Leukocyte migration inhibition of tumor antigen and purified protein derivative reactivity in guinea pigs sensitized to line 10 hepatocarcinoma and BCG.

Agarose microdroplet leukocyte migration inhibition (LMI) assays were performed to measure reactivity against line 10 hepatocarcinoma antigens and purified protein derivative (PPD) with the use of peripheral blood leukocytes from line 10 and/or BCG-sensitized syngeneic guinea pigs. The assay was quite sensitive and detected leukocyte migration inhibition with concentrations as low as 12.6 ng protein/ml of the crude sonicate of the line 10 tumor and 0.1 pg PPD. Specificity was shown by lack of reactivity in leukocytes of line 10 and/or BCG-sensitized animals with antigen preparations of L2C leukemia cells or normal syngeneic liver. Furthermore, leukocytes from normal control guinea pigs failed to react with any antigen. The results also suggested antigen cross-reactivity between line 10 tumor and BCG. Leukocytes from guinea pigs sensitized to only BCG became LMI reactive to the line 10 sonicate as well as PPD. No reactivity was observed with leukocytes of the animals in simultaneous tests with a sonicate of guinea pig L2C leukemia cells. The results demonstrated the usefulness of this microassay in detection of LMI reactivity with low antigen concentrations and small volumes of whole blood.

Indirect leukocyte migration inhibition reactions to a 3-M KCl extract of lung adenocarcinoma by lung cancer patients.

Mononuclear (MN) cells from the peripheral blood of lung cancer patients were tested for their ability to respond to a 3-M KCl extract of adenocarcinoma of lung with the use of an indirect leukocyte migration inhibition (LMl) assay. Antigen-stimulated MN cell cultures were evaluated for leukocyte inhibitory factor production by their ability to inhibit the migration of indicator polymorphonuclear cells from agarose droplets. When supernatants were prepared in conventional round-bottomed tubes (5X10(6) cell/tube), 25 of 44 (57%) lung cancer patients had positive indirect LMl responses to the 7661 antigen as compared to only 2 of the 30 (7%) normal donors. When supernatants were prepared in conical microtubes, with 10 times fewer MN cells, similar results were obtained. Patients with all histologic types of lung cancer had a similar incidence of reactivity, and reactivity of untreated patients did not appear to be related to stage of disease or degree of tumor burden. Surgical removal of the tumor appeared to decrease the incidence of reactivity in the 1- to 12-month postoperative period. These results strongly suggest that the LMl reactivity against lung tumor extracts is lymphokine mediated, inducing cellular responses by the patients against such antigens.

High incidence of migration inhibition reactivity to lung tumor-associated antigen by normal donors in close contact with lung cancer patients or materials.

A high incidence of indirect leukocyte migration inhibition reactivity of normal donors to a 3-M KCl extract from a fresh pleural effusion of a patient with a lung adenocarcinoma (designated 7661) was observed. When these normal donors were classified according to contact with lung cancer patients or materials, 22 of 32 (72%) normal donors in contact with lung cancer patients or materials were reactive with the 7661 extract as compared to only 3 of 76 (4%) who had no contact. Of normal donors involved in the direct care of lung cancer patients, 14 of 20 (70%) were positive, whereas only 2 of 10 (20%) hospital personnel who worked with noncancer patients were reactive. Among laboratory personnel who handled blood and tissue specimens from lung cancer patients, 8 of 11 (73%) were positive with the 7661 extract, whereas none of 5 laboratory workers who worked with cancer materials unrelated to lung cancer were positive. Also, none of 13 personnel working in laboratories adjacent to those where lung cancer tests were performed were reactive with 7661. None of the 16 blood bank donors and none of 11 secretarial and clerical staff who worked in biochemical laboratories were positive. Reactivity was no correlated with a smoking history. Thus development of reactivity appeared to require direct contact with lung cancer patients or materials. The results suggested a horizontal transmission of reactivity against an antigen associated with lung cancer.

Cell-mediated immunity in mice against papain-solubilized histocompatibility and tumor-specific antigens by a macrophage migration inhibition microassay.

