[Cytotoxicity study of high gold content Degutan surfaces of various degrees of roughness with fibroblasts (BALB 3T3) and osteoblasts (hFOB 1.19)]. / Zytotoxizitätsuntersuchung von hochgoldhaltigen Degutan-Oberflächen unterschiedlicher Rauhigkeit mit Fibroblasten (BALB 3T3) und Osteoblasten (hFOB 1.19).

The cytotoxicity of Degutan surfaces with different degrees of roughness, and the effect of surface structures on osteoblast proliferation and differentiation, was investigated with standardised cell culture systems. Fibroblast cell lines (BALB/3T3) and osteoblast cell lines (hFOB 1.19) were used. The number and variability of the cells were determined for assessment of proliferation and alkaline phosphatase activity, collagen I and osteocalcin production were used as parameters for differentiation. In the early phase, the largest numbers of cells and greatest proliferation were measured on polished Degutan surfaces. In the late phase, however, larger numbers of cells and a greater degree of proliferation were to be seen on sandblasted and sandblasted/heat-treated Degutan surfaces. No differences were found for collagen I, osteocalcin production or alkaline phosphatase activity. Neither the osteoblasts nor the fibroblasts revealed a toxic effect of Degutan. The results for osteoblast differentiation correlate with recent studies on identical structured titanium surfaces. In view of the immeasurable amount of ion release, Degutan may be considered an ideal model for an inert material surface.

Electromagnetic fields influence NGF activity and levels following sciatic nerve transection.

Pulsed electromagnetic fields (PEMF) have been shown to increase the rate of nerve regeneration. Transient post-transection loss of target-derived nerve growth factor (NGF) is one mechanism proposed to signal induction of early nerve regenerative events. We tested the hypothesis that PEMF alter levels of NGF activity and protein in injured nerve and/or dorsal root ganglia (DRG) during the first stages of regeneration (6-72 hr). Rats with a transection injury to the midthigh portion of the sciatic nerve on one side were exposed to PEMF or sham control PEMF for 4 hr/day for different time periods. NGF-like activity was determined in DRG, in 5-mm nerve segments proximal and distal to the transection site and in a corresponding 5-mm segment of the contralateral nonoperated nerve. NGF-like activity of coded tissue samples was measured in a blinded fashion using the chick DRG sensory neuron bioassay. Overall, PEMF caused a significant decrease in NGF-like activity in nerve tissue (P < 0.02, repeated measures analysis of variance, ANOVA) with decreases evident in proximal, distal, and contralateral nonoperated nerve. Unexpectedly, transection was also found to cause a significant (P=0.001) 2-fold increase in DRG NGF-like activity between 6 and 24 hr postinjury in contralateral but not ipsilateral DRG. PEMF also reduced NGF-like activity in DRG, although this decrease did not reach statistical significance. Assessment of the same nerve and DRG samples using ELISA and NGF-specific antibodies confirmed an overall significant (P < 0.001) decrease in NGF levels in PEMF-treated nerve tissue, while no decrease was detected in DRG or in nerve samples harvested from PEMF-treated uninjured rats. These findings demonstrate that PEMF can affect growth factor activity and levels, and raise the possibility that PEMF might promote nerve regeneration by amplifying the early postinjury decline in NGF activity.

Evidence for and implications of SV40-like sequences in human mesotheliomas and osteosarcomas.

Pleural mesotheliomas and osteosarcomas develop in hamsters injected intracardially with SV40. Using primers specific for the RB-pocket binding domain of SV40 we analysed with the polymerase chain reaction frozen specimens from 48 human mesotheliomas and 145 human bone tumours. We found that 60% of human mesotheliomas and 33% of human bone tumours contained SV40-like DNA. Immunostaining, Western blot and RNA in situ hybridization experiments revealed SV40 Tag expression in human mesotheliomas. Osteosarcomas were not studied for Tag expression because not enough material was available. Finally, antibodies anti-Tag were detected in the sera collected from patients with mesothelioma. These data indicate that SV40, or a closely related virus, is/are present in human mesothelioma and osteosarcoma.

Simian virus 40 oncogenesis in hamsters.

Simian virus 40 (SV40) is a DNA tumour virus which is highly oncogenic in hamsters. Only specific histologic types of tumours develop in hamsters injected with SV40, and these are influenced by the route of virus inoculation. When SV40 is injected systemically to expose most different cell types to the virus, the animals develop mesotheliomas, osteosarcomas, sarcomas, and lymphomas within six months. When the virus is injected subcutaneously, sarcomas at the site of injection develop. If hamsters are injected intracranially with SV40, they develop ependymomas. These same tumour types have been found to contain SV40.

B lymphocytes secreting IgG linked to latent transforming growth factor-beta prevent primary cytolytic T lymphocyte responses.

