Tube Leukocyte Adherence Inhibition Assay Research Articles

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.

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.

Leukocyte adherence inhibition (LAI) in rats bearing transplantable syngeneic tumors of different immunogenicity

The tube leukocyte adherence inhibition (LAI) assay was used to follow the LAI response in inbred BN rats with subcutaneously (s.c.) transplanted syngeneic liposarcoma (LS 175) tumor and in inbred WAG rats with s.c. transplanted syngeneic colon (CC 531) and skin ( 1618) tumors. Initially, the tube LAI assay was used to follow the LAI reactivity of peripheral blood leukocytes (PBL) from BN rats bearing tumor LS 175. Sporadic LAI reactivity was observed in the PBL of these rats when incubated with a specific crude tumor extract for either 2 or 20 hr. No definite conclusion regarding the lack of significant tumor-specific LAI reactivity could be drawn since LS 175 is a non-immunogenic tumor. Therefore additional LAI studies were performed with the weakly immunogenic tumor CC 531 and the highly immunogenic tumor 1618 in WAG rats. Surprisingly, only sporadic LAI reactivity was observed in the PBL of rats bearing either the CC 531 or the 1618 tumors using both the 2 and 20 hr LAI assays. This absence of tumor-specific LAI reactivity was particularly remarkable in the highly immunogenic 1618 tumor-bearing rats, since it would suggest that the antigen(s) responsible for its high immunogenicity could not evoke any consistent LAI reactivity. Since oxidative metabolites of arachidonic acid have been shown to be the final mediators of chemoattractant-induced LAI, the adherence inhibition effect of leukotriene B 4 (LTB 4) on the PBL of BN and WAG rats was also investigated in order to exclude the possibility that the lack of consistent LAI reactivity was due to a lack of responsive cell population in the PBL. PBL of both BN and WAG rats showed a significant increase in the adherence inhibition in the presence of LTB4, suggesting that the sporadic LAI reactivity was not due to a lack of responsive cell population.

Amplification of lymph node cell tube leukocyte adherence inhibition (LAI) reactivity by leukocyte adherence inhibition factor (LAIF)

This investigation examines the immunologic basis for specific antigen-induced tube leukocyte adherence inhibition (LAI) reactivity of draining lymph node cells (LNC) from dogs with canine transmissible venereal sarcoma (CTVS). CTVS regresser LNC, macrophage-depleted LNC, and enriched T lymphocyte fractions, but not enriched B lymphocyte fractions, were specifically reactive to CTVS antigen extract in direct tube LAI. In addition, regressor LNC amplified tube LAI responses by generating supernatants with leukocyte adherence inhibition factor (LAIF) activity for normal dog indicator LNC and enriched peripheral blood mononuclear cells (PBMC) in an indirect tube LAI assay. However, macrophage-depleted LNC and enriched T lymphocyte fractions failed to generate supernatants with LAIF activity, suggesting that macrophage accessory cells play a central role in the amplification of tube LAI. Interestingly, CTVS regressor peripheral blood leukocytes (PBL) and PBMC, which were specifically reactive in direct tube LAI, also failed to generate supernatants with LAIF activity. These findings demonstrate a distinction between LAIF-mediated amplification and direct tube LAI reactivity, and suggest that leukocyte populations with differing cellular proportions and from different immunologic compartments may participate in tube LAI via different mechanisms.

A Study of the Immune Response to the Organ-Specific Neoantigen of Human Bladder Cancer

The antitumor immune response of monocytes armed with cytophilic antitumor antibody to an organ-specific neoantigen of human bladder cancer was measured by the tube leukocyte adherence inhibition assay. Of 29 patients with Stage A bladder cancer, 27 had positive tests, whereas of 11 patients with Stage B, C and D bladder cancer, 2 had positive tests. If the leukocytes from advanced bladder cancer patients were preincubated briefly with prostaglandin E2, the negative response was converted to positive. Of 189 patients with diseases of other organs, 2 had positive tests. Patients with acute or chronic cystitis had negative tests. The sensitivity of the assay was 88 per cent; specificity was 95 per cent. The bladder cancer extracts prepared from metastatic tissue or the tissue-cultured cell line, T24, had similar activity in the assay. Normal bladder tissue did not share the bladder cancer organ-specific neoantigen nor did cancers of other organs. Bladder epithelial cells expressed the OSN before they acquired invasive properties since patients with dysplastic bladder epithelium or in-situ cancer had positive tests. Antitumor immunity was often detectable before a recurrence became obvious by cystoscopy or cytology of urine and became undetectable quickly after removal of the bladder cancer. Of 100 patients with prior bladder cancer without evidence of exophytic tumor, 18 had positive tests; the possibility must be considered seriously that some had precancerous changes which formed no visible gross abnormalities. The results suggested that antitumor immunity to bladder cancer was a sensitive indicator of precancerous or cancerous changes existing in epithelial cells.

Predictive value of tube leukocyte adherence inhibition (LAI) assay for breast, colorectal, stomach and pancreatic cancer.

