A novel monoclonal human IgM autoantibody which binds recombinant human and mouse tumor necrosis factor-alpha.

Several monoclonal human IgM antibodies to recombinant human tumor necrosis factor-alpha (rhTNF alpha) have been generated and partially characterized. The F78-1A10-B5 monoclonal antibody (mAb) (B5) binds to rhTNF alpha with a titer comparable to three high-affinity neutralizing mouse mAbs, when tested by ELISA. However, the B5 mAb binds relatively weakly to soluble rhTNF alpha. It appears to bind to epitopes on rhTNF alpha distinct from those bound by the mouse mAbs for three reasons. First, preincubation of plate-bound rhTNF alpha with mouse mAbs does not decrease or compete subsequent B5 mAb binding. Second, rhTNF alpha complexed to the mouse mAbs can still be bound by B5 mAb. Third, the mouse mAbs neutralize TNF alpha cytotoxicity whereas the B5 mAb does not. Binding analyses indicate that this human IgM autoantibody binds to both human and mouse recombinant TNF alpha, but not to other antigens commonly recognized by polyreactive natural IgM autoantibodies. The high level of amino acid identity between the human and mouse TNF alpha molecules suggest that the B5 mAb is monospecific for a given epitope shared by these two forms of TNF alpha. This spectrum of characteristics makes B5 a novel mAb.

The B5 monoclonal human autoantibody binds to cell surface TNF alpha on human lymphoid cells and cell lines and appears to recognize a novel epitope.

A human IgM monoclonal antibody (B5) recognizing human TNF alpha was established from peripheral blood lymphocytes by transformation with Epstein-Barr virus and subsequent cell fusion. The B5 monoclonal antibody (mAb) binds to cell surface TNF alpha (csTNF alpha) on human T cells, B cells, and monocytes. In addition, this autoantibody binds to csTNF alpha on a variety of lymphoid and monocyte lineage cell lines of human origin, as well as astrocytomas, a breast carcinoma, and a melanoma. Interestingly, the B5 mAb also binds to chimpanzee lymphocytes and to mouse T lymphoma cell line csTNF alpha. Many neutralizing mouse anti-TNF alpha mAbs do not exhibit comparable binding to csTNF alpha. This is consistent with the previous demonstration that B5 recognizes an epitope on TNF alpha distinct from those recognized by three neutralizing mouse anti-TNF alpha mAbs. B5 binding to csTNF alpha is specific since it can be inhibited by TNF alpha. No inhibition of B5 binding was seen by a neutralizing mouse anti-TNF alpha mAb. The B5 autoantibody appears to recognize the transmembrane form of TNF alpha and most likely also recognizes TNF alpha associated with its receptor. The unique specificity of this B5 autoantibody provides some additional insight into the complex physiology of cell surface-associated TNF alpha.

Cell-type differences in the serum and cyclic AMP-dependent regulation of ornithine decarboxylase activity in two cultured fibroblast types.

Serum re-feeding stimulated ornithine decarboxylase (ODC) activity 8 to 10-fold in FS fibroblasts and 5 to 8-fold in 3T3 fibroblasts. Addition of dibutyryl cyclic AMP or 3-isobutyl-1-methylxanthine at the time of serum re-feeding further stimulated ODC activity in 3T3 fibroblasts but inhibited the serum stimulation of ODC activity in FS fibroblasts. It is suggested that serum and cyclic AMP independently regulate ODC activity in cultured fibroblasts.

A comparison of ornithine decarboxylases from normal and SV40-transformed 3T3 mouse fibroblasts.

Ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) has been purified from 3T3- and SV40-transformed 3T3 mouse fibroblasts by affinity chromatography, and the physicochemical properties of the two enzymes compared. Measured properties include molecular weight of the active species, subunit molecular weight and specific activity of the purified enzymes, kinetic parameters, thermostability, degradation rate in vivo and immunological cross-reactivity. Although crude extracts of the transformant possess more ornithine decarboxylase activity per mg of protein than the parent strain, there is no evidence for the appearance of an altered form of the enzyme in these cells. The results reported in the present paper indicate that the increased ornithine decarboxylase activity in the transformed cells is the result of higher enzyme biosynthesis de novo.

Comparison of collagen and glycosaminoglycan synthesis in attaching control and diabetic human skin fibroblasts.

Cultured fibroblasts derived from normal subjects and juvenile diabetics attach in the absence of serum to plastic culture dishes and secrete macromolecules, including collagenous components, hyaluronic acid, and proteoglycans into the medium and onto the plastic surface where they form a microexudate carpet. Most diabetic fibroblasts examined did not spread as well as normal cells during a 4-hr interval after the initial attachment. There were no significant differences between normal and diabetic cells with respect to proline and lysine incorporation and lysine hydroxylation. The percentage glycosylation of hydroxylysine was marginally higher in the media proteins of diabetic cells, but glycosylation in both normal and diabetic cells was elevated over that typically observed in human skin collagen. Collagenous components were estimated to constitute approximately 15-20% of the microexudate carpet fraction in both normal and diabetic cell strains. Diabetic fibroblasts exhibited a marginally lower ratio of heparan sulfate to chondroitin sulfate in the cell surface to matrix microexudate carpet fraction (trypsinate) than did normal fibroblasts. The hyaluronate and chondroitin sulfate contents of this fraction of diabetic cells were not significantly different from those of normal cells.

The synthesis of macromolecular 3-hydroxyproline by attaching and confluent cultures of human fibroblasts.

