Amelioration of murine dextran sulfate sodium-induced colitis by ex vivo extracellular superoxide dismutase gene transfer.

Although the etiology of inflammatory bowel disease has not been fully clarified, reactive oxygen species is speculated to be involved. Extracellular superoxide dismutase (EC-SOD), an isozyme of SODs, is known to function mainly in body fluids. We investigated the efficacy of an ex vivo EC-SOD gene transfer into dextran sulfate sodium (DSS)-induced colitis mice. Experimental colitis was induced by providing Balb/c mice with DSS in sterile distilled water provided as desired. The syngenic fibroblasts were obtained from Balb/c mice embryos and retrovirally transduced with the hEC-SOD gene. These engineered cells were confirmed to secrete EC-SOD in culture medium by enzyme-linked immunosorbent assay and were inoculated subcutaneously in the backs of DSS-treated mice. Mucosal injury of the colon was evaluated by the disease activity index (DAI: body weight, rectal bleeding, and stool consistency), grading of histologic disease severity, and levels of cytokine (tumor necrosis factor-alpha, interleukin-1beta) production. 8-Hydroxydeoxyguanosine (8-OHdG) levels in the mucosal tissue were assessed by immunohistochemical staining. Malondialdehyde (MDA) was measured using a colorimetric assay. A significant improvement was observed in DAI score and histologic severity as well as in mucosal tissue levels of inflammatory cytokines, 8-OHdG, and MDA of mice treated with the EC-SOD gene as compared with those without gene therapy, not only in a mild colitis model but also in a severe colitis model. Survival of treated mice in these models was significantly prolonged. Ex vivo transfer of the EC-SOD gene was feasible for treatment of DSS-induced colitis.


Hepatitis C virus core protein promotes proliferation of human hepatoma cells through enhancement of transforming growth factor-{alpha} expression via activation of NF-{kappa}B.

Hepatitis C virus (HCV) infection is a major cause of human hepatocellular carcinoma (HCC). The precise mechanism of hepatocarcinogenesis in humans by HCV is currently unclear. It was recently shown, however, that transgenic mice with the HCV core gene often develop HCC, suggesting tumorigenic activity of the HCV core protein. Further, the HCV core protein expressed in HepG2 cells transfected with the core gene was shown to stimulate proliferation of transfectants through activation of nuclear factor-kappaB (NF-kappaB). The downstream target molecule(s) of NF-kappaB activated by the HCV core protein to evoke cell proliferation is not yet identified. Transforming growth factor (TGF) alpha, which is often overexpressed in various tumour tissues such as HCC, has been shown to stimulate hepatocyte proliferation through activation of the mitogen-activated protein kinase or extracellular signal-related protein kinase (MAPK/ERK) cascade. To explore the possibility that TGFalpha might be a target molecule for NF-kappaB activated by the HCV core, and that TGFalpha participates in the growth promotion of the core transfectants in an autocrine manner, activating the MAPK/ERK pathway. A HCV core expression vector was transfected into human hepatoma Huh-7, HepG2 and Hep3B cells. NF-kappaB activity was examined by an electrophoretic mobility shift assay. TGFalpha transcription was assessed by a luciferase reporter assay. TGFalpha protein was determined by immunoblot and ELISA. MAPK/ERK activity was examined by an in vitro kinase assay. Cell proliferation was assessed by a water-soluble tetrazolium salt-1 assay. In the HCV core transfectants, NF-kappaB bound to the kappaB site in the TGFalpha proximal promoter region, resulting in an increase in TGFalpha transcription. Immunoblot as well as ELISA showed increased TGFalpha expression in the HCV core transfectants. SN50, a specific inhibitory peptide for NF-kappaB, cancelled HCV core-induced TGFalpha expression. HCV core protein increased cell proliferation as well as ERK activity of the HCV core transfectants as compared with the mock transfectants. The growth-promoting activity and activation of ERK by the HCV core protein were negated by treatment with anti-TGFalpha antibodies. These results suggest that the HCV core protein promotes proliferation of human hepatoma cells by activation of the MAPK/ERK pathway through up regulation of TGFalpha transcription via activation of NF-kappaB. Our finding provides a new insight into the mechanism of hepatocarcinogenesis by HCV infection.


