Antigen-reactive T cells can be activated buy autologous macrophages in the absence of added antigen.

T cells responsive to macrophages (M phi) in the autologous mixed lymphocyte reaction (AMLR) contain those cells that can be induced to proliferate by soluble antigens. Negative solution (5-bromo-2-deoxyuridine and light) of T cells activated by autologous M phi also removed those cells required for reactivity to Candida albicans and purified protein derivative. Positive selection of T cells responsive to autologous M phi yields a population that is simultaneously enriched in antigen reactivity. Some patients demonstrating cutaneous anergy and diminished in vitro blast transformation in response to soluble antigen also lack T cells responsive to the AMLR to M phi. When considered in conjunction with previously reported data, these findings indicate the AMLR occurring between T cells and M phi in the absence of soluble antigen represents self recognition occurring between antigen-reactive T cells and antigen-presenting M phi.

Factor 8 detection by hemagglutination inhibition: hemophilia A and von Willebrand's disease.

Factor VIII activity was detected immunologically in both the serums and plasmas of 14 normal individuals and 14 patients with hemophilia A. A hemagglutination-inhibition test with rabbit antibody to highly purified (10,000-fold) factor VIII from humans was used. Serums and plasmas from eight patients with von Willebrand's disease showed little or no factor VIII activity in this test, an indication that the test may serve as a specific assay for differentiation between von Willebrand's disease and hemophilia A.

CD8+ lymphocytes can control HIV infection in vitro by suppressing virus replication.

Lymphocytes bearing the CD8 marker were shown to suppress replication of human immunodeficiency virus (HIV) in peripheral blood mononuclear cells. The effect was dose-dependent and most apparent with autologous lymphocytes; it did not appear to be mediated by a cytotoxic response. This suppression of HIV replication could be demonstrated by the addition of CD8+ cells at the initiation of virus production as well as after several weeks of virus replication by cultured cells. The observations suggest a potential approach to therapy in which autologous CD8 lymphocytes could be administered to individuals to inhibit HIV replication and perhaps progression of disease.

Human seminal plasma inhibition of antibody complement-mediated killing and opsonization of Neisseria gonorrhoeae and other gram-negative organisms.

Seminal plasma diluted 1:5-1:1,000 gave marked inhibition of serum antibody complement-mediated bactericidal and opsonic effects against Neisseria gonorrhoeae and other gram-negative organisms. Serum that was bactericidal at a dilution of 1:5,120 was not bactericidal at a dilution of 1:10 when seminal plasma was added. Bactericidal action of immune human or rabbit sera, or purified immunoglobulin (Ig)G or IgM plus complement for six strains of N. gonorrhoeae, serogroups A, B, C, and Y of Neisseria meningitidis, Escherichia coli and other gram-negative rods was inhibited by seminal plasma. Using C8- or C7-deficient sera as antibody and complement sources, opsonization, phagocytosis, and killing of N. gonorrhoeae and E. coli 014-K7 were inhibited by seminal plasma. Opsonization, phagocytosis, and killing of Staphylococcus aureus 502A was not inhibited. For the gram-negative organisms, the early phase of the opsonization process, probably complement activation, appeared to be inhibited rather than the ingestion or polymorphonuclear leukocyte killing steps; addition of seminal plasma yielded a significant reduction in the percentage of polymorphonuclear cells with associated bacteria. Seminal plasma did not prevent attachment of IgG, IgM, or IgA antibodies to gonococci. It reduced serum hemolytic whole complement activity by 25%. The seminal plasma inhibitor was of low molecular weight and was stable at 56 degrees C for 30 min, but inhibitory activity was lost after heating to 100 degrees C for 10 min. It is likely that the inhibitory factor(s) is a low-molecular weight protease or protease inhibitor. Seminal plasma probably has an important role in inhibition of complement and antibody functions in the genital tract. It may enhance pathogenesis of agents of sexually transmitted diseases.

The Wiskott-Aldrich syndrome. Results of transfer factor therapy.

