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Flow cytometry (FC) provides a reproducible investigation of cell surface antigens on platelets. The aim of this study was to elaborate appropriate protocols and to compare them with other techniques that have already been published. (1) Venipuncture with tubes containing citrate was better for the preservation of the antigenicity than using ACD tubes. The isolated platelets could not be completely distinguished from detritus and protein aggregates. Therefore a platelet concentration between 10(7) and 10(8)/ml measurement buffer was necessary to obtain a sufficient resolution by FC. (2) Isolation methods using either differential centrifugation or diluted Ficoll-Hypaque as a flotation medium provided platelets of equal purity. The method with Ficoll-Hypaque resulted in a higher number of isolated platelets than differential centrifugation. The demonstration of platelets and their antigens in whole blood without isolation gave good results provided the platelets were not activated. Activation of platelets with 1 NIH-U thrombin/l resulted in the loss of a part of the highly activated platelets because of their aggregation. (3) Comparing different concentrations of paraformaldehyde in PBS, fixation with 1% for 15 min provided the best antigen preservation for most of the antigens investigated. Isolation induced platelet activation. In order to avoid this effect, the whole anticoagulated blood was fixed with 1% paraformaldehyde for 15 min immediately after venipuncture. Then the platelets were isolated using diluted Ficoll-Hypaque. In this way, systemic activation of platelets can be detected with antibodies against glycoproteins which are translocated from the alpha-granules or lysosomes to the cell membrane. These activation markers can be determined on immediately fixed platelets (already in the whole blood) without any interference due to unspecific activation caused by the isolation procedure. (4) Platelet treatment with citric acid at pH 3, in order to remove the antigenicity of HLA-class I molecules, was sensitive to immediate fixation with paraformaldehyde in the whole blood. Fixation after isolating the platelets made it possible to demonstrate antigen stripping, and the free heavy chain, devoid of the beta 2-microglobulin, could be clearly demonstrated. (5) Using standardization beads, the average number of antigenic sites per platelet could be determined for the investigated specificities. It was shown that antibodies which have been directly conjugated or biotinylated and combined with streptavidin-phycoerythrin yielded similar results in terms of the number of antigenic binding sites while unconjugated antibodies in combination with FITC-conjugated anti-mouse-IgG led to overestimation of antigenic binding sites.

The influence of treatment of platelets with citrate buffer (pH 7.2), chloroquine, or citric acid at pH 3 on the expression of HLA class I antigens and 'thrombocyte-specific' glycoproteins was investigated by means of flow cytometry. After treatment with citric acid at pH 3 and chloroquine, the expression of HLA class I was significantly reduced, while the density of the molecules GPIa/IIa, GPIIb, and GPIIb/IIIa (GP = glycoprotein) carrying 'thrombocyte-specific' antigens was not or only weakly decreased on the surface of the platelets. The use of two monoclonal antibodies (HC-10 and HC-A2) against the native heavy chain of the HLA class I molecule revealed that 'antigen stripping' with chloroquine or citric acid does not affect the entire molecule: only the beta 2-microglobulin is cleaved, or only some epitopes on the heavy chain are altered by this procedure. The treatment with citric acid yielded better results with respect to the removal of HLA class I activity and the preservation of 'thrombocyte-specific' glycoproteins. The presence of the heavy chain of HLA class I molecules on the surface of platelets after treatment with citric acid and chloroquine confirms the hypothesis that platelets--like nucleated cells--bear HLA class I antigens inserted in the cell by a cytoplasmic and a transmembrane domain.

Forty-five patients with myeloproliferative or myelodysplastic syndromes, treated with recombinant interferon-alpha (rIFN-alpha) for a minimum of 1 up to 4 years, were examined for the occurrence of thyroid autoimmunity. During treatment, the rate of thyroid autoimmunity rose to more than 20%. The decrease in severity and frequency of thyroid autoimmunity after withdrawal of IFN shows that this is a potentially reversible side effect. The key determinant for the manifestation of this IFN-related autoimmune phenomenon seems to be a predisposition for autoimmunity, since patients with initially detectable thyroid antibodies are prone to exacerbations of thyroid autoimmunity. Concurrent with thyroid autoimmunity, hypothyroidism occurred but did not correlate with the levels of thyroid antibodies, although severe hypothyroidism in two patients was accompanied by increased levels of thyroid antibodies. This investigation shows that thyroid autoimmunity and consecutively hypothyroidism must be expected in certain patients treated with rIFN-alpha during long periods. Furthermore, it may be assumed that IFN-alpha does not induce the development of autoimmunity, but rather enhances the levels of pre-existent thyroid antibodies.

