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Demonstration of tumor-associated immunity with a leukocyte adherence inhibition (LAI) assay.

A modification of the leukocyte adherence inhibition (LAI) assay of Halliday was used to search for immune reactions against tumor-associated antigens of mouse tumors as well as against embryonic antigens in such neoplasms. Soluble antigen extracts were prepared from transplanted (BALB/c) methylcholanthrene-induced sarcomas and carcinomas, from normal BALB/c embryos taken at 14-18 days' gestation and from kidneys and livers of adult BALB/c mice. Peritoneal cells (PC) from mice immunized against syngeneic tumors gave leukocyte adherence inhibition more commonly when exposed to antigens prepared from the same tumor than did PC from normal (untreated) mice or from mice immunized against a different tumor. However, different tumor antigen preparations varied vastly in their ability to give specific adherence inhibition; some preparations consistently gave a high tumor-specific inhibition, while others did not. This may explain why the degree of reactivity observed was low and its tumor specificity not absolute when data obtained with all different antigen extracts were pooled. PC from multiparous mice gave adherence inhibition when exposed to antigenic extracts from syngenic mouse embryos or from tumors, as compared to PC from virgin mice exposed to the same extracts. Furthermore, PC from tumor-immunized mice reacted more commonly against antigen extracts from mouse embryos than did peritoneal cells from normal untreated mice. Adherence inhibition was not observed when PC from tumor-immunized or multiparous mice were exposed to antigen extracts from adult syngeneic livers or kidneys.

Animals

Binding to idiotypic determinants of large proportions of thymus-derived lymphocytes in idiotypically suppressed mice.

All A/J mice immunized with a conjugate of p-azophenylarsonate groups to keyhole limpet hemocyanin produce antibodies against azophenylarsonate, some of which share a crossreactive idiotype. The appearance of the idiotype can be suppressed, without reducing the response against azophenylarsonate, by injecting rabbit anti-idiotypic antibodies prior to immunization. We have now observed that mice suppressed in this way, or by adoptive transfer of leukocytes from other suppressed mice, and then immunized with the hemocyanin-azophenylarsonate conjugate, possess high proportions (up to 14%) of lymphocytes that form rosettes with A/J erythrocytes coated with Fab fragments possessing the idiotype. Idiotypic specificity was demonstrated by various experiments. Most of all of the rosette-forming lymphocytes appear to be thymus-derived lymphocytes (T cells). Treatment of T cells with trypsin eliminated the capacity to form rosettes, which was restored on standing overnight in medium. Thus, the receptors are synthesized by the cells and are not passively adsorbed. Treatment of mice with anti-idiotypic antiserum without antigenic stimulation did not elicit substantial numbers of rosette-forming cells. The requirement for antigen suggests that antigen-idiotype complexes may be a stimulatory agent. A prolonged rest period after immunization of suppressed mice was required for the induction of high percentages of rosette-forming cells. Rosette formation provides a convenient method for studying factors that induce the formation of idiotype-specific T cells.

Animals

Rosette-forming ability of thymus-derived lymphocytes in cell-mediated immunity. I. Delayed hypersensitivity and in vitro cytotoxicity.

Effector cells in delayed hypersensitivity and in vitro cytotoxicity were studied in lymph node cells from animals immunized with sheep erythrocytes (SRBC) in complete Freund's adjuvant. Delayed hypersensitivity response (DHR) was assayed by the increase in foot pad swelling after the intrafoot pad injection of immune cells plus antigen. Cell-mediated cytotoxicity against SRBC was assayed by a microcytotoxicity test with sheep fibroblasts as target cells. Effector cells were antigen specific, sensitive to anti-theta serum plus complement (C), and insensitive to anti-Ig serum plus C. A nonrosette-forming (non-RFC) small lymphocyte effector T cell and a rosette-forming medium lymphocyte effector T cell were isolated by velocity sedimentation. The small lymphocyte non-RFC required a longer time than the medium lymphocyte RFC effector cell to produce maximum activity. Buoyant density failed to distinguish medium lymphocyte effector cells in DHR and in vitro cytotoxicity.