B lymphocytes secreting IgG linked to latent transforming growth factor (TGF)-beta (IgG-TGF-beta) prevent cytolytic T lymphocyte (CTL) responses to unrelated antigens in mixed lymphocyte cultures (MLC) so long as resting resident macrophages and functional Fc receptors are present. This was shown using IgG-secreting plaque-forming cells (PFC) to sheep erythrocytes (SRBC) obtained from popliteal lymph nodes of mice injected repeatedly in foot pads with SRBC. Remarkably, as few as approximately 300 PFC prevented CTL responses of 5 x 10(5) normal syngeneic spleen cells in MLC. Supranatants of short-term cultures of PFC also prevented CTL responses, and suppression was prevented by eliminating or dissociating IgG and TGF-beta present in supranatants or by antibody against active TGF-beta. Furthermore, the latency-associated peptide of latent TGF-beta was detected in approximately 10% of foci of IgG captured from single PFC, indicating that at least some B lymphocytes secrete IgG-TGF-beta as a complex. Resting resident macrophages (which do not produce latent TGF-beta) and functional Fc receptors were required for suppression, consistent with idea that IgG-TGF-beta is taken up through Fc receptors for IgG and that active TGF-beta, cleaved from latent TGF-beta of the complex, is delivered directly to potentially responding CTL. If CTL responses in man are similarly regulated by B lymphocytes, then an ongoing B cell response in patients with chronic viral infections or bearing immunogenic cancers may prevent effective therapeutic vaccination.

Autoantibodies produced spontaneously by young 1pr mice carry transforming growth factor beta and suppress cytotoxic T lymphocyte responses.

Young MRL/MPJ-lpr (lpr) mice 8-12 wk old challenged with alloantigen had significantly lower specific cytolytic T lymphocyte (CTL) responses than control MRL/MPJ +/+ mice. Serum from lpr mice compared with serum from ++ or normal C3H mice powerfully suppressed CTL responses in mixed lymphocyte cultures (MLC); absorbing lpr serum on protein G, adding antibody against transforming growth factor beta (TGF-beta) to cultures or dissociating immunoglobulin G (IgG) and TGF-beta before additions to cultures prevented suppression. Apparently autoantibody, similar to IgG produced by normal mice in response to immunization, carries TGF-beta which suppresses CTL responses in vivo and in vitro.

A first or dominant immunization. I. Suppression of simultaneous cytolytic T cell responses to unrelated alloantigens.

A first or dominant immunization with one antigen markedly inhibited specific cytolytic T lymphocyte (CTL) responses to a second unrelated alloantigen without suppressing antibody responses to other antigens. Suppression was induced rapidly, became systemic, and could be transferred passively with only serum. Suppression did not result from elimination of cells capable of responding to the second antigen. The mechanisms responsible for this "priority of the first response" may be the same that help protect the fetus during pregnancy, promote renal allograft survival after multiple blood transfusions, and prevent effective CTL-mediated immunity to variants of tumor cells or infectious agents that arise during tumor progression or chronic infections.

A first or dominant immunization. II. Induced immunoglobulin carries transforming growth factor beta and suppresses cytolytic T cell responses to unrelated alloantigens.

Fresh sera from mice immunized by bearing an immunogenic tumor or by repeated injections of allogeneic spleen cells or xenogeneic erythrocytes powerfully suppress cytolytic T cell responses in one-way mixed lymphocyte cultures. Suppression is not antigen specific, though is mediated by immunoglobulin (Ig)G specific for the immunizing antigen. Suppression caused by IgG mimics that caused by active transforming growth factor beta (TGF-beta). IgG associates with or carries latent TGF-beta; however, suppression caused by the complex of IgG-TGF-beta requires macrophages (M phi), whereas active TGF-beta alone does not. Also, IgG dissociated from TGF-beta does not cause suppression, suggesting that M phi may take up Ig-TGF-beta, process the complex, and deliver active TGF-beta to lymphocytes. Indeed, suppression by immune serum was prevented by antibody to Fc receptors, by saturating Fc receptors with heterologous IgGs, and by antibodies against TGF-beta. The overall findings reveal a previously unrecognized regulatory circuit whereby IgG produced in response to one antigen nonspecifically downregulates cytolytic T lymphocyte responses to unrelated antigens. The findings introduce the intriguing possibility that TGF-beta delivered by IgG and processed by M phi may mediate important biological effects in processes such as wound healing, tumor growth, and some autoimmune diseases.

Isolation and preliminary characterization of immunoinhibitory factors from dog erythrocytes and liver.

