A radio (111In) capillary tube leukocyte adherence inhibition assay for the detection of specific tumor-associated immunity

The specific tumor-associated immune response of C3H/HeJ mice was determined at various times after subcutaneous injection with a transplantable mammary adenocarcinoma (H2712) using a radio (111In) leukocyte adherence inhibition (LAI) assay carried out in capillary tubes. Solubilized tumor-associated antigen prepared by a single phase 1-butanol extraction of the specific tumor and other transplantable tumors of different histological origin were used in the evaluation of LAI reactivity. The assay was found to be capable of detecting a significant antitumor response before the subcutaneous tumors became detectable by palpation. The response remained significant until the tumors were greater than 20 mm in diameter.     

Schistosoma mansoni: cell-mediated immunity evaluated by antigen-induced leukocyte adherence inhibition assay

Peripheral blood leukocytes (PBL) from schistosomiasis patients fail to adhere to glass in the presence of soluble egg antigens (SEA) or soluble worm adult antigenic preparation (SWAP). These leukocytes are non-reactive with S. mansoni unrelated antigens (C. albicans and bovine albumin). Supernatants obtained from cultures of mononuclear cells of patients with antigens were able to inhibit granulocyte adherence to glass. The inhibition of antigen-induced adherence (LAI assay) was not observed when PBL or supernatants were obtained from normal subjects or from schistosomiasis patients after chemotherapy. These results show that under the conditions tested, leukocytes appear to react directly with SEA or SWAP thus losing their property of adherence to glass.     

Leukocyte adherence inhibition (LAI) for detecting specific tumor immunity in colorectal cancer

Immunity to colorectal cancer antigen was tested by the tube leukocyte adherence inhibition (LAI) assay, using spent medium of human carcinoma cell lines as a source of antigen. The assay was positive in 18 out of 43 patients (41%) with colorectal tumor in comparison to only 2 out of 34 (5.8%) of colorectal cancer patients clinically free of disease, and to 4 out of 150 (2.6%) healthy subjects. The frequency of positive results correlated negatively with the tumor burden, 16 out of 29 (55.1%) patients with Dukes' A, B and C being positive compared to only 2 out of 14 (14.2%) patients with Dukes' stage D disease. Addition of PGE2 enhanced the sensitivity of the assay without affecting its specificity. The number of positive assays increased from 18 to 32 (41.8% to 74.4%) in the whole group of 43 colorectal cancer patients without altering significantly the frequency of positive results in the control group. The results with all groups of patients were influenced similarly by the addition of prostaglandins, the frequency of positive assays increasing from 55.1% to 82.7% and from 14.1% to 57.1% in early and advanced disease, respectively. These results lend further support to the value of the LAI assay in diagnosis and monitoring of the immune response in human colorectal cancer.     

Leukocyte adherence inhibition: an automated microassay demonstrating specific antigen recognition and blocking activity in two murine tumor systems.

This communication describes an automated micro-adherence inhibition assay. Tumor-specific immunity was demonstrated with B16 melanoma and MCA-38 colon adenocarcinoma, both of which are syngeneic to the same strain of mouse (C57B16/J). Abrogation of the leukocyte adherence inhibition (LAI) response of sensitized leukocytes has been demonstrated in the MCA-38 tumor system by the addition of serum from mice bearing MCA-38 progressively growing tumors, a property not present in normal serum. The sensitivity of the system has also been demonstrated by showing that LAI will change prior to a tumor becoming palpable. This microassay has the advantage of being simple, rapid and reproducible, and involves the use of minimal quantities of antigenic preparations and leukocytes, and in addition is amenable to rigorous statistical analysis.     

Allergen-specific leukocyte adherence inhibition (LAI) assay: sensitivity, specificity and mechanism

An allergen-specific tube leukocyte adherence inhibition (LAI) assay has been developed in order to study the mechanism by which leukocytes lose their normal property of adherence to glass. Peripheral blood leukocytes (PBL) from 27 individuals allergic to Dermatophagoides farinae (DF), 10 with seasonal rhinitis not induced by DF and 49 non-allergic healthy volunteers were challenged in vitro with DF and a non-relevant allergen, Artemisia vulgaris (AV) and then assayed for the ability to adhere to glass tubes. Challenge by DF, but not by AV, resulted in loss of adherence by PBL from patients allergic to DF, but not in those of normal controls. The specific LAI response was dose-dependent and occurred only when a critical dose of 0.5 X 10(3) was employed. Following in vitro challenge with DF, radio-immunoassay using an antiserum to LTC4 detected immunoreactive material in supernatants of PBL from DF-allergic individuals. When highly enriched mononuclear cells from non-allergic individuals were armed with cytophilic allergen-specific IgE and challenged with the specific allergen, they lost the property of glass adherence and released a substance that was immunoreactive with LTC4. The results suggest that the chain of events leading to the LAI response in PBL from allergic individuals involves primary recognition of the allergen by specific IgE antibodies bound to receptors on mononuclear cells. The cells are thus triggered to synthesize cysteinyl-containing leukotrienes which mediate the LAI phenomenon. The results suggest that this assay may be used to study allergen-antibody interaction and the subsequent events leading to the clinical picture of atopic diseases.     

