Suppression of retroviral tumors in hosts immune to viral oncogene product.

Feline sarcoma virus of Snyder-Theilen strain (ST-FeSV) induces sarcomas in Wistar/Ma rats following neonatal virus injection. Induced tumors express the viral oncogene product (P85) and elicit in hosts the specific serum anti-P85 antibody detectable by Western blot analysis. Syngeneic adult female rats were immunized with an ST-FeSV induced sarcoma that was 100% transplantable to syngeneic adult rats. Newborns from immunized rats (vaccinated rats) were found to carry anti-P85 in their sera at birth. Following neonatal injection of the virus to vaccinated and non-vaccinated control rats, tumor incidence was found to be lower and survival time significantly longer in vaccinated rats than in controls (p < 0.01). A nonapeptide known to be thymic hormone (FTS) showed suppressive effects on tumor development. These results indicate that tumors caused by perinatal retrovirus infection may be suppressed by efficient elicitation of cell-mediated immune response against the product of oncogene of the causative virus.

Augmentation with serum thymic factor of suppressive effects on retroviral tumor development in hosts immune to v-onc product.

Inbred adult female rats were immunized against syngeneic ST-FeSV induced sarcoma cells. ST-FeSV was injected subcutaneously into 57 neonates (vaccinated) born from these immunized females and into 60 non-vaccinated syngeneic neonates. Serum thymic factor (FTS) was injected subcutaneously into 10 of vaccinated and 30 of non-vaccinated rats. Sarcomas developed in 40.4% (19/47) of vaccinated (A), 20.0% (2/10) of vaccinated FTS injected (B), 63.3% (19/30) of non-vaccinated FTS injected (C), and 76.7% (23/30) of non-treated (D) rats. By AB immunostaining using antibody to v-fes product (P85), sarcomas developed in 10 of 13 rats of group C tested, and 3 of 6 rats of group D tested were positive, but those in 7 rats of group A and 2 rats of group B tested were all negative. Lung metastasis was observed in rats of all groups except those of B group. All sera of animals that developed sarcomas were positive to P85 in Western blot analysis. These results showed that FTS augmented suppressive effects on sarcoma development in hosts immune to the viral oncogene product.

Induction of differentiation of human myeloid leukemia cells by novel synthetic neurotrophic pyrimidine derivatives.

OBJECTIVE: Some pyrimidine analogues have been found to induce differentiation of several human myeloid leukemia cells. Newly synthesized heterocyclic pyrimidine derivatives promote neurite outgrowth and survival in neuronal cell lines. In this study, the growth-inhibiting and differentiation-inducing effects of these pyrimidine derivatives on human myeloid leukemia cells were examined. MATERIALS AND METHODS: Several myeloid leukemia cells were cultured with novel heterocyclic pyrimidine derivatives. Cell differentiation was determined by nitroblue tetrazolium-reducing activity, morphologic changes, expression of CD11b, lysozyme activity, and hemoglobin production. RESULTS: MS-430 (2-piperidino-5,6-dihydro-7-methyl-6-oxo (7H) pyrrolo [2,3-d] pyrimidine maleate) effectively induced HL-60 cells into mature granulocytes. MS-430 activated the mitogen-activated protein kinase (MAPK) of the cells before causing granulocytic differentiation. MAPK activation was necessary for MS-430-induced differentiation, because PD98059, an inhibitor of MAPK kinase, suppressed the differentiation induced by MS-430. MS-430 also induced monocytic differentiation of THP-1, P39/Tsu, and P31/Fuj leukemia cells, but did not affect erythroid differentiation of K562 or HEL cells. CONCLUSIONS: MS-430 potently induces differentiation of some myelomonocytic leukemia cells. This novel synthesized pyrimidine compound shows promise as a therapeutic agent for treatment of leukemia and as a neurotrophic drug.

Monoclonal antibody to human midkine reveals increased midkine expression in human brain tumors.