The tube LAI assay measures accurately antitumor immunity in patients with early cancer but fails to detect up to 75% of patients with advanced cancer due to excess circulating organ-specific neoantigen (OSN). Substances such as prostaglandin E2 (PGE2) or aminophylline, which increase intracellular nucleotides in leukocytes of patients with advanced cancer reversed this nonreactivity and greatly increased the sensitivity of the assay without any loss of specificity. Antitumor immunity can now be detected in advanced cancer, and a combination of the two assays gives prognostic potential to the assay: a positive test with PGE2 and negative test without indicates the patient has a large tumor burden. The specificity of the assay for each cancer was high and in most instances was greater than or equal to 95%. The PGE2 stimulated assay retained the high specificity. The sensitivity of the regular tube assay was often low, 33-56% because of the many advanced cancer patients tested, whereas the PGE2 stimulated assay showed almost a two-fold increase in sensitivity, 67-93%. The diagnostic value of the assay was estimated by calculating the predictive value for different prevalences of cancer. It was found that at low prevalences of cancer as found in the general population, the assay had a low diagnostic value since few patients with a positive test would have the cancer tested for. With prevalences of cancer of 5% or greater as might be found in a tertiary care clinical setting, the assay would seem to have diagnostic value since one half or more patients with a positive test would have the cancer tested for. Most false positives, but not all, are found in patients who have lesions that are often considered to increase their risk for cancer: severe dysplasia of the breast, colon adenomas, chronic atrophic gastritis and chronic pancreatitis, suggesting that the assay predicts oncogenesis.

Prostaglandin E2 Affects the Tumor Immune Response in Prostatic Carcinoma

Antitumor immunity to prostatic carcinoma was assayed by the tube leukocyte adherence inhibition assay. Peripheral blood leukocytes from 37 patients with prostatic carcinoma and 128 patients without prostatic carcinoma were incubated separately with extracts from prostatic carcinoma and from an unrelated control cancer, and the non-adherence index was calculated. The number of patients with prostatic carcinoma who had a positive response (non-adherence index greater or equal to 30) was small. Nevertheless, the mean non-adherence index (11) of the patients with prostatic carcinoma was statistically different (p less than 0.02) from the mean non-adherence index (2) of the subjects without prostatic carcinoma. Of patients with prostatic carcinoma 14 per cent (5 of 37) had positive leukocyte adherence inhibition responses, compared to 1.5 per cent of control subjects (2 of 128) (p less than 0.05). Sixty patients diagnosed clinically as having benign prostatic hyperplasia were screened by the leukocyte adherence inhibition test and the non-adherence index results then were evaluated with the histopathological diagnosis of the surgical specimen. Of the patients with benign prostatic hyperplasia 3 (5 per cent) had stage A prostatic carcinoma, and 1 of these 3 patients (33 per cent) had a positive leukocyte adherence inhibition test.After in vitro treatment of the peripheral blood leukocytes with 10–6 M. prostaglandin E2 11 of 18 patients with prostatic carcinoma (61 per cent) and 1 of 35 subjects without prostatic carcinoma (3 per cent) had positive leukocyte adherence inhibition responses. The ability of prostaglandin E2 to heighten the leukocyte adherence inhibition response of the peripheral blood leukocytes from patients with prostatic carcinoma suggests that there is a functional defect in the antitumor response of the peripheral blood leukocytes, possibly caused in part by an excess of circulating tumor antigen. In fact, this is supported by the high mean non-adherence index (19) in patients with prostatic carcinoma who had serum prostatic acid phosphatase within normal limits, while patients with an elevated prostatic acid phosphatase had a low mean non-adherence index (0.2). Therefore, patients with prostatic carcinoma are expressing an antitumor response to prostatic carcinoma but this response is weak even when the clinical stage of the disease seems to be early.

A mechanism of suppression of antitumor immunity (LAI reactivity) by surgery

Antitumor immunity assayed by tube leukocyte adherence inhibition (LAI) is suppressed during and for up to 1–3 weeks after surgery. The results of this study suggest that when cortisol is elevated above physiologic levels by the stress of surgery this has a marked suppressive effect on the LAI-reactive cell. Cortisol added to the in vitro tube LAI assay had a two-phase effect. Cortisol at concentrations about two-fold above physiologic levels inhibited the sensitization of the peripheral blood leukocytes with cytophilic IgG antitumor antibody; however, if the cell was already armed, the cortisol did not negate its LAI reactivity. Higher concentrations of cortisol had a direct inhibitory effect on the LAI-reactive cells' ability to react with tumor antigen whether or not the cells were armed. In addition, the in vivo elevation of cortisol by the administration of cortisone acetate to LAI-positive patients inhibited the LAI reactivity of their leukocytes. Stress-induced elevation of cortisol by surgery may have an adverse effect on resistance to tumor growth, and the extent of the immunodepression may be one of the variables in the difference in survival of patients with cancers with similar degrees of invasion.

