Indirect Leukocyte Migration Inhibition Research Articles

Cellular and humoral responses in coeliac disease. 1. Wheat protein fractions

The humoral and cellular immune response of coeliac individuals to various wheat protein fractions was studied using serum antibody ELISA assays and the indirect leucocyte migration inhibition factor (LMIF) assays. Greater migration inhibition factor activity was seen in coeliacs on a gluten-free-diet having low serum antibody titres, and using purified T-cells instead of total peripheral blood mononucleocytes. Gliadin was the most active fraction in both assays. Raised antibodies to low-molecular weight and high-molecular weight glutenin polypeptides was observed, though these proteins had little migration inhibition factor activity. No cellular or humoral response was seen to albumins or globulins. Proteins associated with the granules of well-washed wheat starch are distinct from gluten proteins and had little T-cell activity, correlating with clinical observations that properly prepared wheat starch is devoid of coeliac toxicity. The greater specificity of the humoral response for individual wheat protein fractions in this study, compared with the earlier reports, likely results from cross-contamination in the earlier work of each fraction with gliadin.

Cellular and humoral responses in coeliac disease. 2. Protein extracts from different cereals

The humoral and cellular immune responses to grain protein extracts from coeliactoxic and non-toxic cereals were compared by use of a number of ELISA and immunoblotting methods and the indirect leucocyte migration inhibition factor (LMIF) assay. Both adult and child coeliacs had elevated levels of serum antibody to proteins from the coeliac-toxic cereals, namely bread wheat, durum wheat, rye and barley and low levels of proteins from other cereals. Using protein blotting techniques, antibody binding was greatest to gliadins/low mol mass glutenin subunits and homologous prolamins from rye and barley, consistent with the ELISA findings. Competition ELISA and preabsorption tests indicated that antibody reaction to maize storage proteins did not simply result from cross-reaction of antigliadin antibodies. In LMIF assays, only the wheat extracts had activity in coeliac patients. This is most likely partly due to loss of some of T-cell epitopes from the extraction technique required for these proteins, as well as the relatively small effects seen for even very active fractions in the LMIF assay.

Identification of Reactive Synthetic Gliadin Peptides Specific for Coeliac Disease

Gluten intolerance (coeliac disease) is characterised by the development of a small intestinal lesion following exposure to the gliadin fraction after consumption of wheat and related cereals. Cellular immune mechanisms are thought to be responsible for gliadin toxicity, but the toxic sequence/s within gliadin have not been clearly established. A panel of synthetic gliadin peptides was tested using peripheral blood mononuclear cells from coeliac patients and two assays for cell-mediated immunity. Using the indirect leucocyte migration inhibition factor and the macrophage procoagulant activity assays, gliadin peptides which were located in the aminoterminal or the proline-rich domain of the alpha/beta gliadin molecule were coeliac-active. Peptides predicted by T cell algorithms or on the basis of homology to adenovirus Ad12 Elb protein and which were located in the proline-poor gliadin domains were inactive. Protein sequence studies which indicate significant homology in the proline-poor gliadin domains with a number of non-coeliac-toxic seed proteins also supported the hypothesis that the proline-rich domains may be more important in the pathogenesis of coeliac disease.

Cellular hypersensitivity to a synthetic dodecapeptide derived from human adenovirus 12 which resembles a sequence of A-gliadin in patients with coeliac disease.

The human intestinal adenovirus serotype 12 (Ad12) may be implicated in the pathogenesis of coeliac disease by virtue of immunological cross reactivity between epitopes shared by its early region E1b protein and A-gliadin. In the present study a synthetic dodecapeptide from the corresponding viral epitope (Ad12E1b, residues 384-395) was tested for its effect on peripheral blood mononuclear cells from 22 treated and eight untreated patients with coeliac disease, 22 healthy subjects, 11 patients with ulcerative colitis, and 11 patients with Crohn's disease by an indirect leucocyte migration inhibition assay. In addition, the effect of both the viral and the gliadin synthetic peptides was studied by proliferation and migration assays simultaneously performed in an unselected subgroup of 12 treated coeliac patients and 12 healthy subjects of the study. Coeliac patients with untreated disease showed no response to the viral peptide compared with treated patients (p greater than 0.1). Treated coeliac patients showed a significantly different response from healthy control subjects and control patients with disease (p less than 0.001) which was dependent on the concentration of the viral peptide. In the subgroup of the treated coeliac patients (n = 12) there was a significant correlation between the responses in the migration and the proliferation assay using either the viral (p less than 0.02) or the gliadin (p less than 0.005) peptide at the highest concentration (33.3 micrograms/ml). Furthermore, the responses obtained using viral peptide correlated significantly with the responses obtained with gliadin peptide in both the migration (p less than 0.001) and the proliferation (p less than 0.001) assays. These results show that in coeliac patients there is pronounced cross reactivity at the level of T cell recognition between synthetic peptides derived from the Ad12 and A-gliadin. This antigenic cross reactivity may be involved in the pathogenesis of coeliac disease.