We produced a rat IgG2a monoclonal antibody against the carboxyl terminal region of human midkine (MK), a novel growth factor. This monoclonal antibody was used in immunohistochemical studies to compare the expression of MK, proliferating cell nuclear antigen (PCNA) and p53 protein in 133 primary brain tumors and 21 carcinoma metastases to the central nervous system. Approximately half of the glioblastomas multiforme (GBMs) (19/32), medulloblastomas (8/14), primitive neuroectodermal tumors (PNETs) (5/11), breast carcinoma metastases (Br-Mts) (6/10) and lung carcinoma metastases (L-Mts) (5/11) as well as some astrocytomas (2/14) had tumor cells that expressed MK; however, oligodendrogliomas, ependymomas, schwannomas, meningiomas, and pituitary adenomas did not express MK. The values of the PCNA-labeling index were statistically higher in GBMs, medulloblastomas, PNETs, Br-Mts, and L-Mts that expressed MK than in those that did not (Wilcoxon rank-sum test, p < 0.05). There was no correlation between MK and p53 protein in all tumor types. Normal and non-neoplastic brain tissues were negative for MK, PCNA, and p53 protein. We conclude that primary and metastatic tumors of the brain express MK and that the MK expression in brain tumors may depend, in part, on the proliferating potential.

Radioimmunoimaging of tumors with radioactive antibody against a glycoprotein (GP68) found in developing mouse brain.

The in vivo localization of a polyclonal antibody (pAb) against a glycoprotein with a molecular mass of 68 kDa (GP68), which was found in developing mouse brain, was studied in murine tumor models to evaluate potential applications of this antibody for in vivo radioimmunodetection and/or therapy of cancer. The tissue distribution of 125I-labeled GP68 pAb 3 days after i.v. injection into mice bearing four different kinds of solid tumor revealed a high uptake ratio by adenocarcinoma 755 and Lewis 3LL lung cancer. In contrast, the uptake ratio was low in mice bearing Ehrlich solid tumor and sarcoma-180 (S-180). These uptake ratios accorded well with the in vitro binding activity of this antibody with the tumor cells. In an immunoscintigraphic study, adenocarcinoma 755 was successfully visualized with 67Ga-labeled GP68 pAb. The results of these biodistribution and in vivo radioimmunoscintigraphic studies suggest that GP68 antibody may be applicable to the diagnosis and/or therapy of cancer.

Radioprotective effects of serum thymic factor in mice.

Serum thymic factor (FTS) reduced mortality of mice after total-body irradiation with 7.56 Gy X rays. The radioprotective effect was achieved by daily repeated subcutaneous injections of 3-100 micrograms FTS, while doses higher than 300 micrograms/day/mouse were neither radioprotective nor toxic. Similarly, degeneration of the spleen was moderated by 3-100 micrograms FTS but not by 500 micrograms FTS in sublethally (3.78 Gy) irradiated mice. Histological examination showed that hematopoiesis was enhanced in the spleen by daily injections of 10 micrograms FTS. Spleen cells from the FTS-treated mice incorporated more [3H]thymidine in culture with or without concanavalin A. The treatment with FTS increased the production of colony-stimulating factor in the spleen as well as in peritoneal macrophage-like cells, and caused a significant increase in the number of granulocyte-macrophage colony-forming cells both in the spleen and in the femoral bone marrow. Furthermore, FTS prevented a decrease in circulating neutrophils in the sublethally irradiated mice. Prominent overshoot recovery of myelopoiesis, which occurred occasionally in sublethally irradiated mice, did not occur in the FTS-treated mice. The decrease in blood erythrocytes was also significantly reduced. These observations imply that this thymic hormone has potential as a radioprotector.

MS-430, a synthetic pyrimidine derivative, influences the intracellular signal transduction pathway leading to neuronal differentiation of PC12h cells.

