Bmx tyrosine kinase has a redundant function downstream of angiopoietin and vascular endothelial growth factor receptors in arterial endothelium.

The Bmx gene, a member of the Tec tyrosine kinase gene family, is known to be expressed in subsets of hematopoietic and endothelial cells. In this study, mice were generated in which the first coding exon of the Bmx gene was replaced with the lacZ reporter gene by a knock-in strategy. The homozygous mice lacking Bmx activity were fertile and had a normal life span without an obvious phenotype. Staining of their tissues using beta-galactosidase substrate to assess the sites of Bmx expression revealed strong signals in the endothelial cells of large arteries and in the endocardium starting between days 10.5 and 12.5 of embryogenesis and continuing in adult mice, while the venular endothelium showed a weak signal only in the superior and inferior venae cavae. Of the five known endothelial receptor tyrosine kinases tested, activated Tie-2 induced tyrosyl phosphorylation of the Bmx protein and both Tie-2 and vascular endothelial growth factor receptor 1 (VEGFR-1) stimulated Bmx tyrosine kinase activity. Thus, the Bmx tyrosine kinase has a redundant role in arterial endothelial signal transduction downstream of the Tie-2 and VEGFR-1 growth factor receptors.

Emergence of morphological order in the network formation of Physarum polycephalum.

Emergence in a system appears through the interaction of its components, giving rise to higher order or complexity in the system. We tested for the presence of emergent properties in a biological system using the simplest biological entity of a unicellular organism; the plasmodium of Physarum polycephalum, a giant unicellular amoeboid organism that forms a network-like tubular structure connecting its food sources. We let two plasmodium networks within a single cell interact with each other, and observed how the intracellular interaction affected the morphologenesis of the plasmodium networks. We found that the two networks developed homologous morphology. We further discuss the presence of autonomous and emergent properties in homologous network formation.

Suppression methods of radon emanation from phosphorus fertiliser and diatomaceous earth.

The present paper shows methods to suppress radon emanation from artificial porous materials such as phosphorous fertiliser or diatomaceous earth with a small amount of radium. The basic concept of suppression is making the radon emanation mechanism 'null', which comes from the fact that recoil is the main mechanism of radon emanation at room temperature. Nullifying may be done through removal of water in the pore volume by heating and through removal of pores by melting or filling with sulphur. These radon emanation suppression methods were tried for phosphorous fertiliser and diatomaceous earth with a small amount of radium. The melting method was the most effective of all. Sustainability of these methods was also studied. The melting method was the most sustainable. The heating method was also sustainable for diatomaceous earth in spite of a long-term immersion in water.

Role of fibrin coagulation in protection of murine tumor cells from destruction by cytotoxic cells.

We have previously proposed that fibrin deposition on tumor cells during their migration in the blood could protect them from elimination by natural killer (NK) or other cytotoxic cells. Anticoagulant drugs could prevent fibrin coagulation and increase the efficiency of cytotoxic effector cells in tumor cell elimination. To further investigate the protective roles of fibrin, we studied in vitro the susceptibility of various murine tumor cells to the cytotoxic activity of NK or lymphokine activated killer (LAK) cells in the presence of murine plasma or serum. In the first set of experiments, tumor cells were incubated with plasma (at dilutions of 1:20-1:160) for 30 min before effector cells were added. Similarly, effector cells were first incubated with plasma before mixing with radiolabeled target cells for cytotoxicity assay. In some experiments target and effector cells and plasma were mixed simultaneously. The cytotoxic activity of both NK and LAK cells was inhibited if coagulation occurred around tumor-target or effector cells. Tumor cells were also protected when both target and effector cells were simultaneously mixed and trapped in the fibrin clot. Inhibition of the cytotoxic activity of effector cells against tumor cells was positively correlated with the level of fibrin clot formation. When the larger clot was formed and more radiolabeled tumor cells were trapped in the clot, the higher level of inhibition of cytotoxicity was observed. In contrast, serum did not affect the cytotoxic activity of NK or LAK cells. To exclude possible non-coagulation-related effects of plasma on LAK cells, a cytotoxicity series of experiments was performed using purified fibrinogen and thrombin. When fibrinogen and thrombin were preincubated with tumor cells or LAK cells or all components were admixed simultaneously, substantial protection of tumor cells from destruction by LAK cells was also observed. However, when heparin was added, fibrin coagulation was prevented and cytotoxic activity of LAK cells was restored. Inhibition of LAK cytotoxicity and protection of tumor cells by fibrin coagulation were mostly due to the prevention of tumor-effector cell conjugate formation. Adding plasma at postbinding time periods (15-30 min after mixing effector and target cells) did not affect the ability of LAK cells to kill tumor cells confirming that fibrin coagulation influenced the binding rather than the lytic phase of cytotoxic cell activity.