The role of leukocyte subpopulations in the indirect leukocyte adherence inhibition assay in the mammary tumor virus system.

A modification of the leukocyte adherence inhibition test for the detection of cellular immunologic reactivity of mice to the mammary tumor virus (MTV) has been described. It involves the transfer of the leukocyte adherence inhibition factor (LAIF) produced by spleen cells from immunized animals when cultured with antigen to indicator cells, for which peritoneal exudate cells from normal mice are used. The method proves to be sensitive and highly reproducible. By crude separation of leukocyte subpopulations it became established that for the production of LAIF the following sequence is needed: 1. incubation of adherent cells with MTV;2. transfer of a soluble factor SF1 produced by the adherent cells to T cells; 3. transfer of another soluble factor SF2 released by T cells to the adherent cells.     

Lymphoid source and target of murine leukocyte adherence inhibition factor (LAIF)

The cellular source of murine LAIF detected by an indirect leukocyte adherence inhibition (LAI) assay using capillary tubes was investigated using non-induced peritoneal cells (PC) from normal and spindle-cell sarcoma-bearing A/J mice. Cell populations containing >95% T-cells, B-cells or macrophages were prepared from PC using a series of elimination and enrichment procedures. The in vitro incubation of enriched T-cell populations (<0.05% macrophages) from tumor-bearing mice with the specific soluble tumor antigen did not result in the release of detectable levels of LAIF; however, the addition of 10% normal isologous macrophages to the T-cells resulted in a significant release of LAIF, Enriched B-cell populations did not release LAIF either by themselves or when 10% normal isologous macrophages were added. Cell populations containing both T-cells and B-cells produced significant levels of LAIF, but only when suitable numbers of macrophages were present. Treatment with anti-Thy-1.2 alloantiserum and complement resulted in the abrogation of LAIF production by mixed cell populations. Using Lyt-1.2 and Lyt-2.2 alloantisera and complement to prepare either Lyt-1.2 or Lyt-2.2 depleted T-cell populations, it was found that the Lyt-1.2 subpopulation was responsible for the release of LAIF in this test system. LAIF was found to be effective in reducing the glass adherence of macrophages but not of T-cells or B-cells.     

A microcytotoxicity assay using 51Cr for measuring specific cell-mediated and humoral immune responses.

A microcytotoxicity assay (MCA) for measuring in vitro cell-mediated cytotoxicity (CMC) and humoral cytotoxicity which uses 51Cr-labelled target cells is described. Data is presented to show the uptake and rentention of 51Cr by various viable cultured cells. The CMC determined by 51Cr-labelled target cell retention in microtest II plate wells was verified by microscopic observation. Comparisons between the CMC observed 24 and 48 h after incubation of immune and normal lymphoid cells with 51Cr-labelled target cells were made. The factors, including savings in time and expense, which may make this modified MCA superior to other MCAs in use, are discussed.     

Capillary tube leucocyte adherence inhibition assay for cell-mediated immunity: effects of transient bacterial infection in guinea-pigs.

Adherence of guinea-pig leucocytes to glass, and changes of adherence caused by antigen challenge, were measured during a 3-month period in which an idiogenic, fortuitous bacterial infection passed though the main guinea-pig colony. Expected responses were found in SPF animals, and, at the beginning and end of this period, in stock animals and those immunized with Freund's complete adjuvant with or without added keyhole limpet haemocyanin. During active infection, all non-SPF animals showed enhanced adherence changes whilst SPF animals continued to give expected responses. The implications for use of leucocyte adherence inhibition tests with human subjects are discussed.     

