The impact of the number of occult metastatic lymph nodes on postoperative relapse of resectable esophageal cancer.

Clinical stage II/III esophageal cancer (EC), as defined by the Japanese Classification, relapses at a moderately high rate even after curative resection. The number of lymph node metastases is known to be associated with tumor relapse. Recently, the prognostic significance of occult metastatic lymph nodes (MLNs), as well as that of overt MLNs, has been reported. The aim of this study was to investigate the impact of the total number of MLNs including occult MLNs on postoperative relapse in clinical stage II/III EC. One hundred and five patients with clinical stage II/III EC who underwent esophagectomy accompanied by radical lymphadenectomy at the Department of Surgical Oncology in Osaka City University Hospital between January 2000 and October 2008 were included in this study. Occult MLNs, metastases not detected by hematoxylin-eosin staining, were identified by immunohistochemistry (IHC) using antipancytokeratin antibody AE1/AE3. The clinicopathological features of occult MLNs were compared between the relapse and no relapse groups. A total of 6558 lymph nodes (1357 from two-field dissection and 5201 from three-field dissection) were examined by IHC staining; 362 overt MLNs and 143 occult MLNs were detected. The number of occult MLNs increased in proportion to the International Union Against Cancer pathological (p)N-status and pStage. When the number of occult MLNs was added to the number of pNs, the number of total MLNs was associated with postoperative relapse. With respect to tumor, node, metastasis stage, 6 of 22 patients (27%) who were pathological node-negative converted to node-positive by considering total MLNs. The number of N3 patients with relapse increased markedly with restaging by total MLNs. The number of total MLNs, but not overt MLNs, was an independent prognostic factor on multivariate analysis. These results suggest that occult MLNs were often found, and they were associated with postoperative relapse of resectable esophageal cancer. The total number of MLNs including occult MLNs could contribute to evaluating the precise stage of patients with esophageal cancer.

Combination therapy with short interfering RNA vectors against VEGF-C and VEGF-A suppresses lymph node and lung metastasis in a mouse immunocompetent mammary cancer model.

Cancer metastasis contributes significantly to cancer mortality and is facilitated by lymphangiogenesis and angiogenesis. Vascular endothelial growth factor-C (VEGF-C) and VEGF-A are involved in lymphangiogenesis and angiogenesis. To inhibit metastasis, combination therapy with vector-based small interfering RNA (siRNA) against VEGF-C and/or VEGF-A was conducted on murine metastatic mammary cancer. Syngeneic, inoculated, metastatic mammary cancers received direct intratumoral injection of plasmid siRNA vector targeting VEGF-C (psiRNA-VEGF-C), VEGF-A (psiRNA-VEGF-A), both VEGF-C and VEGF-A (both psiRNA-VEGF-C and psiRNA-VEGF-A vectors injected, referred to as the psiRNA-VEGF-C+A group) or a scrambled sequence (psiRNA-SCR) as control, once a week for 8 weeks. Gene electrotransfer was performed on the tumors after each injection. Tumor volume was significantly lower in the psiRNA-VEGF-A and the psiRNA-VEGF-C+A groups throughout the study. Lymph node metastasis was significantly less frequent in all therapeutic groups, whereas the multiplicity of lung metastases was significantly lower in the psiRNA-VEGF-C+A group only. All siRNA therapeutic groups showed a significant reduction in the number of dilated lymphatic vessels containing intraluminal cancer cells and microvessel density. Our data suggest that specific silencing of the VEGF-C or VEGF-A gene alone can inhibit lymph node metastasis. However, combination siRNA therapy targeting both VEGF-C and VEGF-A inhibits both lymph node and lung metastasis, rendering this combined therapy more beneficial than either alone. The observed anti-metastatic activity of siRNA-expressing vectors targeting VEGF-C or VEGF-A may be of high clinical significance in the treatment of metastatic breast cancer.

Electrogene therapy using endostatin, with or without suicide gene therapy, suppresses murine mammary tumor growth and metastasis.