12 patients with Wiskott-Aldrich syndrome were treated with therapeutic doses of transfer factor in an attempt to induce cellular immunity. Clinical improvement was noted after transfer factor therapy in 7 of the 12 patients treated. Because this disease has a variable course and temporary spontaneous improvement can occur, the observed improvement cannot necessarily be attributed to the transfer factor. However, in two patients repeated remissions consistently followed transfer factor administration on repeated occasions. This included freedom from infections, regression of splenomegaly, and clearing of eczema. An unexpected finding was a decrease in bleeding in 3 of the 10 patients who had bleeding. Conversion of skin reactivity was obtained in all seven patients who clinically seemed to respond to transfer factor. In vitro studies performed after the administration of transfer factor demonstrated that the lymphocytes of the patients now produced migration inhibitory factor in response to appropriate test antigens, but did not undergo increased radioactive thymidine incorporation in response to the same antigens. A defect in the monocyte IgG receptors has been found in certain patients with the disease, and the current study shows that all patients with defective monocyte IgG receptors responded to transfer factor, whereas only one patient with normal receptors showed any response. This test may thus prove to be useful in predicting the results of transfer factor therapy in patients with Wiskott-Aldrich syndrome, although evaluation of a larger series of patients will be necessary to confirm this point. We conclude that cellular immunity can be induced, that there appears to be clinical benefit in certain patients with Wiskott-Aldrich syndrome by the use of transfer factor, and that this mode of therapy warrents trial in these patients and others with defects of cellular immunity.

Lymphocyte proliferative responses to human immunodeficiency virus antigens in vitro.

All HIV seronegative (HIV Ab-) and most HIV seropositive (HIV Ab+) individuals' lymphocytes failed to proliferate in primary cultures in response to purified HIV or to recombinant envelope and core antigens of HIV, even in the presence of recombinant interleukin 2 (rIL-2). Most HIV Ab- and HIV Ab+ individuals' lymphocytes, however, could proliferate or be induced by rIL-2 to proliferate in response to lysates of Escherichia coli or Saccharomyces cerevisiae. These findings indicate selective defects in lymphocyte proliferative responses to HIV antigens before the development of AIDS in which lymphocytes are unable to proliferate in response to any antigens. These defects in cell-mediated immune responses to HIV antigens are likely to play an important role in the pathobiology of HIV infections. Although intact HIV or glycosylated gp120 envelope protein of HIV are involved in these defects, a non-glycosylated recombinant form of the HIV gp120 envelope (ENV2-3) and p25 core proteins did not inhibit antigen- or mitogen-driven lymphocyte proliferation.

Coexistent lymphoid interstitial pneumonia, pernicious anemia, and agammaglobulinemia.

Immunologic factors have been incriminated in the pathogenesis of lymphoid interstitial pneumonia. The discovery of a patient with coexistent lymphoid interestitial pneumonia, pernicious anemia, and common variable hypogammaglobulinemia focused attention on the possible autoimmune nature of this pulmonary disease. Extensive immunologic studies demonstrated a noticeably impaired bonemarrow-dependent (B cell) system and intact thymus-dependent (T cell) system. No evidence of humoral or cellular hypersensitivity to homologous lung determinants was found.

Transcription and replication of human immunodeficiency virus-1 in B lymphocytes in vitro.

We and others have shown that HIV-1 transcription and replication in vitro are increased by the binding of transcriptional enhancer DNA sequences in the HIV-1 long terminal repeat (LTR) to a cellular protein designated Nuclear Factor-kappa B (NF-kappa B), a trans-acting transcription factor which is present in activated but not in resting T cells. Because NF-kappa B is also expressed in resting B cells we suggested that B cells might provide an optimal intracellular environment for HIV-1 transcription. Using a transient transfection assay, we show that the HIV-1 LTR is indeed more efficiently transcribed in the B cell line Raji than in the T cell line Jurkat, and that this effect maps to the transcriptional enhancer (NF-kappa B binding site) of the LTR. Furthermore, we show that Raji cells which have been rendered CD4-positive by gene transfection are susceptible to HIV-1 infection, and that viral gene expression and replication are more efficient in these CD4-positive B cells than in CD4-positive T cells. These findings demonstrate the crucial role of the transcriptional enhancer in regulating HIV-1 expression and, since receptors for HIV-1 are expressed by some Epstein-Barr virus (EBV)-positive B cells, they also suggest that HIV-1 replication could occur in EBV-positive B cells in vivo.

