incubating the cells with 100 mM ethanol. The decrease in cell surface asialoglycoprotein receptors was paralleled by a decrease in total receptor numbers, including intracellular and surface receptors. The internalization of asialoglycoprotein was also diminished, to 44% of that in control cells. The decreases in cell surface receptors and total receptor numbers were partially restored by 2 mM 4-methylpyrazole, suggesting that ethanol metabolites participated in the diminution of asialoglycoprotein receptor expression. However, the steady-state expression of asialoglycoprotein receptor mRNA was increased in ethanol-treated cells and further augmented by a longer ethanol exposure. These paradoxical results, i.e., the decrease of asialoglycoprotein receptor protein and the increase of its mRNA expression, suggest that the reduction in the asialoglycoprotein receptor protein is a post-transcriptional event and that a possible feedback regulatory mechanism may control asialoglycoprotein receptor gene transcription and/or impair the degradation of its mRNA. more...

To explore a mechanism of interleukin (IL)-6–induced hypoferremia in rats, iron metabolism was investigated both in vivo and in vitro. Recombinant IL-6 was intraperitoneally administered to male Wistar rats and the serial change of parameters related to iron metabolism was examined. After administration of IL-6, plasma IL-6 concentration increased rapidly, reached its maximum in 1 hr and thereafter decreased quickly. Plasma IL-6 3 hr after IL-6 injection (50 μg/kg) was 3 units/ml, which is a concentration capable of inducing hepatic 125I–labeled transferrin uptake in vitro using isolated hepatocytes. Plasma iron concentration and transferrin saturation had decreased to approximately one third of the initial level within 3 hr and then recovered. Total iron binding capacity remained unchanged for 6 hr, then began to decrease. Red blood cell count and hemoglobin concentration showed no remarkable changes during this period. By ferrokinetic study with plasma that contained iron 59–labeled transferrin, the plasma iron disappearance half time, calculated from the disappearance curve, was significantly shortened from 55 min to 22 min by IL-6 treatment (p more...

The mechanism of growth inhibition by transforming growth factor (TGF)-beta 1 was investigated. We examined the growth inhibitory effects of TGF-beta 1 on human nasopharyngeal carcinoma (KB) cells which constitutively expressed p53. TGF-beta 1 suppressed the DNA synthesis of KB cells in a dose-dependent manner. It had minimal effect on adenovirus-2-transduced KB cells expressing either adenovirus early region 1B (E1B) or 1A (E1A) product, which respectively binds to p53 or Rb product and inhibits its function, and no growth inhibition at all was observed with KB cells expressing both E1B and E1A products. Dephosphorylation of the p53 was promoted by TGF-beta 1 stimulation in KB cells, but not in E1B-producing KB cells, which sequestrate the function of p53. The growth inhibition of KB cells by TGF-beta 1 was significantly reduced by treatment with okadaic acid. These results suggest that p53 transduces the antiproliferative signal of TGF-beta 1 possibly through its dephosphorylation. more...

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) and erythropoietin (rhE-PO) were used to treat ten patients with myelodysplastic syndromes (MDS). None of the patients showed a favorable response in erythrocyte and platelet counts following 10 weeks' treatment, although favorable responses in neutrophil counts were observed in eight of ten patients (80.0%) and in seven of eight patients (87.5%) following 2 weeks' and 10 weeks' treatment, respectively. However, one patient with refractory anemia had a delayed favorable response in erythrocyte and neutrophil counts at week 14 in spite of the cessation of combination therapy at week 10. These results indicate that combination therapy with rhG-CSF and rhEPO is not beneficial to patients with MDS, based on the presently used protocol. more...

Background. Resistance to chemotherapeutic agents is a major problem in non-small cell lung cancer (NSCLC) chemotherapy, and recent studies have indicated that glutathione S-transferase-π (GST-π) may play an important role in the resistance of cancer cells to alkylating agents that include platinum compound. Methods. GST-π in serum of 121 patients with NSCLC was measured by a sandwich enzyme-linked immunosorbent assay, and the serum concentrations of carcinoembryonic antigen (CEA), squamous cell carcinoma (SCC) antigen, and neuron-specific enolase (NSE) were also examined. Serum levels of these tumor markers were also evaluated in relation to therapeutic response in 69 patients who received combination chemotherapy with platinum compound. Results. Fifty of 121 patients with NSCLC (41.3%) showed elevated serum GST-π levels above a cutoff value of 34.8 ng/ml (mean + two standard deviations in 30 healthy control subjects). The positive rate of GST-π in patients with NSCLC was higher than those of CEA (37.2%), SCC (15.7%), and NSE (14.9%). The mean pretreatment GST-π level was significantly lower in patients with a partial response to chemotherapy than in those with no response (26.9 ± 11.3 ng/ml versus 38.8 ± 16.7 ng/ml; P < 0.003). Among patients with elevated levels of pretreatment serum GST-π, only 13.8% (four of 29) responded to the combination chemotherapy, whereas partial response was observed in 40.0% of patients (16 of 40) with serum GST-π concentration below the cutoff level (P < 0.02). Neither CEA, SCC, nor NSE showed such a relationship. Conclusions. The serum GST-π level may have limited value as a tumor marker for NSCLC. Interestingly, pretreatment serum GST-π levels may be a useful parameter for predicting therapeutic response to combination chemotherapy regimens that include platinum compound. Cancer 1994; 73:1377–82. more...

