Valpha14 NK T cell-triggered IFN-gamma production by Gr-1+CD11b+ cells mediates early graft loss of syngeneic transplanted islets.

Pancreatic islet transplantation is a highly promising approach for the treatment of insulin-dependent diabetes mellitus. However, the procedure remains experimental for several reasons, including its low efficiency caused by the early graft loss of transplanted islets. We demonstrate that Gr-1+CD11b+ cells generated by transplantation and their IFN-gamma production triggered by Valpha14 NKT cells are an essential component and a major cause of early graft loss of pancreatic islet transplants. Gr-1+CD11b+ cells from Valpha14 NKT cell-deficient (Jalpha281-/-) mice failed to produce IFN-gamma, resulting in efficient islet graft acceptance. Early graft loss was successfully prevented through the repeated administration of alpha-galactosylceramide, a specific ligand for Valpha14 NKT cells, resulting in dramatically reduced IFN-gamma production by Gr-1+CD11b+ cells, as well as Valpha14 NKT cells. Our study elucidates, for the first time, the crucial role of Gr-1+CD11b+ cells and the IFN-gamma they produce in islet graft rejection and suggests a novel approach to improving transplantation efficiency through the modulation of Valpha14 NKT cell function.

[Bacteria isolated from surgical infections and its susceptibilities to antimicrobial agents--special references to bacteria isolated between April 2006 and March 2007].

Tendency of isolated bacteria from infections in abdominal surgery during the period from April 2006 to March 2007 were investigated in a multicenter study in Japan, and the following results were obtained. In this series, 474 strains including 23 strains of Candida spp. were isolated from 170 (75.2%) of 226 patients with surgical infections. Two hundred and twenty-six strains were isolated from primary infections, and 224 strains were isolated from postoperative infections. From primary infections, anaerobic Gram-negative bacteria were predominant, followed by aerobic Gram-negative bacteria, while from postoperative infections aerobic Gram-positive bacteria were predominant, followed by anaerobic Gram-negative bacteria. Among aerobic Gram-positive bacteria, the isolation rate of Staphylococcus spp. was higher from postoperative infections, while Enterococcus spp. was higher from primary infections. Among aerobic Gram-negative bacteria, Escherichia coli was the most predominantly isolated from primary infections, followed by Klebsiella pneumoniae, Enterobacter cloacae and Pseudomonas aeruginosa, in this order, and from postoperative infections, P. aeruginosa was the most predominantly isolated, followed by E. coli and E. cloacae. Among anaerobic Gram-negative bacteria, the isolation rate of Bilophila wadsworthia was the highest from primary infections, followed by Bacteroides fragilis and from postoperative infections, B. fragilis was most predominately isolated, followed by Bacteroides caccae, Bacteroides thetaiotaomicron and B. wadsworthia in this order. In this series, we noticed no methicillin-resistant Staphylococcus aureus, nor multidrug-resistant P. aeruginosa. There were three strains of methicillin-resistant coagulase-negative Staphylococcus aureus, but all of them had good susceptibilities against various anti-MRSA antibiotics. We should carefully follow up B. wadsworthia.

Natural killer T-cells participate in rejection of islet allografts in the liver of mice.

A role of natural killer T (NKT) cells in transplant rejection remains unknown. Here, we determined whether NKT cells participate in rejection of islet allografts, using NKT cell-deficient mice. Survival of islet allografts in streptozotocin-induced diabetic CD1d(-/-) mice or Valpha14 NKT cell(-/-) mice was significantly prolonged without immunosuppression when grafted into the liver, but not beneath the kidney capsule, compared with wild-type mice. Acceptance of intrahepatic islet allografts was achieved in CD1d(-/-) mice by a subtherapeutic dose of rapamycin, which was abrogated in conjunction with the transfer of hepatic mononuclear cells from wild-type, but not from CD1d(-/-), mice at islet transplantation. The second islet grafts from a donor-specific, but not from a third-party, strain in CD1d(-/-) mice bearing functional islet allografts were accepted without immunosuppression at 120 days after the initial transplantation. These findings demonstrate that NKT cells play a significant role in rejection of islet allografts in the liver of mice, but that NKT cells are not essential for induction of donor-specific unresponsiveness in this model. The current study indicates that NKT cells might be considered as a target for intervention to prevent islet allograft rejection when the liver is the site of transplantation.