HIV-1 gp120 and anti-gp120 induce reversible unresponsiveness in peripheral CD4 T lymphocytes.

Human immunodeficiency virus type 1 (HIV-1) gp-120 potentially plays an important role in inducing functional suppression and depletion of CD4 lymphocytes following infection with HIV. In order to further understand the mechanisms involved in HIV-induced immunosuppression, we have studied the effects of recombinant HIV-1 gp120/SF2 and anti-gp120/SF2 antibodies on T cell receptor (TCR)-mediated proliferation of peripheral blood mononuclear cells (PBMCs) and isolated lymphocyte subsets from HIV-seronegative donors. In a dose-dependent manner, gp120 significantly reduces the proliferative responses of unfractionated PBMCs and highly enriched CD4 T lymphocytes when they are polyclonally stimulated through the TCR using WT31 (anti-alpha beta Ti chains) and anti-Leu 4 (anti-CD3 epsilon) in the presence of autologous accessory cells. The addition of divalent anti-gp120/SF2 to lymphocytes previously incubated with gp120 further reduces the proliferation to the levels seen after pretreating cells with divalent anti-CD4 (anti-Leu 3a). CD8 T lymphocytes, on the other hand, show no change in TCR-mediated proliferation following preincubation with either anti-CD4 or gp120/anti-gp120. We find no evidence for significant cell death by apoptosis using methods of DNA analysis or flow cytometry and DNA-specific dyes to account for the loss of CD4 lymphocyte proliferation. Interleukin-2 restores the proliferation suppressed by gp120/anti-gp120 suggesting the induction of reversible functional anergy.

Development of a microsphere-based fluorescent immunoassay and its comparison to an enzyme immunoassay for the detection of antibodies to three antigen preparations from Candida albicans.

A sensitive assay for the simultaneous detection of multiple serum antibodies by flow cytometry was developed. Polystyrene microspheres of 5, 7 and 9.3 micron in diameter were used as solid supports for the attachment of three different antigen preparations from Candida albicans. These antigens were a whole cell extract; a cytoplasmic protein extract and a cell wall polysaccharide. Microsphere-associated fluorescence was quantitated by flow cytometry, with the different sized microspheres analyzed separately using electronic volume gating. This procedure allowed for different antigen-coated microspheres with discrete sizes to be analyzed independently for immunofluorescence. The assay detected antibody levels in human serum at dilutions up to 10(-6) and provided complete discrimination, using all three antigen preparations, between antibody levels seen in healthy subjects and those seen in patients suspected of having a systemic Candida infection. A standard enzyme immunoassay (EIA) failed to provide complete discrimination between healthy subjects and patient samples: at least 17% of patient values fell within the healthy subject range using all three antigen preparations. The microsphere assay which allowed for the simultaneous detection of multiple antibodies, has increased dynamic range over EIA and provides for better discrimination of patients from healthy subjects in comparison to EIA. Precise quantitation of antibodies is possible and the rapid analysis of thousands of microspheres markedly enhances the statistical accuracy of the assay. We suggest this assay is likely to have many other important applications in immunologic testing.

Flow cytometric detection and quantitation of immune complexes using human C1q-coated microspheres.