Colorectal Cancer: genetics of development and metastasis.

It has been well documented that there are two major pathways in colorectal carcinogenesis. One is the chromosomal instability pathway (adenoma-carcinoma sequence), which is characterized by allelic losses on chromosome 5q (APC), 17p (p53), and 18q (DCC/SMAD4), and the other is a pathway that involves microsatellite instability. Recent progress in molecular biology, however, has shown that colorectal carcinogenesis is not necessarily clearly divided into these two pathways, but is in fact more complicated. Other routes, including the transforming growth factor-beta/SMAD pathway, the serrated pathway, and the epigenetic pathway, have been reported. Cross talk among these pathways has also been reported. In the invasion and metastasis steps of colorectal cancers, many more genes have now been identified as being involved in proteolysis, adhesion, angiogenesis, and cell growth. Recently accumulated evidence indicates that colorectal cancer is a genetically heterogeneous and complicated disease.


A phase I/II study of nedaplatin and 5-fluorouracil with concurrent radiotherapy in patients with esophageal cancer.

To determine the recommended dose (RD) of cis-diammine-glycolatoplatinum (nedaplatin) when given concurrently with 5-FU and high dose radiation therapy in the treatment of esophageal cancer. The purpose of the phase II trial is to determine efficacy and further define the side effect profile. Twenty-six patients with clinical stage I to IVA squamous cell carcinoma of the esophagus were enrolled in a non-surgical treatment comprised of a fixed dose of fluorouracil (400 mg/m2 administered as continuous intravenous infusion on days 1-5 and days 8-12) plus escalating doses of nedaplatin (40 mg/m2 in level 1, 50 mg/m2 in level 2, or 60 mg/m2 in level 3 on days 1 and 8), repeated twice every 3 weeks with concurrent radiotherapy (60 Gy). Between July 1998 and February 2004, a total of 26 patients entered this trial, all of whom were considered evaluable for toxicity assessment. In phase I of the study, 12 patients were treated in sequential cohorts of three to six patients per dose level. The maximum tolerated dose was reached at level 3 with two grade 4 neutropenia and one grade 4 thrombocytopenia. Thus, the recommended dosing schedule is level 2. Of the 20 patients treated at the RD level 2, including 6 patients of the RD phase I portion, 8 out of 20 patients (40%) had grade 3-4 neutropenia, 5 patients (25.0%) had grade 3-4 thrombocytopenia, 4 patients (20.0%) had grade 3 anemia and 4 patients (20.0%) had grade 3-4 esophagitis. Other toxicities were relatively mild and usually of grade 2 or less. Objective responses were noted in the 26 patients (overall response rate, 88.5%) including 11 (42.3%) complete remissions. The 1- and 3-year survival rates were 65.1 and 37.2%, respectively, with a median survival time of 21.2 months. The combination of nedaplatin and 5-FU with radiation is a feasible regimen that shows promising antitumor activity with an acceptable safety profile in patients with esophageal cancer.


[A case of granulocyte-colony stimulating factor producing small cell carcinoma of esophagus].

The Japanese Journal of Gastro-enterology


Augmentation of Antitumor Effects of p53 Gene Therapy by Combination with HDAC Inhibitor.