Radioisotopic 15Cr-Leukocyte adherence inhibition (LAI) assay. I. Demonstration of anti-tumor immunity in patients with breast carcinoma

a simplified radioisotopic leukocyte adherence inhibition assay (⁵¹Cr-LAI assay) was used to determine tumor-directed immune responses in patients with cancer of the breast. Essential steps in development of this assay are the standardization of conditions for optimal ⁵¹Cr uptake by peripheral blood lymphocytes (PBL) and the inclusion of autologous or normal AB serum in the incubation media. A dextrose salt mixture (GNK) was found to enhance intracellular uptake of ⁵¹Cr significantly (8-fold) without affecting viability of the cells or without causing selective loss of lymphocyte subpopulations. The presence of 10% autologous or normal AB serum prevented non-specific LAI responses to unrelated tumor antigens.In a study of 46 preoperative patients with suspected breast cancer, clear and accurate prediction of the presence of cancer was achieved with this new assay. All patients with localized breast cancer showed significant adherence inhibition in response to allogeneic breast tumor extracts whereas normal control women and patients with benign disease did not respond. Neither patients with cancer nor those with benign breast diseases reacted to extracts of benign breast tissue antigens. LAI reactivities appeared to be directed selectively against tumor-associated antigens (TAA) and reflect specific anti-tumor immunity.This short term (4 h) ⁵¹Cr-LAI assay provides reproducible and specific results analogous to those using tube-LAI assay. The test has the advantages of being accurate, sensitive and free from technical bias.     

Humoral leukocyte adherence inhibition (H-LAI) with basic encephalitogenic protein (BEP) in head and neck cancer

Fourty-five patients with head and neck cancer and 30 healthy subjects were evaluated by a modified humoral leukocyte adherence inhibition assay using basic encephalitogenic protein as antigen. Ninety-three percent of cancer patients gave a positive reaction, whereas every control person remained negative. The results suggest that humoral leukocyte adherence inhibition assay performed with basic encephalitogenic protein may be useful as an early diagnostic tool in head and neck cancer.     

In vitro induction of tumor specific immunity: requirements for T lymphocytes and tumor growth inhibition in vivo

Cortisone-resistant thymocytes, spleen cells, thoracic duct lymphocytes, peritoneal exudate cells and peripheral blood lymphocytes of BALB/c mice were immunized in vitro against syngeneic HPC-108 plasma cell tumor cells. Cocultivation of spleen lymphocytes together with HPC-108 cells generated the highest cytotoxic immune response in comparison to other lymphocyte sources. Cytotoxicity was tested in a 6 hour ⁵¹Cr release assay using HPC-108 cells as target cells. The use of AKR anti-θ C3H serum indicated that thymus-derived (T) lymphocytes are essential to the initiation phase of the immune response to plasma cell tumor cells. Furthermore, evidence is presented that the cytotoxic effector cells in the in vitro tumor immune response are T lymphocytes. Spleen cells activated in vitro against HPC-108 tumor cells were shown to specifically prevent tumor growth from simultaneously injected HPC-108 cells in irradiated recipient mice.     

Effector lymphocyte response to homologous tumor antigens in various stages of malignant disease as monitored by leukocyte adherence inhibition--cell mediated immunity (LAI-CMI)

Leukocyte adherence inhibition-cell mediated immunity (LAI-CMI) studies were performed on leukocytes obtained from patients with various stages of breast cancer, colon carcinoma and lung cancer in order to monitor cell mediated immunity during tumor progression. In the presence of autologous serum, all patients with localized tumors showed positive LAI-CMI indexes (greater than 20%), while significant reduction of homologous tumor antigen recognition as measured by the LAI-CMI responses was observed in nearly all patients with Stage IV breast cancer, Duke C colon cancer and Stage III lung cancer. On substituting autologous serum with normal AB serum, leukocytes from patients with large tumor burdens responded to homologous tumor antigens. These results indicate the existence of organ-specific serum factor(s) which may mask the receptor sites on effector cells for tumor recognition. Patients with such serum blocking factor(s) showed significant increase of IgG immune complexes IgM, IgA and alpha-2-macroglobulins. Application of a protein A affinity column purification resulted in a major reduction of IgG and other immune globulins but not of alpha-2-macroglobulin. The blocking effects of autologous serum, however, were not completely abrogated by filtration on the protein A column, thus suggesting that SBF may be heterogeneous in nature and may occur in other serum protein fractions beside the immune globulins.     

