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Searching for IgG and IgM against the mycobacterial antigen A60 has been recognized as a potential diagnostic tool for pulmonary tuberculosis. The role of detection of anti-A60 IgA in improving diagnostic accuracy of serology is not well known. In this study we measured with ELISA serum levels of both anti-A60 IgG and IgA in 216 subjects. 88 healthy volunteers (44 PPD- and 44 PPD+), 44 patients suffering from nontuberculous lung disease and 15 subjects with healed pulmonary tuberculosis constituted the control population; 69 patients with active pulmonary tuberculosis (35 cavitary forms, 26 productive forms and 8 miliary forms) were examined. The sensitivity of IgG test was 73.9% in pulmonary tuberculosis (77.1% in cavitary forms, 65.4% in productive forms, 87.5% in miliary forms); the specificity of the test was 95.9%. For the IgA test we observed a sensitivity of 72.5% (74.3 in cavitary forms, 69.2% in productive forms, 75.0 in miliary forms) and a specificity of 93.9%. Combination of the two tests increased the sensitivity to 84.0% (+10.1% compared to IgG test, +11.5% compared to IgA test); the specificity decreased to 92.5% (-3.4% vs. IgG test; -1.4 vs. IgA test). In conclusion, the combined use of evaluation of anti-A60 IgG and IgA increases the accuracy of serological diagnosis of pulmonary tuberculosis.

Background: Since the immune system is considered to be a major determinant in the outcome of malignant melanoma, vaccination with BCG (bacillus Calmette-Guérin) or more recently with interleukins is used in this condition, although the effects of this immunotherapy are unclear., Objective: The present study was to elucidate clinical aspects and the prognostic relevance of the cellular and humoral immune responses of melanoma patients treated with two different BCG vaccines., Methods: A subgroup of patients from a multicenter adjuvant trial with BCG in stage I (pT3-4N0M0) high-risk melanoma was prospectively subjected to detailed analysis regarding tuberculin (PPD, purified protein derivate) skin test reactivity, local, regional and systemic reactions to BCG vaccination, PPD antibody response and disease-free survival. Patients were randomized into three arms and received either no adjuvant treatment (22 patients), BCG RIV (40 patients) or BCG Pasteur (44 patients). All patients were followed for up to 10 years (median follow-up 6 years)., Results: Patients treated with BCG Pasteur mounted a stronger antibody response, experienced stronger regional and systemic reactions to vaccination and converted more frequently to positive PPD skin tests, compared to controls and to patients vaccinated with BCG RIV. All BCG-treated patients who developed an antibody response had a longer disease-free interval (p = 0.05), with slightly higher significance for BCG Pasteur-treated patients (p = 0.02)., Conclusions: Cellular as well as humoral immune responses to PPD and BCG thus identify stage I malignant melanoma patients with an overall better prognosis.

Ten hemodialysis patients were treated with intravenous calcitriol (1-1.5 micrograms 3 times per week) for 3 months and parameters reflecting cell-mediated immunity were measured before and at the end of treatment. Peripheral blood CD4+ cells increased from 33.1 +/- 14.2 to 43.8 +/- 5.8% (p < 0.05) causing a comparable increase in CD3+ cells (67.3 +/- 14.3 vs. 77.1 +/- 7.9%, p < 0.05), whereas CD8+ cells did not change significantly (22.2 +/- 5.4 vs. 25.5 +/- 3.0%). Mitogen-induced lymphocyte stimulation responses were normal even before treatment and did not change. Antigen-induced T-cell responses were very heterogeneous before calcitriol therapy; those 5 with initially unmeasurably low serum 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) had a weaker response than the other patients (3,873 +/- 1,528 vs. 22,948 +/- 13,684 cpm, p < 0.05). After calcitriol treatment the patients with pretherapy unmeasurably low serum 1,25-(OH)2D3 had a comparable response to other patients (16,220 +/- 9,674 vs. 22,064 +/- 10,331 cpm). Our study shows that calcitriol therapy restores the depressed antigen-induced T-cell response of the hemodialysis patients most deficient in 1,25-(OH)2D3.