The cell-mediated immune status of B10.D2 (H-2d) mice immunized with spleen cells from a congenic strain, B10.A (H-2a), differing at the H-2 locus and of BALB/c mice immunized with a syngeneic simian virus 40 (SV40)-induced sarcoma (mKSA-TU5) was evaluated by an agarose microassay for migration inhibition factor. The inducing antigens in this experiment were papain-solubilized and partially purified chromatographic preparations of spleen cells from A/J mice (H-2a) and a papain-solubilized antigen extract prepared from a tissue culture-adapted cell line (TU-5), derived from the SV40-induced mKSA tumor. The assay used microliters of normal or immune peritoneal exudate cells (PEC) resuspended in a 2-mul droplet of agarose and cultured in the presence or absence of antigen. Specific migration inhibition of PEC from immunized mice was observed with concentrations of solubilized antigen preparations as low as 2.0 mug/ml (3.67 mug/chamber).

Leukocyte migration inhibition by soluble extracts of MCF-7 tissue culture cell line derived from breast carcinoma.

Studies were conducted to determine whether MCF-7, a tissue culture cell line derived from a pleural effusion of a patient with breast carcinoma, could be used as a source of tumor-associated antigen for direct leukocyte migration-inhibition (LMI) assays. Of 32 patients with breast carcinoma, 27 (84.4%) gave positive migration-inhibition results on their initial tests with a 25-mug protein/ml concentration of a 3 M KCl extract of MCF-7; 1 of 24 (4.5%) normal donors reacted with MCF-7. An intermediate incidence of reactivity (7/16) was observed with the extract when leukocytes of patients with melanoma, lung carcinoma, and Ewing's sarcoma were used. In further specificity studies, leukocytes of patients with breast carcinoma gave a lower incidence of LMl reactivity than did those of patients with Ewing's sarcoma and lung carcinoma with KCl extracts of the appropriate histologic type of tumor. The results indicated that the MCF-7 cells possessed a tumor-associated antigen to which many patients with breast carcinoma are sensitized.

Lymphocytes forming rosettes with sheep erythrocytes in metastatic pleural effusions.

Percentages of lymphocytes forming rosettes with sheep erythrocytes at 29 degrees C were determined in 10 cancer patients with metastases to the pleural cavity. Compared with normal controls, the patients showed a decreased proportion of rosette-forming cells (RFC) in the peripheral blood. The same patients had elevated levels of RFC in their metastatic pleural effusions. However, 2 patients with benign diseases had normal levels of RFC in their peripheral blood and pleural transudates. These observations suggested that in cancer patients some T-cells might migrate from the peripheral blood and accumulate in sites of tumor infiltration such as the pleural cavity.

Use of the leukocyte migration inhibition assay to evaluate antigenic differences in human breast cancers and melanomas.

The leukocyte migration inhibition assay was used to compare the antigenic reactivity of 3 M KCl extracts of human tumors. Many extracts demonstrated strong reactivity with patient leukocytes, whereas others demonstrated weak or no reactivity, Extracts prepared from primary tumors or local recurrent tumors were more antigenic than extracts from involved lymph nodes or pleural effusions. The least reactive preparations were extracts made from specimens of liver metastases obtained at autopsy. A large standard extract tested at a standard concentration was useful for the evaluation of antigenic reactivity of human tumor extracts. It served as a point of reference in simultaneous tests with one blood sample from each individual, thus eliminating the influence of patient variation on extract reactivity.

Immunologic relationship between breast carcinoma and benign breast disease as detected by the leukocyte migration inhibition assay.

Patients with benign diseases of the breast reacted in a migration inhibition assay with extracts of breast cancer and benign breast lesions and a human breast cancer-derived cell line, MCF-7. The incidence of reactivity of the patients with benign breast diseases against these antigens was similar to that of breast cancer patients. In addition, patients with breast cancer reacted to some extracts of benign breast lesions. The reactivity occurred in patients with several different histopathologic types of breast lesions, but was not found in women with no detectable pathologic lesions.

Inhibition of leukocyte migration by tumor-associated antigens in soluble extracts of human malignant melanoma.