Blood stasis and vascular permeability induced by tumor necrosis factor (TNF) in Meth-A tumors transplanted in BALB/c mice were significantly enhanced by hyperthermia at 40 degrees C for 30 min immediately following TNF administration. A dose-dependent, sustained decline in the intratumoral blood flow rate occurred following the administration of TNF alone (i.v.; 5 x 10(3), 5 x 10(4), or 5 x 10(5) JRU/kg) and was enhanced by the administration of hyperthermia in combination with the TNF, even though no decline occurred with hyperthermia alone. The combination of TNF at 5 x 10(5) JRU/kg and hyperthermia resulted in a blood flow ratio (ratio of blood flow after administration to that before) of 0.47 at 1 h compared with a ratio of 0.65 at 1 h after TNF alone. The blood flow in normal skin sites did not decrease in any case. The permeability of the intratumoral vasculature similarly increased in a dose-dependent manner after the administration of TNF alone and was further increased by combination with hyperthermia, even though no increase occurred with hyperthermia alone. The mean permeability in mice receiving TNF alone at 5 x 10(5) JRU/kg was 1.35 times that in untreated mice. In mice receiving TNF at the same dose together with hyperthermia, the ratio was increased to 1.65. The results suggest that TNF selectively suppresses intratumoral blood flow, that this effect is enhanced by mild hyperthermia, and that the mechanism of the suppression by TNF with or without hyperthermia partly involves an increase in blood vessel permeability. more...

The expression and extracellular release of transferrin receptor (TR) was investigated by in vitro model system of erythroid differentiation. Human peripheral blood mononuclear cells were cultured with interleukin- 3 (IL-3) for 7 days, and with erythropoietin (EPO) for an additional 8 days. After EPO stimulation, IL-3-stimulated blastic cells were serially differentiated into mature erythrocytes. [3H]-thymidine incorporation of cultured cells increased linearly from day 0 to 5, followed by a decrease. Flow cytometric analysis showed an increase of TR expression from day 0 to 5, followed by a slight decrease. By metabolic labeling with [35S]methionine and immunoprecipitation, the cell lysate exhibited a 95-kD band corresponding to the intact TR on sodium dodecyl sulfate-polyacrylamide gel electrophoresis/autoradiography at day 5, when polychromatic erythroblasts had their peak. The culture supernatant solubilized by tween-20 exhibited a 95-kD and an 85-kD band on days 5 and 8, which corresponded to the intact and the truncated forms of TR, respectively. The 95-kD band was more intense at day 5 than at day 8. The reverse transcriptase-polymerase chain reaction assay showed that the receptor- mRNA expression was parallel to receptor synthesis. Thus, the synthesis and expression of TR on erythrocytes is associated mainly with cell proliferation in the early phase, and with both cell proliferation and hemoglobin production in the middle to late phases of maturation. Concomitantly, the extracellular release of TR from erythrocytes occurs in the middle to late phases of maturation. These data suggest that polychromatic erythroblasts release soluble TR as both intact and truncated forms and may be an important source of serum TR implicated as an index for erythropoietic activity in the marrow. more...

The asialoglycoprotein receptor (AGPR) is responsible for the catabolism of acute phase proteins. The effects of inflammation-related cytokines on the expression of AGPR were investigated in HepG2 cells derived from a human hepatoblastoma cell line. Binding studies, using a [125I]-labeled asialo-orosomucoid ligand, revealed that AGPR numbers on cell surfaces were increased by interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF). In cells treated with IL-1, IL-6, or TNF, immunohistochemical staining with an anti-AGPR monoclonal antibody demonstrated augmented expression. Pulse labeling analysis, using [35S]-labeled methionine, showed newly synthesized AGPR in both the precursor and the mature forms. When IL-1, IL-6, and TNF were added to the culture medium, total synthesis of AGPR (sum of the mature and precursor forms) was increased. In addition, the relative proportion of the synthesized precursor form of AGPR was higher in cytokine-treated than in untreated cells, suggesting that these cytokines augment the synthesis of AGPR, particularly in the stage prior to glycosylation. more...