Successful islet transplantation to two recipients from a single donor by targeting proinflammatory cytokines in mice.

BACKGROUND: Currently, the inability to achieve successful islet transplantation from one donor to one recipient is a major obstacle facing clinical islet transplantation. We herein determined whether this limitation could be overcome by targeting pro-inflammatory cytokines with the prevention of immediate islet graft loss in association with engraftment in mice. METHODS: Isolated islets were grafted into the liver of streptozotocin-induced diabetic mice and the role of proinflammatory cytokines in the engraftment of islets was evaluated with the use of interferon (IFN)-gamma-/- mice and monoclonal antibodies against proinflammatory cytokines. RESULTS: Hyperglycemia in streptozotocin-induced diabetic mice receiving 200 syngenic islets, which were isolated from a single mouse pancreas, was ameliorated when IFN-gamma-/-, but not wild-type mice, were used as recipients. The treatment with anti-IFN-gamma antibody produced normoglycemia in diabetic wild-type mice receiving 200, but not 100 islets. However, when anti-tumor necrosis factor-alpha and anti-interleukin-1beta antibodies were administered in conjunction with anti-IFN-gamma antibody, wild-type diabetic mice receiving 100 islets became normoglycemic after transplantation. In addition, the favorable effect of the combined use of antibodies was similarly achieved in mice receiving islet allografts when rejection was prevented with anti-CD4 antibody treatment. CONCLUSIONS: These findings clearly demonstrate that successful islet transplantation from one donor to two recipients is feasible by targeting pro-inflammatory cytokines in mice, thus suggesting a potential application in clinical islet transplantation if similar mechanisms of islet graft loss could be mediated in humans.

Balloon-catheter endoscopic retrograde pancreatography-compression study for diagnosis of early-stage pancreatitis.

Endoscopic retrograde pancreatography (ERP) is considered by many as the gold standard imaging method in the diagnosis of chronic pancreatitis (CP). However, conventional ERP usually has a limited ability to accurately diagnose early-stage CP, in which only the branch ducts are involved and the main pancreatic duct (MPD) is unaffected. To visualize precisely the branch ducts, we have developed a more sophisticated ERP method, called balloon ERP-compression study (balloon ERP-CS). In this procedure, a catheter equipped with a balloon at its tip is placed first into the MPD via the papilla with the aid of conventional ERP, followed by the removal of the endoscope, leaving the catheter behind. Then, the balloon is inflated, and the contrast medium is injected slowly. The balloon serves to block the back flow of the injected contrast medium from the MPD to the duodenum, enabling visualization of the branch ducts. The compression study affords further precise pancreatography of the corresponding area. Thus, balloon ERP-CS has now become an essential procedure for diagnosis of pancreatic lesions, including CP. So far (April 1984 to April 2005), we have performed the procedure in 1012 cases, for a total of 1562 examinations. In this study, we focus on the role of balloon ERP-CS in diagnosis of early-stage CP to elucidate its characteristic features in association with histological findings. This presentation will clarify the usefulness as well as the limitations of balloon ERP-CS for the diagnosis of CP, especially cases without the involvement of the MPD.

An endoscopic sphincterotomy of the minor papilla in the management of symptomatic pancreas divisum.

BACKGROUND/AIMS: The efficacy of endoscopic treatment in pancreas divisum remains controversial. This study evaluated the results of an endoscopic sphincterotomy of the minor papilla and temporary transpapillary pancreatic stenting in patients with pancreas divisum. METHODOLOGY: Pancreas divisum was diagnosed in four patients between 1994 and 2004. All patients demonstrated episodes of recurrent upper abdominal and back pain were with a median follow-up period of 14.5 months. One patient was treated by a sphincterotomy of the minor papilla alone, while three others also underwent transpapillary pancreatic stent insertion for seven days. RESULTS: A Sphincterotomy of the minor papilla could be successfully achieved in all patients. There was no instance of bleeding, perforation or sepsis after the procedure. The postoperative serum amylase level in the patients without stent insertion (1352 IU/L) was higher than that the patients with stents (mean level 515 IU/L, range 358 to 680). The dilatated dorsal pancreatic ducts were found to improve after a sphincterotomy in all patients. None of the patients had any further episodes of pancreatitis. In addition, all patients demonstrated a considerable improvement in their upper abdominal or back pain symptoms, which did not require either analgesic medication or hospitalization. CONCLUSIONS: An endoscopic sphincterotomy and temporary transpapillary pancreatic stenting were therefore suggested to be a beneficial treatment modality for patients with pancreas divisum.