Animals

The role of autosensibilization of lymphoid cells in genesis of antitumor resistance.

C3HA mice were inoculated with a mixture of syngeneic transplantable hepatoma 22a cells and syngeneic splenocytes of mice immunized with normal syngeneic tissues or subjected to partial hepatectomy. Control mice were inoculated with a mixture of tumor cells and splenocytes of intact or sham operated mice. A considerable inhibition of tumor growth was observed in the experimental series. Immunization of mice with normal syngeneic tissues as well as partial hepatectomy results in sensibilization of splenocytes not only to normal tissue antigens, but to the antigens of hepatoma 22a cells too. This was shown by the reaction of indirect inhibition of glass adherence of peritoneal cells. The data obtained are considered to be one more possible prove of a special form immune surveillance which controls the state of cytodifferentiation being active in the organism. The involvement of this form of immune surveillance in the genesis of antitumor resistance is discussed.

Animals

Specificity of cell-mediated cytotoxicity against human melanoma lines: evidence for "non-specific" killing by activated T-cells.

The specificity of cell-mediated cytotoxicity against melanoma cells in vitro has been analyzed in a large number of studies with cells both from normal and melanoma subjects. As in a number of other, recent, similar human studies, no evidence for tumour specificity was found. Effector cells in peripheral blood responsible for the cytotoxic raction were examined by cell separation methods based on red cell rosette formation and separation through Hypaque-Ficoll mixtures. The evidence suggests that non-specificity results from killing by cells separating largely in the non-sheep red blood cell rosetting fraction and which have cytotoxic specificity directed broadly to cells with abnormal membranes. Further analysis revealed that the cells were non-phagocytic and did not bear receptors for complement. They appear to be activated into cell division and to bear surface receptors for the Fc portion of IgG. Additional evidence is presented suggesting that the cells mainly responsible are activated thymus-dependent cells present in the circulation of both tumour-bearing and normal subjects.

Antigen-Antibody Reactions

Reticulum cell sarcoma: an effector cell in antibody-dependent cell-mediated immunity.

A transplantable, murine reticulum cell sarcoma is described which exhibits the cytologic, adherence, and phagocytic properties of macrophages. It forms specific rosettes with erythrocytes in the presence of the corresponding anti-serum. The ascites cells mediate antibody-dependent cellular immunity as assayed by release of radioactivity from 51Cr-labeled erythrocytes. The contribution of contaminating host cells in the cytotoxic reaction was ruled out by growing the tumor in F1 mice and removing the host cells by anti-H2 serum and complement. The tumor cells have receptors for IgG2a and IgG2b immunoglobulins. The availability of a pure population of effector cells in the immune system allows study of the biochemical processes pursuant to lysis of foreign cells.

Animals

Early onset of serum blocking in a murine melanoma model.

The early onset of serum blocking of host cell-mediated immunity was investigated in a transplantable murine melanoma employing two in vitro assays. One of these was based on the Takasugi and Klein cytotoxicity test; the other was a new test based on the inhibition of adherence of leukocytes to a plastic surface in the presence of tumor antigen. Both tests yielded similar results, and the adherence inhibition test was more sensitive. Blocking could be detected as early as 1 h after the inoculation of 1 times 10(7) melanoma cells. Early onset of serum blocking was also demonstrable if homogenized of tissue-cultured tumor cells were inoculated. Serum blocking under these circumstances was initially transient, declining at 24 to 48 h, but reappearing by the 4th day after inoculation. Furthermore, a functional immune system was not required for blocking factor development in this system, as evidenced by the onset of a similar pattern of blocking activity in heavily irradiated animals.

Animals

Expression of H-Y antigen in human males with two Y chromosomes.

To determine whether the gene that controls the expression of H-Y ("male") antigen on human cells is Y-linked, we have compared the H-Y antigen level in normal males with that in three males with two Y chromosomes. Leukocytes from one XXYY and two XYY males express more H-Y antigen than leukocytes from normal XY males. We conclude that a structural gene or positive regulatory gene for H-Y antigen is on the human Y chromosome. Testing for the H-Y antigen may be of benefit in patients who have signs of masculinization but who lack an identifiable Y chromosome. Positive results for the H-Y antigen would be tentative evidence that the corresponding region of the Y chromosome was present, perhaps as part of a translocation, despite the absence of a typical Y chromosome.