Granulocyte-colony-stimulating factor (G-CSF), granulocyte/macrophage-colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) were used in combination with PSK, a protein-bound polysaccharide extracted from mycelium of Coriolus versicolor (strain CM101), in myelosuppressed mice. The myelosuppression model consisted of BDF1 mice who received 150 mg/kg 5-fluorouracil (5-FU) intravenously. The peripheral blood leukocyte count during the recovery stage was significantly increased when these cytokines were administered with PSK compared to when the cytokines were used individually. In vitro colony assay revealed that the combination of PSK and any of GM-CSF, IL-3 or stem cell factor (SCF) showed a greater increase in colony numbers than when these materials were administered individually, although G-CSF did not show a synergistic effect with PSK. When bone marrow cells were obtained from mice which had been given PSK or IL-3, the colony assays were made in the presence of PSK or IL-3 in vitro. The greatest increase in the numbers was observed in colonies of the cultured group in the presence of IL-3 after the PSK priming. However, the colony formation potential of PSK was not inhibited by addition of anti-SCF antibody. The above results indicate that the combined administration of PSK with G-CSF, GM-CSF or IL-3 increased the hematological recovery of myelosuppressed mice. Moreover, the phase at which PSK has effects on hematopoietic cells seems to be at a more immature level than with IL-3. The combined administration of PSK and the above cytokines may improve myelosuppression after chemotherapy in patients with malignancy. more...

To explore the mechanism of increased collagen deposition in scirrhous carcinoma of the stomach, an attempt was made to define the role of transforming growth factor beta 1 (TGF-beta 1), secreted from tumour cells, as a possible humoral factor which functions in a paracrine manner to stimulate the production of collagen in regional fibroblasts. Immunohistochemical staining revealed that tumour cells in scirrhous carcinomas were generally stained more intensively than those in other types of carcinomas. On Northern blot analysis the tumour cells established from scirrhous carcinoma (KATO-III, OCUM-1 and HSC-39) exhibited relatively strong signals compared with those from non-scirrhous carcinoma (MKN-28 and MKN-45). In the culture media of scirrhous carcinoma cells, the active form of TGF-beta 1 was detected, while in those of the non-scirrhous carcinoma cells the latent form was demonstrated by both colony and radioreceptor assays. The culture medium from KATO-III showed strong stimulating activity of collagen synthesis in fibroblasts, and this activity was partially neutralised by an anti-TGF-beta 1 antibody. These results suggest that tumour cells in scirrhous carcinoma produce more active-form TGF-beta 1 than does non-scirrhous carcinoma and thus is partially responsible for the observed enhanced collagen deposition in the region. Images Figure 1 Figure 2 more...

症例は70歳女性.微熱及び易疲労感を主訴に近医受診.GOT 532, GPT 359IU/lと上昇し,白血球数60,400/mm3,うち好酸球88%を指摘され紹介入院となる.肝生検にて,肝細胞のballooningと多核巨細胞の出現及び好酸球の浸潤を認め,巨細胞性肝炎と診断した.血液検査上,肝炎ウイルスの関与・免疫異常及び各種ウイルス感染は否定された.約2カ月の経過でGOT・GPTの低下と共に好酸球も減少し,軽快した.経過を追って施行した肝生検では,肝炎の回復と共に好酸球の浸潤は減少していた.また,多核巨細胞の電子顕微鏡的検討では,ウイルス封入体は認められず,核質・核の大きさがほぼ均一で巨細胞の成因として細胞融合が考えられた.巨細胞性肝炎と好酸球増多症の合併例はわずかに欧米で1例に認めるのみであり,その関連性,および巨細胞の成因を考察する上で示唆に富む症例と考えられたので報告する. more...