This study was set in a general, university hospital in the Canary Islands, with the objective to evaluate an enzyme-linked immunosorbent assay (ELISA) using A60 in the serodiagnosis of tuberculosis. IgG antibody to A60 was determined in 205 patients with active disease [149 culture-positive patients with pulmonary tuberculosis (positive sputum smear 94, negative sputum smear 55) and 56 patients with extrapulmonary tuberculosis], 20 patients with inactive disease, 22 patients with lepromatous leprosy, and 51 controls. The mean levels of anti-A60 antibodies were significantly higher in patients with active disease as compared with controls or patients with inactive disease. Differences were also found between tuberculous patients with pulmonary and extrapulmonary disease. In patients with pulmonary disease, significant differences were detected between smear-positive and smear-negative patients. The overall sensitivity of the test (cutoff 240 ELISA units) was 52.2%. The highest sensitivity was found among smear-positive patients with pulmonary tuberculosis (67%) and the lowest among those with extrapulmonary tuberculosis (32.1%). We conclude that ELISA for the measurement of IgG antibody to Mycobacterium tuberculosis antigen A60 could be of interest, specially in smear-negative cases and extrapulmonary tuberculosis.

Elicitation of delayed-type hypersensitivity (DTH) responses is due to the required sequential action of two different antigen (Ag)-specific Thy-1+ cells: early acting, DTH-initiating cells and locally recruited CD4+, alpha beta-TCR+, DTH effector T cells. DTH-initiating cells have an unusual phenotype for Ag-specific cells (Thy-1+, CD5+, CD4-, CD8-, CD3-, sIg-, B220+ (CD45RA+), Mac 1+, IL-2R- and IL-3R+) and act by producing Ag-specific non-IgE factors that sensitize mast cells for release of the vasoactive amine serotonin (5HT) at the local site of elicitation of DTH by challenge. Another mechanism of DTH initiation involves Ag-specific IgE antibodies that also can sensitize mast cells for local serotonin release. Serotonin initiates DTH by activating the local endothelial cells to allow recruitment of DTH effector T cells, and also by activating 5HT-2 receptors on these recruited T cells.

Clinical experience suggests the delayed-type hypersensitivity (DTH) skin test lacks sensitivity in assessing the integrity of systemic cell-mediated immunity (CMI) or the status of recent or remote mycobacterial infections in elderly nursing home residents. In an attempt to clarify this issue, DTH reaction to purified protein derivative (PPD), tetanus toxoid and Candida albicans was compared with circulating thymus-derived lymphocyte (T cell) proliferation (TCP) to stimulation with PPD and anti-CD3 antibody in 24 randomly selected nursing home residents. The DTH reaction and the TCP response correlated reasonably well among the DTH reactors but poorly among DTH nonreactors, suggesting there may be age-related immunologic changes in the skin itself. Also, the DTH skin test to PPD alone was found to be a poor index of the integrity of systemic CMI.

Following repeated BCG vaccination, some people exhibited negative skin tests to tuberculin (PPD) but showed antibody titers similar to those observed in skin test positive people. The purpose of the experiments reported here was to analyze this dissociation observed between cellular and humoral response to tuberculin. Leukocytes from skin test negative people were normally stimulated by various mitogens but not by PPD. After separation of leukocytes into adherent and nonadherent cell populations and in vitro transfer experiments, it was shown that the nonadherent cell population was responsible for the nonresponsiveness to PPD. On the other hand, serum immunoglobulin levels and anti-PPD antibody titers were similar to those observed in skin test positive people. The results show that in skin test negative people, T cells involved in cellular immune response to PPD are ineffective while macrophages, helper T cells and B lymphocytes seem to function normally.