Absorption

Antigen heterogeneity of human B and T lymphocytes.

Rhesus monkeys were immunized with normal human lymphoid cells, cultured lymphoid cells, and chronic leukemic lymphocytes. Antisera were analyzed by cytotoxicity and immunofluorescence techniques to study the antigenic characteristics of human lymphocytes. In an attempt to obtain a reagent specifically reactive with T (thymus-derived) lymphocytes, an antispleen antiserum was absorbed with cellf from five B- (bone marrow-derived) cell lines. After absorption, the antiserum killed 60-75% of peripheral blood lymphocytes and 40-50% of tonsil cells, so that there was a relationship between the percentage of killed cells and the proportion of T lymphocytes. However, when cells after cytotoxic treatment were assayed for rosette formation with sheep erythrocytes (a T-cell marker) 5-20% of viable rosette-forming lymphocytes were found. Therefore, this antiserum was cytotoxic for only 75-90% of T cells. From studies performed with antisera prepared against spleen and B-cell lines, we conclude that lymphoblastoid cells are antigenically different and deficient in comparison to normal B lymphocytes. In addition, cultured B-cell lines appear to be antigenically heterogenous, as shown by the cytotoxic activity remaining in antispleen and anti-B-cell lines sera after absorption with various numbers and types of lymphoid cell lines. After absorption with normal lymphocytes, an antiserum produced against chronic lymphatic leukemia cells had specific activity associated with 12 chronic lymphatic leukemia cells tested. Absorption of the same antiserum with leukemic cells from two patients showed that a certain degree of antigenic heterogeneity also exists among chronic leukemic lymphocytes.

Agammaglobulinemia

Immunologic mimicry between mouse tissue and enterobacterial common antigen.

Organs of Swiss white albino and C57BL/6Ha mice were assessed for an antigen (CRA) which cross-reacts with common enterobacterial antigen (CA). To this end, supernatant fluids (HKS) and ethanol-soluble fractions (ES) of heated homogenates of spleens, kidneys, and livers were examined for their capacities to react with CA hemagglutinins and to engender humoral and cellular events in the rabbit. The immunogenicity of CRA in the rabbit can not be predicted on the basis of CA hemagglutinin neutralization studies alone; although CRA was identified in the liver extracts of both mouse strains, according to this parameter, only the liver fraction of Swiss white albino mice elicited significant numbers of rosette-forming cells (RFC) in the spleens of rabbits. Also, kidney fractions, which primed the rabbits for booster with CA, were less effective in stimulating RFC in the spleens failed to inhibit CA hemagglutination and did not prime rabbits for a CA hemagglutinin response, these same preparations clearly evoked RFC in rabbit spleens. Thus, the antigenicity and immunogenicity of CRA in target organs of mice reflect the mouse strain, extraction procedure, and testing method employed.

Animals

Rheumatoid factor in melanoma patients: alterations of humoral tumor immunity in vitro.

Rheumatoid factors (RF) were associated with alterations of antibody reactions to melanoma cells in vitro by two serologic assays. Removal of RF from melanoma patients' sera by absorption with Cohn's Fraction II coated latex particles enhanced seroreactivity in the Immune Adherence (IA) assay and diminished IgM detection by the Indirect Membrane Immunofluorescence (IMI) assay. The addition of serum with high titers of RF to these assay systems led to diminution of IA reactivity and enhancement of IgM detection by IMI. Since these factors are found in cancer patients' sera and can alter humoral immune reactions directed against antigens on the membranes of tumor cells, their presence should be recognized when performing assays with tumor target cells. RF may be of significance in the host-tumor relationship in vivo.

Antibodies, Neoplasm

Human antibody-dependent cellular cytotoxicity. Isolation and identification of a subpopulation of peripheral blood lymphocytes which kill antibody-coated autologous target cells.