Crude dog liver extract (DLE) inhibits proliferation of dog and human lymphocytes stimulated by phytohemagglutinin and alloantigens. While purifying this activity from dog liver, we observed that dog liver inhibitory factor (DLIF) shared properties with hemoglobin. DLIF migrated with hemoglobin during DEAE cellulose chromatography, and DLIF had oligomeric (61,900) and subunit (17,900 and 15,700) apparent molecular weights (AMW) similar to concurrently analyzed Sigma canine hemoglobin (61,100, 16,700 and 14,900). After separate cross linking, the proteins comigrated on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) gels. We therefore isolated dog erythrocyte inhibitory factor (DEIF) from red blood cells to determine if DEIF was a hemoglobin derivative. DEIF, like DLIF, separated at an isoelectric point of 5.6. DEIF also had similar subunit AMW (17,600 and 15,300) by SDS PAGE, but DEIF had much lower lymphocyte inhibitory activity (LIA = 2.27) than DLIF (LIA = 100.00). We conclude that DLIF and DEIF are similar to each other and hemoglobin, but further studies are needed to determine the function and exact structure of DLIF and DEIF.

Interaction of nerve growth factor with murine alpha-macroglobulin.

The murine nerve growth factor, when injected i.v. or, combined in vitro with plasma, was found largely associated with the mouse alpha-macroglobulin (a homologue of human alpha 2-macroglobulin). The nerve growth factor-alpha-macroglobulin complex produced is sufficiently stable to resist separation by gel filtration in 1.0 M sodium chloride, polyacrylamide gel electrophoresis, and immunoprecipitation by antibodies against alpha-macroglobulin. As determined by equilibrium binding studies and computer generated Scatchard analysis, alpha-macroglobulin apparently possesses two types of binding sites with the apparent dissociation constants of 1.2 x 10(-6) and 2.9 x 10(-9) M, respectively, saturable by 3.7 and 0.03 moles of nerve growth factor. Hence, about one mole of nerve growth factor is bound to each of the four subunits of alpha-macroglobulin. Nerve growth factor can be readily dissociated from alpha-macroglobulin in sodium dodecyl sulfate gel electrophoresis in the absence of a reductant. Procedures that affect the proteinase-binding or methylamine- activities of alpha-macroglobulin do not affect the binding of nerve growth factor, and the binding is unaffected by the presence of zinc ions or EDTA. Hence, nerve growth factor is noncovalently associated with alpha-macroglobulin at a site separate from that of the proteinase-, methylamine-, and zinc-binding sites of alpha-macroglobulin. Mouse alpha-macroglobulin can protect the nerve growth factor from inactivation by trypsin. Even in the presence of trypsin, alpha-macroglobulin-nerve growth factor complexes still can stimulate the neurite outgrowth by dorsal root ganglia of 9-day-old chicken embryos. Since alpha-macroglobulin can specifically and noncovalently carry nerve growth factor, one important role of this alpha-macroglobulin in the circulation and extracellular spaces may be to protect the nerve growth factor from proteinase inactivation.

Macrophage colony-stimulating factor in nerve growth factor preparations.

Following a report that nerve growth factor preparations have granulocyte-colony-stimulating activity, we investigated the presence of colony-stimulating factors in 7s mouse submaxillary nerve growth factor and its subunits. Macrophage colonies were formed in mouse bone marrow cultures after exposure to preparations of 7s nerve growth factor, the gamma subunit, and, to a small extent, the alpha subunit; the beta subunit, which is responsible for the nerve growth function, did not stimulate colony growth. Furthermore, the esteropeptidase activity of the gamma subunit was not detected in preparations of macrophage colony-stimulating factor purified from the giant cell tumor (GCT) cell line. Immunoprecipitation of radiolabeled gamma subunit with a polyclonal antibody to L-cell macrophage colony-stimulating factor showed a protein band that could represent the gamma subunit of nerve growth factor. Separation of the macrophage activity from the esteropeptidase activity of the gamma subunit was accomplished on the basis of molecular size. Thus, macrophage colony-stimulating factor was a contaminant of nerve growth factor produced by the mouse submaxillary gland and copurified with the gamma subunit.

Purification of proteins from polyacrylamide gels, free of detergent or dye.

A two step procedure recovers proteins from sodium dodecyl sulfate polyacrylamide gels. The proteins are eluted by electrophoretic dialysis. The eluent is then passed through an Amberlite CG-400 anion-exchange resin. The recovery of protein is nearly total. The recovered proteins have no detectable sodium dodecyl sulfate contamination. With gels that have been stained with Coomassie Brilliant Blue R, the procedure recovers the proteins free of the dye. We have used this procedure successfully during the purification of epidermal glycoproteins.

Induction of the high-affinity nerve growth factor receptor on embryonic chicken sensory nerve cells by elevated potassium.