[Bacteria isolated from surgical infections and its susceptibilities to antimicrobial agents--special references to bacteria isolated between april 2003 and march 2004].

Tendency of isolated bacteria from infections in abdominal surgery during the period from April 2005 to March 2006 were investigated in a multicenter study in Japan, and the following results were obtained. In this series, 384 strains including 18 strains of Candida spp. were isolated from 161 (70.3%) of 229 patients with surgical infections. One hundred and ninty-five strains were isolated from primary infections, and 171 strains were isolated from postoperative infections. From primary infections, aerobic Gram-negative bacteria and aerobic Gram-positive bacteria were predominant, while aerobic Gram-positive bacteria were predominant from postoperative infections. The isolation rate of aerobic Gram-positive bacteria, such as Enterococcus spp. and Staphylococcus aureus were higher from both types of infections. Among anaerobic Gram-positive bacteria, the isolation rate of Peptostreptococcus spp. was the highest from both types of infections. Among aerobic Gram-negative bacteria, Escherichia coli was the most predominantly isolated from primary infections, followed by Pseudomonas aeruginosa, Klebsiella spp. in this order, and from postoperative infections, E. coli was the most predominantly isolated, followed by Klebsiella pneumoniae and P. aeruginosa. Among anaerobic Gram-negative bacteria, the isolation rate of Bacteroides fragilis group was the highest from both primary and postoperative infections. In this series, we noticed no vancomycin-resistant Gram-positive cocci, nor multidrug-resistant P. aeruginosa. But cefazolin-resistant E. coli producing extended spectrum fl-lactamase was seen in 5.0 per cents. We should be carefully followed up the facts that the increasing isolation rates of B. fragilis group and Bilophila wadsworthia which were resistant to both penicillins and cephems.

MK-1 expression in carcinoma of the ampulla of Vater as a predictor of improved prognosis after surgical resection.

This retrospective study investigated the prognostic significance of MK-1 expression in human carcinoma of the ampulla of Vater (CAV). Expression was examined immunohistochemically using specimens from 38 patients who underwent surgical treatment for CAV. Expression was found in 61% of samples. Thirteen of 15 well-differentiated but only two of eight poorly differentiated adenocarcinoma were positive (P=0.0352). MK-1 positivity tended to show significantly decreasing pT (P=0.0039), pN (P

[Bacteria isolated from surgical infections and its susceptibilities to antimicrobial agents--special references to bacteria isolated between April 2003 and March 2004].

Tendency of isolated bacteria from infections in general surgery during the period from April 2004 to March 2005 were investigated in a multicenter study in Japan, and the following results were obtained. In this series, 645 strains including 17 strains of Candida spp. were isolated from 226 (79.0%) of 286 patients with surgical infections. Three hundred and seventeen strains were isolated from primary infections, and 345 strains were isolated from postoperative infections. From primary infections, anaerobic Gram-positive bacteria and anaerobic Gram-negative bacteria were predominant, while aerobic Gram-positive bacteria were predominant from postoperative infections. The isolation rate of aerobic Gram-positive bacteria, such as Enterococcus spp. and Staphylococcus aureus were higher from both types of infections. Among anaerobic Gram-positive bacteria, the isolation rate of Peptostreptococcus spp. was the highest from both types of infections. Among aerobic Gram-negative bacteria, Escherichia coli was the most predominantly isolated from primary infections, followed by Pseudomonas aeruginosa, Klebsiella pneumoniae and Citrobacter freundii in this order, and from postoperative infections, P. aeruginosa was the most predominantly isolated, followed by E. coli, E. cloacae, and K. pneumoniae. Among anaerobic Gram-negative bacteria, the isolation rate of Bacteroides fragilis group was the highest from both primary infections followed by Bilophila wadsworthia. While the isolation rate of B. fragilis group was also the highest from postoperative infections, the following bacteria were Bacteroides thetaiotaomicron and B. wadsworthia in this order. In this series, we noticed no vancomycin-resistant Gram-positive cocci, but a few strains of moderately arbekacin-resistant MRSA. Carbapenem-resistant P. aeruginosa but not multidrug-resistant was seen in 13.3 per cents. Also cefazolin-resistant E. coli probably producing extended spectrum beta-lactamase was seen in 7.0 per cents. We should be carefully followed up the facts that an increasing isolation rates of B. fragilis group and B. wadsworthia which were resistant to both penicillins and cephems.