Antibody-dependent cellular cytotoxicity (ADCC), has been shown to be independent in vitro of thymus-derived lymphocytes, but the precise nature of the effector lymphocyte has not been fully clarified. To further study the identity of the ADCC effector cell type(s), peripheral blood leukocytes were purified by Ficoll-Hypaque density centrifugation and fractionated into surface immunoglobulin-positive [Ig(+)] and surface immunoglobulin-negative [Ig(-)] populations by chromatographic separation on Sephadex G-200 anti-human immunoglobulin columns. After column fractionations, the ADCC effector activity against antibody-coated autologous lymphocytes was predominantly and consistently found in the Ig(-) fraction. This latter population was then further fractionated, by rosetting techniques, into two subpopulations, The first was depleted by lymphocytes with surface receptors for sheep red blood cells [E(+)]and the second was depleted of lymphocytes with receptors for sheep red blood cell-antibody-complement [EAC-(+)]. Analysis of these populations showed that ADCC effector activity was predominantly a property of the Ig(-) lmyphocytes which are E(-) but EAC(+). These lymphocytes have been referred to as "null lymphocytes" and probably represent a subset of bone marrow-derived (B) cells. In addition, variable and low levels of ADCC activity were observed in some Ig(+) populations (B cells). Further purification of the null cell population by filtration over nylon wool columns to reduce the number of contaminating latex ingesting monocytes did not reduce ADCC effector activity. Isolated null cell ADCC effector activity was inhibited by either rabbit anti-human F(ab)2 or normal pooled rabbit gamma globulin, but not by rabbit F(ab)2 anti-human F)ab)2 or media. This supports the contention previously suggested in studies using unfractionated lymphocyte populations that the ADCC effector cell recognizes the Fc portion of the antibody molecule. The variable and low level of activity noted in the Ig(+) populations is unexplained but possibly due to a variable population of null cell-derived Ig(+) lymphocytes within the whole Ig(+) population. In conclusion, these experiments demonstrate that, in vitro, the major ADCC effector activity of circulating human peripheral blood lymphocytes resides in the Ig(-), E(-), EAC-(+) subpopulation termed "null cells." Since it has been noted that in certain disease states, such as immunodeficiency syndromes, autoimmune disorders, and neoplasms, the percentage of this population of lymphocytes in the peripheral blood is elevated, it is speculated that these cells, perhaps through their ADCC function, may play an important pathophysiologic role in these diseases.

Antigen-Antibody Reactions

Effect of papain on the interaction between human monocytes, erythrocytes, and IgG.

The mechanism by which papain detaches IgG-sensitized erythrocytes from the monocyte surface has been explored in an in vitro assay for the monocyte IgG receptor using red cells quantitatively sensitized with IgG anti-Rh D immunoglobulin. Papain treatment of IgG-sensitized erythrocytes diminished the ability of these cells to bind to the monocyte surface; however, treatment of erythrocytes with papain prior to sensitization with IgG did not inhibit binding, and at papain concentrations is greater than or equal to 38 mug/ml binding was enhanced. IgG receptor activity was not diminished by prior treatment of monolayer cells with papain and was enhanced with high concentrations of papain. These studies suggest that papain detaches erythrocytes from the monocyte surface by virtue of its proteolytic effect on IgG and not by an effect of papain on the D antigen of red cells or the IgG receptor on monocytes.

Animals

Hepatitis in marmosets.

Some species of marmosets are susceptible, not only by parenteral inoculation but also by oral exposure, to human hepatitis A virus present in sera or feces. The stools of animals inoculated parenterally or orally contained fecal antigen during certain times of the incubation period and the early, acute phase of the disease; viruslike particles were present in feces of orally infected animals and such feces were infectious when inoculated into marmosets. The fecal antigen crossreacted both with the fecal virus particles and the immune-adherence antigen (see also papers by Purcell et al and Hilleman et al). The MS-1 and CR-326 strains of hepatitis A appeared antigenically similar or identical whereas the GB strain was antigenically different and may be associated with the recently defined type of hepatitis termed hepatitis C or hepatitis non-A/non-B. On repeated challenge hyperegic responses with diffuse liver cell necrosis occurred in some immune animals and this phenomenon must be taken into account in any future vaccination studies.

Animals