Lymphocytes of tuberculous patients activated in vitro with purified protein derivative of tuberculin (PPD) were examined for rosette formation with human autologous erythrocytes. The percentage of the rosette-forming cell (auto-RFC) was increased when pleural fluid lymphocytes of patients with tuberculous pleurisy and peripheral blood lymphocytes (PBL) from patients with tuberculosis or from tuberculin skin test positive healthy individuals were stimulated with PPD in vitro, whereas no increase of auto-RFC was observed in PBL from tuberculin skin test negative donors. Increased numbers of auto-RFC were shown to form rosettes with sheep erythrocytes but have no IgG Fc receptors on their surfaces. It was also shown that adherent cells were required for the PPD-induced increase of auto-RFC. Depletion of PPD-induced auto-RFC by the density gradient sedimentation technique led to a significant decrease in the degree of the proliferative response to gradient sedimentation technique let to a significant decrease in the degree of the proliferative response to PPD and the number of the auto-RFC after stimulation with PPD. These findings strongly suggest that PPD-induced auto-RFC represents the consequence of an immunological interaction between sensitized T lymphocytes and relevant antigen PPD, and reflects the PPD responsiveness of tuberculosis at the T cell level.

A low molecular weight component, termed counter inhibitor (CI), has been partially purified from human dialyzable leukocyte extracts. Addition of CI to either a direct leukocyte or macrophage migration inhibition system results in reversal of antigen-induced migration inhibition. CI activity requires the presence of antigen for expression, but does not require that the donor of the CI be immune to the antigen used in the migration inhibition assay. Reversal of migration inhibition by CI appears to be a consequence of its ability to prevent PMNs or macrophages from responding to lymphokines which induce migration inhibition.

Dialyzable components of culture filtrate of Mycobacterium bovis were fractionated by gel filtration on Sephadex G-10 into eight fractions, A through H in order of their elution from the column. None of the fractions produced a visible precipitate when reacted with a hyperimmune antiserum. The first five fractions were further investigated. All were capable of inhibiting precipitation of nondialyzable tuberculin. Fraction E, containing the smallest fragments, totally abolished precipitation and fraction A, which contained the largest molecules inhibited the least. Fractions A and B were the only ones that could induce skin reactions in sensitized guinea pigs but did this poorly. In vitro transformation of PPD sensitized lymphocytes was induced only by fraction A. Only fractions A and B could inhibit transformation of lymphocytes by Concanavalin A but only fraction B could inhibit transformation by tuberculin. Dialyzable components injected intracardially 3 hours prior to skin testing with tuberculin caused a reduction in skin reactivity. Since such substances are present in tissues in late stages of tuberculosis investigations were made to determine if they played a role in the induction of the observed accompanying tolerance to skin test cellular hypersensitivity. Guinea pigs treated with dialyzable components prior to and for four weeks post sensitization did not develop tolerance.

Multiple puncture dried tuberculin tests compare favourably with the standard Mantoux test provided the tips of the metal prongs are thinly and uniformly coated with tuberculin. Unfortunately, surface tension, varying viscosity of the acacia solutions, and deeper dipping depths lead to increased quantities of tuberculin materials adhering to the metal prongs which results in thickening and retraction of the materials on the metal. Solutions of tuberculin in buffered phosphate produce thin and uniform coatings provided adequate cleaning of the metal--especially degreasing--is carried out. In the absence of this the coating layer may fragment. However, provided the distal 0.6-0.8 mm is covered, sufficient tuberculin will be deposited intradermally.

Cell-mediated immunity (CMI) was evaluated in 26 patients with lipoid nephrosis (LN), 50 patients suffering from chronic diffuse proliferative glomerulonephritis (CGN) without renal sufficiency and 24 healthy controls. The following parameters were measured: delayed hypersensitivity skin test responses to purified protein derivative (PPD) and candida, circulating lymphocytes. T lymphocytes and T lymphocytes with receptors for the Fc portion of IgG (T gamma cells) or IgM (Tmu cells). Patients with LN in relapse had less mean induration of skin reactivity and a smaller proportion reacting to both antigens as compared with the control subjects. In contrast, the intensity of skin reactivity and the frequency of negative reactions in patients with LN in remission and CGN were similar to those of the control subjects. It was also found that the LN patients in relapse had a significant T lymphocytopenia as well as a significant decrease in absolute numbers of Tmu and T gamma cells, whereas the patients with LN in remission and CGN did not differ significantly from the control population. Thus, the majority of patients with LN in relapse demonstrated an impaired response in a CMI assay system. The disturbed CMI may be secondary to hypoproteinemia and other nutritional factors induced by the nephrotic syndrome.