The synthesis of collagen has been studied during the attachment of freshly trypsinized human fibroblasts to culture vessels by measurement of the incorporation of radioactive proline into macromolecular hydroxyproline. Collagenous protein(s) was found to be a component of a substrate-attached material ('microexudate carpet') synthesized rapidly during cell attachment in the absence of serum. The ratio of 3-hydroxyproline/4-hydroxyproline in the collagenous proteins synthesized during cell attachment was found to be 4-5 fold higher than that of normal type I collagen. The synthesis of 3-hydroxyproline by confluent cultures was diminished by serum deprivation, and was shown to require higher concentrations of ascorbate than the synthesis of the 4-hydroxy isomer.

Enhanced synthesis and extracellular accumulation of hyaluronic acid during stimulation of quiescent human fibroblasts by mouse epidermal growth factor.

The effect of mouse epidermal growth factor (mEGF) on the synthesis of glycosaminoglycans and glycoproteins by human fibroblasts has been studied. The addition of physiological concentrations (10(-9)M) of mEGF to quiescent cultures preincubated in the absence of serum was found to elicit an increased incorporation of 3H-glucosamine into the glycosaminoglycans and glycoproteins of both the cellular and extracellular fractions. Although the growth response to the factor, as measured by DNA replication, was minimal under these conditions as compared with the effect of serum, the mEGF-induced incorporation of glucosamine into these cellular constituents and into the extracellular glycoproteins was comparable to that elicited by serum shift-up. Serum, however, caused a significantly larger incorporation of glucoasimine into extracellular, acid-soluble glycosaminoglycans, which were shown to contain hyaluronic acid as the major component. As previously demonstrated, the growth response to mEGF can be enhanced several fold by an mEGF-binding arginine esterase, which is normally associated with the factor in vivo, and by ascorbate. The esterase was found to increase markedly the mEGF-induced incorporation of glucosamine into extracellular hyaluronic acid, while the addition of ascorbic acid did not significantly alter glucosamine incorporation.

Induction of human fibroblast proliferation by epidermal growth factor (EGF): enhancement by an EGF-binding arginine esterase and by ascorbate.

The effect of mouse epidermal growth factor (mEGF) and an mEGF-binding arginine esterase on the growth of cultured human fibroblasts has been studied. Physiological concentrations (10(-9)-10(-10) M) of the growth factor were found to stimulate DNA replication and cell proliferation in quiescent cultures, and the arginine esterase, which is normally associated with mEGF in vivo, was shown to enhance this growth effect synergistically. The cellular response to mEGF was dependent upon a low, growth-limiting concentration of serum in the extracellular medium. Ascorbic acid, which alone exhibited no growth-promoting effect, could partially replace this requirement, and was found to elicit a rapid and marked increase in proline hydroxylation. Quiescent cultures in serum-free medium containing ascorbic acid were stimulated by the combination of mEGF and the esterase in a manner comparable to that achieved with serum shift-up. The possible requirement of a collagen-containing extracellular matrix for the growth response to mEGF is discussed.

The cytotoxins of cobra venoms. Isolation and partial characterization.

Eight basic proteins which lyse virus-transformed mouse fibroblasts in culture have been isolated from the venoms of six Asian Naja naja subspecies. These cytotoxins appear to represent an homologous series of proteins, all within the molecular weight range of 7000-8000. They have been divided into three arbitrary types on the basis of amino acid composition, electrophoretic mobilities and elution order upon ion-exchange chromatography. The rate at which the toxins effect cell lysis: (1) appears to be a function of the basicity of each toxin; (2) is dependent upon toxin concentration; (3) is temperature dependent; and (4) is inhibited by heparin sulfate. In view of the physical changes, which the cell undergoes during lysis and of the various factors which affect the action of these proteins, it is proposed that interaction of membrane receptors with the toxin, leading to alteration of cell membrane structure, is the principal event which ultimately leads to the disruption of the cell.

Characterization of the binding of 125-I-labeled epidermal growth factor to human fibroblasts.

Epidermal growth factor (EGF) was labeled with 125-I by a lactoperoxidase technique. The unlabeled, monoiodinated and diiodinated species were separated by DEAE-cellulose chromatography and found to possess equivalent biological activities. The binding of monoiodinated epidermal growth factor to human fibroblasts was specific in that unrelated polypeptides did not affect the binding reaction. The binding reaction was a saturable process and was time- and temperature-dependent. A Scatchard analysis of the binding data indicated that each cell was capable of binding approximately 100, 000 molecules of 125-I-EGF. The apparent dissociation constant for the binding reaction was calculated to be 2.7 to 4.3 times 10-minus 10 M. Subsequent to the binding of 125-I-EGF to the fibroblasts, the growth factor was degraded by a cell-mediated proteolysis and [125-I]monoiodotyrosine appeared in the medium. The extent of degradation was reduced by the protease inhibitors, tosyl-L-lysine chloromethyl ketone and the benzyl ester of guanidobenzoic acid. Active binding sites of 125-I-egf appeared to be present in some but not all cell types. These results demonstrated that cells derived from a number of species (human, mouse, rat, and chick) possessed receptors that interacted with this mouse-derived growth factor.

The relationship of DNA replication to the control of protein synthesis in protoplasts of T4-infected Escherichia coli B.

The relationship of DNA replication to the control of early enzyme synthesis and the formation of late proteins was studied in protoplasts of E. coli B cells infected with an amber mutant, T4am130. In this phage-host system the bacterial DNA is not completely degraded, and the onset and the extent of phage DNA synthesis could be regulated by use of 5-fluorodeoxyuridine and thymidine. It was found that DNA replication is necessary for the transcription of "late" regions of the phage genome and, thus, for the synthesis of late proteins. In addition, the sustained synthesis of late RNA and the tail-fiber protein was shown to require the continued replication of DNA. Furthermore, the mechanism regulating the cessation of early enzyme synthesis appeared to become operative shortly after the onset of DNA replication and required a limited amount of DNA synthesis for its full expression.