Randomly controlled study of chemotherapy versus chemoimmunotherapy in postoperative gastric cancer patients.

From September 1979 through March 1981, a total of 302 patients with gastric cancer and undergoing gastrectomy at the Department of Surgery at Chiba University Hospital and its 14 affiliated hospitals was studied for clinical effectiveness of immunotherapy with Nocardia rubra cell wall skeleton. The patients were stratified by gross stage of cancer and degree of operative curability. They were then assigned randomly to either chemotherapy group or chemotherapy plus immunotherapy group. Immunotherapy used was intradermal injection of 400 micrograms of N. rubra cell wall skeleton which was given weekly for the first month and monthly thereafter. After the specimen was examined microscopically, the patients were classified by histological stage of cancer and radicality of surgical intervention into curative or noncurative groups. The patients were surveyed for survival period in December 1981. The postoperative survival rate was compared in patients of histologically curative or noncurative resection cases between the two treatment groups. No statistical difference was detected between the groups in age, sex, or operative procedures that might influence the patient's survival. As a result, statistical intergroup difference in survival rates was not seen in patients of the curative group, probably due to a short observation period. However, the intergroup difference in survival rates was statistically significant in patients of the noncurative group (p less than 0.01). These results indicate the adjunctive effect of N. rubra cell wall skeleton as an immunotherapeutic agent in patients undergoing gastrectomy for gastric cancer.

Endothelial receptor tyrosine kinases activate the STAT signaling pathway: mutant Tie-2 causing venous malformations signals a distinct STAT activation response.

Endothelial receptor tyrosine kinases (RTKs) and their signaling mechanisms are of interest because they may control tumor angiogenesis and thereby tumor growth. In this report we have examined activation of the signal transducers and activators of transcription (STATs) by the three known vascular endothelial growth factor receptors (VEGFR1-3), as well as by the endothelial Tie-1 and -2 receptors. We also studied signaling by the R849W mutant of Tie-2 (MTie-2), which has been shown to cause venous malformations. When overexpressed in 293T cells, MTie-2 activated STAT1 while the other endothelial RTKs failed to do so. In contrast, the three VEGFRs were strong activators of STAT3 and STAT5, suggesting that they activate only a specific subset of these signal transducers. STAT3 and STAT5 were also activated by Tie-2 and, more so, by MTie-2. Tyrosine phosphorylation and DNA binding of STATs correlated with their ability to activate transcription as judged by luciferase assays. When co-expressed with STAT5, VEGFR-1 as well as both the Tie-2 receptor forms increased expression of the cell cycle inhibitor p21. Interestingly, co-expression of the Tie-2 receptors with STAT1 resulted in appearance of a novel, p21 related transcript. Taken together, these findings identify STAT proteins as novel targets for signal transduction by the endothelial RTKs, suggesting that they may be involved in the regulation of endothelial function.

Comparison of VEGF, VEGF-B, VEGF-C and Ang-1 mRNA regulation by serum, growth factors, oncoproteins and hypoxia.

