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A modification of the leucocyte migration inhibition test, in which cells migrate in an agarose layer, was proposed by Clausen (1971). In the present study, this method was applied to PPD and hepatitis B antigen (HBAg), and its advantages were reassessed. From a technical standpoint, the migration in a monolayer facilitated a study of the morphology of the migrating cells. The proportion of the different kinds of migrating leucocytes was calculated and expressed as a function of the distance of migration. On the whole, 92% of the migrating leucocytes were polymorphonuclears and 8% lymphomonocytes. Three to 10% of the incubated cells were shown to migrate in the agarose layer. The surface area of the controls (migration without inhibition) was sufficiently constant and large (on the average 13 mm²) to allow a clear assessment of inhibition when antigen was added. In addition, the small requirement for antigen facilitated the use of this test in experimentation with purified HBAg. Application of the technique to clinical problems revealed that, with PPD as antigen, a highly significant inhibition was obtained in PPD skin test-positive normal individuals (0.0025

Twenty-eight chimpanzees were divided into six groups according to their history of previous immunization or exposure to hepatitis B antigen (HBAg) and studied for delayed hypersensitivity (DH) to HBAg. Purified HBAg derived from a normal human carrier was used for in vivo skin testing and in vitro leucocyte migration inhibition tests. Of seventeen chimpanzees immunized with HBAg in Freund's complete adjuvant (FCA), nine exhibited positive DH reactions to HBAg with good correlation between the in vivo and in vitro responses. Of the seventeen chimpanzees, fourteen also exhibited positive DH reactions to purified protein derivative of tuberculin (PPD) with marked erythema and induration; the other three exhibited only erythema with no induration. None of the seventeen animals exhibited any immediate reactivity to either HBAg or PPD. Intradermal injection of HBAg-negative human serum failed to elicit DH reactions in four animals who showed positive skin test with purified HBAg; the DH response was thus probably HBAg-specific. Nineteen chimpanzees, including six unimmunized animals, three chronic carriers of HBAg and two which had been injected with HBAg without FCA, failed to show DH response to HBAg. Thus, DH to HBAg was observed only in animals hyperimmunized with HBAg in FCA. [ABSTRACT FROM AUTHOR]

Twenty-eight sero-negative young women were vaccinated with RA 27/3 or HPV-77/DE5 strain live attenuated rubella vaccine. Haemagglutination-inhibiting antibody developed in all but one of the subjects. Rubella-specific IgM antibodies were demonstrable 3-6 weeks after vaccination. Complement-fixing and platelet aggregation antibodies were more often detectable alter RA 27/3 than after HPV-77/DE5 vaccination. After vaccination the blood lymphoblast count increased with a peak at 2 weeks. In one subject no humoral antibody response was detectable but even in this case there was a lymphoblast response. A marked increase in blasts was detected in a single case of natural rubella infection. In the vaccinated subjects the tuberculin skin test did not reveal suppression of delayed hypersensitivity 3 weeks after vaccination, nor was there a change in the responsiveness of lymphocytes to PHA in vitro 2 weeks after vaccination. No significant decrease in platelet count was seen in any of the subjects. [ABSTRACT FROM AUTHOR]

Cell-mediated immunity to varicella-zoster antigen was assessed by lymphocyte transformation in fourteen patients with acute herpes zoster and in a group of twelve healthy controls. There was a significant dimunition in cell-mediated immunity to this antigen in the patient group when compared to the controls but no definite difference was observed in the responses to phytohaemagglutinin and PPD measured at the same time. It was suggested that this lack of cell-mediated immunity contributes to the evolution of the disease. [ABSTRACT FROM AUTHOR]

Cell-mediated immunity was assessed in man by summation of positive delayed hypersensitivity (DHS) responses to five test antigens, Candida, mumps, trichophyton, tuberculin and streptokinase-streptodornase (Varidase). The study included forty-four controls comprising healthy persons and hospital inpatients with minor illnesses, forty-five aged persons, twenty-four patients with active chronic hepatitis (ACH), nine with systemic lupus erythematosus (SLE) and fourteen patients with rheumatoid arthritis. For controls and the group with ACH, cell-mediated immunity was assessed also by transformation of blood lymphocytes with phytohaemagglutinin (PHA). The incidence of positive DHS responses to all antigens fell with advancing age, and responsiveness was significantly lower in all three groups of immunopathic disease, particularly ACH. Prednisolone was not associated with significantly depressed DHS reactions, but combined prednisolone and azathioprine were. Weak DHS responses were associated significantly with low titres of natural antibody to flagellin and with lower humoral immune responses to flagellin. Lymphocyte transformation in response to PHA in ACH was significantly less than in healthy controls. Thus certain autoimmune diseases show an immunological imbalance in the form of depressed cell-mediated immunity to extrinsic antigens and 'hyperactive' humoral immune responses to 'self' antigens and selected extrinsic antigens. [ABSTRACT FROM AUTHOR]