Although NGF (nerve growth factor) induces neuronal differentiation of PC12 cells, EGF (epidermal growth factor) acts as a mitogen for these cells. We have studied the effects of a synthetic pyrimidine derivative, MS-430, on the NGF and EGF actions on PC12h cells. We found that MS-430 accelerated NGF-induced neurite extension of PC12h cells and that, in the presence of MS-430, PC12h cells extended neurites in response to EGF. Next, we investigated the tyrosine phosphorylation of NGF receptor TrkA and the EGF receptor (EGFR) as well as mitogen-activated protein kinase (MAPK), which is a key protein in the intracellular signal transduction pathway. It was found that MS-430 prolonged the EGF-induced phosphorylation of EGFR and MAPK compared to that without MS-430. MS-430 also prolonged the NGF-induced phosphorylation of MAPK, but the phosphorylation of TrkA induced by NGF was not affected by MS-430. These results suggest that MS-430 influences the intracellular signal transduction pathway which causes the neuronal differentiation of PC12h cells.

Vascular and stromal changes in irradiated and recovering rat thymus.

To analyze the mechanisms responsible for thymocyte proliferation, maturation and migration in the thymus, the rat thymus just after, and recovering from irradiation was studied morphologically. The vascular structures of the rat thymus after a radiation dose of 6 Gy were found to be destroyed on day 3, but had recovered to almost normal by day 7, suggesting that the abrupt recovery of thymus structure after irradiation was due primarily to this change in vascular structure. Furthermore, the epithelial tissues in the thymic cortex appeared to contribute to this abrupt proliferation, and possibly to the abrupt maturation of thymocytes, while medullary epithelial tissues remained sparse and appeared inactive for a relatively long period. These findings are considered important for understanding the interrelationship between thymic epithelial cells and thymocytes with respect to thymocyte proliferation, maturation and migration.

A newly synthesized neurotropic pyrimidine compound, MS-818, may activate migratory schwann cells in peripheral nerve regeneration.

Following transection of a peripheral nerve in mice, a newly synthesized neurotropic pyrimidine compound, MS-818 was administered intraperitoneally at a dose of 1 mg kg-1 b.wt. day-1. The film model experiments for analyzing the early growth of axonal regeneration suggested that MS-818 activated Schwann cells which migrate from the proximal stump, inducing axonal elongation in vivo.

tGS ganglioside induces peculiar morphological features in grafted dopaminergic cells and promotes motor recovery in rats with unilateral lesions in the nigrostriatal dopamine pathway.

A cell suspension of substantia nigra from fetal rats was introduced into the ipsilateral caudate nucleus of rats with unilateral lesions in the nigrostriatal dopamine pathway, and effects of bovine total ganglioside (tGS) and monosialoganglioside (GM1) treatment on the morphological features of the transplanted cells and recovery from motor imbalance (rotation induced by methamphetamine) were investigated. Gangliosides (30 mg/kg) were administered intraperitoneally once a day for 2 weeks after transplantation to test animals while control animals received saline alone. tGS animals showed definite motor recovery in the 2nd week (P less than 0.05) while control and GM1 animals exhibited slight recovery only. At 6 weeks after transplantation, motor imbalance disappeared in all 3 groups. Tyrosine hydroxylase (TH) immunocytochemical staining revealed that in the 2nd week TH-positive cells in tGS animals had more primary dendrites and more large neurites (meganeurites) than did controls. TH-positive cells of all 3 groups often had spiny processes at that time. In the 20th week, TH-positive cells became more multigonal and had wider dendritic fields in all groups, and had less meganeurites and spines. Motor recovery of each animal was dependent on the number of TH-positive cells and no significant difference was observed in the number of TH-positive cells among the three groups. tGS treatment for 2 weeks without grafting induced immunohistologically no axonal sprouting in the substantia nigra, medial forebrain bundle, accumbens and caudate nucleus when the chemical lesions were complete. Data suggest that tGS induces hypertrophy but not hyperplasia of the transplanted nigral cells, and increases the morphological plasticity. This might be the basis for promotion of recovery in motor function after transplantation.

Effects of neurotropic pyrimidine heterocyclic compound, MS-430, on cultured hepatic parenchymal and stellate cells.