The vascular endothelial growth factor (VEGF) family has recently been expanded by the isolation of two additional growth factors, VEGF-B and VEGF-C. Here we compare the regulation of steady-state levels of VEGF, VEGF-B and VEGF-C mRNAs in cultured cells by a variety of stimuli implicated in angiogenesis and endothelial cell physiology. Hypoxia, Ras oncoprotein and mutant p53 tumor suppressor, which are potent inducers of VEGF mRNA did not increase VEGF-B or VEGF-C mRNA levels. Serum and its component growth factors, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) as well as transforming growth factor-beta (TGF-beta) and the tumor promoter phorbol myristate 12,13-acetate (PMA) stimulated VEGF-C, but not VEGF-B mRNA expression. Interestingly, these growth factors and hypoxia simultaneously downregulated the mRNA of another endothelial cell specific ligand, angiopoietin-1. Serum induction of VEGF-C mRNA occurred independently of protein synthesis; with an increase of the mRNA half-life from 3.5 h to 5.5-6 h, whereas VEGF-B mRNA was very stable (T 1/2>8 h). Our results reveal that the three VEGF genes are regulated in a strikingly different manner, suggesting that they serve distinct, although perhaps overlapping functions in vivo.

Does incineration turn infectious waste aseptic?

Incineration of infectious waste is considered to be biologically safe. We performed basic experiments to confirm that bacillus spores are killed by incineration in a muffle furnace. Biological samples containing 10(6) spores of Bacillus stearothermophilus were placed in stainless steel Petri dishes and then into hot furnaces. The furnace temperature and duration of incineration were 300 degrees C for 15 min, 300 degrees C for 30 min, 500 degrees C for 15 min, 500 degrees C for 30 min and 1100 degrees C for 3 min. We confirmed that all spores of B. stearothermophilus were killed at each of these settings. The effect of incineration seems to be equivalent to that of sterilization, based on the satisfactory sterilization assurance level of 10(-6).

Prospective randomized study of open versus laparoscopy-assisted distal gastrectomy with extraperigastric lymph node dissection for early gastric cancer.

BACKGROUND: Laparoscopy-assisted surgery with extraperigastric lymph node dissection for gastric cancers has been described, but the clinical benefits of these surgeries still are unclear. Short-term clinical outcomes were compared between laparoscopy-assisted distal gastrectomy (LADG) and conventional open distal gastrectomy (ODG) for early gastric cancer in a prospective randomized fashion. METHODS: For this study, 28 patients with early gastric cancers in the lower half of the stomach were randomly assigned to either LADG (n = 4) or ODG (n = 14). Postoperative pain, levels of acute inflammatory responses, and pathologic evaluation of the operative specimens were compared. RESULTS: The LADG group required a significantly shorter period of postoperative epidural anesthesia, showed significantly lower levels of serum interleukin-6 and C-reactive protein, and had no major postsurgery complications. Pathologic examinations showed that surgery was equally radical in the two groups. CONCLUSION: The findings show that LADG with extraperigastric lymph node dissection is a safe and less invasive alternative to the open procedure.

Expression of c-kit mRNA and protein during the differentiation of human hematopoietic progenitor cells.

To investigate how c-kit and c-kit ligand play a role in the function of hematopoietic stem cells, we determined the expression of c-kit in sorted human hematopoietic stem cells, CD34+CD33- cells and CD34+CD33+ cells. CD34+ cells constituted approximately 1% of the population of gated bone marrow cells and contained colony-forming cells. Two-color analysis by a fluorescence-activated cell sorter (FACS) revealed that about one-third to one-half of the total CD34+ cell population were positive for the CD33 antigen. To analyze the relative accumulation of c-kit mRNA in sorted cells, we used the reverse transcription-polymerase chain reaction (RT-PCR) method, followed by Southern blot analysis. There was a linear relationship between the amount of input RNA and products amplified in the range of 10(3) to 10(5) cells. Using this procedure, we carried out an analysis of c-kit mRNA expression in CD34+CD33-, CD34+CD33+, CD34-CD33+, and CD34-CD33- cells. Enhanced expression for c-kit mRNA was observed solely in CD34+CD33- cells. In contrast, flow cytometry shows that c-kit protein was expressed most abundantly in CD34+CD33+ cells. Colony-forming cells were generated on a human stromal cell layer for 5 weeks initiated with CD34+CD33- cells but not with CD34+CD33+ cells. During co-culture with stromal cells, CD34+CD33- cells differentiated into CD34+CD33+ cells. From these findings, it is concluded that CD34+CD33+ cells are direct progenies of CD34+CD33- cells. In this differentiation pathway, the expression of c-kit mRNA decreased and the c-kit protein increased.