Transplantation of human mesenchymal stem cells has been shown to reduce infarct size and improve functional outcome in animal models of stroke. Here, we report a study designed to assess feasibility and safety of transplantation of autologous human mesenchymal stem cells expanded in autologous human serum in stroke patients. We report an unblinded study on 12 patients with ischaemic grey matter, white matter and mixed lesions, in contrast to a prior study on autologous mesenchymal stem cells expanded in foetal calf serum that focused on grey matter lesions. Cells cultured in human serum expanded more rapidly than in foetal calf serum, reducing cell preparation time and risk of transmissible disorders such as bovine spongiform encephalomyelitis. Autologous mesenchymal stem cells were delivered intravenously 36-133 days post-stroke. All patients had magnetic resonance angiography to identify vascular lesions, and magnetic resonance imaging prior to cell infusion and at intervals up to 1 year after. Magnetic resonance perfusion-imaging and 3D-tractography were carried out in some patients. Neurological status was scored using the National Institutes of Health Stroke Scale and modified Rankin scores. We did not observe any central nervous system tumours, abnormal cell growths or neurological deterioration, and there was no evidence for venous thromboembolism, systemic malignancy or systemic infection in any of the patients following stem cell infusion. The median daily rate of National Institutes of Health Stroke Scale change was 0.36 during the first week post-infusion, compared with a median daily rate of change of 0.04 from the first day of testing to immediately before infusion. Daily rates of change in National Institutes of Health Stroke Scale scores during longer post-infusion intervals that more closely matched the interval between initial scoring and cell infusion also showed an increase following cell infusion. Mean lesion volume as assessed by magnetic resonance imaging was reduced by >20% at 1 week post-cell infusion. While we would emphasize that the current study was unblinded, did not assess overall function or relative functional importance of different types of deficits, and does not exclude placebo effects or a contribution of recovery as a result of the natural history of stroke, our observations provide evidence supporting the feasibility and safety of delivery of a relatively large dose of autologous mesenchymal human stem cells, cultured in autologous human serum, into human subjects with stroke and support the need for additional blinded, placebo-controlled studies on autologous mesenchymal human stem cell infusion in stroke. more...

Summary We studied mRNA expression of the cytokine granulocyte-colony stimulating factor (G-CSF), interleukin-1β (IL-1β), IL-6, IL-8 and stem cell factor of stromal cells derived from bone marrows of nine normal volunteers, eight patients with aplastic anaemia (AA) and seven patients with myelodysplastic syndrome (MDS). The proportion of endothelial cells. macrophages. fibroblast-like cells and adipocytes in stromal cells showed no differences between normal volunteers and the patients. Levels of cytokine inKNA expression were determined by reverse transcription-polymerase chain reaction. Spontaneous expression occurred and this was augmented by IPS stimulation in cells of all the normal volunteers and in most patients. When stimulated by LPS. the mean G-CSF and IL-1β mRNA expressions in patients with AA were significantly higher than normal volunteers. but there was one patient showing lower IL-1β. IL-6 and 71,-8 expression with no response to LPS. LPS-induced IL-6 and IL-8 expression of two patients with MDS were significantly higher than normal. The spontaneous and LPS-induced protein concentration of G-CSF, IL-6 and IL-8 in culture supernatants from 15, 10 and four patients, correlated well with the niKNA expression. The correlation coefficients were 0.92. 0.78 and 0.91. respectively. In conclusion, there were a few patients whose aetiology appeared to be reduction of stromal cytokine expression in AA. but most patients with AA and MDS expressed normal or high levels of cytokine mKNA. more...

We prepared various TNF-α derivatives by protein engineering techniques. Mutant 471, in which 7 N-terminal amino acids were deleted and Pro8Ser9Asp10 was replaced by ArgLysArg, had a 8-fold higher antitumor activity against mouse L929 cells than wild-type TNF-α. The additional substitution of Ala156 or Leu157 by more hydrophobic amino acids enhanced the activity of mutant 471. These results suggested that the combinational mutations in the N- and C-terminal regions of TNF-α are effective for the improvement of antitumor activity. more...

Expression of various oncogenes (ras, myc, erbB2, src, fyn, yes and sis) in a high-metastatic clone (MH-02) derived from a murine methylcholanthrene-induced fibrosarcoma A (Meth A) was compared with those of its parent clone (ML-01) by Northern blot analysis. Two oncogenes, fyn, belonging to the tyrosine-kinase family, and sis, belonging to the cellular-growth-factor family, were found to have higher signals (3.6-fold and 1.8-fold respectively) in MH-02 than in ML-01 cells. To explore the possibility that higher expression of these oncogenes is involved in enhanced metastasis of the MH-02 clone, ML-01 was transfected by a fyn vector and the metastatic potential of the transfectant was examined. Mice administered fyn-transfected ML-01 cells had significantly increased metastatic nodules in the lung, as compared with those whose ML-01 cells were transfected with control vector without the fyn gene. The result indicates that the fyn gene is one of the factors governing the metastatic potential of Meth A cells. more...

