Production and characterization of antibody blocking epidermal growth factor: Receptor interactions

Membranes were prepared from the human epithelioid carcinoma cell line A-431 which has approx. 2 · 10⁶ epidermal growth factor receptors per cell. This membrane preparation which retained a high epidermal growth factor binding specific activity was used as an antigen to produce antisera in rabbits. Double-immunodiffusion experiments demonstrated that the immune serum contained precipitating antibodies to several components of detergent solubilized A-431 membranes.The immonoglobulin G fraction of this immune sera inhibited ¹²⁵I-labeled epidermal growth factor binding to receptors in: (1) intact human and mouse cells; (2) membrane preparations from A-431 cells and human placenta, and (3) solubilized A-431 membranes. Inhibition of ¹²⁵I-labeled epidermal growth factor binding was observed with divalent and monovalent fragments of immunoglobulin G prepared from the immunoglobulin G fraction. Also, the immunoglobulin G fraction blocked growth factor binding to membranes at low temperature (5°C).Anti-A-431 antibody blocked the induction of DNA synthesis in quiescent fibroblasts by epidermal growth factor in a manner similar to that of anti-epidermal growth factor antibody. Addition of either anti-A-431 or anti-epidermal growth factor antibodies to fibroblasts at times up to 5 h after the addition of epidermal growth factor completely reversed the hormone's mitogenic potential. At later times (after 12 h) addition of either antibody was without effect on the stimulation of DNA synthesis by epidermal growth factor. Anti-A-431 antibody did not block the induction of DNA synthesis in fibroblasts by fibroblast growth factor or serum.     

Partial purification and characterization of platelet factors stimulating the multiplication of normal human glial cells

Multiplication-stimulating activity for human glial cells was purified from human outdated platelets. By ion exchange chromatography anionic activity was separated from cationic activity. The former could be further separated by Sephadex G-200 gel chromatography into two peaks, whose molecular weights were 40 000 and < 10 000. The cationic activity was partially purified by concanavalin A (ConA) Sepharose chromatography, hydroxylapatite chromatography and SDS-polyacrylamide gel electrophoresis. The cationic activity was heterogeneous as demonstrated by isoelectric focusing (Ip 9.5–10.4), gel filtration on Bio-Gel P-150 and SDS-polyacrylamide gel electrophoresis (mol. wt 26 000–33 000). Less than 50 ng/ml was required of the factor to give a glial cell stimulation corresponding to that afforded by 1 % of human serum. A thymidine-degrading enzyme, present in human platelets and to a low degree also in human serum, was found to interfere with the assay for multiplication-stimulating activity. The enzyme (probably a thymidine phosphorylase) converted [³H]thymidine to [³H]thymine, causing a reduced incorporation of ³H into cellular DNA. This difficulty was circumvented by use of an autoradiographic estimation (per cent labelled nuclei) of the multiplication-stimulating activity.     

Uptake and degdradation of formaldehyde-treated 125I-labeled human serum albumin in rat liver cells in vivo and in vitro

The uptake of formaldehyde-treated ¹²⁵I-labelled human serum albumin in rat hepatocytes and nonparenchymal liver cells was measured in vivo and in vitro. Isolated liver cells were prepared by treating the perfused liver with collagenase. Purified hepatocytes and nonparenchymal cells were obtained by differential centrifugation. Human serum albumin was found to be taken up exclusively or almost exclusively by nonparenchymal cells in vitro and in vivo (after intravenous injection). The maximal rate of human serum albumin-uptake in vitro was comparable to that in vivo. Nonparenchymal cells degraded human serum albumin in vitro as indicated by release of trichloroacetic acid-soluble radioactivity. Degradation started about 20–30 min after addition of human serum albumin to cells and rate of degradation was proportional to rate of uptake. Human serum albumin-degradation could be studied without interference of concurrent uptake by separating cells that had been preincubated with human serum albumin from the medium and then reincubating them with human serum albumin-free medium. The lag phase before human serum albumin-degradation starts and the inhibitory effect of chloroquine on degradation indicate that human serum albumin is degraded in lysosomes. The data obtained show that enzymatically prepared nonparenchymal liver cells retain their endocytic activity in vitro. Denatured human serum albumin should be useful both as a marker for rat liver macrophages and for the study of intracellular proteolysis in these cells.     