Direct leukocyte migration inhibition (LMI) assays were performed to investigate whether cell-mediated immune reactions could be detected in response to tumor-associated antigens of human melanoma. The antigens were 3 M KCl-soluble extracts of different fresh melanomas, other cancers, and benign nevus tissue. A total of 48 of the 79 (61%) blood samples from melanoma patients (64 patients) reacted with extracts of melanoma tissue. Since the subjects were usually tested with two or three extracts, 57/134 (42%) tests with melanoma patients' leukocytes were inhibited by KCl extracts of melanoma tissue, whereas only 3/50 (6%) tests with leukocytes of normal donors and 4/27 (15%) with patients having other cancers gave positive results. No positive reactions were obtained when 13 melanoma patients were tested with a 3 M KCl extract of benign nevus tissue. Likewise, only 2/26 (8%) positive tests were obtained from melanoma patients tested with extracts of other cancers. Individuals in all stages of disease had similar incidences of positive reactions to the soluble melanoma extracts, except for patients with stage-1 disease who exhibited a somewhat higher incidence of reactivity. The highest incidence of reactivity was observed in patients before surgical resection of the tumor, and somewhat decreased reactivity was seen 0-14 days post surgery. The results indicate that the direct LMI assay may be used to measure cell immune reactivity against melanoma-associated antigens. Since many of the positive results were obtained with allogeneic extracts, the results also indicate that different melanomas possess common antigens.

Indirect leukocyte migration inhibition in breast cancer and benign breast disease patients by mouse mammary tumor virus grown in feline kidney cells.

Indirect migration inhibition assays were performed with normal and mammary tumor-bearing C3H/HeN mice and patients with breast disease to assess cellular immunity against three different mouse mammary tumor virus (MTV) preparations grown in feline kidney cell cultures and against a mouse-derived MTV preparation. MTV obtained after passage through feline kidney cells and the mouse-derived MTV were capable of eliciting macrophage migration inhibitory factor production by mouse spleen cells obtained from normal or mammary tumor-bearing C3H/HeN mice, thus demonstrating a similar degree of antigenicity of these preparations. In experiments with human breast cancer patients' leukocytes, leukocyte inhibitory factor (LIF) was produced by 32-50% of these patients in response to the mouse-derived MTV or to three different MTV preparations obtained after passage through feline kidney cells. A significant proportion (31-54%) of benign breast disease patients also reacted with both the mouse-derived and feline-derived MTV preparations. Patients with both malignant and benign breast disease, however, had a significantly different (P less than .05) pattern of reactivity to mouse- and feline-derived MTV preparations from that observed with normal donors. Finally, some LIF activity was also observed (but not statistically significant with the use of nonparametric analysis methods) when feline leukemia virus was used as antigen with these patients. The data suggest that both breast cancer and benign breast disease patients were reactive against antigens largely specific for MTV in the feline cells and, presumably, were not reactive against feline cellular components, although the second possibility cannot be completely ruled out.

Cell-mediated immunity to mouse mammary tumor virus antigens by patients with hyperplastic benign breast disease.

Peripheral blood leukocytes of patients with preoperative breast cancer, benign breast disease, and benign gynecologic disorders and normal healthy females were tested, as blind coded specimens, with murine mammary tumor virus (MuMTV) antigens in the direct and indirect leukocyte migration inhibition (LMI) assays. The incidence of reactivity by patients with breast cancer was low. (From 5 to 35% breast cancer patients reacted, depending on which group of control individuals they were compared to and what antigen was used.) Nonparametric analyses showed no differences between control groups (normal donors and patients with gynecologic disorders) and breast cancer patients with either assay. However, there was a significant difference between benign breast disease patients with hyperplasia and 1) benign breast disease patients without hyperplasia (P less than 0.03) and 2) patients with gynecologic disorders (P less than 0.04) in the direct assay when it was performed blindly with the gp52 antigen. Patients with hyperplasia (benign breast disease as well as breast cancer) had a higher incidence of enhanced migration in the indirect test than breast disease patients without hyperplasia. The enhanced migration to the MuMTV was correlated to enhanced migration to a 3-M KCI extract of the breast cancer cell line MCF-7 in simultaneous tests. Thus the LMI assays with MuMTV antigens do not appear valuable in breast cancer diagnosis, but they may help to identify a small group of benign breast disease patients whose breast pathology is thought to be associated with a high risk for developing breast cancer.

Leukocyte migration inhibition in patients with Ewing's sarcoma by 3-M potassium chloride extracts of fresh and tissue-cultured Ewing's sarcomas.