Shiga toxin activates p38 MAP kinase through cellular Ca(2+) increase in Vero cells.

We examined whether the mitogen-activated protein kinase (MAPK) pathway is involved in Shiga toxin (Stx)-induced Vero cell injury. Consonant with cell injury, Stx caused a transient extracellular signal-regulated kinase1/2 (ERK1/2) and a sustained p38 MAPK phosphorylation. p38 MAPK inhibitors (SB 203580 and PD 169316), but not an ERK1/2 kinase inhibitor (PD 98059), partially inhibited the Stx-induced cell death. BAPTA-AM, a Ca(2+) chelator, reduced both cell injury and p38 MAPK phosphorylation. Antioxidants reduced Stx1-induced p38 MAPK phosphorylation. These data indicate that Stx activates p38 MAPK through an increase in intracellular Ca(2+) and reactive oxygen species, and this signaling is involved in Stx-induced cell death.

Inhibition of peritoneal dissemination of murine colon carcinoma cells by administrating retrovirus harboring IL-2 gene.

We have examined the antitumor effect of the retrovirally expressed interleukin-2 (IL-2) gene in murine colon carcinoma cells (Colon 26) on a disseminated model within the peritoneal cavity. Intraperitoneal injection of IL-2-producing Colon 26, but not wild-type cells, into syngeneic mice did not cause animal death and conferred T-cell-dependent, tumor-specific protective immunity on the inoculated mice. Direct administration of the retrovirus harboring IL-2 gene into the peritoneal cavity of the mice that had intraperitoneal tumor of wild-type cells significantly prolonged the survival of the mice compared with that of the mice that received control retrovirus. In the surviving mice treated with the retrovirus we also observed the induction of protective immunity with tumor specificity. Integrated retrovirus DNA was not detected in any organs examined. Thus, in vivo IL-2 gene transfer by retrovirus enabled tumor-bearing mice to generate antitumor immunity without unnecessary retroviral insertion into host genome.

Role of IA-positive cells in the beneficial effect of donor blood transfusion and induction of suppressor cells in cardiac allotransplantation of rats.

An attempt was made to reveal the role of Ia-positive cells in the beneficial effect of pretransplant donor-specific blood transfusion (DST) and induction of suppressor cells in the cardiac allotransplantation of rats. Mean graft survival time (MST) of F344 (RT1 1v1) hearts transplanted in WKA (RT1k) rats was 6.5 +/- 0.8 days. Pretreatment of recipients with 1 ml DST 10 days before grafting prolonged it to 56.5 +/- 38.1 days. Plasma or erythrocytes did not prolong MST at all, whereas pretreatment with 1 X 10(7) lymphocytes prolonged MST significantly. B enriched cells separated on a nylon-wool column prolonged MST, but T-enriched cells did not. Treatment of lymphocytes with interspecies crossreactive monoclonal anti-Ia antibody and complement eliminated the ability of lymphocytes to prolong MST, suggesting an important role of Ia-positive cells in the beneficial effect of DST. The role of Ia-positive cells in prolonging graft survival, was analyzed by a cell and serum transfer experiment. MST of F344 heart allografts transplanted in sublethally irradiated (500 rads) WKA rats was 17.4 +/- 4.0 days. MSTs of allografts in rats irradiated and treated with serum or spleen cells of blood-conditioned rats were 23.2 +/- 1.6 (P less than 0.05) and 5.2 +/- 0.7 (NS) days, respectively. On the other hand, MSTs of the grafts in rats irradiated and treated with serum or spleen cells harvested from rats that had accepted grafts for 30 days were 18.7 +/- 1.9 (NS) and 63.8 +/- 29.9 (P less than 0.01) days, respectively. The results indicate that the initial role of a serum factor in prolonging graft survival induced by DST containing Ia-positive cells was followed by activating suppressor cells by the presence of the allograft.