We have previously shown that the HDAC inhibitors (HDACI) activate the p53 molecule through acetylation of 320 and 373 lysine residues, upregulate PIG3 and NOXA and induce apoptosis in cancer cells expressing wild and pseudo-wild type p53 genes (Terui T, et al. Cancer Res 2003; 63:8948-54). It has also been reported that expression of the Coxsackie adenovirus receptor and subsequent transfection efficiency of the adenovirus in cancer cells were enhanced by HDACI treatment. In this study, we extended these observations to explore the combination effect of adenoviral vector carrying wild type p53 (Ad-p53) gene therapy with a HDACI, sodium butyrate (SB), on xenografted human gastric cancer cells (KATO-III) and hepatocellular carcinoma cells (HuH7) in nude mice. We first confirmed an increased expression of Coxsackie adenovirus receptors with an associated increment of transgene (X-gal) expression by SB treatment in KATO-III cells. We then injected Ad-p53 into subcutaneous tumors of KATO-III and HuH7 combined with intraperitoneal administration of SB and found a significantly higher growth suppressive effect than single treatments of each. Even a complete regression of tumors was observed in three of five mice treated with this combination while with single treatment no tumor regression was observed. Tumors treated with the combination showed higher numbers of TUNEL positive cells than those treated with a single modality. Moreover, necrotic changes were more evident in tumors treated with the combination than separately, a compatible finding to the observation that vascularity revealed by CD34 staining was poorer in tumors treated with the combination than those treated with p53 gene or SB alone. This was further supported by the finding that BAI-1 (brain specific angiogenesis inhibitor-1), an inhibitor of vascularization, was induced by SB treatment in KATO-III and HuH7 cells transfected with Ad-p53. Thus SB was shown to be an efficient potentiator of p53 gene therapy for cancer.


Human mesenchymal stem cells xenografted directly to rat liver differentiated into human hepatocytes without fusion.

Hepatic transdifferentiation of bone marrow cells has been previously demonstrated by intravenous administration of donor cells, which may recirculate to the liver after undergoing proliferation and differentiation in the recipient's bone marrow. In the present study, to elucidate which cellular components of human bone marrow more potently differentiate into hepatocytes, we fractionated human bone marrow cells into mesenchymal stem cells (MSCs), CD34+ cells, and non-MSCs/CD34- cells and examined them by directly xenografting to allylalcohol (AA)-treated rat liver. Hepatocyte-like cells, as revealed by positive immunostaining for human-specific alpha-fetoprotein (AFP), albumin (Alb), cytokeratin 19 (CK19), cytokeratin 18 (CK18), and asialoglycoprotein receptor (AGPR), and by reverse transcription-polymerase chain reaction (RT-PCR) for expression of AFP and Alb mRNA, were observed only in recipient livers with MSC fractions. Cell fusion was not likely involved since both human and rat chromosomes were independently identified by fluorescence in situ hybridization (FISH). The differentiation appeared to follow the process of hepatic ontogeny, reprogramming of gene expression in the genome of MSCs, as evidenced by expression of the AFP gene at an early stage and the albumin gene at a later stage. In conclusion, we have demonstrated that MSCs are the most potent component in hepatic differentiation, as revealed by directly xenografting into rat livers.


[A case of signet ring cell carcinoma of the stomach discovered by abdominal wall metastasis].

The Japanese Journal of Gastro-enterology


[A family of attenuated familial adenomatous polyposis (AFAP)].


Clinical evaluation of laparoscopy-assisted distal gastrwctomy for early gastric cancer.


Prolonged survival of mice with multiple liver metastases of human colon cancer by intravenous administration of replicable E1B-55K-deleted adenovirus with E1A expressed by CEA promoter.

Liver is the most preferential site for metastasis of colon cancer. We, in the present study, constructed a self-replicable adenovirus in which E1A is driven by a CEA promoter and E1B-55K is deleted from the E1B region (AdCEAp/Rep) and examined its effects on multiple metastases of a human colon cancer cell in a mouse xenograft model. We first showed effective replication of the virus in various CEA-producing human colon cancer cells (M7609, HT-29) and subsequent lysis of the infected cells in vitro. We then demonstrated that a single intratumoral injection of the virus (1 x 10(8) PFU/100 microl) induced a complete regression of subcutaneous tumors (M7609) inoculated into nude mice. Further, we demonstrated that systemic administration of the virus (1 x 10(8) PFU/100 microl) through the tail vein to nude mice, which 1 week prior had been inoculated with tumor cells (colon carcinoma cell line HT-29) via the spleen and showed apparent multiple metastases in the liver, effectively suppressed the metastasis formation. The mean survival time of the treated mice was significantly longer than that of the controls. Thus, the systemic administration of AdCEAp/Rep was considered to be effective on multiple liver metastases of CEA-positive colon cancer in a xenograft model.


Interleukin-11 as an osteoprotegerin-inducing factor in culture medium of blastic cells from a patient with acute megakaryocytic leukemia complicated with osteosclerosis.

