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Results for “Cytotoxicity Tests, Immunologic”

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[The lymphocyte cytotoxicity test in tumor immunology (author's transl)].

The cytotoxic action of lymphocytes on cancer cells in vitro indicates sensitization of the patient against his own tumor. The technical difficulities of this test and possibilities of standardization and simplifying the procedure are discussed. Critical steps are isolation of lymphocytes and culturing target cells without loosing their specific antigenic structure. The need for specificity controls both for lymphocytes and tumor cells is emphasized. Labelling tumor cells with isotopes represents a major improvement in evaluating the result. The role of thymus- and bone marrow-dependent lymphocytes as well as blocking factors in the serum of tumor patients can be analyzed in the cytotoxic assay. A better understanding of these mechanisms may facilitate a therapeutic approach by manipulating the interaction of tumor cells and host.

Animals

Decreased cell-mediated cytotoxicity against virus-infected cells in systemic lupus erythematosus.

Cell-mediated cytotoxicity, directed against virus-infected tissue culture cells, was studied with peripheral blood mononuclear cells from 11 patients with systemic lupus erythematosus (SLE) and 12 matched, normal subjects in a 51Cr release assay. Baseline (preimmunization) levels of cytotoxicity against target cells infected with influenza A/Victoria, influenza B/Hong Kong, Newcastle disease virus, and herpes simplex virus were significantly decreased in patients with SLE compared to normal subjects (P less than 0.001), although serum antibody levels to the respective viruses were similar in both groups. After intramuscular administration of inactivated influenza A/Victoria vaccine, SLE patients failed to generate elevated levels of cytotoxicity against A/Victoria-infected cells, in contrast to normal subjects. SLE patients responded with levels of serum hemagglutination-inhibition antibody which were similar to those of normal subjects. Thus, SLE patients manifest decreased cell-mediated cytotoxicity against virus-infected target cells, although humoral antibody responses appeared to be intact. Studies of SLE patients with influenza may help to define the role of cell-mediated immunity in the pathogenesis of certain viral infections.

Adolescent

Effect of human serum from patients with haematological disorders on mouse pluripotent haemopoietic stem cells (CFU-S).

Sera from 25 individuals (9 healthy subjects, 6 patients with systemic lupus erythematosus, and 10 patients with a variety of haematological disorders) were tested for anti-CFU-S by incubating suspensions of mouse marrow cells with serum and then assaying the cell suspensions for their capacity to form spleen nodules in lethally irradiated mice. The sera from 2 patients (1 with a preleukaemic disorder and the other with a malignant 'histio'-lymphoproliferative neoplasm) had anti-CFU-S activity which behaved like antibodies, rather than like the non-specific cytotoxic activity found in various sera for heterologous tissues, which in the past has been attributed to 'natural antibodies' and which, in recent years, has, in some cases, been found to be related to activation of the alternative pathway of complement. The anti-mouse tissue activity in the sera of the 2 patients may be related either to cross-reactivities between certain mouse and human tissues or to cross-reactivities between exogenous agents such as bacteria and mouse tissues.

Absorption

The role of methylprednisolone in the modulation of cellular cytotoxicity and its relation to renal transplantation.

Inhibition of killer cell generation or suppression of their cytolytic capacity once formed represent fundamental mechanisms by which methylprednisolone may modulate cellular cytotoxicity in the transplant recipient. In vitro studies in mixed lymphocyte culture demonstrated that the presence of therapeutic concentrations of this agent (0.001-1 microgram/ml) during the sensitisation phase resulted in suppression of lymphocyte activation without cytolysis and marked or total inhibition of killer cell generation. A considerable individual variation in sensitivity to methylprednisolone-induced suppression was observed and killer cells once generated were resistant to this agent.

Cytotoxicity Tests, Immunologic

Cell-mediated cytotoxicity for cultured autologous melanoma cells.

Peripheral blood lymphocytes from 32 patients with malignant melanoma were tested for cell-mediated cytotoxicity (CMC) against cultured autologous melanoma cells. Effector cells were prepared from venous blood by defibrination, gel sedimentation, nylon column filtration, and lysis of remaining erythrocytes with NH4Cl. Melanoma cells prelabelled with [3H])proline were used as target cells in a 40-h assay and CMC was evaluated against standards obtained with blood lymphocytes from the least reactive normal donor. Reproducible autologous CMC was detected in 18 of 32 patients in a series of 367 total tests. CMC correlated with tumor volume (5-500 cm3) but not with tumor stage or DNCB reactivity. Preliminary results indicated that autologous CMC was not affected by treatment with DTIC, dexamethasone, intralesional BCG, radiation therapy, or partial surgical excision. Lack of consistent CMC in 14 patients could not be attributed to a measurable decrease in general immune capacity or to increased resistance of the patients' melanoma cells to CMC in general. Fibroblasts were more resistant to CMC than melanoma cells, and therefore of questionable value for defining specificity in direct tests.