Blocking of Lymphocyte-mediated Cytotoxicity for Rat Hepatoma Cells by Tumour-specific Antigen-Antibody Complexes

LYMPHOCYTES from tumour-bearing animals are often cytotoxic in vitro against cultured tumour cells from the same individual^1–4. It is possible that the serum of tumour-bearing hosts may contain circulating factors which interfere with the cell-mediated immune responses concerned in tumour rejection reactions⁵. Evidence has been provided by the demonstration that lymphocyte cytotoxicity against cultured tumour cells could be blocked by first exposing tumour cells to serum from tumour-bearing animals^2,3; similar effects have also been observed in cancer patients^6,7. The blocking factor in tumour-bearer serum has the characteristic properties of 7S immunoglobulins², suggesting the involvement of tumour-specific antibody. Serum blocking activity is rapidly lost, however, in animals rendered tumour free and the activity of tumour-bearer serum can be neutralized by the addition of serum from these animals^8,9. One explanation is that the blocking factor in tumour-bearer serum is antigen-antibody complex and the objective of these studies, using a transplanted rat hepatoma (D23), was to test directly whether such complexes prepared from solubilized tumour-specific antigen and antiserum exhibit blocking activity.     

Cell surface-associated growth inhibitory proteins : Evidence for conservation between mouse and human cell lines

The ability of the normal human fibroblast line (IMR91) to exhibit density-dependent regulation of growth has been examined. The line exhibits density-dependent regulation of growth; saturation density in 15% fetal bovine serum is 2 × 10⁵ cells/cm². Membranes prepared from confluent monolayers of these cells contained growth inhibitory factors to both exponentially growing IMR91 and Swiss 3T3 cells. This factor(s) appears to be similar to a previously described factor found on the surface of Swiss 3T3 cells [14]. The inhibition of DNA synthesis in growing IMR91 cultures by membranes was both time- and concentration-dependent. The effect was reversible by high serum. Specificity experiments utilizing membranes prepared from Swiss 3T3 cells indicated some species specificity for inhibition by membranes, but this specificity was no longer exhibited by solubilized membrane preparations. These results are compatible with the suggestion that both the growth inhibitory factors and their receptors are conserved through evolution.     

Stimulation of ammonium ion excretion in the toad urinary bladder by an extract of human urine

It is well known that ammonium ion excretion is increased during metabolic acidosis in mammals. The purpose of this study was to determine whether we could isolate from human urine during metabolic acidosis a factor that would stimulate NH₄⁺ and/or H⁺ excretion in toad urinary bladder. Extracts of urine from six human subjects collected during NH₄Cl-induced acidosis were prepared. These extracts were tested for their effect on NH₄⁺ excretion in hemibladders mounted between plastic chambers. The extracts significantly increased NH₄⁺ excretion in the toad urinary bladder. We found no effect on H⁺ excretion by these extracts. This ammoniuretic activity was not present in the urine when the same individuals were in metabolic alkalosis. We conclude that during metabolic acidosis a humoral factor is present which stimulates the excretion of NH₄⁺. The factor could act as a permease in the bladder cell or as a stimulator of an NH₄⁺ transport system.     

Cytotoxic effects of normal sera on lymphoid cells. I. Antibody-independent killing of heterologous thymocytes by guinea pig, rabbit, and human sera: role of the alternative pathway of complement activation.

The mechanisms whereby normal sera may cause the death of xenogeneic lymphoid cells in vitro have been reviewed in this study using guinea pig, rabbit and human sera as the source of activity and rat and mouse thymocytes as target cells. In all of the combinations analyzed the cytotoxic reactions were found to be mediated by complement (C) as evidenced by sensitivity of the sera towards either heat inactivation (56 °C, 30 min) or treatment with cobra venom factor or sodium ethylenediaminotetraacetate (EDTA). C activity was provided via the alternative pathway in every instance: (i) both C4-deficient guinea pig serum and C2-deficient human serum displayed cytotoxicity on the target cells; (ii) sera from all three sources were active in the absence of free Ca²⁺, which is required to activate C via the classical pathway; and (iii) GPS incubated at 50 °C for 20 min to destroy the activity of factor B of the alternative pathway lacked significant cytotoxic activity while still able to lyse sensitized sheep red blood cells, a reaction proceeding via the C142 pathway. Two independent lines of evidence appeared to exclude the possible role of antibodies in nonspecific serum cytotoxicity. First, the cytotoxic capacities and the titers of guinea pig and rabbit sera were not significantly affected after absorption with target cells in the presence of EDTA, i.e., in the absence of free divalent cations, a condition which does not interfere with antigen antibody binding. By contrast, the activity was eliminated when absorption was performed in the absence of chelating agents or in the presence of a selective Ca²⁺ chelator, sodium ethyleneglycoltetraacetate, plus excess Mg²⁺ These observations also highlight the Mg²⁺-dependence of the removal of activity by absorption. Second, γ-globulins isolated from a highly cytotoxic guinea pig serum were not toxic for rat thymocytes when tested in the presence of rat C. These results suggest that conventional antibodies, whether of “natural” origin or otherwise, are unlikely to play a role in serum-produced nonspecific cytotoxicity. Furthermore, and since incubation of human serum with rat or mouse thymocytes produced conversion of factor B, “absorption” of cytotoxic activity would seem to be more likely a consequence of the consumption of C activity via the C3 shunt than of the removal of any antibodies.     