A two-step modification of Clausen's technique of leukocyte migration under agarose is described. Blood leukocytes from a subject are incubated for approximately 4 days with or without antigen. The cultures, after reconstitution with antigen followed by repeated washing of the cultured cells, are added to fresh, dextran-separated blood leukocytes, and migration tests are performed in the absence of the antigen. Cells preincubated with antigen give a very marked reduction of the migration of the target leukocytes which must not necessarily be autologous. An inhibition of target cell migration can be seen with cells cultured without antigen for up to 3 days; with longer culture time, this effect is reduced or disappears. The effect of cells cultured with antigen, however, is equally strong irrespective of the culture time up to 1 week, and the most clearcut results will, therefore, be obtained after approximately 4 days culture. Also migration enhancement has been seen. Treatment of cells during the incubation with antigen and cycloheximide gives a dose-dependent blocking of the inhibiting effect. 5 patients were studied both before and after sensitization with DNCB: low concentrations of DNCB gave an inhibition and high concentrations an enhancement of the migration when the patients were sensitized. The technique is more sensitive and reproducible than the original Clausen technique. It is also suitable in studies where shortage of cells is a problem.

The kinetics of lymphocyte recruitment into rat popliteal lymph nodes stimulated with varying doses of allogeneic lymphocytes or purified protein derivative of tuberculin differed markedly. The number of blood-borne lymphocytes entering lymph nodes stimulated with allogeneic lymphocytes was greater and the duration of their entry more prolonged than in lymph nodes stimulated with the soluble antigen purified protein derivative of tuberculin. When individual antigenic doses were compared, lymphocyte recruitment into popliteal lymph nodes was shown to be dependent upon antigenic dose; higher doses of antigen resulting in increased levels of lymphocyte recruitment. In addition it was found that there is no linear relationship between lymph node weight changes and the extent of lymphocyte recruitment.

It was proposed that a study should be made on the specificity of the reactions of 1068 subjects reacting "weakly to moderately" to an intradermal test with 2 units of PPD RT23; 43% were classified as "positives" (greater than or equal to 10 mm) and Palmer type IV indurations or merely erythema were seen in 81%. Three months later, the same people were retested, this time with 2 units of PPD RT23 and, simultaneously, with 0.1 mcg of avian sensitin: 23% of all reactors (13% of all "positives", but none with a Palmer type I of II induration) had completely lost their sensitivity to RT23. Of the 1068 original reactors, 87% reacted to avian sensitin and 69% did so with a reaction greater than or equal to to human RT23. According to the results of this double test, and depending on the strictness of the comparative criterion, 69% to 84% of the original reactors (65% to 78% of the "positives") would be classified as non-specifically infected. There was a high degree of agreement between an induration greater than or equal to 10 mm to PPD RT23 in the second test and classification as specific infection, based on a comparative criterion.

Previous reports studying the immunological and histopathological sequelae following infection with Trichinella spiralis indicated that this nematode can affect the host's immune capability to heterologous antigens. The present investigation was aimed at determining the dose of T. spiralis larvae required for development of maximum delayed-type hypersensitivity (DTH) against heat-killed bacillus Calmette-Guérin (BCG). Mice were injected intraperitoneally with 4 x 10(6) bacteria 28 days after oral intubation with 0, 50, 100, 200, 300 or 400 viable T. spiralis larvae. Groups were tested in vivo for DTH 28 days after administration of nonviable BCG by the footpad swelling assay. Prior infection with 200 nematode larvae resulted in 34 of 42 animals (81%) developing positive 24-hour DTH reactions. When these data were compared to those seen in the other experimental and control groups, the 200 larval dose appeared to effect maximal immunopotentiation in this system.