MS-430 is a novel synthetic pyrimidine derivative that stimulates regeneration of the nerve as a promoter for various growth factors such as epidermal growth factor (EGF) and nerve growth factor, and differentiation of astrocytes. The effects of MS-430 on the liver were tested using hepatocytes and stellate cells in primary culture isolated from rats. MS-430 enhanced EGF-induced DNA synthesis in hepatocytes while it alone failed to increase the basal DNA synthesis. Albumin mRNA expression in the cells and its amount in the medium were not changed by addition of EGF or MS-430 alone or both. Basic fibroblast growth factor (bFGF) increased DNA and but not collagen synthesis by hepatic stellate cells. Addition of MS-430 inhibited DNA synthesis by hepatic stellate cells at either presence or absence of bFGF, and collagen synthesis at the presence of bFGF. However, MS-430 had no effects on basal or bFGF-stimulated TGFbeta mRNA expression in the cells. These results suggest that MS-430 stimulated proliferation of hepatocytes as a comitogen for EGF without affecting albumin synthesis, and suppressed proliferation of activated hepatic stellate cells and their collagen synthesis without affecting TGFbeta expression.

Protective effects of serum thymic factor to Leptospira interrogans serovar Copenhageni infection in Mongolian gerbils.

The susceptibility to Leptospira interrogans serovar copenhageni in Mongolian gerbils treated with 10 micrograms of serum thymic factor (FTS) 1 day before infection was examined. Susceptibility of gerbils treated 5 times with 10 micrograms of FTS was also investigated. Mortality of FTS-treated gerbils was significantly lower than that of controls when small challenge doses were used. To analyse the FTS-induced resistance to leptospiral infection, natural killer (NK) cell activity and macrophage activity were studied. Macrophage activity was unaltered but NK cell activity was enhanced in FTS-treated gerbils, with or without leptospiral infection. Since no side-effects of FTS were observed, this compound should be considered for the treatment of leptospirosis.

Serum valproate concentrations in epileptic children with favourable responses.

The lower limits of the "therapeutic range" for serum levels of sodium valproate (VPA) were evaluated in epileptic children showing a benign clinical course. Twenty-five outpatients, aged 5 to 16 years, whose seizures were well controlled over three years with VPA alone, were studied. Venous blood was taken 1.1 to 5 hrs after the morning dose. Serum VPA concentrations at steady-state after receiving the maintenance doses to control seizures were determined by enzyme immunoassay. The patients were divided into three groups according to the seizure type and the age at onset; A and B: patients with tonic and/or clonic seizures, aged below 3 yrs (n = 11) and 3 to 11 yrs (n = 6), respectively, C: those with absence seizures, aged 4 to 11 yrs (n = 8). The serum concentrations in A (47.8 +/- 21.6 micrograms/ml, mean +/- SD) were significantly (p less than 0.02) lower than those in groups B and C (85.2 +/- 14.0 and 73.0 +/- 17.4 micrograms/ml, respectively). VPA concentrations below 50 micrograms/ml were seen in 6 patients (55%) in group A. It was concluded that many epileptic children, whose ages at onset were below 3 yrs, with tonic and/or clonic seizures could be controlled even with low initial serum concentrations below the "therapeutic range".

Increased midkine expression in hepatocellular carcinoma.

CONTEXT: Midkine (MK) is a novel heparin-binding growth factor whose gene was identified in embryonal carcinoma cells in early stages of retinoic acid-induced differentiation. OBJECTIVE: To examine the overexpression of MK in hepatocellular carcinoma (HCC). METHODS: Seventy-seven primary HCC specimens from patients aged 17 to 72 years (63 men and 14 women) were examined. Histologically, 16 cases of HCC were classified as the well-differentiated type, 50 cases as the moderately differentiated type, and 11 cases as the poorly differentiated type. Immunohistochemical analysis was performed using a rat immunoglobulin G2a monoclonal antibody against the carboxyl terminal region of human MK. In situ hybridization was also performed on 20 HCC samples. RESULTS: We successfully applied this monoclonal antibody against MK to analyze archival paraffin sections. The cancer tissues showed a positive reaction to this antibody, in which there was an intense reaction in their cytoplasm. Approximately one third of the individuals with HCC (26/77) had tumor cells that expressed MK, and these were classified into the following types: moderately differentiated (20/50), well differentiated (3/16), and poorly differentiated (3/11). The in situ hybridization analysis revealed that the signals of MK transcripts were found in the cytoplasm of the cancer cells; the distribution and localization of the MK transcripts' signals determined by in situ hybridization analysis were similar to those obtained by immunohistochemical analysis. CONCLUSIONS: Hepatocellular carcinoma expressed increased MK at the messenger RNA and protein level.