The LEC (Long‐Evans cinnamon) rat is a mutant strain displaying hereditary hepatitis and spontaneous hepatocellular carcinoma, and shows abnormal hepatic copper accumulation similar to that occurring in Wilson's disease. We evaluated the iron metabolism of LEC rats compared to LEA (Long‐Evans agouti) rats. Hepatic iron and ferritin concentrations were remarkably increased depending on age in LEC rats but not in LEA rats. Increased hepatic iron is normally associated with decreased serum transferrin and total iron binding capacity in hepatic iron overload. In LEC rats, however, both serum transferrin and total iron binding capacity increased with increasing hepatic iron. This increase of serum transferrin and hepatic iron may be an additional important factor contributing to liver injury in LEC rats. more...

口腔癌の化学療法効果は, 腫瘍縮小率で判定しているが, 必ずしも縮小率と化学療法効果とは一致しない。そこで, 口腔癌の化学療法による血漿GST-π値の変動を組織学的効果の有効群と, 無効群の2群に分け, 口腔癌化学療法効果判定としてのGST-πを検討した。1. 有効群では, 化学療法前に血漿GST-π値は, 比較的低値を示し, 化学療法後においてはさらに有意に低下した。2. 無効群では, 有効群に比べ, 化学療法前に血漿GST-π値は高値を示し, 化学療法後においても有意に低下しなかった。以上より血漿GST-π値の変動は口腔癌の化学療法組織学的効果を反映しており, 口腔癌の化学療法効果判定として有用と思われる。 more...

A 54-year-old woman with peripheral T cell lymphoma in second complete remission (CR) received an autologous peripheral blood stem cell transplant (PBSCT). Antibiotic-resistant bloody diarrhea, and fever developed 110 days after transplant. Blood and stool cultures were negative. Skin rash was not observed. Barium enema and colonoscopy showed typical features of pancolonic-type ulcerative colitis (UC). Endoscopic biopsies confirmed the diagnosis of UC. Mesalazine and immunosuppressive therapy improved symptoms dramatically. We detected serum antibodies against synthetic tropomyosin (TM) peptide when UC was diagnosed. We postulate that autoimmunity including autoreactive anti-TM antibodies may be involved in the pathogenesis of UC after autologous PBSCT in this patient. more...

Expression of E-cadherin in 21 patients with various histological types of gastric carcinomas was studied by immunoperoxidase staining. Intercellular boundaries of almost all cancer cells in well and moderately differentiated adenocarcinomas stained as deeply for E-cadherin as normal gastric mucosa. However, singly infiltrating cells of those histological types were poorly stained. In poorly differentiated adenocarcinomas, cancer cells forming clusters stained lightly and those infiltrating singly stained even less. In signet ring cell carcinomas, hardly any staining was observed. In each histological type, the staining patterns and intensity at different layers of the gastric wall, were essentially the same. Cancer cells from carcinomatous ascites of gastric adenocarcinomas and pancreatic adenocarcinomas, and those from pleural effusion of lung adenocarcinomas were also studied by immunofluorescence staining. Of 11 specimens, ten were negative and only one from a lung adenocarcinomas was positively stained. By phase-contrast microscopic observations, none of these cancer cells including those from the lung adenocarcinomas, formed obvious cell-cell contacts. Cell aggregation assays confirmed the above results. The molecular weight of E-cadherin of cancer cells of lung adenocarcinomas was less than intact E-cadherin as revealed by Western blot analysis. These results suggest that depressed expression and/or impaired function of E-cadherin in cancer cells, facilitates their liberation from primary sites to infiltrate freely into tissue or fluid. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 9 more...

Background. The authors analyzed the clinical usefulness of glutathione-S-transferase-π (GST-π) as a tumor marker in patients with oral cancer. Methods. GST-π levels in plasma of 61 patients with oral squamous cell carcinomas, 65 patients with benign oral diseases, and 78 healthy subjects were investigated with the sandwich enzyme-immunoassay (EIA) system. Results. Elevated GST-π levels in plasma were observed in patients with oral cancer, but patients with benign oral diseases had normal GST-π levels. More than 70% of patients with Stage III or IV oral cancer and more than 50% of those with Stage I and II disease had elevated levels of GST-π in plasma. Elevated levels of GST-π in plasma were also discovered in most patients with tumor recurring after surgery before recurrence was detected clinically. GST-π also was found to be a useful marker for evaluating the response to chemotherapy, for monitoring postoperative tumor resectability or tumor burden, and for predicting the recurrence of tumor in patients with oral cancer. Conclusions. GST-π is considered to be a useful aid for early diagnosis, predicting tumor extent, and determining parameters of treatment efficacy and prognosis for oral cancer. more...