Intraductal tubular adenoma of the pancreas, pyloric gland type: a clinicopathologic and immunohistochemical study of 6 cases.

The intraductal tubular adenoma (ITA), pyloric gland type, of the pancreas is an uncommon benign tumor, akin to the pyloric gland type adenoma of the gallbladder. We report 6 cases of ITA of the pancreas: 3 male and 3 female aged 50 to 79 years (mean, 63.5 years; median, 65 years); all were examined clinicopathologically. Four patients showed no symptoms, but appetite loss and/or general fatigue presented in two. Grossly, all tumors formed a localized polypoid mass protruding into the lumen of the dilated pancreatic duct. Five of the six tumors were found within the main duct, and the other arose within the branch duct of the pancreas. Microscopically, the tumors were composed of closely packed tubular glands resembling pyloric type glands. They were lined by columnar or cuboidal epithelial cells with foci of mild to moderate dysplastic change. In 2 cases, the adjacent pancreas showed foci of intraductal papillary-mucinous adenoma. Histochemically, the tumors largely showed neutral mucin with a lesser amount of acidic mucin made up mainly of sialomucin. Endocrine cells were found in five tumors. Immunohistochemically, all tumors were labeled with M-GGMC-1 and MUC6, whereas MUC1 and MUC2 stains were negative. Pepsinogen II was positive in 5 tumors; thus, the results displayed a pattern of differentiation similar to those of ordinary gastric pyloric or metaplastic pyloric glands. DPC4 expression was maintained in all tumors and p53-positive nuclei were hardly encountered. All patients are alive with no evidence of disease 3 to 10.5 years after surgical resection.

[Bacteria isolated from surgical infections and its susceptibilities to antimicrobial agents--special references to bacteria isolated between April 2003 and March 2004].

Tendency of isolated bacteria from infections in general surgery during the period from April 2003 to March 2004 were investigated in a multicenter study in Japan, and the following results were obtained. In this series, 455 strains including 14 strains of Candida spp. were isolated from 191(75.2%) of 254 patients with surgical infections. Two hundred and thirty-nine strains were isolated from primary infections, and 216 strains were isolated from postoperative infections. From primary infections, anaerobic Gram-positive bacteria and aerobic Gram-negative bacteria were predominant, while aerobic Gram-positive bacteria were predominant from postoperative infections. The isolation rate of aerobic Gram-positive bacteria, such as Enterococcus spp. and Staphylococcus aureus were higher from both types of infections. Among anaerobic Gram-positive bacteria, the isolation rate of Peptostreptococcus spp. was the highest from both types of infections. Among aerobic Gram-negative bacteria, Escherichia coli was the most predominantly isolated from primary infections, followed by Klebsiella pneumoniae, Enterobacter cloacae and Pseudomonas aeruginosa in this order, and from postoperative infections, E. coli was the most predominantly isolated, followed by P. aeruginosa, E. cloacae, and K. pneumoniae. Among anaerobic Gram-negative bacteria, the isolation rate of Bacteroides fragilis group was the highest from both types of infections. The isolation rate of anaerobic Gram-positive bacteria from primary infections and that of aerobic Gram-positive bacteria from postoperative infections were high in the last several years. In this series, we noticed no vancomycin-resistant Gram-positive cocci, but a few strains of moderately arbekacin-resistant MRSA. Carbapenm-resistant P. aeruginosa was seen in less than 10 per cents. Last year we noticed that there were cefazolin-resistant E. coli producing extended spectrum beta-lactamase, but there was no highly cefazolin-resistant E. coli in this year. In the next series, increase of both anaerobic bacteria and Enterococcus spp. should be carefully followed up.