The neurotrophic pyrimidine heterocyclic compound MS-818 promotes the angiogenesis induced by basic FGF.

The chorioallantoic membrane (CAM) assay is a bioassay used widely for testing angiogenic activities of compounds. In the present study, we used this assay to explore the gross and micropathological effects of 2-piperadino-6-methyl-5-oxo-5,6-dihydro (7H) pyrrolo[3,4-d] pyrimidine maleate (MS-818) in combination with basic fibroblast growth factor (bFGF). Light microscopy revealed that application of 1, 10 and 100 ng/ml bFGF to the CAM induced a concentration-dependent increase in the number of new blood vessels, whereas application of 0.01, 0.1 and 1.0 mM MS-818 did not increase angiogenesis significantly. The combinations of 10 ng/ml bFGF with 0.1, 0.1 and 1.0 mM MS-818 showed significantly greater angiogenic effects than 10 ng/ml bFGF alone. However, 1 and 100 ng/ml bFGF in combination with these concentrations of MS-818 showed no additive effects. Histologically, numerous mononuclear cells became present in the mesodermal stroma, especially around the capillaries, when MS-818 alone and with bFGF was applied to the CAM. The new vessels formed in response to MS-818 plus bFGF were smaller than those formed with bFGF alone. These results suggest that, in the CAM assay system, MS-818 promoted angiogenesis effectively when the concentration or activity of the angiogenic factor (bFGF) was potentiated by MS-818 at a real optimal level.

The effect of MS-818, newly synthesized pyrimidine compound, on fracture repair.

Both periosteal cell proliferation and endochondral ossification accompanied are characteristic of the fracture healing process. This process is regulated by various kinds of soluble factors including prostanoids, cytokines and growth factors. In particularly, basic fibroblast growth factor (bFGF) stimulates the mesenchymal cell proliferation and differentiation in the periosteum and leads to the fracture healing. Recently, newly synthesized pyrimidine compound, 2-piperadino-6-methyl-5-oxo-5,6-dihydro (7H) pyrrolo [3,4-d] pyrimidine maleate (MS-818) has been reported to augment the biological effect of bFGF in vitro. Therefore, we have studied the effect of MS-818 on fracture healing process in which bFGF has been reported to play an important role. In the rat fracture model, 5 mg/kg MS-818 which had been administered intraperitoneally for fourteen consecutive days enhanced the cartilage matrix formation. In the bone defect model, in which we can find only membranous ossification without chondrogenesis, cartilage matrix formation was observed in seven days after 1 microgram of human bFGF containing polymer pellet was embedded in the defect site. Chondrogenesis induced by bFGF was enhanced significantly after 5 micrograms MS-818 containing pellet was implanted with bFGF pellet. These results suggest that MS-818 might promote the fracture healing process through enhancement of the effect of bFGF on endochondral ossification.

Effect of administration of serum thymic factor (FTS) in calves and rabbits infected with bovine immunodeficiency-like virus.