We investigated the effect of recombinant human tumor necrosis factor (TNF) on the lysosomal enzyme activity of various established cell lines in vitro. Incubation of 1 x 10(6) TNF-sensitive mouse tumorigenic fibroblasts (L-M cells) in the presence of TNF (100 U/ml) for 48 h increased the total (the sum of the enzyme activities in the lysosomes and the cytoplasm) acid phosphatase and beta-glucuronidase activities by 3.7- and 4.2-fold, respectively. The same increase was observed even when 1 U/ml of TNF was added to some cultures and no further augmentation occurred at 10 or 100 U/ml. Measurement of total and free enzyme activities showed that TNF stimulation not only enhanced the total intracellular enzyme activity but also accelerated the conversion into free (cytoplasmic) enzyme activity. Addition of a lysosomotropic agent (methylamine) suppressed both the enhancement of lysosomal enzyme activity and the cytotoxicity of TNF. A similar enhancement of lysosomal enzyme activities was also detected in various TNF-sensitive tumor cell lines, and a strong correlation (acid phosphatase: r = 0.836, beta-glucuronidase: r = 0.910) was observed between the enhancement of enzyme activity and sensitivity to TNF. No such increase was detected in TNF-resistant human diploid cells. These results show that TNF induces the activation and release of lysosomal enzymes in TNF-sensitive cells, and suggest that such events may play an important role in TNF-mediated cytotoxicity. more...

We previously reported that recombinant human tumor necrosis factor (rhTNF) and hyperthermia had a synergistic effect against tumors, in vitro and in vivo. We have now investigated the mechanism of this synergy by measuring the lysosomal enzyme activity and hydroxyl radical production of L‐M cells treated with rhTNF and/or hyperthermia. A synergistic activation of lysosomal enzyme and the induction of hydroxyl radical production in L‐M cells treated with both rhTNF and hyperthermia was observed. A synergistic cytotoxic effect was observed when rhTNF and hyperthermia were combined, and was inhibited by the addition of a reactive oxygen scavenger, dimethyl sulfoxide or bipyridine. The results show that the augmenting effect of hyperthermia on lysosomal enzyme activation and induction of hydroxyl radical production by rhTNF plays an important role in the synergistic cytotoxic effect. more...

Based on findings that the cytotoxic effects of tumor necrosis factor (TNF) are closely related to levels of intracellular oxygen radicals, and on the results of TNF gene transfection studies, the hypothesis was made that endogenous TNF (enTNF) acts as a protective factor against exogenous TNF by inducing inhibitors or scavengers of oxygen radicals. In order to test this hypothesis, we investigated the intracellular levels of manganous superoxide dismutase (MnSOD) and glutathione (GSH) in L-M(pNTnF) cells carrying a TNF gene induced by dexametha- sone (DM). When L-M(pNTnF) cells were treated with DM they expressed enTNF, and acquired resistance to exogenous TNF. There was no change in the GSH concentration after enTNF induction, but a 1.9- to 3.9-fold increase in MnSOD levels was noted. Our findings suggest that enTNF exerts its protective function against the cytocidal effect of exogenous TNF by inducing MnSOD production. more...

A case is presented of an adult chylous cyst of the mesentery that was preoperatively diagnosed to be a pancreatic cystadenoma. A 66-year-old asymptomatic male was followed up for 15 months under the diagnosis of a benign pancreatic cyst. On October 1997, computed tomography showed a 45 x 40 mm cystic mass in the upper abdomen which came in contact with the pancreas. Endoscopic ultrasonography revealed a multilocular mass with a 7 x 4 mm elevated lesion. Endoscopic retrograde cholangiopancreatography and magnetic resonance cholangiopancreatography revealed the cystic mass to be unrelated to the pancreatic duct. The preoperative diagnosis was a pancreatic cystadenoma or cystadenocarcinoma. A laparotomy showed a 50 x 40 mm cystic mass containing chylous fluid, that arose from the mesentery of the upper part of the jejunum. The pathological diagnosis was a chylous cyst of the mesentery. The preoperative diagnosis in this case was very difficult because the chylous cyst appeared to be attached to the pancreas and this phenomenon is considered to be extremely rare. more...