Culture medium with elevated K+ has been shown to enhance the survival of neurons isolated from several different regions of the nervous system. Nerve growth factor binds to binding sites on sensory and sympathetic neurons through two sites, one of high-affinity (Kd1 approximately 3 X 10(-11) M) and the other of low-affinity (Kd2 approximately 2 X 10(-9) M). Equilibrium binding data generated on dissociated cells derived from E9 chicken embryo dorsal root ganglia, has shown that there is a two-fold increase in the number of high affinity (type I) receptors, with no effect on the affinity, when cells are incubated for 2 hours in buffer containing 59 mM K+. There does not appear to be a significant change in the affinity or the number of low-affinity binding sites. This two-fold increase in type I receptors is dependent on temperature, Ca+2, and active protein synthesis. There does not appear to be an intracellular pool of the type I receptor sufficient to account for this increase. The induction is not observed on sensory nerve cells cultured in 59 mM K+ for 24 hours, either in the presence or absence of nerve growth factor. Additionally, the induction in the number of type I receptors requires that both nerve growth factor and K+ be present simultaneously. Taken in total, this data suggests that there may be a critical period in which the sensory neurons require nerve growth factor exposure to respond. Evidence is presented which indicates that nerve growth factor responsive cells are able to elicit neurites after an acute exposure to nerve growth factor of as little as 4 hours. Finally, there is an approximate two-fold decrease in the concentration of nerve growth factor needed to elicit maximal fiber outgrowth, consistent with the two-fold increase in the number of type I receptors.

Characteristics of partially purified nerve growth factor receptor.

Receptors for the nerve growth factor protein (NGF) have been isolated from three cell types [embryonic chicken sensory neurons (dorsal root sensory ganglia; DRG), rat pheochromocytoma (PC12) and human neuroblastoma (LAN-1) cells] and have been shown to be similar with respect to equilibrium dissociation constants. The present results demonstrate that there are multiple molecular weight species for NGF receptors from DRG neurons and PC12 cells. NGF receptors can be isolated from DRG as four different molecular species of 228, 187, 125, and 112 kilodaltons, and PC12 cells as three molecular species of 203, 118, and 107 kilodaltons. The NGF receptors isolated from DRG show different pH-binding profiles for high- and low-affinity binding. High-affinity binding displays a bell-shaped pH profile with maximum binding between pH 7.0 and 7.9, whereas low-affinity binding is constant between pH 5.0 and 9.1, with a twofold greater binding at pH 3.6. At 22 degrees C, the association rate constant was found to be 9.5 +/- 1.0 X 10(6) M-1 s-1. Two dissociation rate constants were observed. The fast dissociating receptor has a dissociation rate constant of 3.0 +/- 1.5 X 10(-2) s-1, whereas the slow dissociating receptor constant was 2.4 +/- 1.0 X 10(-4) s-1. The equilibrium dissociation constants calculated from the ratio of dissociation to association rate constants are 2.5 X 109-11) M for the high-affinity receptor (type I) and 3.2 X 10(-9) M for the low-affinity receptor (type II). These values are the same as those determined by equilibrium experiments on the isolated receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding constants of soluble NGF-receptors in rat oligodendrocytes and astrocytes in culture.

The neuronotrophic factor NGF binds to peripheral neurons of the dorsal root ganglion and the sympathetic nervous system. NGF binds to a cell surface receptor, NGFR, on these cells and displays Kd's of 10(-9) and 10(-11)M. NGF receptors have also been reported for basal forebrain magnocellular neurons. In addition, NGF specifically binds to NGFR on Schwann cells although the biological significance of this binding is not known. Here we report that NGF binds in a saturable and specific fashion to receptors on cultured isolated populations of rat astrocytes but not to oligodendrocytes. The binding to astrocytes in culture displayed a Kd of 2.7 +/- 1.0 nM with 36,000 receptors per cell.

Receptors for nerve growth factor on rat spleen mononuclear cells.

Considerable evidence is mounting to support the concept of a modulatory role for the brain and neuroendocrine system on the immune response. This neuroimmunomodulation occurs in part through the interaction of specific neurosubstances with receptors on lymphocytes and monocytes. Nerve growth factor (NGF) is a neuronotrophic factor necessary for the development and maintenance of sympathetic and embryonic sensory neurons. This trophic effect is initiated through binding of NGF at specific cell surface receptor sites on NGF-responsive cells. Several recent studies suggest that NGF may interact with cells of the immune system and may play a role in the regulation of some immunologic reactions. In this study we report on the presence of specific receptors for NGF on the surface of mononuclear cells from rat spleens. The NGF-binding sites are of the low-affinity type with Kd's in the 10(-9) M range. These receptors migrate on SDS-PAGE as two molecular species of approximately 190 and 125 kilodaltons. Our findings of receptors for NGF on lymphocytes and accessory cells support other evidence that NGF may influence immunoreactivity in vivo.