Syngeneic inoculated metastatic mammary cancers received direct intratumoral injection of a plasmid vector containing either endostatin (pEndo) with or without a suicide gene (pHSVtk), pHSVtk alone or control vector once a week for 8 weeks. We applied electrogene transfer to the tumors after each injection and administered ganciclovir (GCV) to pHSVtk-transfected mice using an osmotic minipump. Anticancer efficacy was monitored using a variety of parameters, namely tumor volume, intratumoral microvessel density and DNA synthesis, number of mice with metastasis, and number of sites of metastasis per mouse. Tumor volume was significantly lower in all therapeutic groups, with the most effective growth suppression in the pEndo+pHSVtk/GCV group. Lymph node metastasis was significantly less frequent in all therapeutic groups, whereas the multiplicity of lung metastases was significantly lower only in the pEndo and pEndo+pHSVtk/GCV groups. All therapeutic groups showed significantly lower intratumor microvessel density and DNA synthesis. The pEndo and pEndo+pHSVtk/GCV groups also showed a significant reduction in the numbers of dilated lymphatic vessels containing intralumenal tumor cells. Our data suggest that endostatin electrogene therapy alone or in combination with pHSVtk/GCV suicide gene therapy is more beneficial than suicide gene therapy alone. The observed antimetastatic activity of endostatin may be of high clinical significance in the treatment of metastatic breast cancer.

Disseminated strongyloidiasis in nephrotic syndrome.

Strongyloides stercoralis is endemic in the southwestern islands Amami and Ryukyu in Japan. Systemic strongyloidiasis occurs in immunocompromised hosts. We report here on a 60-year-old patient with minimal-change nephrotic syndrome (MCNS) without eosinophilia or HTLV-I infection. She was treated with corticosteroid for MCNS and died of disseminated strongyloidiasis. The patient developed systemic purpura, ileus, respiratory distress, malabsorption, pancytopenia, pulmonary hemorrhage and sepsis due to Escherichia coli before death. Massive infestation with Strongyloides stercoralis was disclosed by autopsy, and the larvae was considered as a pathomechanism or exacerbating agent of nephrotic syndrome in endemic areas.

Antitumor effects of Marginisporum crassissimum (Rhodophyceae), a marine red alga.

Marginisporum crassissimum (Yendo) Ganesan, a marine red alga found in the ordinal coastal sea around Japan, revealed antitumor (antimetastatic) effects in vitro and in vivo. In in vitro experiments, extracts of this alga inhibited not only the growth of several tumor cell lines, such as B16-BL6 (a mouse melanoma cell line), JYG-B (a mouse mammary carcinoma cell line) and KPL-1 (a human mammary carcinoma cell line), but also invasion of B16-BL6 cells in a culture system. In in vivo experiments, the lung metastasis of B16-BL6 cells inoculated to the tail vein of B57BL/6J mice was inhibited by intraperitoneal administration of an extract from the alga. In addition, life prolongation of B57BL/6J mice inoculated with B16-BL6 cells was also observed by the intraperitoneal administration of the extract. An effective substance showing B16-BL6 growth inhibition in vitro was partially purified by filtration and hydrophobic column chromatography, and was revealed to be sensitive to trypsin-digestion and heat-treatment. The molecular weight of the substance was greater than 100 kDa. This is the first study demonstrating antitumor (antimetastatic) effects of M. crassissimum.

Elevated serum IP-10 levels observed in type 1 diabetes.