Prognostic utility of a glycoprotein tumor-associated antigen (TA90) specific immune complex assay in patients with cancer

This review summarizes the development and applicability of an assay to detect an immune complex (IC) of a 90kD glycoprotein tumor-associated antigen (TA90). The antigen is immunogenic in the cancer host and expressed by a variety of solid tumors. In circulation, it induces formation of endogenous antigen-antibody complexes. The presence of TA90-IC has been detected in significantly greater proportions of serum samples from cancer patients compared to normal controls. The postoperative presence of TA90-IC in circulation strongly correlated with subsequent development of clinically detectable recurrence within an average interval of 3 to 19 months. The TA90-IC status after surgery but before initiation of adjuvant therapy consistently correlated with disease-free survival and overall survival. Because of its ability to accurately detect subclinical metastasis and predict survival 1 to 3 months after surgical resection of early-stage cancer, TA90-IC represents a novel and feasible prognostic marker to select candidates for adjuvant therapies and to stratify patients in clinical trials. Expression of TA90 by melanoma, colon cancer, lung cancer, breast cancer and other solid neoplasms makes TA90-IC an ideal marker in the management of early-stage cancer. Its specificity can be significantly enhanced if positive samples are subsequently analyzed by a panel of tumor markers characteristic for a particular type of cancer, such as CA15-3 for breast cancer and carcinoembryonic antigen (CEA) for colon cancer. TA90-IC could be used in conjunction with these site-specific markers as part of a cancer screening program to identify high-risk individuals for closer surveillance.     

~3H-TdR、~(51)Cr、~(125)I-UdR释放试验测定NK活性的比较研究

用~3H-TdR、~(51)Cr、~(125)I-UdR 分别标记YAC-1细胞,测定小鼠 NK 细胞活性 ,三种方法所获得的结果呈高度相关性,~3H-TdR 分别与~(51)Cr、~(125)I-UdR 相比以及~(51)Cr与~(125)I-UdR 相比的相关系数依次为 0.9917(P<0.001),0.9725(P<0.001),0.9478(P<0.005),表明三种释放试验均可用于检测细胞毒活性。用收集器收集残存的~(125)I-UdR 标记细胞,不仅其特异性释放率比常规测上清液的释放率要高10％左右,而且制样快速、简便。本文对三种方法的优缺点作了比较,认为~3H-TdR 释放试验作为检测细胞毒是目前国内切实可行的方法。     

Cell mediated immunity to herpes simplex in man. IV. A correlation of lymphocyte stimulation and inhibition of leukocyte migration.

The cell mediated immune response to herpes simplex virus was assessed by a modification of the leukocyte migration inhibition test, developed to increase its sensitivity and correlated with the in vitro stimulation of lymphocytes. The introduction of a preincubation step allowed us to demonstrate significant inhibition of leukocyte migration in 15 out of 24 subjects susceptible to recurrent herpes labialis. This was not present in any of the 10 subjects without such infections. However lymphocytes from all 24 subjects demonstrated stimulation in response to herpes virus antigen. The degrees of lymphocyte stimulation and migration inhibition in the susceptible subjects were well correlated and do not support the suggestion that there is a focal defect in this aspect of the cell mediated immune response to herpes simplex virus in those patients.     

Studies on cellular immunity against bile proteins in primary biliary cirrhosis by the leukocyte migration inhibition test (microdroplet method)

Cellular immunity against human bile proteins was investigated by the leukocyte migration inhibition test (LMIT) with 13 primary biliary cirrhosis (PBC) patients, 10 chronic aggressive hepatitis (CAH) patients and 21 healthy adults. Hepatic bile taken from patients operated on for lithiasis of the biliary tract was fractionated into five fractions with Sepharose 6B gel. A subtoxic dose of each fraction was determined in the healthy adults, and used as the antigen for LMIT. Out of the 5 fractions, only the third fraction led to an LMIT positive response in 8 out of 11 (73%) PBC patients and in 1 out of 10 (10%) CAH patients. The difference between PBC and CAH was significant (p less than 0.005). The remaining 3 PBC patients with LMIT negative responses were all under D-penicillamine treatment. Antibody to each fraction was prepared in rabbits. Using the antibodies after absorption with human serum, the localization of the antigens which were present in each fraction was investigated immunohistochemically using human liver sections. The antigen to the anti-first fraction antibody was detected specifically in the epithelial cells of the bile ducts and the ductules, and the antigen to the anti-third fraction antibody was detected specifically on the membrane of the bile canalicules. The third fraction was fractionated into three fractions by Sephadex G-200 gel. Only the first of the 3 fractions showed an LMIT positive response in 3 PBC patients, and its molecular weight was determined to be about 500,000. It is concluded that PBC patients develop cellular immunity against canalicular-antigen-containing fractions but not ductal-antigen-containing ones.