Autoimmune MRL mice develop a poor delayed-type hypersensitivity (DTH) after BCG cell wall (BCG CW) immunization. To test the possible participation of suppressor cells on DTH induction, MRL/MpJ-lpr/lpr (MRL-lpr) and MRL/MpJ-+/+ (MRL-+/+) mice were pretreated with cyclophosphamide (Cy) 4 days before the immunization. The footpad reaction to the purified protein derivative of tuberculin (PPD) was enhanced remarkably by Cy pretreatment in both MRL-lpr and MRL-+/+ mice. In addition, the lymph node cells obtained from nonpretreated MRL-lpr and MRL-+/+ mice at 7 days after BCG CW immunization suppressed the DTH induction of Cy-pretreated MRL-+/+ mice. On the other hand, a positive proliferative response to PPD in vitro was seen only in the cells from Cy-pretreated MRL-+/+ and younger MRL-lpr mice, but not in the cells of nonpretreated MRL-+/+ or both pretreated and nonpretreated older MRL-lpr mice. Removal of B220-positive cells from the lymph node cell populations of Cy-pretreated MRL-lpr mice restored PPD responsiveness, but the same treatment had no effect on the cells from nonpretreated MRL-lpr mice. The results suggest that DTH responses to PPD in MRL mice immunized with BCG CW are regulated by Cy-sensitive suppressor cells. These suppressor cells occur in MRL mice irrespective of whether they carry the lpr gene. And it is also demonstrated that MRL-lpr mice have TDTH precursor cells in a sufficient number for TDTH production even after expressing lymphadenopathy.

The Electrophoresis-Mobility-Test is a modified Macrophage-Electrophoretic-Mobility-Test by using sheep erythrocytes, tanned and stabilized by sulfosalicyl acid. The test is used to demonstrate specific sensibilised T-lymphocytes in cell culture after having them isolated from human venous blood and after stimulation with antigen. 34 tests were done by stimulation with antigen PPD, the lymphocytes having been taken from patients with positive Tuberculin-Tine-Test. All tests were bacteriologically and cytologically controlled and only 9 could be used finally as a base for statistical control by using x2- und t-tests and histogram. A special attention was payed to the aggregation of monocytes and their morphological changes in cell culture. Every single test is not statistically significant, but the results of the whole lot give an attest of the concept of the EM-Test, which can be reproduced with a probability of about 60%.

Despite the importance of the guinea pig as a model test animal for studying the humoral immune response in comparison to the development of cellular immunity after BCG or tuberculosis infection, no precise and sensitive method for the determination of such antibodies is available. We developed an indirect solid-phase radioimmunoassay on microtiter plates for this purpose. All assay conditions have been optimized such as selection of suitable plastic material, use of carrier substances, concentrations and amount of the antigen as well as the serum and second antibody, buffer, incubation times and all steps of the procedures for coating, washing, and measuring. The assay gives a linear relationship between antibody concentration in the serum to be tested and binding of the radiolabelled second antibody. A comparison with four conventional antibody titration methods reveals a 128- to 512-fold higher sensitivity of the RIA. Favorable aspects of this RIA as opposed to the methods hitherto used are discussed.

Examination of medical student's and nursing student's sensitivity to PPD-Y, PPD-G and PPD-B before and after BCG vaccination revealed that some students who were not reactive prior to vaccination became strongly reactive to one or more of the atypical tuberculins after vaccination. This result is not due to BCG vaccine since it was not seen in guinea pigs or in children. This evidence of the interaction of multiple sensitivities after infections with different mycobacteria has significance in the interpretation of anti-tuberculosis immunity in BCG trials.

