Serological detection of mixed lymphocyte culture identity between cells that differ by one HL-A haplotype.

Antibody mediated cell dependent immune lympholysis (ABCIL), an extremely sensitive serological technique for detecting tissue sensitization, was used in a family segregation study. Two serums capable of demonstrating ABCIL were used to identify members of the family who differed by one HL-A haplotype but whose cells did not stimulate in mixed lymphocyte culture. Absorption study of one serum indicated that the ABCIL reaction of that serum was directed against a factor, independent of HL-A, that might be responsible for lymphocyte stimulation in mixed lymphocyte culture. Thus the ABCIL technique may be used to detect lymphocyte-defined gene products.

Thyrotropin receptor epitopes recognized by graves' autoantibodies developing under immunosuppressive therapy.

Abnormal modulation of the immune system is a prerequisite for the expression of Graves' disease. Thus, when hyperthyroidism developed in a renal transplant recipient under long term immunosuppression with cyclosporine A and prednisone, we carefully evaluated the basis for her hyperthyroidism and her state of immunosuppression. Immunosuppression was confirmed by finding markedly deficient lymphocyte responses to common mitogens. Lymphocyte phenotype frequencies were those previously found in Graves', i.e. elevated frequencies of CD3/DR, CD5/26, and CD3/25 lymphocytes. There was also reversal of the CD4/CD8 ratio due to increased CD8 frequency; this is not a typical finding in autoimmune hyperthyroidism, but has been seen in the intrathyroidal lymphocyte populations of some Graves' patients and is associated with other forms of autoimmunity. The patient's serum contained a broad spectrum of TSH receptor autoantibodies (TSHRAbs) characteristic of Graves' disease. To determine whether these were an unusual population of autoantibodies, we determined their functional epitopes before and for nearly 1 yr after radioiodine therapy. Stimulating TSHRAbs that increase cAMP levels were human receptor (TSHR) specific and consistently recognized functional epitopes located on TSHR residues 90-165. Stimulating TSHRAbs that increased arachidonate release and inositol phosphate levels recognized residues 25-90, as did TSH binding inhibitory Igs present in the patient. These data demonstrate that Graves' disease with a wide array of TSHRAbs can develop in a patient despite adequate immunosuppression. More importantly, they show that the cAMP-stimulating TSHRAb associated with disease expression in this patient had a homogeneous subtype dependent on TSHR residues 90-165. As persistence of this type of TSHRAb over time has been associated with resistance to methimazole therapy in Graves' patients, we speculate that the development and persistence of TSHRAb with this homogeneous epitope may be linked to resistance to immunosuppressive therapy.

False positive 51Cr release tests during post-transplant immunological monitoring due to recipient serum radioactive contamination.

The occurrence of false positive 51Cr release test results in post-transplant immunological monitoring of human kidney graft recipients due to 131I carryover from renograms is described. False positivity was detected in 7 instances in 4 recipients, and suspected in 12 instances in 7 recipients, in a series of 46 consecutive transplant recipients. Technical methods and controls to detect and prevent such false positivity are described.

Renal and testicular agenesis in a patient with Darier's disease.

Darier's disease is a familial disorder of the skin that has been associated with corneal, bone, pulmonary, and urogenital abnormalities. This report describes a novel urogenital anomaly, namely renal and testicular agenesis, in a patient with Darier's disease. Detailed study of the kindred demonstrated an autosomal dominant pattern of inheritance for Darier's disease and also revealed the presence of autoimmune thyroiditis in several family members. Thyroid involvement ranged from isolated goiter to hypothyroidism. Tissue typing for HLA-A, B, C, and DR antigens did not reveal a specific haplotype common to all the carriers of the cutaneous or thyroid disorder. It is concluded that patients with Darier's disease should be carefully evaluated for the occurrence of systemic diseases, especially urogenital abnormalities and thyroid disorders.

T-lymphocyte activation in adult-onset idiopathic hypoparathyroidism.

