Leukocyte Adherence Inhibition Research Articles

Humoral leukocyte adherence inhibition response in patients with oral cancer.

Antitumor immunity was assessed in a group of 10 patients suffering from squamous cell carcinoma of the oral cavity using the humoral leukocyte adherence inhibition assay (H-LAI). A tumor extract from a person with cancer of the tongue was used as antigen. Sera from five patients with carcinoma of the tongue were all positive in the test. Of the other five patients, three had cancer of the lips while in two patients the type was unspecified. All patients except one with cancer of the lips gave a positive response in the test. Thus, of the 10 patients with carcinoma of the oral cavity, nine responded. Among nine controls, one person gave a positive response.

Measurement by Leukocyte Adherence Inhibition of Autosensitization of Cancer Patients to Myelin Basic Protein

In vitro cell‐mediated immunity was assayed by leukocyte adherence inhibition (LAI) to determine the extent of autosensitization to myelin basic protein (MBP). Leukocytes from 123 cancer patients, 16 patients freed of cancer, 135 patients with benign disease, and 26 patients with destruction of nervous parenchyma were tested. Most patients with cancer reacted to MBP: 92%, 93%, 82%, 78%, 75% and 62% for pancreatic, colonic, esophageal, lung, ovarian and breast. Few patients with benign diseases reacted to MBP. Patients with multiple sclerosis (MS) were sensitized to MBP, but patients with other nervous tissue injury did not react to MBP. Cancer patients did not remain sensitized to MBP once they were freed of their cancer. The LAI assay is a straightforward method of measuring cellular autosensitivity to MBP. In the population of patients tested, autosensitivity to MBP was confined, except for MS, principally to cancer patients.

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 (> 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 fitration 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.

Effect of aggregated IgG on mononuclear and polymorphonuclear cell cooperation.

The leukocyte adherence inhibition (LAI) test was used to determine the response of different leukocyte subpopulations in rheumatoid arthritis (RA) patients and normal donors to heat-aggregated gammaglobulins (HAGGs). The adherence of polymorphonuclear cells of (PMNs) could be inhibited either directly by high concentrations of HAGGs or indirectly by soluble factors secreted by RA mononuclear cells incubated with low amounts of HAGGs. The stimulatory site of the IgG was located on the Fc fragment. Since the LAI reaction was also observed with material recovered from rheumatoid synovial fluid, tissue, and nodules, these results could have clinical relevance in RA.

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 × 10 3 was employed. Following in vitro challenge with DF, radioimmunoassay using an antiserum to LTC 4 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 LTC 4. 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.

Automated leucocyte adherence inhibition testing in patients with colorectal cancer.

This paper details our initial experiences with a semi-automated leucocyte adherence inhibition (SALAI) assay in patients with colorectal disease. Two assay systems were used. Leucocytes from blood donors and patients with different colorectal diagnoses were tested for sensitization to colorectal tumour extracts, and leucocytes from healthy blood donors were assayed with serum from blood donors or patients to determine whether the serum itself contained factors which would react with the non-sensitized leucocytes in the test system. The sensitivity of the SALAI assay using patients' leucocytes was 64%, and the specificity was 68%. Discriminant analysis did not affect the sensitivity of the assay for colorectal cancer (64%), although the specificity was increased for all patients except those with malignant disease other than colorectal cancer. The sensitivity of the SALAI assay using patients' serum was 50% but the specificity was 74%. Discriminant analysis increased the sensitivity of this assay to 80% but specificity was reduced to 61%. Thus, the SALAI assay with patients' serum, although potentially more advantageous than the assay using patients' leucocytes in the clinical setting, was less specific. Furthermore, samples from patients with early colorectal cancers were less reactive making the serum assay unsuitable for screening asymptomatic patients. The SALAI assay using patients' leucocytes, however, has a higher sensitivity than most reported variations of the assay but a slightly lower specificity. It is suggested that the SALAI assay is preferable to other methods for leucocyte adherence inhibition (LAI) testing.