Primary and secondary proliferative responses to hapten (TNP)-modified autologous cells as well as to allogeneic cells, antigen (purified protein derivative) and mitogens were used for in vitro studies of the radiosensitivity of human T cells. To be able to compare data obtained from the various assay systems, optimal responding/stimulating cell ratios were used and the post-irradiation period kept constant. With these precautions, no difference in radiosensitivity of the various T cell responses could be found whether they were induced by mitogen, antigen, allogeneic cells or TNP-modified autologous cells. Neither could, on a per-cell basis, any difference in radiosensitivity between primed and non-primed cells be detected. At 400 rad, about 10% of the response was recovered in either case. The immunocompetence of the T cells surviving irradiation, as estimated by the ability to give a TNP-specific response, was not impaired.

We compared the accessory cell function of human alveolar macrophages (AM) to that of human blood monocytes (Mo) obtained by bronchoalveolar lavage and venipuncture from normal volunteers. Graded numbers of either AM or Mo were added to autologous peripheral blood T lymphocytes that were stimulated with a purified protein derivative of tuberculin (PPD). Either AM or Mo were cocultured with allogeneic T lymphocytes in mixed lymphocyte reaction (MLR) experiments. Both AM and Mo supported the PPD-induced T lymphocyte proliferation and allogeneic MLR at low ratios of AM or Mo to T lymphocytes with similar efficiency. However, AM showed marked suppressive effects at higher ratios of AM to T lymphocytes (1:1). PPD-pulsed AM, but not AM killed by physical treatments (heat, freeze-thaw, sonication), induced T lymphocyte proliferation. An indirect immunofluorescent study demonstrated that most AM express HLA-DR antigens. Furthermore, AM synthesized DR antigens with molecular weights of 33,000 and 29,000-31,000 daltons. When AM were treated with both anti-DR monoclonal antibody and complement, PPD-induced T lymphocyte proliferation and MLR were diminished. These results suggest that human AM function as accessory cells in the antigen-induced T lymphocyte proliferation and DR antigens on AM play an important role in the accessory cell function.

The development of tuberculin sensitivity in groups of guinea pigs immunized with living BCG vaccine or with heat-killed Mycobacterium tuberculosis suspended in oil (TB) was measured by skin tests and lymphocyte transformation (LT) tests with tuberculin PPD. LT tests on lymphocytes from peripheral blood (PBL) and lymph nodes (LNL) and skin tests were carried out on different groups of guinea pigs for 1 year following immunization. Three ways of expressing the LT results were considered, i.e. as stimulation index, delta cpm and uncorrected cpm values. Since neither of the first two methods were equally applicable to all of the data, we decided to express the data as geometric means of cpm values for stimulated and control cultures, respectively. For both immunogens the sensitivity measured by PBL LT and skin tests showed a closely parallel development; LNL reactivity appeared earlier and remained at a higher level than PBL reactivity. Antibody levels in the guinea pigs were measured using a radioimmunoassay technique, were found to be dependent on the immunization period and not correlated to the cellular immunity.

Dual skin tests with tuberculin purified protein derivative (PPD) and with one of the following antigens prepared from mycobacteria other than M. tuberculosis, such as PPD-Y, PPD-G, PPD-B, PPD-F, etc., are performed to establish the frequency of "atypical" mycobacterial infection in a given population. However, most of this work has been done in areas where BCG vaccination was rarely implemented, if at all. The purpose of this study was to assess the significance of a dual skin test using PPD and PPD-B in a population which had been vaccinated with BCG. It was found that in a population with a high level of sensitivity to PPD, median skin reaction 12 mm, only 7% of the subjects tested gave a greater response to PPD-B than to PPD, whereas in a population with a low level of sensitivity to PPD, median skin reactions 3 mm, 36% of the subjects gave a greater response to PPD-B than to PPD. It would appear, therefore, that in a population vaccinated with BCG and with a low level of sensitivity to PPD, a relatively high percentage of positive reactors, those with an induration of 5 mm or more to either PPD or PPD-B, gave a larger skin reaction to PPD-B than to PPD. To avoid erroneous conclusions as to whether or not there is a high frequency of atypical infection in a given population, the authors suggest that the BCG status of those tested with PPD and PPD-B be taken into consideration when interpreting the results of an epidemiological survey that includes an atypical antigen such as PPD-B.