Guinea-pigs were immunized with an emulsion of human thyroglobulin (Tg) and BCG vaccine in Freund's incomplete adjuvant. Peripheral blood lymphocytes binding Tg and purified protein derivative of mammalian tuberculin (PPD) labelled with 125I were demonstrated with an autoradiographic technique. The percentage of these cells was compared with the size of the delayed hypersensitivity (DH) skin reactions to Tg and PPD, and Tg antibody titres at intervals up to 35 days after immunization. The individual responses to Tg showed the time course of primary immune reactions although they did not parallel each other closely. By contrast, a depression of cutaneous DH to PPD was observed in spite of the presence of substantial numbers of 125I-PPD-binding peripheral blood lymphocytes. In non-immune guinea-pigs, approximately 2-8% of peripheral blood lymphocytes bound 125I-PPD whereas <0-l% bound 125I-Tg. The significance of antigen-binding lymphocytes in the immune response is discussed, and it is suggested that the presence of a substantial pool of antigenbinding lymphocytes in unprimed animals may be of importance in the induction of DH. [ABSTRACT FROM AUTHOR]

The development of cell-mediated immunity to paternally-derived foetal transplantation antigens in parous mice has been assessed by two techniques: skin grafting in an H-2 incompatible mating and a local graft-versus-host assay in an H-2 compatible mating. (1) Lymphoid cells from BALB/c females, parous one to seven times to C3H/Bi males, were injected i.p. into syngeneic males or females which were then grafted with skin from the paternal strain. A slightly accelerated rejection in the adoptively immunized recipients was observed. (2) Lymphoid cells from CBA females, parous one to five times to AKR males, were injected into the feet of young F1 hybrid recipients. The degree of popliteal lymph node enlargement was significantly greater in recipients of cells from parous than from virgin donors. Results using both techniques demonstrated that the development of cell-mediated immunity to paternal transplantation antigens is a regular feature in parous mice and that the degree of immunity increases with successive interstrain pregnancies. [ABSTRACT FROM AUTHOR]

The macrophage migration inhibition test was employed to show reactivity of maternal peripheral blood leucocytes to placental antigens. Four to 5 days after delivery, all of forty-one postpartum women tested had leucocytes in their blood that reacted with pooled antigens from five placentas. When antigens from a single placenta were used leucocytes from only the woman who donated that placenta and one of nine unrelated postpartum women were reactive. Leucocytes from seventeen-control subjects, including nulligravidous women, were non-reactive with either the single or the pooled placental antigens. The results indicated that the mothers may be specifically sensitized, as far as the cell mediated immune response is concerned, by the placenta during pregnancy. [ABSTRACT FROM AUTHOR]

The ability of peripheral lymphocytes from patients with. Hashimoto's disease to destroy target cells coated with thyroid-related-autoantigens has been demonstrated. Direct cytotoxicity was demonstrable against mouse mastocytoma target cells coated with human thyroglobulin or microsomal antigen from thyrotoxic thyroid glands. No correlation was noted between circulating antibody and the ability of these cells to be cytotoxic. The significance of the in vitro expression of cell-mediated immunity in this method is discussed. [ABSTRACT FROM AUTHOR]

The experiments reported here were concerned with determining the effects of lymphocyte activation products (LAP) on lymph node architecture, and with assaying afferent lymph for evidence of the peripheral release of LAP during the induction of an immune response. Intralymphatic inoculation of purified homologous LAP into guinea-pigs resulted in increased weight and cellular content of the draining node. Histologically these nodes showed paracortical distension and dense aggregations of lymphoid cells or 'cellular plugs' in the paracortical sinuses. It was suggested that one effect of LAP may be to cause cellular retention in the paracortex of lymph nodes by regulating the rate of cell exit via the sinuses of the node. The peripheral lymph of rabbits was assayed for its ability to inhibit macrophage migration and to accelerate lymphocyte DNA synthesis after stimulation with three different antigens. The antigens were chosen to give a spectrum which ranged from a primarily humoral response (erythrocyte stimulation) through a mixed humoral and cell-mediated response (diphtheria toxoid stimulation) to a predominantly cell-mediated type of response (skin contact sensitization to fuorodinitrobenzene-FDNB). Paracortical distension with lymphoid cell sinus plugging, similar to that observed in the guinea-pig nodes following intralymphatic injection of LAP, were common features of both the diphtheria toxoid and FDNB responses. It was concluded that the development of this type of sinus plugging and paracortical distension might be related to multiple activities of LAP getierated and released either at the peripheral antigen depot or within the draining node. [ABSTRACT FROM AUTHOR]