OBJECTIVE: Although most patients with type 1 diabetes are considered to have T-cell-mediated autoimmune disease, a method of measuring of pancreatic beta-cell-specific T-cell function in cases of type 1 diabetes has yet to be established. Here, we focused on interferon-inducible protein-10 (IP-10), a chemokine that promotes the migration of activated T-helper 1 (Th1) cells and measured serum IP-10 levels in patients with human type 1 diabetes, which is regarded as a Th1-mediated disease. RESEARCH DESIGN AND METHODS: Serum samples were obtained from diabetic patients, and the levels of autoantibodies (GAD and insulinoma-associated protein-2 [IA-2]) and IP-10 were measured. Diabetic patients positive for either or both of the autoantibodies were classified as Ab+ type 1, and those negative for both were classified as Ab type 1. To evaluate islet antigen-specific responses, peripheral blood from patients stimulated with or without GAD was used, and intracellular cytokine staining for flowcytometry was performed. RESULTS: The Ab+ and Ab- type 1 groups both showed a significantly higher serum IP-10 level than the healthy subjects (P < 0.001 and P < 0.05, respectively), and the IP-10 level in the recent-onset Ab+ subgroup was significantly higher than that in the established (longstanding) Ab+ subgroup (P < 0.002). Furthermore, there was a significant positive correlation between the serum IP-10 level and the number of GAD-reactive gamma-interferon-producing CD4+ cells in the Ab+ type 1 group (P < 0.007). CONCLUSIONS: Our findings demonstrate that measurement of serum IP-10 concentrations is useful in patients with type 1 diabetes.

Lipid profile of nutrition students and its association with cardiovascular disease risk factors.

OBJECTIVE: To describe the lipid profile and to verify its relationship with cardiovascular disease risk factors in students at a public university in São Paulo. METHODS: After obtaining clinical, anthropomorphic, and lipid profile data from 118 students, variables of the lipid profile were related to other risk factors. RESULTS: The mean age of the students was 20.3 years (SD = 1.5). The risk of cardiovascular disease was characterized by a positive family history of ischemic heart disease in 38.9%; sedentariness in 35.6%; limiting and increased total and LDL-C cholesterol levels in 17.7% and 10.2%, respectively; decreased HDL-C levels in 11.1%; increased triglyceride levels in 11.1%; body mass index > 25 in 8.5%, and smoking in 6.7% of the subjects. Students' diet was found to be inadequate regarding protein, total fat, saturated fat, sodium, and fiber contents. A statistically significant association between cholesterol and contraceptive use, between HDL-C and contraceptive use, age and percent body fat, and triglycerides and percent lean weight was observed. CONCLUSION: A high prevalence of some risk factors of cardiovascular disease as well as the association between these factors with altered lipid profiles was observed in the young population studied.

Proximal symphalangism with "coarse" facial appearance, mixed hearing loss, and chronic renal failure: new malformation syndrome?

A 25-year-old man is described with short stature, moderate mental retardation, an abnormal facial appearance, a webbed neck, skeletal abnormalities including proximal symphalangism of bilateral second through fifth fingers, mixed hearing loss, and slowly progressive, sclerosing nephropathy. He was large at birth with generalized edema, more pronounced around the jaw, neck and the upper part of the body, but became short with increasing age, and currently measures 143 cm (-4.9 SD). He had intermittent proteinuria and slowly progressive deterioration of the renal function. A biopsy of the left kidney showed global glomerular sclerosis with interstitial fibrosis. He was placed on maintenance peritoneal dialysis at age 17 years, and now on hemodialysis. His skeletal abnormalities included, in addition to proximal symphalangism, stenosis of the cervical canal, scoliosis, brachydactyly of the hands, hypoplastic hip joints, and pes valgus. Other abnormalities noted were a communicating defects of the diaphragm (surgically corrected), bilateral inguinal hernia and cryptorchidism. These clinical manifestations indicate a hitherto undescribed combination of manifestations and nephropathy.

Factors that affect absorption behavior of cyclosporin a in gentamicin-induced acute renal failure in rats.