Leukocyte migration inhibition (LMI) assays were performed to detect cell-mediated immune reactions against tumor-associated antigens (TAA) of Ewing's sarcoma. With the use of crude antigen preparations obtained by 3M KCl extractions of fresh Ewing's sarcoma or of tissue culture cells derived from a pleural effusion of a Ewing's sarcoma patient, assays were performed with leukocytes from these patients, patients with other cancers, and normal donors. The results demonstrated approximately 60% or greater positive LMI reactivity in Ewing's sarcoma patients, as compared to less than 10% reactivity of normal donors, with the use of extracts of either fresh or tissue-cultured Ewing's sarcoma cells. A lower proportion of positive reactivity was observed in patients with breast and lung cancer. Further specificity tests indicated that a smaller proportion of patients with Ewing's sarcoma had LMI reactivity with KCl extracts of tissue-cultured cells derived from breast cancer of fresh lung cancer cells than did patients with the homologous disease. The results indicate that many patients with Ewing's sarcoma have cell-mediated immunity toward TAA on Ewing's sarcomas. Inasmuch as all the LMI assay were performed with allogeneic extracts, the data also suggested that different Ewing's sarcomas possess common antigens and that some breast and lung cancers may share some TAA with Ewing's sarcoma.

Cell-mediated immune responses of breast cancer patients to autologous tumor-associated antigens.

Lymphocyte proliferation assays with autologous tumor material in mixed leukocyte-tumor interactions (MLTI) were employed to monitor tumor-associated cell-mediated immune responses of peripheral blood lymphocytes from patients with carcinoma of the breast. In addition, leukocyte migration inhibition (LMI) assays were employed to compare reactivity to autologous breast-tumor extracts versus allogeneic breast-tumor extracts. Positive lymphoproliferative responses to tumor-associated antigens (TAA) were observed in the MLTI assay with the use of either intact autologous tumor cells or crude extracts (in mug and ng quantities) in 12 of 34 (35%) breast cancer patients studied. Positive reactivity to tumor, but not to normal tissue of reactive patients, was observed in repeated assays. Finally, patients demonstrating positive MLTI responses to autologous tumor extracts likewise responded in LMI assays to these same autologus extracts as well as to allogeneic breast-tumor extracts, but not to non-breast-tumor extracts. Thus breast tumors appeared to possess common TAA among both male and female patients.

Human cell-mediated immunity to tuberculin as assayed by the agarose micro-droplet leukocyte migration inhibition technique: comparison with the capillary tube assay.

Direct and indirect agarose microdroplet migration inhibition assays were performed with log dilutions (50--5 X 10(-8) microgram/ml) of soluble purified protein derivative (PPD) of tuberculin and leukocytes (4 X 10(5)/droplet) from PPD skin test positive or negative individuals. Some tests were run in parallel with the capillary tube method. Inhibition of migration of leukocytes from 9/11 PPD skin test positive donors studied was observed in direct tests when employing PPD doses ranging from 1--50 microgram/ml PPD. Inhibition of migration of leukocytes from some PPD skin reactive donors was obtained with as little as 5 X 10(-3)--5 X 10(-7) microgram/ml (i.e., 5 nanograms to 0.5 picograms). Some inhibition of leukocyte migration was seen with skin test negative donors (presumably toxicity) with the higher doses of PPD (50 microgram/ml), but generally little inhibition was observed with these donors at lower doses. Comparative experimetns of the agarose micromethod and the capillary tube technique indicated that the agarose assay was more sensitive in that it could detect reactivity with 2--4 logs lower antigen concentration. Indirect assays using supernatants of cultures of PPD triggered mononuclear cells and indicator granulocytes in agarose droplets demonstrating that a lymphokine (presumable leukocyte inhibitory factor) was being produced and suggested that cell-mediate immune (CMI) reactions were operative. The results indicate the usefulness of this technique in the sensitive-detection of CMI against such antigens as soluble PPD. The assay should prove useful in quantitative assessment of cell-mediated reactivity by using a wide range of antigen concentrations and the leukocytes from as little as 2--5 ml of blood.

Brief communication: technical modifications of the human agarose microdroplet leukocyte migration inhibition assay.

Direct leukocyte migration inhibition assays using the capillary tube technique can be used to demonstrate cell-mediated immunity in vitro. Unfortunately, the cumbersome nature of this technique makes it time consuming and difficult to perform. Similar results have been obtained using the direct agarose microdroplet leukocyte migration inhibition assay. In this paper, modifications of the agarose technique are outlined which insure standardization of droplets and ease of performance of the assay. Additionally a technique is described to reduce the time required for calculation of results.

