Bovine glucagon, a polypeptide of 29 amino acids, was immunogenic in guinea pigs. The immunologic determinants of glucagon were investigated using isolated tryptic peptides of the hormone. Antibodies from virtually all of more than two dozen animals had specificity primarily for the amino-terminal heptadecapeptide (NM) and showed little or no binding with the carboxy-terminal undeca- and dodecapeptides (C). The smallest synthetic peptide of a series initiated at residue 16 which measurably bound antibody comprised residues 5–16 of glucagon. In cellular immune assays, both NM and C elicited delayed cutaneous reactions and inhibited the migration of peritoneal cells from immune animals. However, only intact glucagon and its C fragment stimulated lymphoid cells to synthesize DNA. While glucagon was somewhat more active than C, the addition of NM to C did not enhance its transforming activity. The smallest synthetic carboxy-terminal peptide with discernible transforming activity comprised residues 19–29 of glucagon. In both native and synthetic C peptide preparations, the undecapeptide was generally more active than the dodecapeptide, although cells from different animals gave different response patterns. The difference between the two is the presence of arginine at the amino-terminus of the peptide chain. Thus, the recognition specificity of populations of antigen-reactive cells from different animals displays a variation which is at least superficially analogous to that of populations of antibody molecules. In limited experiments using NM and C peptides as immunogens, neither gave rise to delayed hypersensitivity or to glucagon-binding circulating antibody, following a regimen which invariably provoked these responses when glucagon itself served as the immunogen. These results indicate that glucagon was cleaved by trypsin along functional lines into two parts, one of which housed the major antigenic determinant and the other of which carried the major immunogenic determinant, and they are highly compatible with a two-cell mechanism of immune induction. An apparent dissociation between the capacity to provoke delayed hypersensitivity reactions and to transform antigen-reactive cells in culture was observed.

We have tested the hypothesis that tumor necrosis factor (TNF), by binding to and activating granulocytes, may contribute to the pathogenesis of gram-negative sepsis and the adult respiratory distress syndrome (ARDS). Buffy coat granulocytes incubated with as little as 0.5 ng/mL of recombinant TNF (rTNF) showed a dose-related increase in nitroblue tetrazolium dye reduction, in granulocyte polarization, in superoxide anion release, and in visually apparent aggregation. Purified lipopolysaccharide (1 microgram/mL) caused polymorphonuclear (PMN) aggregation and activation that was neutralized by polymyxin B. The release of superoxide was augmented by preincubation of the PMNs with gamma-interferon. The effect of TNF was neutralized by TNF- specific murine monoclonal antibodies but not by polymyxin B. Scatchard analysis of 125I-rTNF binding to granulocytes revealed about 1,200 receptors per cell with a Kd of 4.9 X 10(-10) mol/L. These results suggest that the release of TNF by mononuclear phagocytes contributes to granulocyte activation and aggregation during inflammation.

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We have selected a thioguanine-resistant lymphoblastoid cell line (LTR228) that forms human-human hybrids with high efficiency. Fusions with peripheral B cells consistently yield one colony per 105 cells plated. To produce antitetanus monoclonal antibodies, we withdrew blood from persons who had recently received booster injections of tetanus toxoid. T cells were separated from peripheral mononuclear cells by 2-aminoethylisothiouronium bromide-induced rosette formation, given 1,500 rads (1 rad = 0.01 gray), and cultured in a 1:1 ratio with nonrosetting cells. (6,7), and fusion with transformed lymphoblastoid cell lines (8, 9), or combinations of the above (10,11).We have developed an EBV-positive splenic lymphoblastoid cell line, LTR228, that forms stable human-human hybridomas with high efficiency. Here we describe the production and characterization of antitetanus human monoclonal antibodies.MATERIALS AND METHODS Cell Line. LTR228 is a lymphoblastoid cell line that originated in spleen cell culture (12). We selected a spontaneous 6-thioguanine (20 ,g/ml)-resistant mutant for its high fusing capacity. Our line is mycoplasma-free and doubles every 16 hr when grown in Iscove's Dulbecco's minimal essential medium (DME medium) (GIBCO) with 15% fetal calf serum. LTR228 has a typical lymphoblastoid nuclear EBV nuclear antigen staining pattern.Preparation of B Cells. Volunteers were vaccinated intramuscularly with tetanus toxoid (Wyeth, Philadelphia). Peripheral blood (80 ml) was drawn in heparin 9 days after vaccination. Lymphocytes were separated by Ficoll-Hypaque density gradient centrifugation. The lymphocytes were washed twice in Hanks' balanced salt solution and the T cells formed rosettes with 2-aminoethylisothiouronium bromide-treated sheep erythrocytes as described (13). The rosetted T cells (6 X 106) were exposed to 1,500 rads (1 rad = 0.01 gray) in a Gammacell 40 gamma-irradiator (Atomic Energy of Canada) and subsequently mixed with 6 x 106 nonrosetting B cells. The mixed cells were cultured at a density of 106 per ml in Iscove's DME medium/ 15% fetal calf serum/1% pokeweed mitogen (GIBCO). After 3 days the cells were again separated by Ficoll-Hypaque to obtain a population of primarily B-cell blasts. Preliminary experiments showed that B-cell blasts fused better than unstimulated lymphocytes. The irradiated rosette-forming T cells gave much less suppression than unfractionated lymphocytes.Fusion Protocol. Cells were combined and fused according to the protocol of Brahe and Serra (14) with slight modification. Cells were fused with 40% polyethylene glycol 4,000 (BDH)/ 10%0 dimethylsulfoxide/poly(L-Arg) (5 ,ug/ml) (Sigma) in Hanks' balanced salt solution without calcium and with magnesium. By using this technique, we consistently observed low cell death and formation of 3-8% heterokaryons. Cells were cultured 24 hr after fusion prior to addition of hypoxanthine (100 ,uM)/azaserine (8 ,g/ml) in Iscove's DME medium with 15% fetal calf serum. Fusions were plated at a density of 105 cells per microtite...

Antibody titer, lymphocyte stimulation and leukocyte migration inhibition with chlamydial antigens were determined repeatedly over many months on human subjects. The volunteers were retrospectively placed into four groups on the basis of clinical, laboratory and epidemiologic criteria. Group A consisted of persons with proven or probable chlamydial infection, including an illness confirmed by chlamydial isolation or seroconversion, or a clinically compatible illness with positive serologic results. Group B were sexual partners or close contacts of group A individuals. Group C were laboratory workers with prolonged exposure to viable chlamydiae or their antigens. Group D included persons of comparable age as those in groups A and B, but lacking a history of symptomatic chlamydial infection or of contact with chlamydiae. Individual cases illustrated the rise of antibody and some cell mediated immunity reactions (CMI) with active chlamydial infection. By contrast, laboratory exposure resulted in elevation of CMI but not of antibody. Statistical analysis of the results in 46 volunteers tested repeatedly indicated a strong association of specific antibody with lymphocyte stimulation, but not with leukocyte migration inhibition. Regression analysis suggested that the type of exposure markedly influenced the relationship between antibody and lymphocyte stimulation. Measurement of immunotype-specific antibody titer by microimmunofluorescence (or an equally sensitive method) remains the best laboratory indicator of past chlamydial infection. Neither antibody nor CMI can, as yet, be definitely related to resistance to re-infection in humans.