Renal deposition of soluble immune complexes in mice bearing B-16 melanoma. Characterization of complexes and relationship to tumor progress.

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.

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.

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.

Monoclonal antibody analysis of responder and stimulator cells in the human autologous mixed lymphocyte reaction.

Responder and stimulator cell subpopulations in the autologous mixed lymphocyte reaction (AMLR) were determined with the OK series of monoclonal antibodies. Mitomycin-C-treated, monocyte-enriched cell populations were used as stimulator cells in the AMLR. Treatment of these monocytes with either OKM and/or OKI monoclonal antibodies and complement resulted in a marked loss of ability of these cells to act as stimulators in the AMLR. Removal of OKT3+ and OKT4+ cells diminished the proliferative responses of AMLR cultures. Interaction of T cells with autologous monocytes resulted in generation of cells capable of suppressing both MLR and AMLR cultures. The suppressor activity of these cells was diminished by treatment with OKI , OKT4 or OKT8 monoclonal antibodies. No cytotoxic activity to autologous or allogeneic monocytes was observed. Additional studies showed an increased number of OKT9 + and OKI + as well as OKT8+ T cells in the AMLR responder cell population. This study indicates that cultures of T lymphocytes with autologous monocytes yield T cell subset(s) which suppress MLR and AMLR reactivity.

Phenotype of the hairy cells of leukemic reticuloendotheliosis defined by monoclonal antibodies.

Hairy cell populations of greater than 90 purity were prepared from six samples obtained from four patients with leukemic reticuloendotheliosis (LRE). These then were analyzed for surface immunoglobulin (SIg) and antigens specified by a panel of monoclonal antibodies. The hairy cells from all patients displayed SIg and antigens reactive with OKIa-1 and OKM-1 antibodies; these markers often were expressed simultaneously. T-cell-specific antigens were not displayed on the surface of hairy cells. The simultaneous expression of SIg and OKM-1 which ordinarily are unique for B cells or myeloid cells, respectively, suggests that hairy cells may represent an aberrant form of either cell type with defective regulation of antigen expression. It alternatively suggests the possibility that B cells and myeloid elements develop along a common pathway and that the hairy cell is a component of such a pathway.

Ultrasonic diagnosis of abdominal wall desmoid tumor.

Abdominal wall desmoid tumor is a rare, locally invasive proliferation of mature fibrous tissue which does not metastasize. It usually presents as an asymptomatic mass, and occurs most frequently in young women after childbirth. All three of our patients showed an echo-free mass deep in the abdominal wall with poor sonic transmission. Ultrasound cannot only accurately localize the lesion, but allows the radiologist to suggest the correct diagnosis.

Hydatidiform mole with co-existent fetus.

Co-existence of hydatidiform mole and fetus is a rare condition, but one in which the radiologist, through ultrasound, can play an important role in diagnosis. The typical patient, with no pre-existing hypertension or renal disease, develops severe pre-eclampsia in the second trimester with elevated gonadotropin levels. Ultrasound shows a large placenta containing varying numbers of sonolucent spaces, associated with a live or dead fetus. This condition appears to be a separate entity from classical hydatidiform mole both pathologically and prognostically. It should also be differentiated from simple hydatidiform swelling which does not have the associated clinical picture.

Ultrasonography in the diagnosis of thyroid lymphoma.

Lymphoma of the thyroid is a rare condition, classically occurring in elderly caucasian women, presenting as a rapidly enlarging, symptomatic mass. Almost all examples are due to non-Hodgkin's lymphoma, and the lesion is commonly superimposed on pre-existing Hashimoto's thyroiditis. Prognosis is best if the disease is diagnosed early and while confined to the thyroid region, there being a favourable response to radiotherapy alone. Unfortunately, lymphoma is rarely considered in the differential diagnosis of thyroid masses, often leading to unnecessary, excessive surgery. Ultrasonography of five patients was analysed to determine if any common diagnostic features were present. All had a similar pattern, with a large, circumscribed, solid hypoechoic mass. Although nonspecific, this appearance, with awareness of a rapidly growing mass, should alert the radiologist and attending clinician to the possibility of lymphoma.

Differential regulation of the immune response to SRBC by monoclonal antibodies to interferon-gamma.

Three monoclonal antibodies against different epitopes of interferon-gamma (IFN-gamma) were used to assess its role as a normal immunomodulatory molecule. Two of these antibodies were able to reduce significantly the primary antibody response to sheep erythrocytes in an in vitro culture system. One of these two antibodies has been reported to suppress both the antiviral and macrophage activation factor activities of IFN-gamma by binding to its carboxyl terminus. These findings indicate that IFN-gamma is an important lymphokine for the maximum expression of the immune response and that it acts via the carboxyl terminus of the molecule. This antibody suppressed the immune response only when added at the initiation of culture, suggesting that the action of IFN-gamma is on an early component of the response. The third monoclonal antibody, which binds to the amino end of IFN-gamma, did not suppress the in vitro response. However, it was able to block the effects exerted by an immunosuppressive dosage of exogenous IFN-gamma on in vitro antibody production. These results indicate that the immunosuppression vitro antibody production. These results indicate that the immunosuppression induced by the addition of IFN-gamma to a primary antibody response and the role that it plays in that response are mediated through different sites on the molecule and, therefore, probably by different mechanisms.