Alpha-Galactosylceramide (alpha-GalCer) is a potent CD1d ligand that activates natural killer like T-cells (NKT), leading to the production of helper T (Th) 1 and Th2 cytokines that mediate various immunemodulatory and antitumor effects. Here, we determined whether the administration of adenovirus-vector-encoding mouse interleukin-2 (AdmIL-2) can augment the antitumor effect of alpha-GalCer on subcutaneous and metastatic tumors in mice. Mice were intraperitoneally injected with alpha-GalCer on days 7, 10 and 13 after tumor inoculation, with or without intratumoral injection of AdmIL-2 on day 7. alpha-GalCer treatment increased the serum levels of interferon-gamma, while intratumoral injection of AdmIL-2 elevated serum IL-2 levels. A combination of alpha-GalCer and AdmIL-2 (alpha-GalCer/AdmIL-2) inhibited the in vivo tumor growth and improved the survival of tumor-bearing mice, as compared to the use of a single agent. Experiments on spontaneous metastasis models revealed that alpha-GalCer/AdmIL-2 reduced lung metastasis and prolonged survival, as compared to control groups. In addition, the splenic and liver mononuclear cells from mice treated with alpha-GalCer/AdmIL-2 showed enhanced cytolytic activity against NK-sensitive YAC-1 and NK-resistant 3LL tumors. Moreover, alpha-GalCer/AdmIL-2 treatment expanded the absolute numbers of lung and liver NK, NKT and T-cells as well as the TNF-related apoptosis-inducing ligand (TRAIL) expression of these cells. This study shows the efficacy of alpha-GalCer/AdmIL-2 immunomodulatory therapy, and provides a cellular mechanism on how it exerts the antitumor effects. more...

When considering surgery for branch duct-intraductal papillary mucinous neoplasms (BD-IPMNs) with suspected malignancy, it should be recognized that these lesions are frequently multifocal and are usually found in elderly patients with potential comorbidities that could affect the outcome of surgery. Clinical trials of chemoprevention have been conducted for a wide variety of malignancies. Twenty-two BD-IPMN patients participated in the trial at our institution from June 2004 to January 2007. Ten of the 22 patients who rejected surgical therapy although their lesions or clinical symptoms met the criteria for surgical resection of the International Association of Pancreatology guidelines were assigned to the treatment group. Sulindac (150 mg twice daily) and omeprazole (20 mg once daily) were administered to these patients for 18 months. The remaining 12 patients comprised the control group. Branch duct diameter and mural nodule heights were monitored by either magnetic resonance cholangiopancreatography (MRCP) or computed tomography (CT) and by endoscopic ultrasonography (EUS). Both branch duct diameter and mural nodule height of BD-IPMNs in the treatment group were significantly reduced, while those in the control group were unchanged. Immunohistochemical staining for cyclooxygenase-1 and -2 was negative in hyperplasia, adenoma and carcinoma portions of resected pancreatic specimens but was clearly positive for glutathione-S-transferase π (GST-π), suggesting that GST-π is a putative target molecule for sulindac. Although a larger scale randomized controlled study is needed in future, the present results suggest the promise of chemoprevention of carcinoma derived from BD-IPMNs by sulindac. more...

A case of unresectable hepatocellular carcinoma which responded favorably to combined therapy with tumor necrosis factor (TNF) and local hyperthermia is reported. A 58-year-old man was admitted to our hospital in June 1988 for treatment of hepatocellular carcinoma affecting S4 and S8. After three sessions of transcatheter arterial embolization (TAE) therapy, the serum alpha 1-fetoprotein level decreased, and a reduction in the size of the lesions was also noted. Thereafter, the patient received local hyperthermia once a week (60 minutes of irradiation from a Thermotron-RF8 at 1,100W), but the alpha 1-fetoprotein level increased again in February 1989. On examination, enlargement of the S8 lesion and a new nodule in S7 were recognized. Since TAE was contraindicated due to liver dysfunction, human recombinant TNF (1 x 10(6)U) was given by intravenous infusion together with local hyperthermia once a week. Eight sessions of the combined therapy reduced the serum alpha 1-fetoprotein level markedly (7,512.0 to 2,782.0 pg/ml) and after eighteen sessions, 58.1% regression of tumor size (partial response) on computed tomography scans was observed. This anecdotal case supports previous experimental evidence suggesting that TNF plus hyperthermia may be effective for treating unresectable hepatocellular carcinoma. more...