Various studies in the United States and Europe have established human leucocyte antigens (HLA) Cw3, DR2 and DR4 as risk factors for manifest Borrelia burgdorferi infection or the development of chronic courses of Lyme disease. Other studies failed to confirm these findings. In the present study the frequencies of HLA A, B, Cw and DR were analysed in 283 persons from Austria and Germany with manifest B. burgdorferi infection. No statistically significant differences were found between patients and the control groups with regard to the frequencies of particular HLA antigens, nor were differences in antigen frequencies in the patients with manifestations of different stages of disease significant. Furthermore, a statistical re-evaluation of all the European studies failed to confirm particular HLA antigens as risk factors for B. burgdorferi infections to become manifest or chronic.

Corneas used for transplantation are typically obtained from donors up to 48 hr post mortem. By this time, standard HLA typing usually is impossible because of the lack of viable lymphocytes in spleen and peripheral blood. To increase the number of HLA-typed corneas, we developed a method in which the retinal pigment epithelial (RPE) cells of the donor eye are isolated, cultured in the presence of 1000 IU/ml interferon-gamma (IFN-gamma), and, after 4 d, are typed for HLA Class I and Class II antigens in the standard NIH cytotoxicity assay. Sixty five donors were typed simultaneously using peripheral blood lymphocytes and RPE. One hundred and sixteen out of 120 HLA-A antigens, 125/127 HLA-B, 106/108 HLA-C, and 92/100 HLA-DR antigens were identical using the same technique. Donor age, sex, cause of death, time of enucleation post mortem, and time of RPE preparation post mortem, as well as duration of culture period prior to stimulation with IFN-gamma did not correlate with the results of HLA typing. These data show that RPE cells can substitute for lymphocytes in post mortem HLA typing. Consequently, every donor with corneas suitable for transplantation can be prepared for matched transplantation.

A total of 52 nonrelated Caucasian patients (21 male, 31 female, age at onset of the disease: 7-74 years, average: 31.6 years) suffering from intermediate uveitis were tissue typed for 56 different HLA-A, -B, -C and -DR-antigens. No significant association between any specific HLA-antigen and the disease, both when comparing the total patient group with a large, healthy control population, or any of the different subgroups (male vs. female, early vs. late onset, mild vs. severe course) could be demonstrated.

The etiology of autoimmune diseases is multifactorial with genetic factors being an important prerequisite. There are two clinical manifestations of autoimmune thyroiditis: the goitrous form (Hashimoto's thyroiditis) and the atrophic variant, which is characterized by hypothyroidism (primary myxoedema). Different genetic markers were assumed to be predisposing factors for the distinct clinical presentation. In the present study, we determined HLA A,B,C,DR,DQ alloantigens serologically and HLA-DQ by gene analysis in patients with nongoitrous autoimmune thyroiditis and randomly chosen controls. To verify the exact classifications, thyroid volume (median 5.85 ml) was measured by ultrasonography. HLA-DR5 was found in 16 of 36 (44%) patients with nongoitrous autoimmune thyroiditis and in only 26 of 175 controls (15%) (Pc = 0.0018). There was a tendency towards a lower frequency of HLA-DR7 with 6% positivity in patients vs. 29% in controls (Pc = 0.052). Regarding HLA-DQ, DQ7 was found in 17 of 35 patients (48%) vs. 21 of 98 controls (21%) (Pc = 0.028) (relative risk 3.5). No other association was found with HLA-A,B,C and HLA-DR and -DQ. Our data indicate that the genetic susceptibility to autoimmune nongoitrous thyroiditis is closely associated to HLA-DR5 and DQ7 and not distinct from goitrous disease. We conclude that factors other than genetic ones explain the different immunological and clinical manifestation of chronic lymphocytic thyroiditis.