Optimal serum trough levels of FK506 in renal allotransplantation of the beagle dog.

The present study was performed to estimate the optimal serum trough levels of FK506 (FK) for prophylactic use and for the treatment of acute rejection in renal allotransplantation of the beagle dog. The serum trough levels of an immunosuppressive dose of FK 1.0 mg/kg p.o. ranged from 0.1 to 0.4 ng/ml. The data indicate that the effective serum trough level is about 100 times lower than that of cyclosporine, as was already observed in previous in vitro studies. Combining treatment with a nonimmunosuppressive dose of cyclosporine of 2.5 mg/kg could lower the effective trough levels of FK. By the combining treatment, 2 out of 5 renal recipient dogs survived with well-functioning grafts as long as 60 days with the trough levels between 0.04 and 0.07 ng/ml. High-dose 5-day i.m. FK treatment of 0.5 or 1.0 mg/kg was effective in the reversal of acute rejection, with peak serum trough levels during successful rejection therapy ranging between 0.28 and 3.7 ng/ml. Two dogs died of malaise or pneumonia with peak trough levels of 2.25 and 2.78 ng/ml. Among the wide range of the effective trough levels for successful acute rejection therapy, those above 2.0 ng/ml seem to be toxic in some renal-transplanted dogs.

Effects of rapamycin in experimental organ allografting.

The immunosuppressive effect of rapamycin (RAPA), a macrolide antibiotic produced by Streptomyces hygroscopicus, was tested using allografts in the rat and dog. It was immunosuppressive at a dose of 3.0 mg/kg/day for 8 days in skin allografts and 0.1 mg/kg/day for 11 days using cardiac allografts in the rat. It prevented rejection of renal allografts in beagle dogs at a dose of 0.3 mg/kg; however, emaciation and gastrointestinal toxicity resulted in some deaths in dogs. Mucosal necrosis and vasculitis in the submucosal layer were the predominant findings at autopsy in dogs receiving RAPA. Serum amylase values rose soon after commencement of RAPA treatment. Combined drug treatment with CsA at a nonimmunosuppressive dose of 3.2 mg/kg in the rat or 2.5 mg/kg in the dog was synergistic, with a low dose of 0.1 mg/kg/week RAPA. Combined therapy of CsA and RAPA also inhibited the frequency of vasculitis and emaciation in dogs. The present data suggest that RAPA is immunosuppressive in organ allografting and that the combination of CsA and RAPA would be effective clinically.

Effects of combination treatment with FK506 and cyclosporine on survival time and vascular changes in renal-allograft-recipient dogs.

In our previous experiments studying the effects of FK506 on renal allografting in the dog, we encountered two major problems. One problem was anorexia and the other problem was vascular changes mainly in the recipient heart. Anorexia was generally dose dependent, but the vascular changes were seen to be more prominent at lower doses rather than at higher immunosuppressive doses. The present study was undertaken to study these two problems. A nonanorexic, vascular change-related, nonimmunosuppressive dose of FK506 was combined with a low dose of cyclosporine or prednisolone in beagle dogs after renal allografting. Treatment with either FK506 alone at a dose of 0.32 mg/kg or cyclosporine alone at 2.5 mg/kg was not effective in prolonging renal recipient survival. The recipient dogs died of rejection, and a variety of vascular changes were observed in the hearts of both groups. Combined treatment with FK506 and cyclosporine at these same doses resulted in statistically significant prolongation of the survival time of the renal recipient (P less than 0.01), and histologic studies showed that the frequency and severity of the vascular changes were suppressed in the recipient receiving the combined treatment. The combination of FK506 and prednisolone at 0.5 mg/kg was not effective in prolonging survival. Furthermore, the extent of vascular changes was similar to those found in recipients receiving FK506 alone. The data suggest that combined treatment with low doses of both FK506 and cyclosporine acted synergistically in prolonging canine renal allografts and that the vascular changes frequently seen at low doses of FK506 were reduced by additional immunosuppression with a low dose of cyclosporine.

Studies of the effects of FK506 on renal allografting in the beagle dog.