Demonstration of competence and progression activities for human fibroblasts

Human embryonic lung fibroblasts (HEL) completed 4.5 population doublings in 6 days when maintained in DMEM supplemented with 10% human whole blood serum (WBS), plasma-derived serum (PDS) or defibrinogenated plasma containing 10 mM CaCl₂. Plasma in the absence of additional calcium promoted less growth. Sera and plasma chromatographed through carboxymethyl Sephadex (CMS) supported only one population doubling. Increased growth resulting in three doublings was observed in CMS-treated WBS or PDS supplemented with commercially prepared platelet-derived growth factor (PDGF). The magnitude of this PDGF response was dependent on serum concentration. A significant increase in the proportion of cells incorporating [³H]thymidine was observed in confluent cultures exposed to PDGF prior to incubation in WBS-CMS or PDS-CMS indicating competence and progression activities for human fibroblasts. In contrast, cells maintained in the presence of plasma-CMS failed to grow in response to PDGF. Factors bound to CMS columns restored growth-promoting activity to PDGF-supplemented WBS-CMS, PDS-CMS and plasma-CMS. However, growth-promoting CMS-bound components from plasma were lost during dialysis through membranes excluding materials above 12000 MW.     

Permeabilization of Ehrlich ascites tumor cells by human serum

Human serum rapidly permeabilized Ehrlich ascites tumor cells to inorganic cations such as Rb⁺ and Ca²⁺; serum from several other species showed little or no activity. The effect of human serum was not reversed by washing the cells. Human serum, deficient in specific complement proteins, had no activity, but was reactivated by the addition of the missing complement component. Since Ca²⁺ was not required for the permeabilization, the alternative pathway of complement activation was implicated. Human serum deficient in Factor B of the alternative pathway was ineffective, but permeabilizing activity was restored by addition of Factor B. Rb⁺ uptake of several other cells was not inhibited by human serum. We conclude that an interaction between human complement and Ehrlich ascites tumor cells is responsible for the membrane lesion observed.     

Nerve growth factor binds to serum alpha-2-macroglobulin.

Nerve growth factor labelled with ¹²⁵I and incubated with mouse serum became associated with a serum protein that eluted in a high molecular weight position on Sepharose 6B. The binding protein for the nerve growth factor was purified to homogeneity and characterized as the murine counterpart of the human α₂-macroglobulin. The nerve growth factor was quantitatively bound to highly purified mouse α₂-macroglobulin after a few hours of incubation. The interaction displayed little species specificity in as much as human and equine α₂-macroglobulin strongly bound murine nerve growth factor.     

In vitro Proliferation of Human Bone Marrow Mesenchymal Stem Cells Employing Donor Serum and Basic Fibroblast Growth Factor

A method for the in vitro proliferation of human bone marrow mesenchymal stem cells (MSCs) employing a medium not containing fetal calf serum (FCS) was developed for a regenerative medicine of cartilage using MSCs. Without using density-gradient centrifugation, the bone marrow aspirate was poured into a dish (6.0 \times 10⁵ nucleated cells/cm²) with DMEM medium containing 10% serum (FCS or donor serum) and basic fibroblast growth factor, and incubated at 37 °C under a 5% CO₂ atmosphere. The density of adhesive cells incubated with the medium containing human serum and basic fibroblast growth factor (10 ng/ml) almost reached confluence at 19d and was 1.4-2.7 times that in the medium containing only FCS. The density of cells incubated with the medium containing only human serum was 0.1-0.6 times that in the medium containing only FCS. The content of CD45^- CD105⁺ cells among the cells harvested after a 19-d incubation in the medium containing human serum and basic fibroblast growth factor was higher than 90%. This high content and chondrogenic activity, which was confirmed by pellet cultivation and staining with Safranine O, were maintained even after further subcultivation in the medium to 17 population doubling levels. Consequently, this method might be applicable to in vitro proliferation of MSCs for the regeneration of cartilage.     