Recognition of a Purified <italic>M</italic><sub>r</sub>-40,000 Organ-Specific Immunogen of Human Lung Cancer by Class II-Restricted T-Cells<xref ref-type="fn" rid="FN1">2</xref>

An Mr-40,000 polypeptide (p40) was purified from a lung cancer cell line on the basis of its antigenicity with leukocytes from lung cancer patients in an in vitro immunological assay of leukocyte adherence inhibition. Here, the cells and mechanism responsible for recognizing the purified p40 organ-specific cancer neoantigen (OSN) were studied. Buffy coat leukocytes from lung cancer patients showed a bell-shaped dose response with a peak response at 0.75 micrograms/assay. Mononuclear cells showed a similar response pattern, whereas pure T-cells were unreactive. Monoclonal antibodies (MAbs) to T-cell differentiation antigens T3 and T4 inhibited the response in a dose-dependent fashion, whereas anti-T8 had no effect, indicating T helper cells and their T3-antigen receptor complex recognized the antigen. MAbs to class II major histocompatibility complex (MHC) antigens also inhibited the response, whereas MAbs to class I MHC had no effect, indicating an important role for class II MHC antigens of monocytes. None of the MAbs inhibited the response to OSN on membrane fragments, which is mediated by antibody-dependent monocytes. Trypsin- or cyanogen bromide-cleaved p40 OSN triggered a response at the same concentrations as the intact molecule. The p40 OSN incubated with live leukocytes showed less than 30% proteolytic digestion. The results indicate that class II-restricted T-cells recognize, via their antigen-specific cell surface receptors, contiguous sequences within the immunogenic tumor molecule in the context of the molecule or peptides binding to class II transplantation antigens.

Leukocyte adherence inhibition: History, biological basis and clinical usefulness

Leukocyte adherence inhibition: History, biological basis and clinical usefulness

Inhibition of sensitized leukocyte's in vitro reactivity by circulating immune complexes in prostate cancer.

Circulating immune complexes in the sera of patients with confirmed histological diagnosis of carcinoma of the prostate, were found to interfere in the sensitized leukocyte's in vitro reactivity to prostate cancer associated antigen as evaluated by tube leukocyte adherence inhibition assay, thereby suggesting an inhibitory role of such serum factors in host's anti tumor cell mediated immune responses.

An animal model for evaluation of antigen-specific dialyzable leukocyte extracts therapy of osteosarcoma

The effects of human osteosarcoma (OS)-specific dialyzable leukocyte extracts (DLE) in hamsters bearing human OS were investigated. The DLE used in this investigation was prepared from rabbits immunized with human osteosarcoma-associated antigens (DLE-OSAA). Tuberculin (DLE-PPD) and control DLE were prepared from rabbits injected with tuberculin or 0.85% NaCl (DLE-NaCl). DLE was administered subcutaneously into inbred hamsters (each injection contained DLE derived from 10 7 rabbit leukocytes). Four groups of animals were studied: group 1, amputation alone; group 2, amputation plus DLE-OSAA; group 3, amputation plus DLE-PPD; group 4, amputation plus DLE-NaCl. Of the DLE-OSAA-treated animals (group 2), 60% were still alive at 300 days postamputation; whereas in animals in groups 1, 3, and 4, all died within 90 days postamputation. In separate experiments, we found that 100% of the animals in groups 1, 3, and 4 developed pulmonary metastases within 30–60 days post-amputation, whereas only 20% of the animals in group 2 developed metastases at the same time; indeed 40% of the DLE-OSAA-treated animals were free of metastases in 240–300 days postamputation. Both the leukocyte adherence inhibition assay (LAI) and lymphocyte DNA synthesis assay (LDS) were used to monitor the transfer of antigen-specific cell-mediated immunity in each group of tumor-bearing hamsters. All surviving hamsters in group 2 had high LAI and LDS activity. Our results suggest that DLE-OSAA is effective in preventing pulmonary metastases and death of OS-bearing hamsters (after amputation) as compared with amputation alone, amputation plus DLE-NaCl, and amputation plus DLE-PPD, and that its effect is via an antigen-specific mechanism.

Cell-populations involved in the humoral leukocyte adherence inhibition reaction. The T8-suppressor/cytotoxic cells are both effectors and responders

In an attempt to identify the cell subpopulation involved in the humoral leukocyte adherence inhibition (H-LAI) reaction, mononuclear leukocytes were fractionated and the response with the different cell populations was measured in the H-LAI assay using sera from lung cancer patients and a lung cancer associated antigen. The monocytes were found to exhibit a suppressive effect on the H-LAI response. The B-lymphocytes seemed not to take an active part in the reaction. Depletion of the T-lymphocytes abolished the reaction. Subsequent studies on the T-lymphocyte subpopulations revealed that removal of the T 4-cells had little effect while removal of the T 8-cells resulted in complete abrogation of the H-LAI response. Direct blocking by monoclonal antibodies against the T 4 and T 8 surface determinants gave additional support for the T 8-cell dependency of the H-LAI assay. The cells that specifically were induced to loose adherence in the H-LAI assay upon incubation with serum from a lung cancer patient and lung cancer associated antigen were also analyzed. Compared to the total mononuclear leukocyte population, the relative content of monocytes and B-cells were lower in the non-adherent population. An increase was found in the relative number of T-cells. While the relative number of T 4-cells decreased, the content of T 8-cells was significantly increased. Furthermore, a direct correlation was found between the loss of adherence of the T 8-subpopulation and the index of the H-LAI response. The present study indicates the T 8-subpopulation of the T-lymphocytes to be involved both in the effector and responder side of the H-LAI reaction and gives implications for an immunological reaction behind the response of the H-LAI assay.