A solid phase human C1q-binding fluorescent immunoassay for the measurement of immune complexes in human serum was developed. The solid phase used was 5 micron diameter polystyrene microspheres. Serum immune complexes bound to the C1q-coated microspheres were measured by flow cytometry using fluoresceinated anti-human IgG, and heat-aggregated human IgG as a standard. Patient samples were assayed and results compared to a standard fluoroimmunometric C1q-binding immune complex assay. Greater differences in circulating immune complexes were observed between the healthy control group mean and the mean of the patient values in the microsphere-flow cytometric method than were seen in the standard assay. In the microsphere-flow cytometric assay, the mean patient value was 7.5 times greater than the control mean, whereas in the standard assay the mean patient value was 2.8 times the control mean. Preliminary results suggest greater sensitivity of the microsphere-flow cytometric method over the other method.

The numerical development of lymphoid cells during human embryogenesis.

We have studied the cellular growth rates in the fetal thymus, spleen, and bone marrow of 12 human fetuses ranging in gestational age from 7 to 20 weeks. In all instances growth rates were linear when plotted logarithmically. However, the spleen had a biphasic growth curve. These data are useful in predicting the cellular yields which can be obtained from human fetal lymphoid organs which are to be used for transplantation in immune deficiency diseases and other clinical conditions.

Ontogeny of human T cells.

Current knowledge of human T cell ontogeny is reviewed in terms of appearance of T cells in central and peripheral lymphoid organs, maturation of T cell markers, and development of immune functions. Extrapolation of growth curves derived from cell counts from fetal thymus, spleen, and bone marrow indicates the appearance of lymphocytes at 3.5 weeks gestation. E-rosette-forming cells are present in thymus at 11 weeks and in peripheral organs 15 to 16 weeks gestation. beta-2-Microglobulin is associated with all lymphoid cells by 13 weeks gestation. Lymphocyte responses to the T cell mitogen phytohemagglutinin (PHA) are first detected in thymus at 10 weeks and in spleen and blood 3 to 4 weeks later. Allogeneic responses in mixed lymphocyte reactions develop at about 7.5 weeks in fetal liver and later in thymus and peripheral organs. Lymphocytotoxicity for xenogeneic cells is a property of bone marrow cells and not thymocytes. Several aspects of development of a suppressor T cell in human neonates is discussed and related to similar findings in the mouse. These studies indicate a relatively high degree of maturation of human T cells during fetal life.

A phase I study of HGP-30, a 30 amino acid subunit of the human immunodeficiency virus (HIV) p17 synthetic peptide analogue sub-unit vaccine in seronegative subjects.

HGP-30-KLH vaccine in alum at doses of 10, 25, 50, and 100 micrograms/kg administered intramuscularly at weeks 0, 4, and 10 appear well-tolerated clinically. Local pain at the injection site, appears to be the main clinical toxicity. Laboratory parameters are not affected by administration of the vaccine candidate except for perhaps mild urinalysis abnormalities at the highest dose. This vaccine candidate has no apparent immunotoxicity and does not appear to affect lymphocyte populations or T-cell functional studies. Low levels and transient antibodies develop in a minority of subjects early after immunization with the vaccine candidate. These responses were observed in the lowest dose range. Higher doses, and longer follow-up will be needed to confirm this observation. T-cell proliferative responses to KLH and KLH-HGP-30 are consistent and may not be dose dependent, but the proliferative responses are variable and more data need to be accumulated. Preliminary, there appears to be an HGP-30-induced CTL response of HGP-30-coated EBV-transformed autologous B cell lines. This study was approved under an IND for the California Department of Health Services' Food and Drug Branch. They have provided excellent support and regulatory guidelines for this project. Future work will extend and confirm these initial observations.

Immunocompetence of murine placental lymphocytes. In-vitro response to mitogens and to allogeneic cells.