Purification and Some Properties of a Fungal Lipase Preparation Used for Milk Flavouring

Intracellular lipase of a strain of Rhizopus fungus which is effective for producing a milk flavor was purified and fractionated into two components, I and II, by DEAE Sephadex A-50 column chromatography. They both proved homogeneous by electrophoresis and ultracentrifugal analysis. The sedimentation coefficient was respectively calculated to be 5.8×10^?13 for lipase I, and to be 2.2×10^?13 for lipase II. From substrate specificity, it was found that lipase I was an ordinary lipase hydrolyzing olive oil and tributyrin favourably, while, II, rather, a special lipase having a high affinity towards tricaprylin. They, also, respectively had an apparent phospholipase activity on soy-lecithin and, clearing activity on chylomicron prepared from olive oil and human serum. Their mode of action, and the effect of metals and emulsifying agents on their activity are also presented.     

Rapid purification of cobalamin-binding proteins using immobilized aminopropylcobalamin

Aminopropylcobalamin (AP-Cbl), prepared from 3-chloropropylamine and cob(I)alamin, was immobilized on CNBr-activated Sephacryl beads. The product, Sephacryl-aminopropylcobalamin, contained ca. 1 μmol of AP-Cbl/ml of beads. Cobalamin-binding proteins in biological fluids were adsorbed selectively and quantitatively by Sephacryl-aminopropylcobalamin. After being washed to remove extraneous protein, the beads were photoirradiated to release the cobalamin-binding proteins as their aquacobalamin complexes. The latter could be converted to labeled cyanocobalamin complexes by treatment with [¹⁴C]KCN. The efficacy of this affinity chromatographic method is illustrated by the purification to near homogeneity and in high yield of three representative proteins: transcobalamin II from rabbit serum, intrinsic factor from human gastric juice, and R binder from human saliva.     

Molecular feature of the nerve growth factor in human serum

High molecular weight binding components which bind [¹²⁵I] mouse β nerve growth factor exist in human serum. The binding of β nerve growth factor to the serum components was inhibited at alkaline condition. After gel filtration of human serum on a Sephadex G-150 column at neutral condition, the nerve growth factor-like immunoreactivity was observed in only one peak, differing from the high molecular weight serum components. However, at alkaline condition two peaks with nerve growth factor-like immunoreactivity appeared; one was almost at the position observed at neutral pH, and the other was a new peak eluted approximately to the column volume. these results suggest that there are at least two nerve growth factor-like molecules in human serum and most of the nerve growth factor in the serum exists in a complex form associated with serum components with high molecular weight.     

Isolation and Purification of Human Serum Alpha-1 Acid Glycoprotein

Abstract

Alpha-1 acid glycoprotein (or orosmucoid) was obtained in a pure state from normal human serum by ion exchange chromatography followed by curtain electrophoresis and a final ion exchange chromatography step. Pure α₁ acid glycoprotein (α₁A) has a sedimentation coefficient of 3.1 S and a diffusion coefficient of 5.2 × 10^?7cm² sec^?1, which yields a molecular weight of 44,680 Daltons and an asymmetry factor of 14.6. The αA prepared in the manner here described appears less denatured than the same protein isolated by the Cohn fractionation method.^1,2 ' Alpha-1 A acts as a depressant of phagocytosis³ and is one of the constituents of Mowbray's serum fraction,″which induces a prolongation of skin homografts.     

Effect of serum on prostaglandin production during the co-culture of human thyroid cells and peripheral blood mononuclear cells

Prostaglandin generation by human peripheral blood mononuclear cells is enhanced during co-culture with human thyroid cells. The objective of the present study was to determine the influence of various sera on this process. Human thyroid adenoma cell monolayers were cultured with normal human peripheral blood mononuclear cells for three days in the presence of a variety of sera, or serum fractions. Prostaglandin E (PGE) in the medium was measured by bioassay or by radioimmunoassay. Significantly more PGE was generated in cultures containing fetal calf serum than in those containing human serum. This difference was not abolished by dialysis of the human serum. When the 50% (NH₄) ₂SO₄ precipitate of the serum was used, PGE generation was similar to that in fetal calf serum, indicating the presence of an inhibitory factor in human serum. The degree of this inhibitory activity was similar in autologous and heterologous human serum, as well as in normal subjects and patients with Graves' disease. Gel filtration and ion-exchange chomatography of human serum showed the inhibitor to co-migrate with albumin. Evidence presented suggests that the inhibitor is not albumin itself but is, instead, a factor tightly bound to albumin. Inhibitory activity was also found in rabbit, goat, rat and cow serum. Prostaglandins are potent modulators of immune-cell function. These data indicate that this process may be modulated by a factor in mammalian serum. The relative absence of this factor in fetal serum may have important implications in regard to the profound changes which occur in the immune system after birth.     