Adult

Cytotoxicity to isolated rabbit hepatocytes by lymphocytes from children with liver disease.

A test of lymphocyte cytotoxicity for isolated adult rabbit hepatocytes has been performed using lymphocytes from 40 children with acute or chronic liver disease. Positive cytotoxicity was not observed in 26 children without liver disease and rarely in 13 children with disease affecting primarily the biliary tract. Temporarily positive tests were found in those with acute hepatocellular disease, but tests remained positive in patients with chronic active hepatitis, while liver function tests remained abnormal. Persistently positive test occurred in those with liver disease associated with alpha-antitrypsin deficiency. Such altered immunoresponsiveness could be an important pathogenic mechanism leading to chronic liver disease in childhood.

Acute Disease

Immunological diagnosis of rejection in human renal allotransplanted patients--a prospective study.

The object of this study has been to evaluate the recipient's immunological reactivity towards donor lymphocytes in relation to rejection episodes. All recipients (20) of local necrokidneys during 1976 were immunologically monitored immediately before transplantation and subsequently twice weekly for donor-specific complement dependent lymphocytotoxic (CDC) antibodies, antibody dependent cell-mediated cytotoxicity (ADCC) and cell-mediated lympholysis (CML). Experiments were performed until graft removal or dismissal (approx. 1100 patient days). Clinical diagnosis of rejection was made independently of immunological results. All clinically suspected rejection episodes, except one, were checked by microscopy. A positive CML-test accompanied 9 out of 11 rejection episodes; the test was negative on all other occasions. Positive CDC and ADCC tests exhibited no obvious correlation with rejection episodes: positive ADCC may be more frequent in clinically uncomplicated phases. Positive CML did not generally precede clinical graft rejection. Positive CML before transplantation was observed in two cases and was followed by irreversible, accelerated acute rejections. The CML-test may prove a reliable tool in rejection diagnosis and may yield results comparable with graft biopsy without inflicting any risk on the patient.

Antibodies

Spontaneous human lymphocyte-mediated cytotoxicity against tumor target cells. IV. The suppressive effect of normal pregnancy.

The effects of pregnancy and other donor characteristics on spontaneous lymphocyte-mediated cytotoxicity (SLMC) were studied. Sex, age, and stage of the menstrual cycle had no significant effect on the SLMC of the control donors, the SLMC was significantly lower in pregnant donors in the second or third trimester. However, this lowered level of activity was still within the 95 per cent confidience limits of the normal population and parity had no cumulative effect. At delivery, fetal cord blood lymphocytes possessed substantial cytotoxic activity, further evidence that the fetus is immunologically competent at birth. The relevance of these data to the immunologic coexistence of the mother and fetus is discussed.

Adult

Natural killer and tumor recognizing lymphocyte activity in tumor patients.

Several mechanisms can lead to the killing of a target cell by lymphocytes. In order to study immunologically specific phenomena it has to be ensured that the natural killer (NK) effect does not operate in the system. Using targets which are sensitive to NK, the effector populations have to be depleted of lymphocytes with such potential. The blood lymphocytes of tumor carrying patients often have reduced NK activity. In two assay systems anti-tumor autoimmune reactivity has been demonstrated (the majority of tested patients had lung carcinomas or osteosarcomas). The tests were: 1. Induction of blastogenesis in blood lymphocytes by in vitro confrontation with autologous biopsy cells. 2. Lymphocyte mediated killing of autologous biopsy cells in short term in vitro assay. Cross reactivity between patients was rare which indicates either that the putative tumor antigens are individual specific or their recognition is restricted by histocompatibility.

Animals

Immunopathologic studies of rheumatoid arthritis. I. Absence of complement-dependent cytotoxicity of rheumatoid sera for rheumatoid synovial cell cultures.