We came across a rare case of acute megakaryocytic leukemia, the clinical course of which was relatively chronic and nonaggressive. This case was complicated with generalized severe osteosclerosis (OS). The medium in which blastic cells from the patient were cultured showed a strong activity to enhance the expression of an osteosclerotic cytokine, osteoprotegerin (OPG), as revealed by real-time quantitative RT-PCR and Western blot analysis. The OPG-inducing activity of the culture medium was neutralized by the anti-interleukin-11 (IL-11) antibody. These results indicate that IL-11 produced by the blasts was a causative factor of the OS observed in this patient.


Glutathione S-transferase P1-1 protects aberrant crypt foci from apoptosis induced by deoxycholic acid.

Aberrant crypt foci, precursors of colonic adenoma, are frequently positive for glutathione-S-transferase P1-1. Because deoxycholic acid is an apoptosis-inducing xenobiotic in the colon, we examined the possibility that aberrant crypt foci, through the cytoprotecting function of glutathione-S-transferase P1-1, resist deoxycholic acid-induced apoptosis, thereby surviving to become adenomas and subsequently cancer. Glutathione-S-transferase P1-1 or cyclooxygenase-2 expression and the percentage of apoptotic cells in aberrant crypt foci were examined by immunohistochemistry and by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, respectively. Glutathione-S-transferase P1-1 was transfected into colon cancer cells (M7609) and human lung fibroblasts, and deoxycholic acid-induced apoptosis was evaluated by a dye-uptake assay and flow cytometry. Binding of deoxycholic acid to glutathione-S-transferase P1-1 was analyzed by circular dichroism and immunoprecipitation. Caspase activities were determined by colorimetric protease assay, and sulindac binding to glutathione-S-transferase P1-1 was determined by inhibition assay of glutathione-S-transferase P1-1 activity. Aberrant crypt foci showed positive immunostaining for glutathione-S-transferase P1-1 but negative staining for cyclooxygenase-2. The percentage of apoptotic cells in aberrant crypt foci was significantly lower than in healthy epithelium, and the difference became more apparent with deoxycholic acid treatment. The impaired sensitivity of aberrant crypt foci to deoxycholic acid was restored by the glutathione-S-transferase P1-1-specific inhibitor gamma-glutamyl-S-(benzyl)cysteinyl-R-phenylglycine diethylester. By transfection of glutathione-S-transferase P1-1, M7609 cells became more resistant to deoxycholic acid-induced apoptosis than mock transfectants. Direct binding of glutathione-S-transferase P1-1 to deoxycholic acid was proven by circular dichroism and by immunoprecipitation. The aberrant crypt foci in adenoma patients treated with sulindac, which was shown to bind to glutathione-S-transferase P1-1, underwent apoptosis in 4 days and mostly regressed in 2-3 months. Glutathione-S-transferase P1-1 protects aberrant crypt foci from deoxycholic acid-induced apoptosis and may play a pivotal role in early colon carcinogenesis.


Fas-mediated apoptosome formation is dependent on reactive oxygen species derived from mitochondrial permeability transition in Jurkat cells.

Generation of reactive oxygen species (ROS) and activation of caspase cascade are both indispensable in Fas-mediated apoptotic signaling. Although ROS was presumed to affect the activity of the caspase cascade on the basis of findings that antioxidants inhibited the activation of caspases and that the stimulation of ROS by itself activated caspases, the mechanism by which these cellular events are integrated in Fas signaling is presently unclear. In this study, using human T cell leukemia Jurkat cells as well as an in vitro reconstitution system, we demonstrate that ROS are required for the formation of apoptosome. We first showed that ROS derived from mitochondrial permeability transition positively regulated the apoptotic events downstream of mitochondrial permeability transition. Then, we revealed that apoptosome formation in Fas-stimulated Jurkat cells was clearly inhibited by N-acetyl-L-cysteine and manganese superoxide dismutase by using both the immunoprecipitation and size-exclusion chromatography methods. To confirm these in vivo findings, we next used an in vitro reconstitution system in which in vitro-translated apoptotic protease-activating factor 1 (Apaf-1), procaspase-9, and cytochrome c purified from human placenta were activated by dATP to form apoptosome; the formation of apoptosome was markedly inhibited by reducing reagents such as DTT or reduced glutathione (GSH), whereas hydrogen peroxide prevented this inhibition. We also found that apoptosome formation was substantially impaired by GSH-pretreated Apaf-1, but not GSH-pretreated procaspase-9 or GSH-pretreated cytochrome c. Collectively, these results suggest that ROS plays an essential role in apoptosome formation by oxidizing Apaf-1 and the subsequent activation of caspase-9 and -3.