Murine lymphoid cells from late (16-20 day) gestational placentae were studied for their ability to respond to mitogens and adult allogeneic cells in the mixed lymphocyte reaction (MLR) and the cell-mediated lympholysis test. Placental lymphocytes from Balb/c mice expressed a weak to moderate proliferative response to concanavalin A (Con A), phytohemagglutinin (PHA), and pokeweed mitogen when compared to the mitogenic responses of adult spleen cells. The stimulatory effects of Con A and PHA were abrogated after depleting the T cells from the placental lymphocyte preparation. Lipopolysaccharide-induced reactivity was similar for both cell populations. In MLR Balb/c placental lymphocytes did not undergo significant DNA synthesis after in-vitro exposure to irradiated C75BL/6 spleen cells, nor were cytotoxic lymphocytes generated against H-2b alloantigens. Lack of an MLR response was not associated with the presence of a suppressor lymphoid-cell population in the placenta. In marked contrast, placental trophoblastic cells were refractory in their responsiveness when cultured with either mitogens or allogeneic cells. These data show that although there is a selective deficiency in the placenta of cells reactive toward alloantigens, limited differentiation of lymphoid cells does occur which may offer immunologic protection to the fetal allograft during embryogenesis.

AUR Memorial Award 1991. Immunogenicity of gadolinium-based contrast agents for magnetic resonance imaging. Induction and characterization of antibodies in animals.

To evaluate the immunogenic potential of gadolinium-based magnetic resonance imaging (MRI) contrast agents, Sprague-Dawley rats were sensitized with gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA) dimeglumine and with Gd-DTPA covalently linked to either human serum albumin, dextran, or polylysine. IgG antibodies directed against Gd-DTPA were detected in immune sera by an enzyme-linked immunosorbent assay (ELISA), and were confirmed by competitive inhibition of antibody binding using free Gd-DTPA dimeglumine. Antiserum induced by immunization with human serum albumin-(Gd-DTPA) was characterized by a monophasic competition curve with 50% inhibition (IC50) = 5.5 x 10(-4) M when Gd-DTPA dimeglumine was used as both the well-coating and the displacing agent in a competition ELISA. Antiserum induced by Gd-DTPA dimeglumine alone was characterized by a biphasic competition curve with IC50 = 6.5 x 10(-7) M and 7.9 x 10(-4) M. Antisera obtained after exposure to either dextran-(Gd-DTPA) or polylysine-(Gd-DTPA) were of insufficient titer for characterization. The detection of antibodies specific for Gd-DTPA suggests in vivo protein binding with formation of hapten-carrier conjugates. This hypothesis is supported by increased relaxivity values observed when Gd-DTPA dimeglumine is incubated in serum rather than in water. Gd-DTPA dimeglumine and albumin-(Gd-DTPA) are immunogenic in rats under idealized experimental conditions. Additional studies will be necessary to determine the potential for immunologic response in humans to gadolinium chelates under conditions of exposure inherent in clinical use.

Gadolinium-ethoxybenzyl-DTPA, a new liver-directed magnetic resonance contrast agent. Absence of acute hepatotoxic, cardiovascular, or immunogenic effects.

RATIONALE AND OBJECTIVES: Gadolinium-ethoxybenzyl-DTPA (Gd-EOB-DTPA) is a recently introduced experimental magnetic resonance (MR) contrast agent for hepatic imaging. Although liver enhancement has been investigated in a number of animal models, tolerance evaluations of Gd-EOB-DTPA injection have been limited. METHODS: The authors investigated acute hepatotoxicity in an isolated perfused rat liver model, cardiovascular effects in the anesthetized rat, and potential immunogenicity of Gd-EOB-DTPA using detection of specific antibodies. RESULTS: Using perfused rat liver model, no significant deviation could be observed for functional parameters, liver enzymes, or potassium release, comparing Gd-EOB-DTPA to a control, but there was a significant choleresis (+250% bile flow). Hemodynamic effects of Gd-EOB-DTPA were observed after femoral bolus injection, but only with relatively high dosages (0.3-0.5 mmol/kg, 10-fold the likely clinical dose in humans). Experimental conditions, idealized for antibody induction, failed to cause an IgG immune response to Gd-EOB-DTPA in the intact rat. CONCLUSIONS: The results further support preliminary conclusions that Gd-EOB-DTPA is a well-tolerated MR contrast agent.

Differential actions of progesterone and cortisol on lymphocyte and monocyte interaction during lymphocyte activation--relevance to immunosuppression in pregnancy.