Extraction of immunogenic murine mammary tumor antigens with 1-butanol.

Solubilized tumor associated antigens (TAA) were prepared by a single phase 1-butanol extraction of a transplantable murine mammary adenocarcinoma (H2712), other syngeneic non-mammary tumors, normal mammary tissue, and embryonic tissues. The crude butanol extract (CBE) of H2712 was found to have tumor-specific activity both in vitro in the leukocyte adherence inhibition (LAI) assay and in vivo in an immunoprophylaxis assay. The failure to detect any of the major mammary tumor virus (MTV) envelope glycoproteins or internal core protein in CBE-H2712 and the failure of tumor-bearing mice to give a LAI response to CBE prepared from embryonic tissues suggests that the CBE antigens which are responsible for LAI activity are not viral or oncofetal antigens. Enzymatic treatment of the CBE-H2712 indicated that a sialated glycoprotein formed the antigenic determinants recognized in CBE by the LAI assay. Gel filtration of LAI-reactive CBE-H2712 on a Sephadex G-75 column isolated both the in vitro and in vivo tumor-specific activity in a fraction pool. Polyacrylamide gel electrophoresis revealed the presence of a 67 kD subunit as the major species in the active fraction.

4666834 Leukocyte adherence inhibition assay: Julius G Bekesi, James F Holland, Peter Tsang assigned to Mount Sinai School of Medicine of the City University of New York

4666834 Leukocyte adherence inhibition assay: Julius G Bekesi, James F Holland, Peter Tsang assigned to Mount Sinai School of Medicine of the City University of New York

Tumor-specific idiotopes on suppressor factors and suppressor cells revealed by monoclonal anti-idiotope antibodies

Two monoclonal anti-idiotope antibodies, previously found to induce tumor-specific cellmediated immunity in mice, were examined for their relationship to tumor-associated suppressor factors (SF), produced in culture by spleen cells from tumor-bearing mice or present in sera from such mice. A leukocyte adherence inhibition assay was used to detect cellular immunoreactivity to tumor antigens and its inhibition by SF, using peritoneal cells from mice bearing tumor or sensitized with anti-idiotope antibody. The SF were specifically absorbed by the corresponding anti-idiotope antibodies coupled to a solid phase and were recovered by elution. They were also specifically neutralized by the addition of the respective antibodies to the assay system. Antiidiotope antibody, used with complement to pretreat spleen cells from tumor-bearing mice, prevented these cells from producing SF in culture. Tumor antigen-reactive effector cells, suppressor cells, and SF thus share similar idiotopes, permitting their respective functions to be modulated by appropriate anti-idiotopes.

Immune reactivity to Thomsen-Friedenreich antigen in patients with lung cancer detected by superoxide assay leukocyte adherence inhibition test

The immune reactivity to partially purified Thomsen-Friedenreich antigen was investigated in patients with lung cancer. The modified method of original leukocyte adherence inhibition test, termed superoxide assay-leukocyte adherence inhibition test, was used to detect the reactivity. The coded peripheral mononuclear cells from 34 of 50 (68%) patients with lung cancer showed a positive response to the antigen whereas in only 3 of 19 (16%) patients with benign pulmonary disease was there a reaction to the antigen. The same experiments were performed using the 3 m KCl extract of lung tumors as an antigen. In this case in 39 of 50 (78%) patients with lung cancer but in only 4 of 24 (17%) with benign pulmonary disease and in none of the breast cancer patients (0 of 17) was there a reaction to the antigen. These results strongly suggest that patients with lung cancer are sensitized to both Thomsen-Friedenreich antigen and tumor-associated antigens expressed in cancer cells of lung tissue origin.

A comparison of the macrophage migration inhibition (MMI) assay and the semi-automated leucocyte adherence inhibition (SALAI) assay.