Successful treatment with prostaglandin E1 of ischemic colitis complicated by colonic stricture.


A case of Gardner syndrome with a mutation at codon 1556 of APC: a suggested case of genotype-phenotype correlation in dental abnormality.

A 25-year-old man with suspected Gardner syndrome was introduced to our hospital by a dentist who, during examination of the patient, had found dental dysplasias and multiple osteomas of the jaw. Radiographs, endoscopy and biopsies revealed adenomatous polyposis of the colon. Genetic analysis of peripheral lymphocytes revealed a one-base deletion at codon 1556 in exon 15 of APC, which caused a frame shift and a premature stop at codon 1564. The pedigree analysis demonstrated five patients in his family who presented with dental abnormality and osteomas in addition to adenomatous polyposis of the colon. Although the relationship between the location of APC mutations and dental abnormalities remains controversial, this case supports the hypothesis that a mutation at around codon 1556 of APC is closely associated with dental abnormality and osteomas.

European Journal of Gastroenterology & Hepatology


Gamma/delta T-cell lymphoma of duodenal bulb: a case report.


Inhibition of type I procollagen production by tRNAVal CTE-HSP47 ribozyme.

We suggest applicability of this ribozyme as a new modality for antifibrosis therapy.


[A case responding to weekly paclitaxel (TXL) therapy as third line chemotherapy for scirrhous type gastric cancer].

A 67-year-old female was admitted to our hospital in May, 2001 for examination. She was diagnosed with advanced gastric cancer that was inoperable due to peritoneal dissemination. Seventeen courses of sequential MTX and 5-FU therapy, and 2 courses of TS-1 plus CDDP were carried out. A partial response (PR) and prolonged NC were obtained after these chemotherapies. However, pleural effusion and ascites appeared again, and we diagnosed progressive disease. As a third line chemotherapy for this patient, paclitaxel (TXL) was administered. Treatment consisted of two 3-week courses of paclitaxel 70 mg per m2 on day 1 of each week, with a 1-week break between the courses. Two weeks after the start of this therapy, pleural effusion and ascites had completely disappeared. Paclitaxel is considered to be promising for advanced gastric cancers, as second or third line chemotherapy with paclitaxel for patients with inoperable gastric cancer seems to be effective in improving QOL.

Japanese Journal of Cancer and Chemotherapy


[A case of irinotecan-resistant colon cancer responding to chronotherapy with oxaliplatin, 5-FU, l-LV].

We report a case of irinotecan-resistant colon cancer responding to chronotherapy with oxaliplatin (L-OHP), 5-FU, l-LV (l-Leucovorin). A 72-year-old man was examined at a certain hospital because of constipation and appetite loss. Chest computed tomography (CT) revealed lung metastases, and abdominal CT revealed liver metastases. He was then referred to our hospital. A colonoscopy revealed type 2 tumor in the colon, at the hepatic flexure. We diagnosed adenocarcinoma of the colon with metastases to the liver and lung. Resection of the primary lesion was performed, and chemotherapy consisting of systemic administration of CPT-11, 5-FU and l-LV was performed. After 2 courses of combined treatment with CPT-11/5-FU/l-LV, CT revealed considerable reduction of the metastatic tumors. However, after 3 courses of combined treatment, progressive disease was observed and new brain and bone metastases were detected. We imported and used a non-approved/pending drug, oxaliplatin from the Remedy and Health Corporation, with informed consent from the patient and his family and our clinical ethics committee. Chronotherapeutic schedules have been performed, from which the safety and activity of oxaliplatin against advanced colorectal cancer was reported. Our patient received a 5-day course of chronomodulated 5-FU and l-LV (750 and 300 mg/body/day, respectively; peak delivery rate at AM 4:00 hours) with L-OHP on the first day of each course (100 mg/body, as a 6-hour infusion). Each course was again repeated every 21 days. A partial response was observed in the liver and lung metastases. These results indicate that chronomodulated 5-FU and LV with L-OHP therapy could be an effective regimen for cases of irinotecan-resistant colon cancer.