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.

Suppression of cell-mediated immunity in experimental African trypanosomiasis.

Adult New Zealand white rabbits were experimentally infected with a parasitic African hemoflagellate, Trypanosoma congolense, and were subsequently tested for in vivo and in vitro aspects of cell-mediated immune function. Chronically infected rabbits were sensitized to mycobacterial protein and skin-tested with purified protein derivative; all infected animals demonstrated much milder skin-test responses to antigen than control groups. Similarly, peripheral blood lymphocyte responses in vitro to purified protein derivative and, as well, to phytohemagglutinin were markedly suppressed. Supernatant fluids of antigen-stimulated lymph node cell cultures from T. congolense-infected rabbits failed to demonstrate migration inhibitory factor activity but did possess normal levels of blastogenic factor activity. An active infection was necessary for demonstration of suppressed immune responses, and components present in infected rabbit serum were apparently not responsible for the observed abnormalities. Suppression of T-lymphocyte subpopulations may well explain the occurrence of numerous immunological aberrations arising during human and animal infections with the African trypanosomes.

The influence of cigarette tobacco smoke products on the immune response. The cellular basis of immunosuppression by a water-soluble condensate of tobacco smoke.

The immunosuppression exhibited by a water-soluble condensate of tobacco smoke (WSC) has been studied in vivo and in vitro. When multiple sublethal doses of WSC were injected into C57Bl/6 mice, their ability to respond to immunization with sheep erythrocytes by the formation of plaque-forming cells was severely inhibited. In addition, spleen cells from WSC-treated mice were unable to mount a primary response to SRBC in vitro. Studies on the cellular basis of the immunosuppression induced by WSC showed a decrease in T lymphocytes in the spleens of WSC-treated mice. Additional experiments were conducted in which isolated populations of T cells, B cells and macrophages from WSC-treated or normal mice were combined and then tested for responsiveness to SRBC in vitro. Results of these experiments also indicated that T cells were particularly susceptible to WSC exposure. T cells from WSC-treated mice were unable to co-operate with normal B cells and macrophages in the response to SRBC. A less marked suppression of B-cell function was noted in condensate-treated mice. While B cells from such animals were able to co-operate with normal T cells and macrophages to give a detectable primary response to SRBC, the response was depressed. In contrast, macrophages from WSC-treated animals enhanced the response of normal T and B cells to SRBC.

Generalized monocyte deficiency in leukaemic reticuloendotheliosis.

Examination of the blood of 40 patients with leukaemic reticuloendotheliosis (LRE) revealed a drastic reduction in monocyte numbers (mean 74/microliter) when compared to a normal group of 33 (mean 442/microliter). Repeated blood studies in 8 LRE patients were made over periods of 2 months to 5 years and monocytopenia was observed to be a persistent phenomenon. Similarly, tissues from 29 patients showed an overall decrease in numbers of monocytes/ histiocytes when compared with those of 73 control subjects. Skin window examinations in all 11 patients so studied showed marked reduction or absence of monocyte response to inflammation. 2 exceptional cases to this trend are noted. In one, circulating monocyte levels remained normal over a 14-month period while examinations of blood and marrow in this case showed few hairy cells. In another case, severe and persistent monocyte deficiency was noted during a 2-year period of unsuccessful treatment. When he eventually responded to low dose chlorambucil and his Hb rose from 40-140 g/l and WBC became normal, the number of circulating monocytes also became normal.

The prickly lettuce agglutinin. I. Isolation from leaves of the prickly lettuce plant (Lactuca scariole).

An agglutinin has been isolated from leaves of the prickly lettuce plant (Lactuca scariole). The prickly lettuce agglutinin (PLA) agglutinates erythrocytes of the mouse and rat, and binds to, but does not agglutinate erythrocytes of other species. PLA can be precipitated from crude plant extracts by cold acetone and ammonium sulfate treatment, is sensitive to proteolytic enzyme activity, and contains carbohydrate residues, suggesting that it is a glycoprotein. The agglutinin has a molecular weight of approximately 84,000 as determined by SDS-PAGE studies. The receptor for PLA has not been elucidated but may involve, in part, sialic acid residues.

Tumoristatic effects of nonimmune BALB/c peritoneal macrophages on syngeneic lymphoma cells in vitro.

Peritoneal macrophages from unstimulated nonimmune BALB/c mice exerted nonspecific cytostatic effects of tumor cells in vitro. Incubation of syngeneic B40 lymphoma or allogeneic B16 melanoma cells with peritoneal macrophages or with macrophage culture supernatant fluids (SF) resulted in decreased target cell DNA synthesis and growth; in contrast, nontumorigenic syngeneic 3T3 fibroblasts were stimulated by macrophages but SF exerted no significant effects. Lipopolysaccharide activation of macrophages did not induce cytotoxicity for any target cell tested, and did not enhance the observed tumoristatic effects. The level of tumor inhibition associated with macrophage culture SF was dependent upon the concentration of fetal calf serum present in the medium as well as upon the numbers of macrophages cultured. Inhibitory SF could be harvested continuously from macrophage cultures for at least 7 days with no appreciable loss of activity.

Release into culture medium of membrane-associated, tumor-specific antigen by B-16 melanoma cells.

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.