The present study was undertaken in order to compare simultaneously the semi-automated leucocyte adherence inhibition (SALAI) assay with the two-stage macrophage migration inhibition (MMI) assay in patients with colorectal disease. Eighty-six patients were assayed simultaneously in the MMI assay and the SALAI assays using patients' leucocytes and 83 patients were assayed simultaneously in the MMI assay and the SALAI assay using patients' serum. The SALAI assay using patients' leucocytes was more sensitive (70% vs 40%) and more specific (87% vs 73.3%) than the MMI assay. The SALAI assay was simple to perform, allowed many replicate samples and was more specific and sensitive in relation to the detection of colorectal cancer than other more commonly advocated tests. It may be useful as a screening test for colorectal cancer and it is suggested that further studies should be undertaken in this area.

Leukocyte adherence inhibition in patients with nonmalignant disorders of the colon and colorectal cancer.

The tube leukocyte adherence inhibition (LAI) technique was used to investigate the antitumor immunity in two groups of patients generally considered to be at "high risk" of developing colorectal cancer. The first group comprised 21 patients with colorectal polyps and the second 12 patients with various forms of colitis. Also 29 patients with histologically confirmed colorectal cancer were tested. The tube LAI assay was performed using peripheral blood leukocytes from individual patients and crude extracts of colorectal and breast cancers. Positive LAI reactions were observed in 18 out of 29 (62%) patients with colorectal cancer, in 1 out of 21 (5%) patients with colorectal polyps and in 1 out of 12 (8%) patients with colitis. The results indicate that in confirmed cases of malignancies, sensitization to colon tumor-associated antigens could be detected in the tube LAI test, whereas, premalignant sensitization to these antigens in "high risk" groups of patients could not be demonstrated.

An isolated enriched organ-specific cancer neoantigen of human lung cancer for leukocyte adherence inhibition assays

Until now, measurement of human antitumor immunity to organ-specific cancer neoantigen (OSN) by the leukocyte adherence inhibition (LAI) assay depended on using crude extracts of cancer. In this study, a new method is presented to generate and to isolate a highly enriched OSN from spent medium of a lung cancer cell line, NCI-H69, grown in chemically defined medium. Production of large quantities of OSN with minimal contamination by extraneous proteins was possible. Four physicochemical steps were used to give a 1000-fold enrichment of OSN activity: anion-exchange and molecular-sieve chromatography; Blue Sepharose affinity chromatography; and finally anion-exchange high-pressure liquid chromatography. The enriched OSN isolates showed dose-response antigenicity when tested in LAI assay with leukocytes from lung cancer patients but had no antigenicity with leukocytes from control subjects or patients having malignant melanoma, colon cancer, or pancreatic cancer. Cross-reactive antigenicity was observed with leukocytes from patients with breast cancer and slight reactivity with leukocytes from bladder cancer patients. The final isolate from the four-step separation procedure as well as the isolates produced using additional separation techniques consistently had antigenicity at less than 10 ng in blocking LAI and 500 ng in the direct assay and showed components with molecular weights of about 62,000 +/- 3,000 (SD) (p62), 40,000 +/- 3,000 (p40), and 25,000 +/- 1,000 (p25) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The OSN isolates on two-dimensional gels showed p40 to have microheterogeneity (seven spots), with a pl from 6.2 to 7.6, and p62 and p25 as even more basic streaks. The polypeptide bearing the antigenic determinant was not purified, although we tried to separate p62, p40, and p25 to determine whether they carried the OSN determinant. The results of this study are important in showing that an isolate of an organ-specific tumor antigen containing 5 to 13 components, as determined by highly sensitive silver stains and radiolabeled patterns on single and two-dimensional gels, can be used successfully in LAI to measure tumor immune responses.

Purification from a human lung cancer cell line of a water soluble molecule mediating leukocyte adherence inhibition for patients with lung cancer

Purification from a human lung cancer cell line of a water soluble molecule mediating leukocyte adherence inhibition for patients with lung cancer

New micro-glass-tube leukocyte adherence inhibition assay assessing cell adherence of mononuclear cell subpopulations defined by monoclonal antibodies

A new micro-glass-tube leukocyte adherence inhibition (LAI) assay which is appropriate for detecting delayed type hypersensitivity in vitro has been developed for human leukocytes. Enumeration of adherent cells is replaced by a cellular radioimmunoassay determining antibody binding of the monoclonal reagents, OKT4, OKT8, and OKM1, to glass-adherent cells, fixed by glutaraldehyde or formaldehyde. An LAI reactivity to purified protein derivative of tuberculin (PPD) was detectable in donors giving a positive PPD skin test with OKT4 reagent, but not with the other two reagents.