Esophageal cancer complicated by cytomegalovirus esophagitis during chemoradiotherapy: case report.


Argon plasma coagulation for successful treatment of early gastric cancer with intramucosal invasion.

APC is a safe and effective modality for treatment of early gastric cancer with intramucosal invasion untreatable by surgical resection or EMR. However, further observations are necessary to determine the long term prognosis of patients undergoing this treatment.


A case of true malignant histiocytosis: identification of histiocytic origin with use of immunohistochemical and immunocytogenetic methods.

We report here an autopsy case of true malignant histiocytosis. The patient was a 67-year-old woman who exhibited fever, wasting, hepatosplenomegaly, and progressive pancytopenia. The bone marrow aspiration disclosed hemophagocytosing cells, which resembled histiocytes. The molecular analysis did not show the clonal gene rearrangement of T-cell receptor or immunoglobulin heavy chain. Although the patient had been started on methylprednisolone pulse therapy and chemotherapy with etoposide, she died from cerebral hemorrhage. The autopsy specimens of spleen and liver showed extensive infiltration of atypical cells, for which histiocytic origin was identified with an immunohistochemical method using monoclonal antibodies against CD11c, CD68, macrophage colony-stimulating factor (M-CSF), M-CSF receptor, lysozyme, antitrypsin and alpha1-antichymotrypsin. Recent investigations have disclosed that in most cases diagnosed as malignant histiocytosis, hemophagocytosis was reactive and not evoked by histiocytic malignancy. True malignant histiocytosis, for which histiocytic origin is confirmed, is extremely rare.


Primary intestinal lymphangiectasia successfully treated with octreotide.

A 21-year-old man with diarrhea and edema was admitted to our hospital and diagnosed with protein-losing enteropathy caused by primary intestinal lymphangiectasia. He was placed, in turn, on a low-fat diet, an elemental diet, and, subsequently, fasting therapy with total parenteral nutrition (TPN) support. However, his symptoms were not relieved, but, rather were exacerbated. On the 45th day of hospitalization, octreotide therapy was initiated. After 2 weeks of treatment, his clinical symptoms, as well as hypoproteinemia and hypoalbuminemia, gradually became alleviated. The improvement was confirmed in terms of scintigraphy, endoscopy, and histology of the duodenum. The patient remained healthy until 6 months after the commencement of octreotide treatment, when he discontinued octreotide at his own discretion, at which point the symptoms recurred. Resumption of the drug, however, again brought about remission, which has continued until the present, March 2000. Thus, octreotide therapy is one modality which may be useful for refractory primary intestinal lymphangiectasia.


Synergistic suppressive effect of double transfection of tumor necrosis factor-alpha and interleukin 12 genes on tumorigenicity of Meth-A cells.

Tumor necrosis factor-alpha (TNF-alpha) and interleukin 12 (IL-12), both potent antitumor cytokines, are known to be involved in the host's antitumor immune surveillance in tumor bearers, via different mechanisms. The former enhances the activities of dendritic cells, natural killer / lymphocyte-activated killer (NK / LAK) and cytotoxic T lymphocyte (CTL), while the latter induces Th1-type cellular immunity and enhances the activities of natural killer T (NKT), NK / LAK and CTL. In the present study, in the expectation of synergistic actions of these cytokines in stimulating the host's immune responses, we investigated the feasibility of a cancer vaccine involving double transfection with both genes in a murine model. The expression of major histocompatibility complex (MHC) class I, class II and B7.1 on the surface of the double transfectants was enhanced as revealed by FACS analysis. A significant decrease in tumorigenicity was observed in mice inoculated with the double transfectants. Cytotoxicity assay revealed that the activities of NK / LAK and CTL from spleens of mice bearing the double transfectants were enhanced. The induction of tumor-specific immunity was confirmed by rechallenge with parental Meth-A cells in mice that had rejected the double transfectants. Thus, double transfection of TNF-alpha and IL-12 genes was considered to bring about synergistic suppressive effects on the tumorigenicity of transfectants through the activation of killer cells by produced cytokines and the enhancement of expression of MHC class I, II and B7.1 molecules.