Isolation of breast cancer tumour antigen from serum and urine.

AbstractA blocking tube leukocyte adherence inhibition assay (tube LAI) was used to detect and monitor the isolation of blocking factor from the serum and urine of advanced breast cancer patients. The results of the present study indicate that the blocking factor in serum and urine was soluble breast cancer TSA. In the serum, TSA chromatographed in the region of about 80,000 to >150,000 mol. wt and was shown not to be IgG. By the technique of polyanion precipitation, the TSA was shown to be lipoprotein in nature and co‐isolated with high‐density lipoproteins. In the urine, the TSA chromatographed in the region of about 48,000 mol. wt and this suggested that the serum TSA had undergone enzymatic cleavage prior to filtration. By immuno‐adsorbent affinity chromatography with xeno‐antisera directed to the xenoantigenic determinant of HLA, blocking factor (TSA) was isolated from sera from patients with metastatic breast cancer. The specificity of binding of TSA to the immuno‐adsorbent columns and the immunologically specific abrogation of LAI reactivity were clearly shown. Breast cancer TSA was also isolated from serum by immunoadsorbent affinity columns with human cytophilic IgG anti‐tumour antibody to breast cancer. The tumour‐specific antigenic determinant of human breast cancer appeared either to be associated with the major histocompatibility complex or to be a modified HLA antigen. The present study has defined the features of breast TSA present in serum and urine and it is possible that, from these sources and with these methods, sufficient quantities of pure TSA can be isolated.

An overview: Antitumor immunity in breast cancer assayed by tube leukocyte adherence inhibition

The adherence to glass of human peripheral blood leukocytes (PBL) incubated with tumor antigen in vitro, is specifically inhibited if the PBL are sensitized to the antigen. The presence of leukocyte adherence inhibition (LAI) to tumor extracts indicates the presence of systemic antitumor immunity. By the tube leukocyte adherence inhibition assay (tube LAI), it was shown that 85% (191 of 223) Stage I and II, 45% (15 of 34) Stage III and 29% (30 of 103) Stage IV breast cancer patients had LAI reactivity. LAI responsiveness diminished with an increased tumor burden and most patients with advanced cancer exhibited no LAI reactivity. When LAI reactivity was monitored for 1 to 6 months after surgery, 13 of 25 Stage I and II breast cancer patients were negative on the first repeat assay. In general, 7 months after mastectomy most patients clinically free of cancer showed no LAI reactivity. Of thirty-five patients tested between 7 and 18 months after mastectomy, 6 were positive and 4 of the positives had local recurrence. The phenomenon of tube LAI appears to be mediated by monocytes armed with cytophilic antitumor antibody. The serum of patients whose leukocytes responded in the tube LAI assay had free cytophilic antitumor antibody that "armed" or sensitized normal leukocytes to respond in the LAI assay. Serum arming paralleled leukocyte reactivity before and after surgery. Patients with advanced cancer whose leukocytes failed to react in the LAI assay had serum blocking factors (excess tumor antigen) that abrogated the LAI reactivity of leukocytes from reactive patients.

Tube leukocyte adherence inhibition assay for the detection of anti-tumor immunity. I. Monocyte is the reactive cell.

Peripheral blood leukocytes (PBL) from breast cancer patients with early or localized cancer fail to adhere to glass in the presence of breast cancer extract. The same leukocytes do not react to unrelated tumour extracts. Enrichment and depletion of certain PBL populations from patients with apparently localized breast cancer indicated that the indicator and/or reactive cell manifesting non-adherence in the presence of appropriate tumour antigen was phagocytic, glass adherent in the absence of tumour antigen and had cell surface Fc-receptors. The cell involved, therefore, appears to be the circulating monocyte. These results show that in the tube LAI assay, the peripheral blood monocyte appears to react directly with the tumour antigen resulting in a loss of its property of adherence to glass.

Tube leukocyte adherence inhibition assay for the detection of anti-tumour immunity. II. Monocyte reacts with tumour antigen via cytophilic anti-tumour antibody.

The peripheral blood monocyte is the reactive cell in the tube LAI assay. The monocyte loses its properties of adherence to glass upon exposure to specific antigen. Two different experiments to determine if lymphocytes, when they reacted with tumour, released mediators that were responsible for inhibiting monocyte glass adherence, gave negative results. The mechanism wherby the specific tumour antigen appeared to be recognized was the binding of cytophilic IgG antitumour antibody to receptors on the cell surface of the monocyte. The results of the experiments indicate that normal peripheral blood monocytes could be made specifically reactive ("armed") to the tumour extract by incubating normal peripheral blood leukocytes with serum from a reactive cancer patient. IgG isolated from "arming" sera was shown to have the capacity to sensitize normal leukocytes. Patients with breast cancer or malignant melanoma with limited tumour burdens had free cytophilic anti-tumour antibody in their serum, whereas the serum of patients with large tumour burdens (metastatic cancer), whose leukocytes did not react in the tube LAI assay, did not "arm".