Cell-mediated immunity (CMI) was evaluated in 11 patients with idiopathic membranous nephropathy (MN), 50 patients suffering from chronic proliferative glomerulonephritis (CGN) without renal insufficiency and 24 healthy controls. The following parameters were measured: delayed skin reactivity to purified protein derivative (PPD), circulating lymphocytes, lymphocyte cell-surface markers (En and EAC rosettes) and functional markers (mitogenic responses to Con A and PHA). The MN patients with nephrotic syndrome (NS) had less mean induration of skin reactivity and a smaller proportion reacting to the PPD antigen as compared with the control subjects. In contrast, the intensity of skin reactivity and the frequency of negative reactions in MN patients in remission and CGN were similar to those of the control subjects. During the nephrotic stage of MN the proportion of T lymphocytes decreased with simultaneous increase of the proportion of B lymphocytes. It was also found that the MN patients with NS showed impaired lymphocyte reactivity with lower Con A and PHA responses compared to the normal controls. Conversely, the mean mitogenic responses to the antigens in patients with MN in remission and CGN were similar to those of the control subjects. Thus, the majority of MN patients with NS demonstrated an impaired response in a CMI assay system. The possible significance of these phenomena in the pathophysiology of MN is discussed.

We found that optimal conditions for the beryllium transformation test required the use of purified lymphocyte suspensions in 20% human serum, with beryllium sulphate at concentrations of 10(-6) to 10(-7) M. Using these conditions, all 15 chronic beryllium patients tested gave a stimulation index of more than 2. Despite the above, 10(-5) and 10(-6)M, BeSO4 inhibited spontaneous thymidine incorporation and lymphocyte transformation to PHA and PPD in normal subjects. Alternative methods, using whole blood, venous and thumb-prick samples, gave fewer and less reproducible positive results.

The effect of supplemental B lymphocytes on the antigen-induced in vitro proliferative response of highly purified lymph node T cells was studied in two immune systems of complete Freund's adjuvant (CFA) and testicular antigen (TA) sensitized guinea pigs. When the T lymphocytes obtained from animals 2 weeks after CFA sensitization, though unresponsive to purified protein derivate of tuberculin (PPD) by themselves, were cultured together with mitomycin C-treated autologous B (Bm) lymphocytes (1:0.5 ratio), they responded well to PPD. The enhancing effect of Bm lymphocytes was less than that of mitomycin C-treated macrophages (M phi m) from the same animals. Analogous helper effect of Bm lymphocytes on the T cell proliferation was observed also in TA-sensitized animals. Additionally, it is noteworthy that the PPD-induced proliferation of T lymphocytes at 2 weeks after CFA sensitization was absolutely dependent on M phi and B lymphocytes, whereas T lymphocytes at 10--14 weeks exhibited a low but significant response by themselves, although their response to PPD was markedly augmented with supplemental M phi m or Bm lymphocytes.

Pig peripheral blood lymphocyte subpopulations were characterized by their adherence to nylon wool. Thus whilst B cells, defined by surface Ig and Fc receptor, were predominantly adherent, thymus-dependent Null cells and the T cells forming rosettes with sheep red blood cells in dextran (DS+) were predominantly non-adherent. There was a direct relationship between the size of the non-adherent fraction and the proportion of the total DS+ cells which were in the non-adherent fraction. Using nylon wool and/or DS rosette separation, lymphocytes from normal and BCG sensitized animals were stimulated in vitro with phytohemagglutinin (PHA), concanavalin A (Con A) and tuberculin (PPD). The cells responding to PHA and Con A (T cell mitogens in other species) were DS+ lymphocytes. Non-rosetting, adherent (B cells) and non-adherent (thymus dependent Null cells) lymphocytes were unresponsive, even in the presence of homologous alveolar macrophages at levels which greatly enhanced the PPD response. Cells responding to PPD were present in the DS+ and DS-, adherent and non-adherent fractions but their response was apparently controlled by macrophage dependence and suppression. Techniques are described for the preparation and storage in liquid nitrogen of alveolar macrophages resulting in high numbers of viable cells, capable of helping in vitro responses, even after 3 years of storage.