Gann : Japanese Journal of Cancer Research


[Severe hemolysis and SIADH-like symptoms induced by vincristine in an ALL patient with liver cirrhosis].

An 11-year-old boy was diagnosed as having acute lymphoblastic leukemia (ALL, L1) in 1987 and underwent treatment with an ALL high-risk protocol (prednisolone, vincristine (VCR), daunorubicin, 1-asparaginase), which resulted in complete remission. In 1990 he developed chronic hepatitis C and received interferon therapy. In December 1994, ALL recurred, and the patient was treated with VCR. He subsequently developed severe hemolysis (Hb 12.5 g/dl-->6.8 g/dl) with increases of indirect bilirubin, AST, and LDH. Furthermore, symptoms resembling a syndrome of inappropriate secretion of ADH (SIADH) and DIC developed. Upon incubation of the patient's red blood cells with VCR in vitro, extreme deformity of the cells was observed. These findings suggested that splenomegaly, due to liver cirrhosis which had developed rapidly from chronic hepatitis C while the patient was in an immunosuppressed state induced by anticancer drugs, had trapped the deformed red blood cells and resulted in severe hemolysis. The patient died on the 165th day after admission due to liver failure.

The Japanese Journal of Clinical Hematology


In vivo gene delivery to tumor cells by transferrin-streptavidin-DNA conjugate.

To target disseminated tumors in vivo, transgenes [beta-galactosidase gene, green fluorescence protein (GFP) gene, herpes simplex virus thymidine kinase (HSV-TK)] were conjugated to transferrin (Tf) by a biotin-streptavidin bridging, which is stoichiometrically controllable, and Tf receptor (Tf-R) affinity chromatography, which selects Tf conjugates with intact receptor bindings sites from reacting with the linker. Tf-beta-galactosidase plasmid conjugate thus constructed was specifically transfected to human erythroleukemia cells (K562) via Tf-R without the aid of any lysosomotropic agents. The transfection efficiency of the conjugate was superior to those of lipofection (1% staining) and retroviral vector (5%) and slightly lower than that of adenovirus (70%). The high level of expression with our conjugate was confirmed using other tumor cells (M7609, TMK-1) whereas in normal diploid cells (HEL), which express low levels of Tf-R, expression was negligible. When GFP gene conjugates were systemically administered through the tail vein to nude mice subcutaneously inoculated with tumor, expression of GFP mRNA was found almost exclusively in tumors and to a much lesser extent in muscles, whereas GFP revealed by fluorescence microscopy was detected only in the former. To exploit a therapeutic applicability of this method, suicide gene therapy using Tf-HSV-TK gene conjugate for massively metastasized k562 tumors in severe combined immune-deficient mice was conducted, and a marked prolongation of survival and significant reduction of tumor burden were confirmed. Thus, this method could also be used for gene therapy to disseminated tumors.


[Pure red cell aplasia induced by clomipramine hydrochloride in a patient with SLE].

A 48-year-old woman, who had been suffering from systemic lupus erythematosus (SLE), developed normochromic normocytic anemia after receiving clomipramine hydrochloride. Her reticulocyte count was low, and a bone marrow aspirate revealed erythroid hypoplasia without involvement of other cell lines. Thus a diagnosis of pure red cell aplasia (PRCA) was made. The anemia gradually resolved following withdrawal of the drug. Although several drugs are known to cause PRCA, this is the first time that clomipramine hydrochloride has been reported to have such an effect. The underlying SLE in this case suggested the possible immunological pathogenesis of drug-induced PRCA.

