John H. Wallace

Assessment of a method for immunochemical detection of antigen on nitrocellulose membranes.

Immunoblotting techniques are widely used for detection of antigen immobilized on nitrocellulose membranes. There are many immunolabeling methods and staining methods available to disclose the presence of antigen in such techniques. Five common staining methods each for alkaline phosphatase and horseradish peroxidase were examined. The staining methods with the highest sensitivity and the lowest background were selected for studies comparing five immunological labeling methods using human IgG as a model antigen. Results were evaluated on the basis of the least amount of detectable antigen and background staining. The most sensitive dot-blot method was then tested for its applicability to Western blots. For both dot-blots and Western blots, the immunoalkaline phosphatase methods are more sensitive than the corresponding immunoperoxidase methods. The use of biotinylated secondary antibodies and an avidin-enzyme conjugate is recommended. Disclosure of alkaline phosphate is best achieved with naphthol AS phosphate as substrate and fast blue BB as chromogen. Peroxidase is best stained using H2O2 and diaminobenzidine (DAB). Potential endogenous enzyme activities are demonstrable by blotting methods but can be inhibited by including levamisole in the disclosure reaction medium for calf intestinal alkaline phosphatase indicators, or by incubation of blots with sodium azide and hydrogen peroxide before immunolabeling when using horseradish peroxidase indicators.

Treatment of murine immune complex glomerulonephritis with prostaglandin E2: Dose-response of immune complex deposition, antibody synthesis, and glomerular damage

A model of immune complex glomerulonephritis (ICGN) produced in mice by the daily injection of apoferritin was employed to study the effect of treatment with various doses of prostaglandin E2 (PGE2) on glomerular damage, immune complex deposition, proteinuria, and serum anti-apoferritin antibody. Administration of PGE2, 200 micrograms twice daily, resulted in a significant decrease in glomerular damage and immune complex deposition, prevented the development of proteinuria, and significantly reduced serum levels of anti-apoferritin antibody. PGE2, 100 micrograms twice daily, resulted in a decrease in immune complex deposition as assessed by immunofluorescence microscopy, but this dosage did not significantly alter glomerular damage, proteinuria, or antibody levels. PGE2 dosages of 50 and 25 micrograms twice daily had no effect on any of these parameters. The protective effect of PGE2 on the development of ICGN occurred only at dosages that were associated with decreased anti-apoferritin antibody.

Modulation of the human in vitro antibody response by human leukocyte interferon preparations

Abstract The ability of human leukocyte Interferon to modulate the plaque-forming-cell response of human peripheral blood leukocytes to horse red blood cells was examined. Human peripheral blood mononuclear cells were cultured in vitro with the addition of varying doses of human leukocyte interferon 24 hr prior to, simultaneously with, and 24 hr after sensitization of the cultures with horse red blood cells. Plaque-forming-cell responses were measured 5 days after sensitization with antigen using poly- L -lysine-coupled horse red blood cell monolayers. When human leukocyte interferon preparations were added 24 hr prior to sensitization with antigen, a significant enhancement of the plaque-forming-cell response was observed. When the interferon was added simultaneously with antigen, the plaque-forming-cell response was significantly suppressed. Therefore, human leukocyte interferon appears to have a time-dependent immunomodulatory activity. The kinetics of immunomodulation appear to be different from those of previously described mouse models.

Regulation of oxygen radical release from murine peritoneal macrophages by pharmacologic doses of PGE2

The ability of pharmacologic doses of PGE2 to alter the release of superoxide (O2-) and hydrogen peroxide (H2O2) from elicited peritoneal macrophages (M theta) was studied. Twice-daily administration of 200 or 100 micrograms of PGE2 to mice during accumulation of peritoneal M theta resulted in a significant reduction in M theta recovery and in the triggered release of H2O2, but not O2-. Cultivation of elicited M theta from normal mice with concentrations of PGE2 in excess of 10(-7) M for 24-48 h resulted in a significant reduction in the triggered release of H2O2, but not O2-. Cultivation for shorter periods of time or with lower concentrations of PGE2 failed to alter H2O2 release. This effect of PGE2 was reproduced by the phosphodiesterase inhibitor theophylline. The ability of PGE2 to inhibit H2O2 release in the presence of normal production of O2- was not prevented by the addition of superoxide dismutase. Cultivation of peritoneal M theta with 10(-5) M PGE2 for 48 h failed to increase intracellular catalase, although increased H2O2 scavenger activity was demonstrated. The inhibition of extracellular release of H2O2, but not O2-, by pharmacologic doses of PGE2 may be one mechanism for the anti-inflammatory action of this compound.

Release into Culture Medium of Membrane-Associated, Tumor-Specific Antigen by B-16 Melanoma Cells

Summary 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 (C57B1/6J). Similar supernatant fluids derived from an unrelated cell line (L-929) or from normal C57B1/6 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.