The effect of serum thymic factor (FTS) administration in bovine immunodeficiency-like virus (BIV)-infected calves and rabbits was examined. We previously found that some of the macrophage functions were depressed and humoral immune responses against foreign proteins were delayed in BIV-infected calves compared to uninfected calves. After FTS administration, however, no delay of antibody responses against foreign proteins was observed in BIV-infected calves. Though the chemiluminescence (CL) responses of macrophages in BIV-infected calves were significantly depressed (p < 0.05), FTS administration resulted in the recovery of the CL responses in the BIV-infected calves comparable to those in the control calves. Antibody responses against foreign proteins in BIV-infected rabbits were significantly depressed (p < 0.025) as compared with those in uninfected rabbits, though the depression became no significant after FTS administration.

Activation of canine monocytes and polymorphonuclear cells by serum thymic factor (FTS) in vivo.

The effect of serum thymic factor (FTS) was evaluated from the immunoresponse augmented in canine monocytes and polymorphonuclear cells (PMN) using the chemiluminescence technique. FTS did not affect the number of leukocytes and differential count of leukocytes. CL activity of the whole blood was significantly elevated by FTS from 72 hr to 120 hr after administration (p < 0.05), and that at 96 hr after administration was about 3-fold higher than that before the administration. The CL response of PMN was significantly elevated by FTS administration from 24 hr to 96 hr after administration (p < 0.05), and that at 48 hr after administration was about 7-fold higher than prior treatment. FTS also significantly elevated the CL response of monocyte from 24 hr to 96 hr after administration (p < 0.01), and the CL count of monocyte in 24 hr and 48 hr was about 100-fold higher than that before FTS administration. These findings suggested that FTS may be efficacious and useful immuno-potentiator for canine monocytes and PMN.

Peripheral nerve regeneration in a silicone tube: effect of collagen sponge prosthesis, laminin, and pyrimidine compound administration.

Regeneration of transected peripheral nerve with a 10-mm gap encased in a silicone tube was evaluated in the presence of collagen sponge with or without laminin, or with systemic administration of a pyrimidine compound, MS-818. The sciatic nerve of 20 adult rats was transected and the proximal and distal nerve stumps were fixed in a silicone tube. The lumen of the silicone tube was empty, or filled with a collagen sponge alone or with a laminin-soaked collagen sponge. Also, a pyrimidine compound was injected intraperitoneally after implantation of the empty silicone tube. Three weeks later, the contents of the silicone tubes were processed for histological examination of regenerated nerve fibers. Other animals were observed 6, 12, and 18 months after surgery to examine the long-term effects of the collagen sponge on nerve regeneration. All animals had regenerated tissue within the tube 3 weeks after nerve transection. The diameter of the tissue decreased toward the distal stump in the empty tube, but was the same throughout the full length in the collagen sponge-containing tube. Immunohistochemical studies revealed that the nerve fibers extended beyond the midline of the regenerated tissue in animals treated with a laminin-containing collagen sponge or receiving a pyrimidine compound. Long-term observation showed the regenerated nerve was thick as the proximal stump and many neurofilament- and peripheral myelin-positive fibers were observed around the collagen sponge. Collagen sponge assists the progress of regenerated tissues in silicone tubes, and laminin-containing prostheses and administration of a pyrimidine compound enhance peripheral nerve regeneration.

Expressions of a 68kDa-glycoprotein (GP68) and laminin in the mesodermal tissue of the developing mouse embryo.

Immunohistochemistry revealed initial expression of the stage-specific glycoprotein, GP68, in various mesenchymal tissue substructures of mouse embryos. During the 11-15th days of gestation, GP68 was localized in the primitive meninges, chondroblasts and perichondrium of pre-cartilaginous vertebral bodies and ribs, connective tissue cells of the dermis, the epicardium and endocardium of the heart, the epimysium and perimysium of skeleton musclature, and the basement membranes of splanchnic organs. Double staining for laminin expression indicated coincidental expression in identical tissue substructures. However, laminin was expressed in days 10-18 embryos and the neonate. Therefore, GP68 is coincidentally expressed with laminin in mesenchymal tissues between the 11th and 15th day of gestation, and may play a role as a laminin-associated protein. In the light of these results, a hypothesis concerning the relationship between these two proteins and the mechanisms of non-integrin laminin-associated proteins during normal embryogenesis is discussed further.