Demonstration of an antibody against platelet-derived growth factor

An antibody was raised in a rabbit against platelet-derived growth factor (PDGF), purified from human platelets. It recognized specifically ¹²⁵I-labelled PDGF, as demonstrated by gel electrophoretic analysis of precipitated radioactivity. Addition of immunoglobulins from the immune serum to cell cultures together with PDGF inhibited the multiplication-stimulating activity of the growth factor. Further, multiplication-stimulating activity in crude platelet lysate was retained upon passage over a column of Sepharose-bound anti-PDGF; it was recovered after elution of the column with acid. A radioimmunoassay for PDGF was developed, permitting the determination of 5 ng PDGF/ml. Platelet factor 4 and β-thromboglobulin, both being proteins of the platelet α-granule, did not cross-react in the radioimmunoassay for PDGF. The radioimmunoassay was utilized to examine the immunological relationship between PDGF and some other growth factors. The anionic growth factors previously demonstrated in pletelets were found to be antigenically distinct from cationic PDGF. Similar results were obtained with epidermal growth factor (EGF) or fibroblast growth factor (FGF). In contrast, osteosarcomaderived growth factor, a PDGF-like growth factor previously known as a product of cultured human osteosarcoma cells, showed some cross-reactivity in the radioimmunoassay for PDGF. It was concluded that osteosarcoma-derived growth factor (ODGF) has both physical-chemical and immunological properties in common with PDGF.     

Kinetics of conversion ofNeisseria gonorrhoeae to resistance to complement by cytidine 5′-monophospho-N-acetyl neuraminic acid

Freshly isolated gonococci upon subculture are readily lysed by normal human serum although a few strains remain inherently resistant to the complement activity. The sensitive gonococci can be converted to serum resistance by incubation with a host derived factor referred to as cytidine 5-monophospho-N-acetylneuraminic acid (CMP-NANA). These gonococci resist complement mediated killing due to their sialylation of an epitope structure on a component of lipo-oligosaccharide (LOS). In the present study, the kinetics of conversion to serum resistance by the action of sialyltransferase (STase) inNeisseria gonorrhoeae was followed with very low concentrations of CMP-NANA. This conversion could not be perceived at 2×10^–3 nmol.ml^–1 but was fully attainable from 8×10^–3 to 2×10^–2 nmol.ml^–1 CMP-NANA. When pretreated up to 100 min in presence of the very low concentration of 2×10^–3 nmol.ml^–1, a potentiating effect on the conversion of gonococci by 2×10^–2 nmol.ml^–1 was observed in relation to the time of preincubation. This action was abolished after exposure to a subinhibitory concentration of chloramphenicol (0.5 µg.ml^–1). The gonococci recovered their ability to convert to serum resistance following adequate washing. The potential for increase in STase activity should be of interest for understanding the conversion from a serum sensitive to a serum resistance state.     

Studies on esterolytic activity of alternative complement component factor B

Alternative complement component factor B was purified from human plasma and its esterolytic activity on various α-naphthylester derivatives was examined. Of these substrates, LeuAlaArg-naphthylester was the most susceptible. The K_m value of factor B for this substrate was about 5·10⁻⁴ M. 6-Amidino2-naphthyl-4-guanidinobenzoate inhibited the esterolytic activity of factor B. After activation of factor B, the esterolytic activity did not increase and the decayed form, Bb, cleaved the substrate to the same extent as factor B.     

A platelet-derived immunoregulatory serum factor with T cell affinity

A factor in mouse and human serum which enhances immune responses to SRBC and TNP-Ficoll in mice has been described. This factor completely reverses immunosuppression induced by syngeneic lymphoma (RCS) or allogeneic lymph node cells. T lymphocytes bind this factor, since the activity can be removed from serum by absorption with T cell containing spleen cells or cloned cytotoxic T lymphocytes. Furthermore, the factor is active only in the presence of mature T cells. Platelet alpha-granules seem to be the source of this serum factor because 1) the activity is lacking in plasma, in serum prepared in the absence of platelets, and in serum prepared from a patient lacking platelet alpha-granules and 2) isolated mouse or human platelets release an immunostimulatory factor with very similar properties upon incubation with thrombin.