Incorporation of 3H-Thymidine into PHA-Stimulated Rabbit Peripheral Blood Lymphocytes. Kinetics of the Response

Summary Rabbit PBL were shown to incorporate predictably high levels of 3H-TdR following stimulation with PHA. The conditions necessary for optimal stimulation of rabbit PBL were: (a) separation of lymphocytes from whole blood by the Hypaque-Ficoll technique; (b) 2 × 106 PBL per culture in RPMI-1640 containing 5–10% heat-inactivated, autologous plasma; (c) 48 hr culture period; (d) 5 μg/ml PHA-P; and (e) 1 μCi/ml 3H-TdR added to cultures 22 hr before termination. Under these conditions, individual and group variations in the response to PHA were slight, and PBL responsiveness to other mitogens as well as to antigen were good.

Alterations in serum antibody and peripheral T-lymphocyte subsets resulting from treatment of murine immune complex glomerulonephritis with PGE2

The effects of treatment with 16,16-dimethyl prostaglandin E2 (DMPGE2) on histologic damage, glomerular immune complex deposition, serum total IgG subclass levels, anti-apoferritin IgG levels, and peripheral blood T-lymphocyte subsets were determined in apoferritin-induced immune complex glomerulonephritis of mice. The results demonstrate that doses of DMPGE2 ranging from 2.5 to 10 micrograms twice daily significantly reduced the degree of glomerular damage in a dose-dependent manner. Similarly, these doses of DMPGE2 reduced the amount of immunoglobulin deposition along peripheral capillary loops. Total IgM, IgG1, IgG2a, and IgG2b were unaffected by DMPGE2 administration. Serum anti-apoferritin IgG levels were significantly reduced in mice receiving DMPGE2 at doses of 5 and 10 micrograms twice daily. Nephrotic mice had significantly reduced peripheral blood total T lymphocytes (Lyt-1+) and a reduction of T-suppressor (Lyt-2+) cells. Administration of DMPGE2 at doses of 5 and 10 micrograms twice daily prevented these T-lymphocyte alterations. These studies indicate that treatment of mice receiving apoferritin with DMPGE2 may prevent glomerulonephritis by altering both cellular and humoral immune responses.

Characterization of Suppressor Cells in Mice with a Transplantable Malignant Melanoma

C57BL/6 mice with progressive B-16 melanoma develop a generalized immunosuppression, as measured by their lack of response to SRBC in vivo and in vitro. The severity of immunosuppression increases with the progress of the tumor, and is due to the generation of an antigen-nonspecific suppressor cell. Suppressor cells were first demonstrable 15 days after the appearance of the tumor, and their appearance correlated with the induction of immunosuppression in vivo. The suppressor cell generated by the growth of B-16 melanoma was a T lymphocyte since it was nonadherent to plastic and nylon wool, unaffected by passage through Sephadex G-10, and sensitive to treatment with rabbit antimouse brain serum and complement.

Cultivation and characterization of macrophages from murine embryonic skin. Are they Langerhans cells

Abstract We have observed a population of trypsin-resistant adherent cells in long-term primary cultures of murine embryonic skin. These cells were subsequently demonstrated to share a variety of characteristics with cells of the monocyte/macrophage lineage. The trypsin-resistant adherent cells stained positively for nonspecific esterase, exhibited surface receptors for Fc-IgG, and complement components as well as strong phagocytic activity. Additionally, these cells exhibited membrane ATPase enzyme activity and a large proportion of the cells expressed la antigens as detected by cytotoxicity and membrane fluorescence. The possible relationship between these trypsin-resistant adherent cells and Langerhans cells of the skin is discussed.

The association of suppressor cells with mononuclear cell aggregates in spleens of tumor-bearing mice☆

Abstract Spleen cell suspensions from mice with progressive B-16 melanoma consistently contained significant numbers of aggregates of mononuclear cells (MN-Agg), when compared to spleen cell suspensions from normal mice or mice in the early stages of tumor growth. Histological, histochemical and immunological characterization of the cells involved in MN-Agg from tumor-bearing mice indicated that aggregates were composed of macrophages and T and B lymphocytes. The formation of MN-Agg was dependent upon the macrophage content of the spleens of tumor-bearing mice since the appearance of MN-Agg correlated temporally with an increase in the number of splenic macrophages demonstrable in tumor-bearing animals. An antigen nonspecific suppressor cell was identified in the spleens of mice 15 days following the appearance of palpable B-16 tumor, and the appearance of the suppressor cell population closely correlated with the appearance of MN-Agg. Additionally, fractionation of MN-Agg-containing cell suspensions demonstrated that fractions highly enriched in MN-Agg were concomitantly enriched for suppressor cells. The suppressor cell associated with MN-Agg was a T lymphocyte since suppressor activity of MN-Agg could be abolished by treatment of MN-Agg with a rabbit anti-mouse brain serum and complement. It is proposed that the generation of suppressor cells in mice with B-16 melanoma may require specific interaction between macrophages and lymphocytes which is manifested in the spleens of tumor-bearing mice by the formation of MN-Agg.