Factors that affect the absorption of cyclosporin A (CsA) were examined in gentamicin-induced acute renal failure (ARF) rats. In ARF rats, the area under the blood CsA concentration-time curve after oral administration was significantly decreased in comparison with that of control rats; 5.81 +/- 0.55 vs 11.30 +/- 1.59 mg h mL(-1)(mean+/-s.e.m.), respectively, and the relative bioavailabilities in ARF and control rats after oral administration were 15.2% and 43.4%, respectively. The flow rate of bile and the amount of bile acids in ARF rats were markedly decreased to about 61% of control, and 41% of control, respectively. The amount of CsA uptaken into the evened sac of jejunum, transferred to serosal side, and metabolized in tissues was significantly decreased in ARF rats without verapamil, while with 0.3 mM verapamil, the amount in ARF rats recovered to the levels of control rats. The absorption clearance of CsA in ARF rats was significantly decreased, however it was significantly improved by adding bile or bile acid. Adenosine triphosphate released from enterocytes in ARF rats was significantly decreased in the presence of 2.0 microM CsA, 0.3 mM verapamil, or both, in comparison with control rats. From these findings, we concluded that a reduction of CsA bioavailability during ARF is caused by depression in bile excretion and renal function-dependent depression of uptake from intestinal tract via maybe P-gLycoprotein in enterocytes. They are main two factors that reduce the absorbed fraction of CsA in ARF rats.

Telomere length, telomerase activity and telomerase RNA expression during mouse mammary tumor progression.

To investigate the roles of telomere length (mean length of the terminal restriction fragments; TRFs), telomerase activity (TA) and telomerase RNA (mTR) expression in relation to mouse mammary tumor progression, we examined a pregnancy-dependent mouse mammary tumor line (TPDMT-4) and its four autonomous sublines (T4-OI320: non-metastatic; and T4-OI165, -OI96, and -OI145: artificial metastatic) of DDD/1 mouse origin, and an autonomous growing mammary tumor (JYG-MC) showing spontaneous lung metastasis developed in BALB/c mice infected with a Chinese feral mice (Sub-Jyg)-derived mouse mammary tumor virus (JYG-MTV). Compared with normal (pregnant) mammary tissue, the TA was elevated in the TPDMT-4 tumor and in the non-metastatic subline tumor (T4-OI320) (x10 fold, respectively), and was further increased (x13-15 fold) in parallel with the acquisition of metastatic potential (T4-OI165, -OI96, and -OI145). The mTR level was upregulated (x2.7-2.8 fold) in all autonomous growing tumors compared to the normal counter-part, but not in TPDMT-4. The TRF was shorter in accord with tumor progression (normal mammary tissue, 48 kb; TPDMT-4, 45 kb; T4-OI320, 37 kb; T4-OI165, -OI96 and -OI145, mean 37.7 kb; and JYG-MC, 21 kb). These results suggest that the activation of TA occurs as an early event at the stage of hormone-dependent tumorigenesis, followed by the up-regulation of mTR expression in accordance with the acquisition of autonomous growth, and then further activation of TA occurs when the tumor acquires metastatic potential. The TRF shortening was in parallel with the tumor progression.

Inhibition of tumor growth and metastasis by angiogenesis inhibitor TNP-470 on breast cancer cell lines in vitro and in vivo.

Antitumor and antimetastatic activity of the angiogenesis inhibitor O-(chloroacetyl-carbamoyl) fumagillol (TNP-470), a semisynthetic analogue of fumagillin, was evaluated in breast cancer cell lines. In an in vitro MTT assay, after 72 hrs continuous exposure to TNP-470, growth inhibition was observed in all seven cell lines of murine (JYG-A, JYG-B, DD-762, and BALB/c-MC) or human (KPL-1, MDA-MB-231, and MKL-F) origin, in which the 50% inhibitory concentrations (IC50) at 72 hrs treatment were 4.6, 4.4, 4.6, 10.1, 35.0, 25.3, and 33.4 micrograms/ml, respectively. In an in vivo assay using JYG-A, JYG-B, KPL-1, and MDA-MB-231 cells by orthotopic (right thoracic mammary fat pad) transplantation in female nude mice, TNP-470 at 30 or 50 mg/kg body weight was injected s.c. every other day from the day of tumor cell inoculation until the end of the experiment. The inhibitory effect on primary tumor growth was obtained in all four cell lines in a dose-dependent manner. In the 50 mg/kg TNP-470-treated group, the reductions in tumor weight of the JYG-A, JYG-B, KPL-1, and MDA-MB-231 cells with respect to the controls were 50%, 30%, 4%, and 49%, respectively. Metastasis was seen in the JYG-A, JYG-B, and KPL-1 cells. The numbers of mice bearing pulmonary metastases of JYG-A and JYG-B cells and regional axillary lymph node metastases of KPL-1 cells were reduced, and TNP-470 at the 50 mg/kg dose to KPL-1 cells significantly reduced lymph node metastases compared with the control. Although the weight gain was retarded in the TNP-470-treated mice, weight loss was not seen. TNP-470 was highly effective in the treatment of breast cancer cells. These results suggest that the clinical use of TNP-470 may be a promising treatment for breast cancer patients.