We have evaluated the radioimmunoassay for type I procollagen carboxy terminal peptide (type I C-peptide), which is liberated from type I procollagen during its conversion to collagen, in the serodiagnosis of scirrhous carcinoma of the stomach. The mean (SD) serum concentration of type I C-peptide in 39 normal subjects was 41.7 (19.7) ng/ml. The mean serum values and the positive ratio of type I C-peptide in 11 patients with stages II and III scirrhous carcinoma of the stomach were 91.2 (41.9) ng/ml and 54.5%, respectively. In 10 patients with other types of gastric carcinoma, the mean type I C-peptide values were not significantly different from the normal value. Serum type I C-peptide values reflected the clinical course of scirrhous gastric carcinoma in five patients who underwent either operation or chemotherapy. The measurement of serum type I C-peptide concentrations could provide a useful way of diagnosing and monitoring scirrhous carcinoma of the stomach. more...

We prepared several TNF mutants by protein engineering techniques and compared their biological properties with those of the wild-type TNF. The mutant that lacked 7 N-terminal amino acids had higher cytotoxicity and higher binding activity to receptors on tumor cells. In contrast, the mutagenesis of Arg32 or Ala84, in combination with the deletion of 7 N-terminal amino acids, eliminated the cytotoxicity and the receptor binding. These mutants also lacked acute lethal toxicity in normal mice. Therefore, we concluded that the N-terminus, Arg32 and Ala84 of TNF might be concerned with binding to the TNF receptor. It was also suggested that the receptor molecules on tumor cells bound to the same or neighboring sites on TNF molecules as normal cell receptors. more...

血中GST-πは近年種々の悪性腫瘍マーカーとして注目されるようになった。しかし口腔癌患者におけるGST-πの臨床的応用に関しては, 検討されていない。本研究は口腔癌患者44例, 健常人49例の血漿GST-πをわれわれの開発したEIA法により測定し, 検討を行った。さらに口腔癌の腫瘍マーカーとして知られているSCCとも比較, 検討した。結果1. 口腔癌患者と健常人の血漿GST-π値は各々59.0±18.6ng/ml, 18.2±6.7ng/ml (P more...

血清中にinterleukin 6(IL-6)活性を認めたimmunoblastic lymphadenopathy (IBL)類似薬剤アレルギーの1例を報告する.症例は, 43歳男性で,解熱鎮痛剤服用後に発熱,全身リンパ節腫脹,皮疹,肝脾腫が出現した.また,著明な溶血性貧血,血小板減少,多クローン性高γ-グロブリン血症,各種自己抗体の陽性化,細胞性免疫能の低下など, IBLと同様の検査所見を呈した.しかし,摘出リンパ節の病理像は,反応性リンパ節炎の所見であり,また,薬剤のリンパ球刺激試験が強陽性を示したことから, IBL類似の臨床症状を呈した薬剤アレルギーと診断した.病態解析のため行った血清および摘出リンパ節培養上清中のIL-6活性測定では通常,血清中に検出されないIL-6活性が認められ,さらに培養上清中でも高値を示し,病態を考えるうえで興味深い症例と考えられた. more...

We established five clones (ML-01, ML-02, MH-01, MH-02, MH-03) from murine 3-methylcholan-threne-induced fibrosarcoma A (Meth A), and investigated their experimental metastatic potentials in relation to their platelet-aggregating activities. A clone with a high metastatic potential (MH-02) showed a characteristic biphasic pattern of platelet aggregation, of which the first peak was not present in the aggregation patterns of the clone with low metastatic potential (ML-01). The first peak was eliminated by treatment of the cells with apyrase, indicating that adenosine diphosphate (ADD was the causative substance of this particular peak. The metastatic potential of clones correlated well with the ADP concentration of the culture media. These results suggest that the increased ADP production and consequential enhancement of platelet-aggregating activity are closely related to the increment of pulmonary metastatic potential of MH-02 clone. more...

A 65-year-old male, who had been diagnosed with melanoma of stage IIB and treated by chemotherapy since 2003 at the Dermatology Department, was referred to our department for liver metastasis of melanoma that had become resistant to chemotherapeutic agents. In 2006, he started receiving hepatic arterial infusion of CDDP. He was admitted to the hospital on an emergency basis for general fatigue the next May. Blood tests revealed anemia and thrombocytopenia. Contrast CT showed aggravation of liver metastasis. Contrast ultrasonography revealed nodular contrast enhancement at the margin of the tumor. On the basis of image findings and blood test results, DIC due to intratumoral hemorrhage was diagnosed. CDDP arterial infusion with DSM resulted in improved DIC, and he was able to be discharged. Taken together, attention has to be paid to the potential for emergency complications of DIC due to liver metastasis of melanoma with intratumoral hemorrhage. Moreover, it was shown that arterial infusion with DSM was effective for liver metastasis of melanoma. more...