Lymph node cells from guinea pigs sensitized by BCG elaborated two chemotactic factors for lymphocytes when stimulated with purified protein derivative in vitro 4 weeks after immunization. Production of the factors occurred within 24 h of incubation. The two factors were termed alpha- and beta-lymphocyte chemotactic factors (alpha-LCF, beta-LCF), relative to their elution order on gel chromatography. The molecular weights were about 160,000 and 27,000, respectively. alpha-LCF was labile to heating at 56 degrees C for 30 min. Both factors were sensitive to digestion by trypsin, and were effective for T cells but not for B cells.

Preparations of gliadin, extracted from wheat gluten, were shown to stimulate a proliferative response by lymphocytes of normal donors. Not all donors responded to gliadin. In all but one case responder or non-responder status remained consistent on retesting. There was no association of ability to respond with HLA A or B type.

Antigen-specific Ts against DTH to BCG were induced in vitro by culturing the splenic T cells of C3H/He mice together with the lined macrophage, SL-1, pulsed with PPD. The suppressor activity was determined by the suppression of DTH in CY-treated syngeneic recipients which had received the cultured cells before BCG sensitization. SL-1 cells express I-A and I-J antigens on the surface, while SL-4 cells, which originated in the same mouse strain as SL-1, do not express any Ia antigens. When I-A antigens on SL-1 were blocked by anti-I-A antibody without complement before PPD-pulsing, the induction of Ts was rather enhanced. When I-J antigens on SL-1 were blocked by anti-I-J alloantiserum, no Ts were induced. Instead, these I-J-blocked and PPD-pulsed SL-1 cells induced effector cells of DTH in vitro. Ia-negative lined macrophage SL-4 cells did not show any activity either in suppressor or in effector cell induction. From these results, we propose a model for the immune modulation led by APC.

Administration of cyclosporin A (CsA; 25 mg/kg) orally to guinea pigs from the time of immunization with ovalbumin (OVA) in complete Freund's adjuvant, followed by drug withdrawal 4 days later, resulted in marked potentiation of classical, tuberculin-like delayed-hypersensitivity skin responses to OVA. However, no such augmentation of delayed-type hypersensitivity (DTH) to purified protein derivative (PPD) was demonstrated. The enhancing effect of CsA was also dependent on the dose of OVA used for both immunization and skin testing and on the interval between drug withdrawal and the elicitation of DTH. A single intraperitoneal injection of CsA (200 mg/kg) given 2 days before immunization also had an augmentary effect on 14-day responses to OVA. Similar treatment protocols, however, did not enhance Jones-Mote (cutaneous basophil) hypersensitivity to OVA or contact sensitivity reactions to dinitrofluorobenzene. Longer courses of CsA (25 mg/kg per os) between sensitization and skin testing severely depressed all three categories of type IV hypersensitivity reactions. Our observations may have important cautionary implications for the prospective management of immunologically mediated diseases of intermittent activity, including certain autoimmune disorders, where short courses of CsA might be contemplated.

Specificity of plasmacellular infiltration was studied using a guinea pig peritoneal inflammation model. Acute and chronic inflammations were induced by repeated injections of either of two non-crossreacting antigens (DNP-BSA and PPD). With an enzyme-immunohistochemical sandwich procedure allowing quantitation of DNP-BSA-specific plasma cells, specificity of plasmacellular infiltration could be demonstrated. DNP-BSA-specific antibody-forming cells were found not to enter inflammatory reactions elicited by PPD. Our data support the hypothesis that virtually all plasma cells in a chronic inflammatory exudate release antibodies specific for antigens that are locally available, and that such antigens are likely to play a central role in the perpetuation of chronicity.