The indirect leucocyte migration inhibition assay--an endotoxin-sensitive chemokinetic assay. Part II.

Depletion of agarose for endotoxins resulted in a low spontaneous migration of polymorphnuclear cells (PMNC). Re-addition of endotoxin, in casu lipopolysaccharide from E. coli 026:B6 (LPS), enhanced the spontaneous PMNC migration in a two-phased dose-response pattern, reaching maximum migration with LPS 1 x 10(-7) g/ml. Thus, the migration of PMNC under agarose seems to be a chemokinesis. Leucocyte migration inhibition factor (LIF), induced by PPD 50 micrograms/ml at endotoxin-free conditions, significantly reduced the PMNC migration compared to supernatants from control cultures, however not compared to the conventional limit of significance, MI = 0.80. With increasing PMNC migration there was an insignificant decrease in the MI. Addition of LPS, 1 x 10(-9) g/ml, during LIF induction caused a significant increase in LIF production, an effect which overshadowed the effect of PPD. Thus, the application of the conventional limit of significance, MI = 0.80, may result in false-negative or false-positive conclusions, depending upon the endotoxin contamination. A standardization of the endotoxin content in both steps of the indirect leucocyte migration inhibition assay seems mandatory in order to obtain a reliable and reproducible bioassay.

Immune Response to BCG Vaccination in Children

90 children attending a medical center were administered the BCG vaccine after initial negative tuberculin reaction with 1 tuberculin unit (TU) of PPD-S. 50 children were tested for post vaccination allergy (PVA) at 1 year of whom 15 children were also tested at 6 months. 90% of the 50 children showed negative reaction to PPD-S (5mm) at 1 year only 10% had a positive reaction. All the 50 children had positive response to 24 dinitrochlorobenzene (DNCB) indicating normal delayed cutaneous hypersensitivity. 14 children who were negative reactors after BCG showed normal response to phytohemagglutinin (PHA) in the lymphocyte transformation test (LTT) indicating normal cell medicated immune response (CMI). They all had significant response to PPD and BCG in indirect leucocyte migration inhibition test (ILMS). The stimulation index in the lymphocyte proliferative response to PPD was 2.8 +or- 2.24 in these children. Thus in vitro tests indicate that the T lymphocytes were sensitized to PPD and BCG after BCG vaccination even though tests for post vaccination allergy were negative. This was significant as 8 nonvaccinated tuberculin negative children did not show evidence of lymphocyte sensitisation to PPD and BCG. (authors)

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.

EBNA-specific LIF production of human lymphocyte subsets

Using the indirect leukocyte migration inhibition technique T cells have been identified as being responsible for Epstein-Barr virus nuclear antigen-induced specific leukocyte migration inhibitory factor production. The response was dependent on the presence of macrophages or their product, T-lymphocyte activating factor.

Stimulation humaner und boviner Lymphozyten durch verschiedene Streptokokken-Species

Concerning the question of species specifity of lymphokines in the presented examination the effect of the leukocyte migration inhibition factor (LIF) secreted by human and bovine lymphocytes was tested using PMN leukocytes of man and different animal species as indicator cells. The method was the so called indirect leukocyte migration inhibition test according to Clausen. As antigens for stimulating the lymphocytes cell wall preparations of group A- and B-Streptococci and as a mitogen Concanavalin A was used. From the results the following facts can be stated: — Human LIF has no inhibiting effect on the migration of leukocytes from cattle, sheep and pigs — independent of the way of stimulation by specific antigens or by the mitogen. — A corresponding factor to human LIF can be demonstrated from bovine lymphocytes which, however, inhibits only the migration of bovine indicator cells. This phenomenon is also independent of the kind of stimulation. — Migration inhibiting substances from bovine lymphocytes are only produced after stimulation with group A- and B-streptococci cell walls if the donor animals have been sensitized with these antigens. — The interaction between LIF and indicator leukocytes obviously is dependent on host specifity and not on a peculiar relation of macro- and microorganisms.

Advances in the diagnosis of tuberculosis.

Several advances in the laboratory diagnosis of tuberculosis have been described recently. Refinements in collection of specimens, methods of staining and culture techniques have been described. Several newer immunological tests such as ELISA, SAFA, and leucocyte migration inhibition test are useful in the diagnosis of sputum negative pulmonary tuberculosis. Indirect immunoflouresence test and leucocyte migration inhibition test are useful in determining the duration of chemotherapy. Detection of mycobacterial cellular products such as tuberculostearic acid in sputum are helpful in diagnosing sputum negative cases. This article describes briefly the principle and usefulness of these tests in diagnosis of tuberculosis.