PURPOSE: Patients with adult-onset idiopathic hypoparathyroidism (AOIH) often have antibodies against the parathyroid glands and other tissues, suggestive of immune activation. The purpose of this study was to determine whether T-cell activation is also a component of the endocrine disease. PATIENTS AND METHODS: We identified eight patients with idiopathic hypoparathyroidism diagnosed after the age of 30 years at two tertiary care centers and evaluated peripheral blood lymphocyte subset phenotype frequencies using monoclonal antibodies and flow cytometry. Control subjects were 13 patients with Graves' disease (five thyrotoxic and eight euthyroid) and 110 healthy volunteers. In two of the patients with AOIH, we also determined the mitogenic response to parathyroid cell membranes in peripheral lymphocytes. RESULTS: Patients with AOIH had higher than normal frequencies of the following phenotypes (p less than 0.05 versus controls, one-way analysis of variance): CD4, helper T cells; CD29/CD4, inducer of helper T cells; CD16 and CD56, natural killer cells; and CD3/DR, activated T cells coexpressing DR. Patients with Graves' disease had significantly higher than control frequencies of CD25 (T cells bearing the interleukin-2 receptor), CD3/DR, and CD26 (also a marker of T-cell activation); whereas the frequency of CD29/CD4 was significantly less than the control frequency. Neither of the two AOIH patients tested showed lymphocyte proliferation in response to parathyroid or thyroid cell membrane fractions. CONCLUSIONS: Generalized T-cell activation represents a novel feature associated with AOIH. Although we could not demonstrate parathyroid-specific lymphocyte clonal expansion, these data are suggestive of a generalized immune disturbance possibly related to autoimmunity, in which one of the manifestations is hypoparathyroidism.

Lymphocytic interstitial pneumonitis in autoimmune thyroid disease.

Four patients are described who were found to have autoimmune thyroid disease associated with lymphocytic interstitial pneumonitis. The patients were not receiving any medications known to cause lymphocytic interstitial pneumonitis. Their response to steroid therapy and the relapse of their clinical symptoms after steroid withdrawal support an underlying immunologic dysfunction. It is proposed that lymphocytic interstitial pneumonitis may be yet another manifestation of immune dysfunction in autoimmune thyroid disease.

Differences in immunoglobulin subclasses between dye exclusion and 51Cr release complement-dependent lymphocytotoxicity assays.

Paradoxical differences previously noted between lymphocytotoxicity detected by dye exclusion at room temperature (CDCE) or by 51Cr release (CDC51Cr) at 37 degrees C in maternal antipaternal complement-dependent lymphocytotoxicity have suggested that CDCE and CDC51Cr at 37 degrees C, but not at 20 degrees C, may detect different immunological antibody-antigen interactions. Reactions in the two test systems against the same target cells were compared in sera from known immune dialysis patients, secondary aborting women, and refractory platelet recipients before and after heat treatment of sera, absorption with solid-phase heparin, anti-light-chain augmentation, and the addition of murine monoclonal anti-IgG subclass antibodies. The results demonstrate significant differences between the two tests using the same target and sera. Further, the results imply the presence of an inhibitor and an inhibitor of inhibitor in sera. The involvement of different immunoglobulin subclasses was shown in the two tests. These data demonstrate the necessity for further study of the nature of the differences in the mechanisms of these clinically important antibody-detecting systems.

Autosomal recessive type of whistling face syndrome in twins.

A report of concordant monochorionic, diamnionic like-sex twins with whistling face syndrome is presented. The diagnosis was based on the characteristic facies with prominent supraorbital ridge, sunken eyes, telacanthus, short nose and colobomata of the nostrils, long philtrum, high narrow palate, and marked microstomia with puckered lips and an "H"-shaped cutaneous dimpling on the chin. The hands showed symmetrically clenched fingers with camptodactyly and ulnar deviation. The feet demonstrated mild bilateral talipes equinovarus. HLA studies to determine the zygosity of the twins showed discordance in HLA haplotypes, which indicated dizygosity. The pedigree analysis showed normal nonconsanguineos parents, and no other family members had the syndrome. The findings support the existence of an autosomal recessive type of whistling face syndrome.

Characterization of immunoglobulin class and subclass responses in secondary aborter sera.

Sera from secondary (2 degrees) aborters exhibit persistent, high-titred cytotoxicity against paternal as well as HLA dissimilar non-paternal lymphocytes. The majority of antipaternal complement-dependent cytotoxicity (CDC) and complement-independent antibody dependent cellular cytotoxicity (ADCC) was recovered in IgG enriched fractions following ion-exchange chromatography of 2 degrees aborter sera. The IgG subclasses mediating antipaternal reactivity were determined using murine anti-human IgG subclass specific monoclonal antibodies and Protein A affinity chromatography (SPA). Inhibition of paternal CDC by the anti-subclass reagents showed 75-90% of the reactivity mediated by maternal IgG1 antibodies. Anti-IgG3 inhibited 15-30% whereas anti-IgG2 produced little inhibition. SPA chromatography of 2 degrees aborter IgG supported the monoclonal antibody results in that greater than 80% of the CDC activity was recovered in the IgG1, 2, and 4 containing eluate and 20% was found in the IgG3 enriched effluent. Although the anti-IgG subclass specific monoclonals did not inhibit antipaternal ADCC, IgG3 did not appear to mediate this cytotoxicity as the ADCC was recovered in the eluate and not the effluent following SPA chromatography of 2 degrees aborter IgG enriched serum fractions. These data indicate that the humoral antipaternal and polyspecific CDC immune reactivities of 2 degrees aborters are due to the production of IgG1 and IgG3 antibodies.