Gallbladder cancer treatment using adenovirus expressing the HGF/NK4 gene in a peritoneal implantation model.

Gallbladder cancer cells are stimulated by hepatocyte growth factor (HGF) in vitro and in vivo. We constructed an adenovirus vector, AdCMV.NK4, carrying the HGF antagonist HGF/NK4 (NK4) and evaluated whether or not this vector can suppress the peritoneal implantation of gallbladder cancer in a novel peritoneal injury mouse model. A human gallbladder cancer cell line (GB-d1) and human peritoneal mesothelial cells infected with the adenovirus vector produced a substantial level of NK4 protein. An invasion of GB-d1 cells was determined by a coculture with AdCMV.NK4-infected human mesothelial cells in vitro. Both the invasion and migration of GB-d1 cells were dramatically inhibited by this vector in a multiplicity of infection (MOI)-dependent manner. GB-d1 cells were intraperitoneally injected into the nude mice with peritoneal injury, followed by either AdCMV.NK4 or a control vector (AdCMV.LacZ). The incidence and the size of the metastatic tumor drastically decreased by AdCMV.NK4 (MOI 100: n=4, P<.0001). Real-time PCR analysis revealed a transient elevation of mouse HGF mRNA expression at the peritoneal injury sites. AdCMV.NK4 has been suggested to induce the inhibition of the implantation and growth of gallbladder cancer cells in vivo through its anti-HGF activity, and the use of NK4 gene transfer could be an effective modality for preventing peritoneal metastasis of gallbladder cancer.

Beneficial effects of costimulatory blockade with anti-inducible costimulator antibody in conjunction with CTLA4Ig on prevention of islet xenograft rejection from rat to mouse.

BACKGROUND: Costimulatory signals have been reported to play an important role in islet-xenograft rejection, although the precise mechanisms remain unknown. The aim of the present study was to determine a role of a novel costimulatory molecule, inducible costimulator (ICOS), in rat islet-xenograft rejection in conjunction with CTLA4Ig with respect to cellular as well as humoral immune responses. METHODS: Isolated rat islets were transplanted into the liver of streptozotocin (180 mg/kg) induced diabetic mice. Cellular immune responses to islet xenografts, and productions of anti-rat antibody in mice were examined by flow cytometry (FACS) after transplantation. RESULTS: Intrahepatic rat islet xenografts were rejected in mice within 8 days after transplantation. FACS analysis revealed an expansion of CD8(+) T cells in the liver as well as a production of anti-rat antibody in recipient mice in association with rejection. The treatment with anti-ICOS antibody in conjunction with CTLA4Ig produced a marked prolongation of islet-xenograft survival with neither expansion of CD8(+) T cells nor production of anti-rat antibody, whereas, in contrast, those treated with anti-ICOS antibody or CTLA4Ig alone did not have prolonged survival, and CD8(+) T cells were expanded. CONCLUSION: These findings demonstrate that cellular rather than humoral immune responses are considered responsible for islet-xenograft rejection from rat to mouse and that the blockade of costimulatory signals with anti-ICOS antibody in conjunction with CTLA4Ig has a favorable effect on prevention of islet xenograft rejection.

Acceptance of islet allografts in the liver of mice by blockade of an inducible costimulator.

BACKGROUND: An inducible costimulator (ICOS) has been found to be a novel costimulator for T-cell activation, although its precise role in transplant immunobiology remains unclear. This study determined whether ICOS plays an essential role in rejection of intrahepatic islet allografts in streptozotocin-induced diabetic mice. METHODS: Mononuclear cells in the liver of mice were isolated and examined by flow cytometry with respect to expression of ICOS in association with rejection, and the effects of in vivo treatment with an anti-ICOS antibody on survival of intrahepatic islet allografts were determined. RESULTSFlow cytometric analysis of mononuclear cells in the liver of normal untreated mice revealed that ICOS is expressed on CD4+CD3int natural killer T cells. The expression of ICOS was up-regulated on CD4+CD3bright T cells and expanded CD8 T cells in the liver in association with rejection. Posttransplant short-term administration of anti-ICOS antibody alone produced a significant prolongation of islet allograft survival. Administration of the antibody in conjunction with a subtherapeutic regimen of FK506 prevented rejection, leading to the acceptance of islet allografts. CONCLUSION: ICOS has an essential role in rejection of intrahepatic islet allografts and the blockade of ICOS interaction might be a novel approach for preventing islet allograft rejection.