Inhibitory Effect of the Angiogenesis Inhibitor O-(Chloroacetyl-carbamoyl)fumagillol(TNP-470) on Tumor Growth and Metastasis of Murine Mammary Tumor Cell Line (JYG-B)Inoculated in Female Nude Mice.

The effect of the potent angiogenesis inhibitor O-(chloroacetyl-carbamoyl) fumagillol(TNP-470), a semisynthetic analogue of fumagillin on tumor growth and metastasis was studied using murine mammary tumor cells (JYG-B) inoculated into female nude mice. Injection of TNP-470 at 10 and 30 mg/kg doses, 3 times a week, until the end of the experiment (41 days)inhibited the s.c.inoculated primary tumor growth in a dose-dependent manner (45%and 65% in volume, respectively). TNP-470 at 30 and 60 mg/kg doses, 3 times a week, until the end of the experiment (35 days), reduced the weight of the i.p. inoculated total tumor mass of abdominal dissemination in a dose-dependent manner (54% and 76%, respectively). Pulmonary metastasis was found in all mice but TNP-470 significantly reduced the lung weight reflecting the total volume of the metastatic foci, and the numbers of metastatic foci in the liver and kidneys was reduced by TNP-470 treatment. These findings show that the angiogenesis inhibitor (TNP-470)has a strong inhibitory effect on tumor grwoth and metastasis in vivo.

Multiple metastases of mammary carcinoma cell lines isolated from feral mouse.

Two established cell lines (JYG-A and B) isolated from mammary carcinoma tissues of M. m. musculus Sub-Jyg (a chinese wild mouse) showed multiple metastasis in the lung (100%), liver (40-60%), kidney (40-80%), lymph node (20-60%) and other organs of the nude mice inoculated with these cells subcutaneously or intravenously. In addition, 100% brain metastasis or infiltration was observed only when inoculated with JYG-B cells intravenously.

Mouse Mammary Tumor Virus Proviral Integration in the DD/Tbr Mice.

The patterns of mouse mammary tumor virus (MMTV) integration in the DNA of spontaneous-mammary tumors, salivary glands and livers of DD/Tbr mice were examined using MMTV env, int -1c and int-2c probes. The MMTV env probe revealed 1 to 7 new proviral insertions in all mammary tumors. MMTV integration into int-1 was observed in 10 of 18 mammary tumors, whereas that into int-2 was seen in only 2 of 18 tumors. Of the 13 salivary glands examined, only 3 showed new MMTV proviral integrations, but rearrangement in int-1 or int-2 loci by MMTV was not observed. Immuno-collidal gold electron microscopy revealed the presence of MMTV particles both in mammary tumors and in salivary glands, but no tumors were found to be developed in salivary glands. Taken together these results suggest that salivary glands support MMTV replication, but the virions thus produced may not lead to salivary gland tumorigenesis. It is suggested that the salivary gland is the source of horizontally transmitted MMTV in DD/Tbr mice.

Hormone-dependent human tumors express the fms gene-product.