Partial purification of soluble products of guinea-pig lymphocyte activation (LAP) was undertaken by fractional precipitation with ammonium sulphate, ion-exchange and Sephadex chromatography, and by immune precipitation of inducing antigen and of contaminating serum protein. During these purification steps the activity of macrophage migration-inhibition factor (MIF) was concentrated up to 1300-fold and separated from inducing antigen and serum protein. An endpoint assay was devised for expressing antigen-induced MIF activity of LAP fractions as weights of material giving 30% inhibition of migration (MI30 doses). MIF activity precipitated between 50% and 80% saturated ammonium sulphate and eluted from DEAE-cellulose at pH 7-9 at intermediate salt concentrations (0-03-0-2 M phosphate). On Sephadex gel filtration MIF activity was concentrated in fractions of molecular weight range 56,000-82,000 with a smaller amount of activity eluting from 20,000-56,000. After immune precipitation of extraneous protein and elution from DEAE-cellulose, LAP material was found to have an MI30 dose of 0-4 /μg. Materials representative of antigen and serum protein-depleted MIF were selected for intralymphatic injection in order to determine whether MIF-rich LAP fractions were able to induce paracortical distension in guinea-pig lymph nodes (see following paper). [ABSTRACT FROM AUTHOR]

Recipient guinea-pigs pretreated with Freund's complete adjuvant alone show depressed 4 hr (Arthus) reactions following passive transfer of antiserum to bovine γ-globulin and human serum albumin. Recipient outbred guinea-pigs and inbred rats also show depressed 24 hr delayed reactions following passive transfer of immune peritoneal exudate cells and serum. This indicates that Freund's complete adjuvant depresses certain inflammatory responses. Donor guinea-pigs and inbred rats pretreated with FCA alone and then immunized with BGG in FCA transfer smaller 24 hr reactions to normal recipients than comparable donor animals which have not been pretreated with FCA. This suggests that pretreatment with FCA depresses the central state of delayed hypersensitivity which normally follows immunization with BGG in FCA. These two findings may explain why pretreatment with FCA depresses the 4 and 24 hr skin reactions which otherwise follow immunization with antigen in FCA. [ABSTRACT FROM AUTHOR]

A modification or the macrophage or leucocyte migration inhibition, assay usable in a murine model has been developed. The system operates with mixed spleen-thymus cell populations from mice sensitized. to soluble protein or to transplantation antigens. In order to obtain significant inhibition it was found necessary to preincubate the sensitized cells with specific antigen for 24 hr prior to the migration assay. The macrophage inhibitory factor (MIF) has been found to determine the rate of migration inhibition, in the mouse system whereas the role of humoral antibody is negligible. The murine MIF is not strain specific and operates in the absence of specific antigen. Spleen-thymus cells from St/a mice sensitized to a first set C3H skin allograft were inhibited by approximately 30% while cells sensitized to a second set graft showed a 50% inhibition when migrating against C3H cells or MIF produced by confrontation between sensitized St/a lymphoid cells and C3H antigen. [ABSTRACT FROM AUTHOR]

The immune response to a protein antigen, keyhole limpet haemocyanin, was studied in fourteen normal subjects and twenty-one patients with solid tumours. Imm- unological responsiveness was assessed by intracutaneous skin testing, by haemagglutinin titres and by in vitro blast transformation. No significant difference was found in the kinetics or magnitude of the immune response among subjects immunized with 0.01, 0.10, or 5.0 mg. Delayed hypersensitivity to keyhole limpet haemocyanin developed in thirty-two of thirty-four skin tested; a positive antibody titre occurred in all; and thirty-one of thirty-five had positive in vitro responses. Patients in good general condition (Group 1) had significantly greater delayed hypersensitivity and antibody responses than the normals but similar in vitro responses. All immunological parameters were depressed in the patients with advanced neoplastic disease (Group 2). Although only skin test positive subjects bad positive in vitro responses, no direct correlation was found between the degree of delayed hypersensitivity and the decree of in vitro blast transformation. Excellent correlation was demonstrated, however, between the in vitro response and the haemagglutinin titre (correlation coefficient +0.52, standard error ±0.09). [ABSTRACT FROM AUTHOR]

Human blood, lymphocytes from donors sensitized by skin grafting were inhibited their migration from capillary tubes by antigens from the skin graft donor. Lymphocytes from non-sensitized human subjects showed no or a very slight inhibition with similar antigens. When confronted with cell antigens from individuals, which did not possess any of the detectable HL-A specificities of the donor, the sensitized lymphocytes exhibited a marginal degree of inhibition (15.3±2.0%). The degree of migration inhibition increased to an average of 24.9±2.0% when sensitized lymphocytes were confronted with one HL-A antigen similar to that of the immunizing donor. When there were two and three HL-A antigens involved, inhibition increased to 29.4±4.1%, and 37.8±4.6%respectively Lymphocytes from two patients who had received kidney allografts exhibited a small but significant degree of inhibition when tested against cells sharing one antigen with the allograft. Lymphocytes from these patients maintained strong reactivity to tubercle bacilli and streptococcal antigens despite prolonged immuno-suppressive therapy. [ABSTRACT FROM AUTHOR]