Pharmacological characterization of the excitatory innervation to the guinea-pig urinary bladder in vitro: evidence for both cholinergic and non-adrenergic-non-cholinergic neurotransmission.

1 Field stimulation of strips of guinea-pig isolated urinary bladder with 5 s trains at 0.1 to 15 Hz resulted in frequency-dependent, reproducible contractions. 2 At concentrations of 1 and 4 x 10(-7) M and 1 x 10(-6) M, atropine produced a variable, partial inhibition of contractions at all frequencies but was most effective at frequencies of 3 Hz or more. 3 Tetrodotoxin (TTX), 5 x 10(-7) M, inhibited contractions at all frequencies by 80 to 90%. 4 Physostigmine, 2 x 10(-6) M, significantly enhanced the contractile response to frequencies of less than 10 Hz but did not enhance responses resistant to inhibition by atropine. Hexamethonium, 1 x 10(-4) M, slightly enhanced the contractile response to frequencies of 4 Hz or greater. 5 (+/-)-Propranolol (5 x 10(-6)M), guanethidine (1 x 10(-6)M), phentolamine (5 x 10(-6)M) and clonidine (3 x 10(-8)M) each enhanced the contractile response to field stimulation. 6 Contractile responses obtained in the presence of atropine (4 x 10(-7) M) and guanethidine (1 x 10(-6) M) increased with time and were inhibited 60 to 80% by TTX (5 x 10(-7)M. 7 It is concluded that the cholinergic nervous system contributes, in part, to electrically-induced excitatory contractions of the isolated urinary bladder of the guinea-pig. Concomitant sympathetic stimulation appears to serve an inhibitory role. In addition, a major portion of the contractile response appears to be due to a non-cholinergic non-adrenergic, as yet unidentified, substance.

Cell-mediated immunity to melanoma-associated antigens in patients with ocular malignant melanoma.

We tested 11 patients with choroidal melanomas and 32 control subjects for cell-mediated immunity to melanoma-associated antigens by an in vitro leukocyte migration inhibition assay. Five of seven patients with choroidal melanomas, who received two melanoma extracts used in this series of experiments, had significant leukocyte migration inhibition as compared with none of 17 normal subjects. Four of five melanoma patients who received a third melanoma extract had significant leukocyte migration inhibition as did three of four normal controls, indicating that the extract showed nonspecific inhibition. These data agreed with the concept that patients with choroidal melanomas have cell-mediated immunity to common melanoma-associated antigens.

Disabled workers' risk of hospitalization and death.

Data from the 1982 New Beneficiary Survey (NBS) were matched with 5 years (1984-88) of Social Security and Medicare data to analyze disabled workers' probability of death and inpatient care. Fifteen percent of the disabled workers died within 18-24 months of initial eligibility; 34 percent died within 5 years. Older disabled workers had higher probabilities of death and hospitalization. Males were two times as likely to die as females, but no more likely to be hospitalized. Black persons also had a higher risk of death but no greater risk of hospitalization than other races. Additional health insurance had no influence on survival, but was differentially associated with inpatient care. Married males were more likely to survive. Physical functioning capacity had no influence on survival or hospitalization. Respiratory, circulatory, and digestive disorders increased the probability of hospitalization and mortality.

Health of retired workers: survival status and Medicare service use.

Data from the Social Security Administration's 1982 New Beneficiary Survey and Master Beneficiary Record were matched with 1984 data from the Medicare Automated Data Retrieval System to study the effects of self-reported health on subsequent health service usage and survival. Proportionately, more new retired workers who reported poorer health in 1982 were decreased by December 1984. Functionally dependent beneficiaries as determined by the Functional Capacity Limitation Index had death rates four to five times greater than those who reported no limitations. The health status of retired workers who received Social Security benefits before age 65 was no better than beneficiaries 65 or over. Decedents were more likely than survivors to incur Medicare charges, and to have substantially higher median charges--$8,834 compared with $285.

Surveying board and care homes: issues and data collection problems.

National information is seriously lacking concerning the number and types of board and care homes, their residents, and the quality of services provided. Shortcomings in current living arrangement classification procedures used in federal surveys are critically examined. A definition and classification system is proposed that would transcend state and local government differences caused by differences in licensure and regulations.