Inhibition of immunoglobulin (Ig)G-Fc-mediated cytotoxicity by seminal plasma IgG-Fc receptor III antigens.

OBJECTIVE: To determine if the presence of soluble immunoglobulin (Ig)G-Fc receptor III (Fc gamma RIII) antigens in human seminal plasma interfere with IgG-Fc-mediated effector functions. DESIGN: An antibody-dependent cellular cytotoxicity (ADCC) assay was used as a model for IgG-Fc-mediated effector functions. Human red blood cells (RBC), labeled with 51Cr were sensitized with rabbit anti-RBC and used as targets for peripheral blood leukocyte (PBL) effector cells. Cytotoxicity was measured by assessing the release of 51Cr from RBC. INTERVENTIONS: (1) Seminal plasma was added at different concentrations to the ADCC, and inhibitory effects were measured. (2) The level of seminal plasma interaction in ADCC was studied by comparing ADCC results in the presence and absence of seminal plasma with findings of target and effector cells that had been preincubated with seminal plasma. (3) The role of seminal plasma Fc gamma RIII in inhibiting ADCC was studied by coincubating seminal plasma with monoclonal antibodies (MAs) Leu 11b that block Fc gamma RIII binding sites. Two isotype-matched MA controls were used at identical concentrations. RESULTS: (1) Seminal plasma dose dependently inhibits ADCC. (2) Seminal plasma inhibition of ADCC occurs at the level of IgG-Fc interaction with effector cell Fc gamma receptors. (3) Inhibitory effects of seminal plasma on ADCC can be specifically blocked by coincubating seminal plasma with MAs Leu 11b that block Fc gamma RIII binding sites. CONCLUSIONS: Seminal plasma Fc gamma RIII antigens interfere with IgG-Fc-mediated effector functions. This mechanism could play a beneficial role in controlling potentially harmful antipaternal immune responses.

Clinical, immunologic, and genetic definitions of primary and secondary recurrent spontaneous abortions.

Fifty couples with repeated pregnancy failures were grouped by clinical history as being primary (no children) or secondary (abortions subsequent to having children or stillbirths) aborters. Human leukocyte antigen (HLA) sharing between husband and wife and maternal antipaternal immunity were studied in both groups. Analyses showed significantly more HLA-A, B, and DR locus antigens shared in the primary abortion group than in the secondary abortion group (P = 0.01). The number of couples sharing two to five HLA antigens was higher for those couples with primary abortions than for secondary abortions (P = 0.009). Antipaternal immunity as measured by visual complement-dependent lymphocytotoxicity (CDCE), by 51Cr release (CDC51Cr), and by complement-independent, antibody-dependent cell-mediated cytotoxic (ADCC) assays was different between the two groups. CDCE was not present in the primary aborters' sera but was found in 13 of the 15 secondary aborters' sera (P = 0.0001). Similarly, ADCC was not apparent in primary aborters but was present in 14 of the 15 secondary aborters (P = 0.0001). CDC51Cr was detected in 3 of the 11 primary aborters and in 13 of the 15 secondary aborters (P = 0.008). Such differences in HLA sharing and immune reactivities between these two groups strengthen the idea that either inadequate or inappropriately vigorous maternal antipaternal immunity can be related to spontaneous abortions. These results uphold a central role for immunology in human reproduction and provide further support for the use of immunotherapy to prevent pregnancy losses in certain abortion-prone women.

Inhibitors of complement-mediated cytotoxicity in normal and secondary aborter sera.

Sera from patients with secondary (2 degrees) spontaneous abortions contain complement-dependent cytotoxic (CDC) antibodies with specificity for paternal lymphocytes. These lymphocytotoxins are not anti-HLA (human lymphocyte antigen) as shown by their polyspecificity on HLA select cell panels and by their removal following absorption with HLA-negative trophoblast membranes. They are predominantly IgG and have been designated as trophoblast-lymphocyte cross-reactive (TLX) antibodies. Normal and homologous 2 degrees aborter sera contain a CDC inhibitor that does not bind to paternal cells and must be present when complement is added to antibody. The inhibitor does not manifest anticomplement effects and appears to be species specific. Inhibitory capacity is increased by heating (56 degrees C for 30 min) and by absorption with heparin. When chromatographed on G-200 Sephadex, inhibitor appears in the void volume, suggesting a molecular weight of more than 250,000. It can be isolated from diethylaminoethyl cellulose into an euglobulin fraction that does not contain IgG, but does contain IgM, though no studies indicate the inhibitor to be IgM. We suggest that the inhibitor is under the control of a regulator molecule, probably an inhibitor-of-inhibitor, and that in 2 degrees aborter sera the equilibrium is unbalanced between antibody, inhibitor, and regulator.