A sensitive complement-dependent chromium release cytotoxicity assay was used to determine whether sera from rheumatoid arthritis (RA) patients contain antibody specific for an antigen on rheumatoid synovial cell cultures. Two hundred eight RA sera-RA synovial culture combinations were studied employing 21 sera and 16 synovial membranes; control combinations were derived from 5 normal sera and 10 degenerative joint disease synovial membranes. Anticomplementary activity of some rheumatoid sera was overcome using an increased complement concentration. The percent cytotoxicity of RA serum-RA culture combinations, both homologous and autologous, was not significantly greater than that of RA serum-control culture combinations. No correlation between duration of disease or duration of cell culture and percent cytotoxicity was found. Thus a unique antigen on cultured rheumatoid synovial cells was not recognized by rheumatoid serum antibody by use of this cytotoxicity assay.

Adolescent

Colony formation of cytolytic T cells in semisolid medium.

Addition of supernatant from concanavalin A-stimulated spleen cells to in vitro primed cytolytic T lymphocytes in semisolid medium stimulated the growth of colonies of cytolytic lymphocytes. Optimal results were obtained using a peritoneal adherent cell underlayer where a 10% plating efficiency (greater than or equal to 4 cells per colony) was achieved when between 5000 and 100 000 cells were plated per dish. Individual colonies were harvested and tested in a short term (5h) 51Cr release microassay, employing 200 target cells. The frequency of lytic colonies varied from 46--67%. The observed lytic activities were specific for the relevant allogeneic target cells.

Animals

Influenza virus-specific cytotoxic T cells in man; induction and properties of the cytotoxic cell.

Human peripheral blood lymphocytes have been sensitized in vitro to influenza virus antigen. After an induction period of 4--14 days, cytotoxic cells which lyse autologous influenza virus-infected lymphoid cells could be demonstrated. The cytotoxic cell is a T lymphocyte which shows specificity for sensitizing influenza virus type A or B. It cannot distinguish between major subtypes of influenza A virus. The use of virus-infected normal lymphoid cells as target cells overcame the difficulties of nonspecific killing encountered with some transformed cells.

Cytotoxicity Tests, Immunologic

High nonspecific reactivity of normal lymphocytes against mycoplasma-infected target cells in cytotoxicity assays.

Several rat tumor cell cultures were deliberately infected with three species of mycoplasma commonly found as contaminants of cell lines grown in vitro, and the effect of mycoplasma infection on the results of cytotoxicity assays was examined. Lymph node cells and spleen cells from normal animals showed an apparently high spontaneous cytotoxic activity against tumor cells infected with either M. arginini or M. hyorhinis, but the reactivity against cells infected with M. orale was not significantly higher than that against uninfected cells. The high reactivity towards tumor cells infected with M. arginini and M. hyorhinis bore a close resemblence to natural cell-mediated immunity in that spleen cells were much more reactive than lymph node cells, spleen cells from nude mice were as effective as spleen cells from normal mice, and the reaction crossed both strain and species barriers. However, closer examination revealed that the cytotoxic effects were directly caused by depletion of arginine or other essential nutrients from the medium. These findings imply that a cautious approach should be taken when interpreting certain aspects of spontaneous cell-mediated cytotoxicity, and that the greatest care be taken to ensure that the cells used as targets in any cytotoxicity test are mycoplasma-free.

Animals

Lymphocyte cytotoxicity against autologous tumour biopsy cells in humans.

By the application of separation techniques in a stepwise manner to mechanically prepared cell suspensions from human tumour biopsies it has been possible to isolate tumour cells having high viability and low contamination with host cells. These tumour cells have been used as targets in 51Cr release cytotoxicity assays using autologous lymphocytes from blood or lymph node as effectors. It has been possible to investigate this reactivity in 30 patients with malignant and four patients with benign tumours. Significant autologous cytotoxicity in the blood lymphocytes was detectable in two of 12 lung tumours, nine of 18 other carcinomas and sarcomas but in none of the benign cases. Cytotoxicity was not limited by the presence of obvious residual disease at the time of operation although reactivity was less frequently detectable in patients with secondary involvement. Reactivity in the tumour-draining lymph nodes largely paralleled that in the blood in the limited number of patients examined. The technical problems encountered during this study and the role of natural cytotoxicity in the interpretation of these data are discussed.

Animals

Tumor-associated humoral cytotoxicity in patients with acute myelogenous leukemia before and after chemotherapy.

Sera of eight unselected adult patients with acute myelogenous leukemia obtained before and after chemotherapy were repeatedly tested for specific complement-dependent cytotoxicity against autochthonous peripheral white blood cells from the acute leukemia stage and from the remission stage, respectively. Complement-dependent cytotas demonstrated in all of the eight patients, while none of three patients' sera were reactive against white blood cells from the remission stage tested in parallel. The cytotoxicity was increased after chemotherapy, also in those patients in whom remission was not achieved.

Adult