The immunosuppressive activities of a newly discovered macrolide extracted from Streptomyces tsukubaensis, FK506, were examined using 38 renal allografts in the beagle dog. The median survival time was 15.5 days in dogs without treatment, 61 days with a dose of 0.08 mg/kg/day and 176 days with a dose of 0.16 mg/kg/day of intramuscularly administered FK506. Prolongation of survival was statistically significant when compared with controls (P = 0.02, 0.0044, respectively). None of 6 recipient dogs receiving the agent at a dose of 0.16 mg/kg/day encountered rejection during the treatment course. Three of them survived over 200 days. Oral administration of FK506 at a dose of 0.32 mg/kg/day did not prolong the median survival time (20.5 days) compared with the placebo treated control (16.5 days), but oral treatment with 1.0 mg/kg/day resulted in all of the recipient dogs surviving over 130 days. Histological studies of 7 kidney graft biopsy specimens of the dogs surviving over 3 months revealed no cell infiltration or only some degree of reversible interstitial cell infiltration, but vascular and glomerular changes were not observed in any of the specimens. Irregularity of nuclear shape and cytoplasmic vacuolation of the pars recta of the proximal tubules were observed in one dog each. Liver biopsy specimens showed no consistent evidence of hepatocellular damage. Three dogs died of intussusception 2-3 weeks posttransplant. The dogs treated intramuscularly with 0.32 mg/kg/day suffered from anorexia. Two dogs receiving oral treatment at a dose of 1.0 mg/kg developed papilloma of the skin around day 60, but the tumors disappeared by day 120. We conclude that FK506 is a powerful immunosuppressant in the dog with tolerable side effects.

Reduced tumorigenicity of human gastric carcinoma cells engineered to produce IL-2 in SCID mice reconstituted with peripheral blood cells from cancer patients.

We have examined the validity of a humanized immune system with an animal model to assess cytokine gene therapy for cancer patients. For that purpose, we prepared hematologically-reconstituted severe combined immunodeficiency mice by transferring patient's peripheral blood cells containing CD34+ cells. These animals were inoculated subcutaneously with human gastric cancer lines transduced with cytokine genes. Tumorigenicity of interleukin-2-producing cells was significantly reduced in reconstituted but not in non-reconstituted mice, whereas that of wild-type and interleukin-6 producer cells was not affected irrespective of the reconstitution status. An inability to induce protective immunity in the reconstituted mice, which had rejected interleukin-2-producers, suggested that the effector cells mediating the antitumor response were non-T cells of donor origin. The experimental system presented in this study seems to be a feasible model to investigate applicable cytokines for patients.

Inhibition of peritoneal dissemination of colon carcinoma in syngeneic mice immunized with interleukin-2-producing cells.

We have examined the antitumor effect of murine colon carcinoma cells engineered to produce human interleukin-2 (IL-2) in syngeneic mice. Subcutaneous inoculation of retrovirally-transduced cells with IL-2 gene formed small tumors, but they became regressed spontaneously. Consequently, the inoculated mice showed prolonged survival. Histological examination of the tumors derived from IL-2-producers revealed predominant infiltration of macrophages around tumor necrotic masses. Thus, inoculation of IL-2-producing cells could protect the mice from subsequent subcutaneous or intraperitoneal challenges with wild-type cells, suggesting the induction of acquired immunity due to the effect of tumor vaccination.

Antitumor effect induced by the expression of granulocyte macrophage-colony stimulating factor gene in murine colon carcinoma cells.

Murine colon carcinoma cells which secrete several kinds of cytokine after retroviral transduction with corresponding genes, were examined for their antitumor effects in syngeneic mice. The mice inoculated with granulocyte macrophage-colony stimulating factor (GM-CSF) producer cells showed not only prolonged survival but also reduced tumorigenicity. The antitumor effect caused by the expression of interleukin-4 was less than that of GM-CSF, and interleukin-6 producer cells did not show any effects on the survival of the host animals. Histological examination of the GM-CSF-producing tumor revealed predominant infiltration of neutrophils and necrotic change of the tumor. The present study indicates the feasibility of cancer gene therapy with the expression of GM-CSF gene in tumor cells.