Human mammary and thyroid tumors as well as peripheral blood cells of the same patients were examined for the amplification of oncogene fins and the expression of its product. Of the 7 mammary tumors analyzed, amplification of fms was observed in 6 (86%) mammary tumor DNAs, while no amplification was seen in 5 thyroid tumors and 7 peripheral blood cell DNAs tested. None of the mammary and thyroid tumors showed any rearrangement of the fms gene. Investigations of the expression of the product of fms in various human tumors were carried out following the method of immunohistochemical staining in which polyclonal antibody to the fms gene product was used. The incidence of the expression of fms gene product in the tumors of mammary and thyroid glands, prostate and ovary, the hormone-dependent organs, was 38-97%, whereas fms protein was found to be present only in 20 and 42% of the tumors from hormone-independent organs, the brain and the bladder. Expression of the fms gene product was not detectable in normal tissue surrounding the tumor tissue in any of the cases examined. These results suggest that the expression of the product of fms may be associated with the development of some tumors of hormone responsive organs, especially the breast and thus the fms gene product may be a valuable marker for human mammary tumors.

Polyclonal and monoclonal antibodies monospecific to MMTV LTR orf protein produced in E. coli.

Monoclonal and polyclonal antibodies specific to an open reading frame of the mouse mammary tumor virus long terminal repeat were generated using an open reading frame-beta-galactosidase fusion protein produced in E. coli. Both antibodies reacted with the open reading frame-beta-galactosidase fusion protein but not with beta-galactosidase alone using an immunoblotting technique. It is concluded that these antibodies were specific for the protein encoded by the open reading frame of the mouse mammary tumor virus long terminal repeat.

Genetic analysis of susceptibility to radiation-induced apoptosis of thymocytes in mice.

Genetic control of thymocyte susceptibility to radiation-induced apoptosis was investigated in BALB/cHeA, STS/A and five other strains of mice by counting pyknotic cells in a selected area of thymic cortex on histological specimens after whole-body X-irradiation. Number of dead cells increased almost linearly with doses (range 0.25-0.75 Gy) in BALB/cHeA and STS/A mice. However, dead cell counts in BALB/cHeA mice were more than twice those in STS/A mice at each dose. Of five other strains of mice, C57BL/6N and B10.BR mice exhibited a sensitive phenotype similar to BALB/cHeA mice, while C3H/HeAMsNrs and NFS/N mice showed a resistant phenotype similar to STS/A mice. A/J mice seemed to be rather resistant. A sex difference was not recognized in BALB/cHeA and STS/A mice. Resistance was dominant over susceptibility in the progenies of reciprocal crosses between the two strains, indicating an autosomal inheritance and no maternal effect. Segregation ratio of susceptible phenotype to resistant one in the backcrosses of female (BALB/cHeA x STS/A)F1 mice with male BALB/cHeA mice was not significantly different from 1:1 and all backcrosses of female (BALB/cHeA x STS/A)F1 mice with male STS/A mice exhibited a resistant phenotype. Results suggested that thymocyte susceptibility to radiation-induced apotosis is controlled by one major autosomal allele.

Carcinogenicity study in rats of phytic acid 'Daiichi', a natural food additive.

The carcinogenicity of phytic acid 'Daiichi' (PA), a natural food additive, was examined in Fischer 344 rats of both sexes. PA was added to the drinking-water of groups of 60 male and 60 female rats at levels of 1.25 or 2.5% for 100-108 wk. There was a dose-dependent reduction in the mean final body weights of rats treated with PA. Necrosis and calcification of the renal papillae were observed in PA-treated rats, but not in the controls. The incidences of necrosis (calcification) were as follows: one (three) out of 57 males given 2.5% PA; one (none) out of 59 males given 1.25% PA; 10 (17) out of 55 females given 2.5% PA; six (six) out of 58 females given 1.25% PA. Renal papillomas occurred in three of the high-dose male rats, four of the high-dose female rats, and three of the low-dose female rats. The development of papillomas seemed to be related to calcification and necrosis of the renal papillae induced by PA. While many other tumours developed in all groups, including the controls, the organ distribution of these neoplasms and their histological characteristics did not differ significantly from those known to occur spontaneously in this strain of rats.