Reduced expression of heterogeneous nuclear ribonucleoprotein B1 in adult T-cell lymphoma/leukemia.

It is considered that hnRNP B1 expresses similarly in the various types of tumor cells. Recently, we demonstrated high B1 expression in B-cell lymphoma and carcinoma. To evaluate the difference of B1 expression between B and T-cell lymphoma, we immunologically studied the B1 expression in 22 cases with nodal T-cell lymphoma, comprising adult T-cell leukemia/lymphoma (ATLL; n=15) and angioimmunoblastic T-cell lymphoma (AILD; n=7), using an anti-hnRNP B1 monoclonal antibody, 2B2. In ATLL cases, scattered large transformed lymphoma cells demonstrated strong B1 expression, while the medium-sized lymphoma cells were negative. On the one hand, lymphoma cells in AILD diffusely expressed B1. The mean B1 expression rate in ATLL was 22%, which was significantly lower than that in AILDs (45%), B-cell lymphomas (44%), and metastatic carcinomas (53%) (p<0.01). Our result might suggest that process of hnRNP B1 expression in ATLL differs from those in other lymphoid neoplasms and carcinoma.

Overexpression of heterogeneous nuclear ribonucleoprotein B1 in lymphoproliferative disorders: high expression in cells of follicular center origin.

It is reported that overexpression of hnRNP A2 and B1 proteins is useful for detecting early cancers, and that B1, a splicing minor isoform of A2, is more specific than A2. The B1 expression is still undetermined in human lymphoid tissues. We quantitatively studied the B1 expression in 85 lymph node specimens, comprising reactive lymphoid hyperplasia (RLH; n=8), B-cell lymphoma (n=23), T-cell lymphoma (n=22), and metastatic carcinoma (n=32). Immunostaining and immunoblotting analyses with an anti-B1 monoclonal antibody, 2B2 were performed, and the two sets of results correlated with each other (p<0.05). In RLH specimens, B1 expression rate was significantly higher in follicular centers (FC; 44%) than in mantle zone (MZ; 15%) and paracortex (16%) (p<0.01). B1 expression was statistically higher in B-cell lymphoma than in T-cell lymphoma (p<0.01). In B-cell lymphomas, B1 expression rates were 51% in diffuse large B-cell lymphoma (DLBL; n=5) and 45% in follicular lymphoma (FL; n=16), and they were almost the same as that of the FC. Especially in DLBLs, CD10+ FC-origin lymphomas expressed greater amount of B1 than CD10- non-FC-origin lymphomas. B1 expression rate was low in mantle cell lymphoma (MCL; n=2) and similar to that of MZ in RLH. These results suggest that B1 expression is associated with differentiation in lymphoid tissue rather than transformation. B1 expression increases during the process of B-cell differentiation in the FC, and that high B1 expression is maintained in B-cell lymphomagenesis, especially in cells of FC-origin DLBL.

Expressions of two adenomatous polyposis coli and E-cadherin proteins on human colorectal cancers.

Mutations in the adenomatous polyposis coli (APC) gene contribute to the progression of colorectal tumorigenesis. Despite the importance, few studies regarding the localization of this protein on surgically resected human colorectal cancer specimens using immunohistochemistry have been reported so far because of the unavailability of the antibodies for this use. The goal of this study has been to provide the APC protein expression and to validate the APC molecular studies. We took advantage of an immunohistochemistry procedure of applying the unique detergent-mediated antigen retrieval technique to frozen sections and examined the expressions of one amino (N)-terminal (AC4) and one carboxy (C)-terminal APC antibody (HG2). Further, we compared the stainings of APC antibodies with those of the E-cadherin antibody using a quantitative image analysis. E-cadherin is a critical morphogenetic regulator during embryogenesis and recent evidence strongly suggests that downregulation of E-cadherin expression in cancers is associated with a high rate of invasion and metastasis. The analysis indicated statistically that normal epithelia showed stronger staining than cancer cells ( P<0.05). Further, in normal epithelia, the amino (N)-terminal APC antibody (AC4) showed a positive correlation with another carboxy (C)-terminal APC antibody (HG2). E-cadherin showed no positive correlation with other APCs in either the normal epithelia or cancer cells. This study verified reduced expressions of APCs and E-cadherin proteins in colorectal cancer cells. This suggests that the normal APC and E-cadherin protein expressions in benign epithelium are progressively and independently lost in the sporadic colorectal cancers.

