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Serum-free supernatant fluids from monolayer cultures of B-16 mouse melanoma cells were found to contain a soluble membrane associated tumor-specific antigen. The 100,000 g supernatant of the culture fluid induced an antibody response to the B-16 cells both in rabbits and in the mouse strain of origin (C57Bl/6J). Similar supernatant fluids derived from an unrelated cell line (L-929) or from normal C57Bl/6 fibroblasts did not contain the B-16 specific material. Preliminary results indicate that the B-16 specific material is a protein of low molecular weight which is released into the culture fluid chiefly by living cells and, to a lesser extent, by autolysing cells.

A radioimmune assay for the detection and quantitation of circulating immune complexes has been developed which employs the L1210 murine leukemia cell. The assay is based upon the binding of immune complexes to the L1210 through its Fc receptor followed by quantitation of the complexes with an 125I-labelled anti-IgG. The radioactivity of the cell pellet is referred to a standard curve generated by incubating the L1210 with known amounts of heat aggregated IgG (AIgG). 7S IgG of three species (human, canine, murine) do not bind significantly to the L1210 in contrast to the respective AIgG. The assay readily distinguishes between sera of healthy individuals and sera of individuals (human and canine) with diseases known to be associated with circulation immune complexes (i.e., systemic lupus erythematosus, HBAg positive acute hepatitis). The L1210 radioimmune assay is capable of detecting as little as 5 micrograms of immune complexes per ml of serum in all three species tested. The assay possesses several advantages over those currently employed, the most notable being the ability of the L1210 cell to detect immune complexes irrespective of their complement fixing properties.

The L1210 enzyme-linked immunoassay (ELIA) for circulating immune complexes, previously developed for use in humans, has been adapted for use in mice. The assay detected elevated levels of immune complexes in 100% of NZB/W female mice (6-10 months old) with age-related immune complex-mediated glomerulonephritis. Application of the assay to the serum of one year old C57Bl/6 male mice failed to demonstrate elevated levels of serum immune complexes in spite of the routine finding of renal deposition of IgG plus proteinuria in aged mice of this strain. Since the L1210 ELIA detects all IgG-containing immune complexes, regardless of size or complement fixing capacity, these results suggest that the renal IgG deposits seen in aged C57Bl/6 mice are not derived from circulating IgG immune complexes.

The frequency of rheumatoid factor (RF) seropositivity and the frequency of association of RF with immune complex (IC) elevation were determined in 72 patients with a variety of solid tumors, in 78 patients with IC-associated diseases, 27 patients with non-IC-associated diseases, and in 31 normal healthy controls. The frequency of RF positivity in cancer patients (31%) was virtually identical to that in patients with IC-associated diseases (28%), and did not differ significantly from that seen in patients with non-IC-associated disease (15%). Whereas IC elevation was clearly related to tumor progress, RF was not. RF and IC status also appeared to be unrelated to each other, not only in cancer patients, but in patients with other diseases as well. The results do not support the concept that RF reflects progressive tumor growth or an immune response to IgG-containing immune complexes.

A sensitive, quantitative, non-complement dependent enzyme-linked immunoassay (ELIA) for human serum immune complexes has been developed utilizing the L1210 murine leukemia cell and peroxidase-conjugated anti-IgG. At end-point O-dianisidine dihydrochloride method was developed which detects as little as 16.45 x 10(-5) purpurogallin units of horse radish peroxidase. The L1210 ELIA requires only five microliters of serum and reproducibly quantitates as little as 55 nanograms of an artificial immune complex standard (heat aggregated IgG). The validity of using aggregated IgG as a standard was demonstrated by the fact that it produced a dose dependent inhibition of binding of a known immune complex (peroxidase-anti-peroxidase) to the L1210 cells whereas monomeric IgG showed no inhibition. Results of the L1210 ELIA are similar to those of the L1210 radioimmune assay (RIA) which we have previously reported. There was no significant difference in mean immune complex levels between the ELIA and the RIA when serums from normal healthy blood donors were tested. A strong correlation (rs equals +0.787) of immune complex levels as determined by the ELIA and the RIA was found in 17 serum samples from patients with disease associated with elevated levels of circulating immune complexes.

The influence of pregnancy, pregnancy plasma, and pregnancy-associated lhormones on the response of peripheral blood lymphocytes to two phytomitogens (PHS and PWM) was examined in a group of 17 healthy women during pregnancy and the postpartum period. No consistent disparity between pregnancy, postpartum, and control tests was noted in either intrinsic lymphocyte responsiveness or in the effect of autologous plasma or four pregnancy-associated hormones (estriol, progesterone, hPL, and estradiol-17 beta) on lymphocyte responsiveness.

Immune complexes (ICs) were determined by the non-complement-dependent L1210 radioimmune assay on 132 serum samples collected from 53 patients with a variety of cancers. Both the mean IC levels and frequency of positive tests were significantly greater in cancer patients (mean = 96 +/- 100 microgram/ml, 46% positive) than in a control group of 67 normal healthy blood donors (mean = 39 +/- 15, 3% positive). When cancer patients were assorted into groups by disease progress, those with large or progressing tumors had significantly higher mean values (136 +/- 129) and frequency of positives (75%) than those with small or regressing tumors (58 +/- 18, 22% positive). In lung cancer patients, IC levels showed a strong inverse correlation (rs = -0.903) with survival time in patients who died, and appeared to be a better prognostic indicator than performance status (Karnofsky scale) at time of diagnosis. Serial IC measurements taken on several patients showed a decrease in levels concomitant with a favorable response to cytoreductive therapy, sustained normal levels during periods of prolonged remission, and a rise to elevated levels with (and sometimes preceding) documentation of new metastases.