Immunotherapy of primary immunological aborters: rationale for the use of pooled cryopreserved purified normal peripheral blood mononuclear cells.

Patients with recurrent spontaneous abortions have been successfully treated in many centers with third-party immunization directed to a putative TLX antigen system. This immunotherapy requires the screening of a large number of donors to match the patients' red blood cell (RBC) phenotype and has the potential risks associated with transfusions from 30 to 50 donors. Our modified approach to third-party immunization is to use irradiated frozen-stored purified lymphocytes pooled from five normal donors. Mononuclear cells from normal donors are obtained in a cell separator. After sedimentation and Ficoll-Hypaque separation, the cells are stored in liquid N2. The RBC depletion of the final preparation is of the order of 5 to 6 logs, theoretically decreasing the need for RBC phenotyping except for the Rh system. Using a highly sensitive fluorescence-activated cell sorter technique and an ADCC assay, we found that ABH, Rh, Fya Fyb, Jka Jkb, MNS, and Kell antigens are either not expressed by cryopreserved human mononuclear cells, or, if so, they are below the level of detection of these highly sensitive assays. We conclude that the use of pooled frozen mononuclear cells is an adequate alternative for immunotherapy. It decreases the transfusion risks associated with exposure to a large number of donors and the need for RBC phenotyping, making this modality of treatment more accessible.

Immunological responses to ultraviolet light B radiation in Black individuals.

Immunological factors are important participants in the pathogenesis of experimental skin tumors. We therefore studied cutaneous immune responses in subjects with either low natural incidence (Black individuals), or a high frequency rate (White individuals) of skin cancer. We performed whole body irradiation with a low dose of ultraviolet light B (UV-B) and evaluated peripheral lymphocytes. UV-B irradiation was associated with small but significant changes in lymphocyte phenotype frequency. In White subjects this consisted of an increased number of CD19 (B cells) and CD 4/29 (inducer of helper T cells); Black subjects had a slight decrease in CD3 (T cells). Natural killer activity, not affected by UV-B in White subjects, increased significantly in Black subjects. UV-B was devoid of immunological effects in vitro for any of the parameters tested. As expected, the low UV-B dose used in this study induced increases of serum vitamin D3 concentrations in White subjects, with lack of response in the Black subjects. We conclude that Black individuals selectively exhibit an increase in Natural Killer activity in response to irradiation with low dose UV-B. This race group-specific immune response to ultraviolet radiation appears to require mediation by the skin. Enhanced Natural Killer activity could underlie at least partly the resistance in Black individuals to the development of photodependent skin cancer.

Immunological consequences of exposure to pentachlorophenol.

Evaluation of lymphocyte phenotype frequencies, functional responses, serum immunoglobulin levels, and autoantibodies was completed for 38 individuals (i.e., 10 families) who were exposed to pentachlorophenol (PCP) in manufacturer-treated log houses. Comparison of subjects with controls revealed that the exposed individuals had activated T-cells, autoimmunity, functional immunosuppression, and B-cell dysregulation. Autoimmunity was evidenced by elevation of TA1 phenotype frequencies and a 21% incidence of anti-smooth muscle antibody. Functional immunosuppression was evidenced by the significantly reduced responses to all mitogens tested and to allogeneic lymphocytes in the mixed lymphocyte culture test. There was a significant elevation of CD10, and an 18% increase or decrease in serum immunoglobulins was noted. A striking anomaly was the enhanced natural killer activity found in exposed females but not in males.

Immune alterations in humans exposed to the termiticide technical chlordane.

Lymphocyte phenotype frequencies and in vitro functional assays were studied in 27 individuals who had been exposed to technical chlordane in their homes or at their places of work. A control group consisted of 118 individuals who were similar to the exposed group with respect to age and sex distribution, and who had not knowingly experienced exposure to technical chlordane, was chosen for study. A significantly increased frequency of cortical thymocytes in the circulation (CD1) (p less than .001) and a decreased frequency of the suppressor-inducer phenotype CD45RA/T4 (p less than .01) were noted in the exposed group. Both kappa and lambda light-chain frequencies were elevated (p less than .01). Proliferative responses to the three mitogens tested, PHA, CONA, PWM, and to allogeneic lymphocytes in the mixed-lymphocyte culture assay were significantly lower than in controls (p less than .01). Responses in assays of the natural killer function were not significantly different from those of controls, but Fc receptor-associated K cell function was significantly greater than responses in controls. Of 12 individuals tested for evidence of autoimmunity, 11 demonstrated some increased titer of a form of autoantibody. This cluster of significant findings demonstrates the emergence of aberrant peripheral T and B cell regulation and a potential for autoimmune activation, detectable up to 10 y after exposure to technical chlordane.