Progesterone and glucocorticoids share important anti-inflammatory and immunosuppressive properties. Both hormones have potent anti-proliferative effects in MLR, mitogen activation and cytotoxic T-cell generation. We investigated the cellular target of this in vitro anti-proliferative activity by comparing the effects of progesterone and cortisol on lymphocyte-monocyte interaction in concanavalin (Con A) induced human T-cell activation. Three different in vitro systems for assessing monocyte dependent T-cell activation by Con A were used: (1) limiting concentration of monocyte, (2) preincubation of isolated populations of monocytes and T cells with steroids and (3) role of steroid on action of Interleukin-1 (IL-1) activity. Monocytes separated from human peripheral blood leukocytes by flotation gradients and adherence to plastic were cultured at concentrations of 0.5-10% with constant numbers of isolated autologous T cells. Inhibition of Con A activation in cortisol (0.1-10 micrograms/ml) treated cultures was inversely proportional to percent monocytes, whereas in progesterone (2.0-20 micrograms/ml) treated cultures, inhibition was independent of monocyte concentration. Separated monocytes preincubated with progesterone and cultured with fresh T cells supported normal (108 +/- 7% control) levels of activation, but progesterone treated T cells and fresh monocytes responded at about 60% control levels. Similar experiments with cortisol (1 or 10 micrograms/ml) revealed significantly reduced responses when either cell population was preincubated with steroid. IL-1 induced by LPS stimulation of monocytes was blocked in its ability to stimulate Con A induced T cell proliferation with either steroid present during the assay of IL-1. These data provide additional support for local immunosuppression by steroids in the placenta during pregnancy. They suggest that progesterone selectively blocks T cell activation by a direct effect on T cells, whereas cortisol interferes with both monocytes and T cells.

Suppression of murine allogeneic cell interactions by sex hormones.

Investigations have been carried out on the action of several steroid hormones on lymphocyte functions in inbred strains of mice. The recognitive, proliferative and effector phases of allogeneic cell interactions in vitro were assessed using a mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML). In MLR containing Balb/c (responder) and C57bl/6 (stimulator) splenocytes DNA synthesis was markedly reduced in the presence of progesterone, cortisol or estradiol. In CML, progesterone and estradiol (1-5 microgram/ml) blocked in vitro generation of cytotoxic lymphocytes, while cultures with cortisol were partially inhibited. None of these hormones suppressed the cytotoxic activity of previously sensitized effector cells generated in vitro. Cultures containing testosterone expressed both normal DNA synthesis in MLR and cytotoxic activity in the CML test. These findings suggest a selective pattern of immunosuppression by sex hormones which may be important in preventing graft rejection or graft-versus-host interactions which may arise as a consequence of fetal engraftment during pregnancy.

Effects of psychosocial treatment in prolonging cancer survival may be mediated by neuroimmune pathways.

Research has provided growing evidence of links between the social environment and cancer progression. Indeed, social support in the form of marriage, frequent daily contact with others, and the presence of a confidant may all have protective value against cancer progression. Furthermore, retrospective data suggest that major stressful life events are more prevalent in patients with relapse or malignancy, and thus may contribute to cancer morbidity. Initial studies of the effects of psychosocial intervention with cancer patients have provided some promising results. In three randomized prospective trials, protective effects of psychosocial interventions on cancer progression have been confirmed, while one matching and one randomized study showed no survival effect after psychosocial treatment. Though more research is clearly needed in this area, this body of evidence suggests that psychosocial factors have potentially powerful modulating effects on the course of disease. Here we review evidence of one possible mechanism whereby psychosocial factors may influence disease-resistance capabilities: the neuroimmune connection. Suppressive effects of stress on immune function are well documented, and these effects have been shown to be modulated by social support. Thus, it is reasonable to hypothesize that supportive social relationships may buffer the effects of cancer-related stress on immunity, and thereby facilitate the recovery of immune mechanisms that may be important for cancer resistance. Data addressing this hypothesis are reviewed.