A 20,000 g pellet obtained from the homogenate of CD1 mouse spleen cells has measurable peroxidase and bactericidal activities. Whole body x-irradiation stimulates the in vitro peroxidative and bactericidal activities of the spleen cells. These cells do not phagocytize. However, addition of polystyrene latex particles to spleen cells from non- or x-irradiated mice, increases glucose oxidation. The extent of this stimulation (three- to fourfold) is less than that of PMN (six- to tenfold). Interaction between particles and lymphocyte surface may be the cause of this stimulation. The increased bactericidal activity due to x-ray is attributed mainly to an increase in the peroxidase activity of the lymphocytes. Surface (membrane) action rather than phagocytosis may be involved in the bactericidal process. The spleen lymphocyte peroxidase is distinct from myeloperoxidase in several respects.

A patient with chronic membranoproliferative glomerulonephritis is presented whose serum contains a monoclonal IgG3 cryoglobulin. The presence of persistent hypocomplementemia suggested the possibility that the cryoglobulin, upon cold-induced precipitation, was capable of activating the complement system. Because visible cryoprecipitation commenced in vitro at 30 degrees C, the patient's serum and normal serum had been added the isolated cryoglobulin were repeatedly cooled to 30 degrees C and rewarmed to 37 degrees C. This reproduction of the in vivo counterpart of blood circulating through an extremity exposed to the cold resulted in activation of C3-proactivator (properdin factor B), C3 cleavage, and a 78% reduction in total hemolytic complement. This study demonstrates that IgG is capable of activating complement via the alternate pathway and reveals a mechanism through which this can occur in vivo; namely, by means of temperature dependent polymerization. In addition, we postulate that episodic complement activation initiated by the cryoglobulin contributed to the development of glomerulonephritis in this patient.

Histologic and immunofluorescence studies of the kidneys of mice bearing a progressive melanoma show a proliferative glomerulonephritis associated with immune complex deposition in the mesangium and along the glomerular basement membrane This immune complex disease is distinct from the age-associated disease of the C57BL/6J host strain and the complexes can be shown to consist of soluble tumor antigen and antitumor antibody. Furthermore, the intensity of IgG complex deposition correlates directly with tumor progress (size and metastases) and inversely with mononuclear leukocyte infiltration of the tumor. In vitro assays for lymphocyte cytotoxicity and humoral antibody were found to be less reliable indicators of tumor progress. The possible role of circulating soluble tumor antigen in modifying the immune response to tumors is discussed.

Circulating immune complexes (ICs), assayed by the L1210 enzyme-linked immunoassay, were detected in 48% of patients with hemophilia. A, 50% of patients with von Willebrand's disease, and in none of our patients with hemophilia B. Eighty-five % of the hemophilia A and B patients had mild to moderate disease with only one patient demonstrating a circulating inhibitor. No correlation was found between IC levels and hepatitis B infection, SGOT, disease severity, total quantity of factor VIII or IX infused, time interval from list infusion, or rheumatoid factor positivity. Although the nature of the ICs is not known, the similarity of IC levels between hemophilia A and von Willebrand's disease is discussed with regard to antibodies generated to non-procoagulant portions of the factor VIII molecule.

A patient had spurious thrombocytopenia resulting from a mechanism not previously described. Whereas in prior reports the in vitro phenomenon of platelet clumping has been effected by either EDTA-dependent or temperature-dependent antibodies capable of direct platelet agglutination, neither the IgG nor the IgM fractions of this patient's serum demonstrated such activity. However, agglutination was produced by incubating allogeneic platelets with the IgG fraction followed by a room temperature incubation with the rheumatoid factor-positive IgM fraction. The data support a new mechanism for spurious thrombocytopenia resulting from the interaction of a cold-reactive rheumatoid factor with antiplatelet antibody.

Platelet-associated IgG (PAIgG), circulating IgG-containing immune complexes (ICs), plasma beta-thromboglobulin (beta-TG), and platelet factor 4 (PF-4) levels were assayed in 33 consecutive patients with culture-proved bacteremia. Thrombocytopenia occurred in 18%, and no correlation was seen between platelet count and PAIgG, beta-TG, or PF-4. In contrast, when patients were assigned to groups on the basis of rising or stable counts vs falling counts, a significant increase in the frequency of elevated PAIgG was seen in those with falling platelet counts. Elevated ICs did not correlate with PAIgG, platelet count, beta-TG, or PF-4. The results of this study of patients with uncomplicated bacteremia, although clearly demonstrating intravascular platelet activation and suggesting a pathologic role for elevated PAIgG, do not support the hypothesis that platelet-bound serum ICs are responsible for either phenomenon.

1. MPO, H2O2 and C1- form a complex that undergoes intramolecular rearrangement yielding the chlorinium ion. 2. The chlorinium ion can interact with MPO, bacteria and amino acids. 3. The reaction can occur at a wide range of pH, H2O2 concentration and C1- concentration. 4. The chlorinium ion can attack different protein molecules to cause structural changes. 5. Preincubation of MPO with bacteria results in greater bactericidal activity. 6. Diffusible bactericidal agents are also produced by the MPO-H2O2-C1- system.