The Japanese Journal of Clinical Hematology


[Spontaneous internal drainage of pancreatic pseudocyst with fistula to the common bile duct].

The Japanese Journal of Gastro-enterology


[Granulocyte colony stimulating factor (G-CSF) producing renal cell carcinoma].

A 88-year-old male patient with G-CSF producing renal cell carcinoma is reported. The patient was admitted to our hospital complaining of macrohematureia. The laboratory examination showed marked leukocytosis of 18,200/microliter (neutrophil 92%) in the peripheral blood and high levels of G-CSF (120 pg/ml) in the serum. An abdominal CT scan revealed a right renal tumor. The neutrophil count rose to 38,700/microliter with increasing of tumor size. Histopathological diagnosis was renal cell carcinoma (Grade 3) and immunohistochemical staining using Histo anti-G-CSF antibody demonstrated cancer cells produced G-CSF. This is the second case of G-CSF producing renal cell carcinoma diagnosed by immunohistochemical staining in the literature.


Augmented antitumor effects of killer cells induced by tumor necrosis factor gene-transduced autologous tumor cells from gastrointestinal cancer patients.

The purpose of this study was to determine the feasibility of a vaccine therapy using tumor necrosis factor (TNF) gene-transduced autologous tumor cells for the treatment of human gastrointestinal cancers, which tend to have lower immunogenicity than other cancers such as melanoma and renal cell carcinoma. We succeeded in establishing primary cultured tumor cells from 12/54 carcinomatous effusions (4 liver cancer patients, 5 gastric cancer patients, 1 pancreatic cancer patient, and 2 colon cancer patients) and in transducing the TNF gene to the tumor cells by using the retrovirus vector MFG-TNF. Even after irradiation, TNF production (0.3-3.5 U/ml per 10(6) cells per 72 hr) was confirmed for 10 of 12 transfectants, and the other two transduced cells were found to have approximately one TNF gene copy. In 7 of the 12 patients, the cytotoxic activity of killer cells to nontransduced autologous tumor cells incubated with these TNF gene transfectants was augmented. This activity was blocked with anti-HLA class I antibody or BrefeldinA (BFA), suggesting that the killer cells were cytotoxic T lymphocytes (CTL) and tumor antigens are presented with HLA class I molecules. Indeed, enhanced expression of HLA class I and/or ICAM-1 molecules on the surface of the TNF gene-transduced tumor cells were observed by fluorescence-activated cell sorting (FACS) analysis. Furthermore, natural killer (NK) and/or lymphokine-activated killer (LAK) activities determined by using K562 or Daudi cells as targets were also enhanced in some of these cases when they were incubated with TNF gene-transduced tumor cells. These findings indicate the feasibility of using TNF gene-transduced tumor cells as a vaccine in gastrointestinal cancer patients.


Efficacy of TNF-alpha gene-transduced tumor cells in treatment of established in vivo tumor.

The therapeutic effect of TNF gene-transduced mouse fibrosarcoma cells (Meth-A: C5) on pre-inoculated parental cells (Meth-A: M0) was studied. Subcutaneous (s.c.) transplantation of M0 cells into one flank of syngeneic BALB/c mice was followed by s.c. injection of irradiated MO or C5 into the opposite flank 1 week later. The initial M0 tumor (T-MO) completely regressed in C5-vaccinated mice, whereas in M0-vaccinated mice continuous growth of T-M0 was observed. When a similar experiment was carried out in SCID mice, no regression of T-MO was observed, suggesting that the tumor regression in BALB/c mice was not due to direct anti-tumor activity of TNF secreted from C5, but to systemic immunity. Regression of the rechallenged M0 tumor was observed in mice which had shown T-MO regression by C5 vaccination, but rechallenged Colon 26 cells (syngeneic to BALB/c mice) continued to grow, indicating a specific immunity to Meth-A cells). The systemic immunity evoked in C5-vaccinated mice was directly demonstrated by enhanced killer activities of LAK and CTL with a proliferation of T-cell population in their splenocytes. Abrogation of the therapeutic effect of C5 vaccination with anti-Thy 1 and anti-Lyt 2 also demonstrates the involvement of cellular immunity in tumor regression.