Effect of Nma on growth inhibition by TGF-betaa in human gastric carcinoma cell lines.

Non-metastatic gene A (nma) has a homologue DNA sequence to a gene of bone morphogenetic proteins and activin membrane-bound inhibitor (BAMBI), which negatively regulates TGF-beta signaling. In this study, we analyzed the functional homology between Nma and BAMBI in human gastric carcinoma cell lines. Various levels of nma mRNA expression were detected by the RT-PCR technique in all human gastric carcinoma cell lines. Then, Nma antisense and sense S-oligodeoxynucleotide (ODN) were used to analyze the response of TGF-beta to cell growth and invasion gastric carcinoma cell lines. The cell growth was inhibited by TGF-beta in Nma antisense S-ODN treatment gastric carcinoma cell lines, MKN28, MKN1, MKN74 and TMK1. TGF-beta reduced cell growth and invasive activity of MKN28 treated with Nma antisense S-ODN in a dose and time-dependent manner. Furthermore, lysates of Nma sense or antisense S-ODN treated MKN28 cells were immunoprecipitated with anti-TGFbetaR-I or anti-TGFbetaR-II antibody. The 29 kDa signal considered as Nma appeared in sense S-ODN treated MKN28 cells immunoprecipitated with anti-TGFbetaR-I. These results indicate that Nma negatively regulates TGF-beta signaling, consequently playing an important role as one of the escape mechanisms from TGF-beta-mediated growth control similarly to BAMBI, and induce cell growth and invasion in human gastric carcinoma cell lines.

Effect of human noxa on irinotecan-induced apoptosis in human gastric carcinoma cell lines.

BACKGROUND/AIMS: A protein BH3-only member bcl-2 family, Noxa is a proapoptotic mediator for p53-induced apoptosis. We analyzed the effect of Noxa on p53-induced apoptotic gastric carcinoma cell lines. METHODOLOGY: The expressions of human Noxa (hNoxa) mRNA on human gastric carcinoma cell lines were assessed with RT-PCR. Further, hNoxa antisense and sense S-oligodeoxynucleotide (ODN) were used to analyze the effect of hNoxa on p53-induced apoptotic gastric carcinoma cell lines. RESULTS: Various levels of hNoxa mRNA expression were detected in all gastric cell lines. MKN45 that has wild-type p53 showed severe inhibition by irinotecan compared with MKN28, which has mutated p53. Cell growth under hNoxa antisense S-ODN treatment did not differ from that under sense S-ODN treatment in MKN28. On the other hand, the suppression of cell growth in MKN45 decreased with hNoxa antisense S-ODN treatment as compared to hNoxa sense S-ODN treatment. MKN45 cells exhibited DNA fragmentation clearly after 24 hr of 3 mM hNoxa sense S-ODN treatment. The DNA fragmentation in MKN45 was inhibited by hNoxa antisense S-ODN treatment. CONCLUSIONS: It seems that hNoxa plays an important role in induction of apoptosis on p53 wild type gastric carcinoma cell lines.

Gamma interferon gene transfection efficiently inhibits proliferation of neurofibroma cell lines in vitro.

Primarily isolated neurofibroma cell lines and a human dermal fibroblast cell line were transfected with human gamma interferon gene in vitro, and changes in the cell proliferation rates were investigated. The proliferation rates of both cell lines were remarkably suppressed after the gene transfection. In particular, the neurofibroma cell lines almost stopped proliferating five days after gene transfection. The tritium thymidine uptake of the fibroblast cell line was almost abolished three days after gene transfection. The culture media from both of the gene-transfected cell lines contained a measurable gamma interferon concentration as late as five days after transfection, this was detected by an enzyme-linked immunosorbent assay. These data suggest that gamma interferon gene therapy might be a possible treatment for intractable or inoperable neurofibromas in patients with von Recklinghausen's disease in the future.