One-step indirect migration inhibition (LIF) assay

A simple, 1-step indirect leukocyte migration inhibition (LIF) assay is described. Advantages of this method are as follows: the mitogen does not directly influence the migration of polymorphonuclear leukocytes, relatively few cells are needed, and results are obtained within 24 h. LIF is simultaneously produced by Con A-pretreated lymphocytes in the migration test system. The lymphokine-producing cells are in the test plate well, in the culture medium surrounding the agarose droplets containing migrating cells. The LIF production can be inhibited by the protein synthesis inhibitor puromycin and by prostaglandin (PGE 1). In this test system the LIF production is not influenced by endogeneous prostaglandins. This method can be used for complex immunopharmacological investigations because effects of agents on the random migration of polymorphonuclear leukocytes and on LIF production of lymphocytes can be tested in the same system.

Cell-Mediated Immunity to Mouse Mammary Tumor Virus Antigens by Patients With Hyperplastic Benign Breast Disease<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

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.

Use of cryopreserved lymphocytes for the indirect leukocyte migration inhibition assay

Production of leukocyte migration inhibitory factor (LIF) by fresh and cryopreserved lymphocytes from the same donors was detected by indirect leukocyte migration inhibition (LMI) assay. The same results were obtained when fresh and frozen lymphocytes were tested in parallel. This indicates that cryopreservation does not impair the ability of lymphocytes to produce LIF.

Indirect Leukocyte Migration Inhibition Reactions to a 3-<italic>M</italic> KCI Extract of Lung Adenocarcinoma by Lung Cancer Patients<xref ref-type="fn" rid="fn2">2</xref>

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.

Immunity to herpes simplex virus type 2 (HSV-2): I. Development of virus-specific lymphoproliferative and leukocyte migration inhibition factor responses in HSV-2-infected guinea pigs

The development of virus-specific cell-mediated immune (CMI) memory and effector response was studied in strain 13/N guinea pigs infected with herpes simplex virus type 2 (HSV-2) (G). The indirect leukocyte migration inhibition factor (LIF) and the lymphocyte transformation (LT) assays, chosen as probable indicators of effector and memory responses, respectively, were performed simultaneously on spleen cells (SC) obtained at varying times after infection and cultured in the presence of uv-inactivated HSV-2 (G) antigen. Kinetic and dose-response analyses revealed: (i) a time-dependent increase in the magnitude and antigen sensitivity of the LT response as well as a time-dependent decrease in the in vitro “doubling time,” both suggestive of immune maturation, and (ii) a biphasic pattern of LIF production in vitro consisting of an “early” component generated within the first 24 hr in culture, and a “late” component detected between 3 and 6 days in culture. “Late” LIF production correlated well with the lymphoproliferative response and appeared to require the presence of glass-adherent cells and active cell division.

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.

Hyporesponsiveness to Virus Antigens in Rheumatoid Synovial and Blood Lymphocytes Using the Indirect Leucocyte Migration Inhibition Test

Mononuclear cells (MNC) from rheumatoid synovial tissue and peripheral blood were tested plasma pneumonia by the indirect leucocyte migration inhibition test. MNC from the eleven rheumatoid synovial tissues tested had deficient leucocyte inhibitory factor production against all antigens tested for, and this was also the case in the peripheral blood of seven juvenile rheumatoid arthritis patients (JRA). In the peripheral blood of eight rheumatoid arthritis (RA) patients there was also generally low reactivity. However, significant differences in migration indexes were found with rubella viral antigen and with PPD at 5 micrigram/ml when zero-hour and overnight incubations of the culture were compared. In contrast, MNC of peripheral blood of control donors had significant responses to PPD (19/19), mumps virus (7/11), rubella virus (10/19), cytomegalovirus (4/11), and herpes simplex type 1 virus (4/11) antigen after zero-hour culture, and no differences was seen after overnight incubation.

Leukocyte Migration Inhibition Factor in Atopic Dermatitis: Induction by Concanavalin A in Vitro

Assays for the production of leukocyte migration inhibition factor (LMIF) activity by cultured lymphocytes in the presence of the T-cell mitogen concanavalin A (ConA) have been shown to be highly sensitive for detecting decreases in cell-mediated immunity in patients with malignant diseases and primary and secondary immunodeficiency diseases. We have applied the leukocyte migration inhibition test in agarose using supernatants from ConA-stimulated lymphocyte cultures (indirect leukocyte migration inhibition test in agarose for studying patients with atopic dermatitis. Only 5 of 14 lymphocyte cultures from atopic dermatitis patients produced LMIF when stimulated with ConA, as compared with 34 of 34 controls. This difference is highly significant.