It has been known for a long time that there is an increased incidence of Graves' disease and immune thyroiditis in certain families. Genetic research of this disease has shown that it is most probably transmitted in a multifactorial way, i.e. that environmental as well as genetic factors play a role in the genesis of the diseases. This hypothesis is supported by the fact that the rate of concordance is maximally 50% in identical twins. The following model of threshold values is conceivable for the formal genetics of Graves' disease and immune thyroiditis: the diseases break out whenever the sum of environmental (viruses, bacteria, iodine excess, hormones, stress) and genetic (MHC and non MHC-restricted) factors is higher than a given threshold. One of the major genes influencing the genesis of the diseases seems to be HLA-DR3 (or a closely linked gene in strong linkage disequilibrium). If this gene is present, fewer environmental factors are possibly needed.

Clinical, electrophysiologic and biopsy findings as well as studies of blood group markers in a family with hereditary neuropathy with liability to pressure palsies (HNPP) are reported. There was an autosomal dominant trait without genetic linkage between the HNPP gene and blood group markers controlled by chromosome 1. Reduced motor and sensory nerve conduction velocity was found in clinically affected and unaffected nerves. Characteristic morphological changes in sural nerve biopsy including tomaculous swelling were present.

The basis and efficiency of paternity testing using conventional markers (alloantigens or electrophoretically defined polymorphisms in blood) for individuals involved in cases of affiliation are shown. A possible use of DNA polymorphisms in paternity cases is discussed. Due to the fact that the formal genetics of DNA polymorphisms have not been fully validated by the analysis of large numbers of informative meioses, it is not yet possible to include these systems in routine paternity testing. As the vast majority of cases can be resolved by conventional markers, the inclusion of DNA polymorphisms is not usually necessary; in some cases, however, they can provide additional information.

The results of the serological typing and the patterns of restriction fragment length polymorphisms for the detection of HLA-DR gene products are compared. The data shown demonstrate that the use of DNA typing gives a clearer definition of the HLA-DR antigens and that the HLA-DR polymorphism is greater than detected by serology.

The HLA system encompasses approximately one thousandth of the human genome and contains a series of closely linked loci coding for molecules which provide the context for the recognition of antigens by T lymphocytes. Within the HLA system, several phenotypically expressed and genomic polymorphisms can be distinguished. These polymorphisms are described and the main objectives for the future research are discussed.

Peripheral blood lymphocytes transformed significantly to denatured type I collagen in 22 of 38 patients with rheumatoid arthritis (58%), 1 of 9 patients with Reiter's disease (P less than 0.05), and 9 of 32 controls (28%) (P less than 0.01). Determination of HLA antigens revealed significant differences between 24 rheumatoid arthritis (RA) patients and a control population for HLA-DRw4 (75% versus 23%). No correlation was observed between lymphocyte transformation to collagen and antibodies to collagen, rheumatoid factor, stage or duration of RA, or HLA antigens.

One hundred sixty-eight patients with rheumatoid arthritis treated with chloroquine (n = 87), gold salts (n = 133) and/or penicillamine (n = 77) were investigated for possible associations between HLA antigens and toxic reactions. Patients with 2 or more side effects to gold and/or penicillamine had a significantly increased frequency of antigens HLA-B8 and DR3 compared to patients with one or without adverse reactions. Proteinuria to gold or penicillamine was significantly associated with HLA-B8 (relative risk [RR] 4.2) and DR3 (RR 14.0) whereas nonnephrologic side effects to gold or penicillamine were associated with B7 and DR2 (RR 3.5 and 2.8). Patients with skin reactions to gold had a significantly greater frequency of HLA-B7. We found no correlation between chloroquine side effects and any HLA antigen. The results suggest a genetic predisposition to toxic reactions to gold or penicillamine based on an immunologic dysregulation.

Due to the absence of vessels and lymph drainage, human cornea is the tissue grafted with the greatest success. From an immunological point of view, this advantage is lost when the recipient cornea has pronounced vascularization and is sensitized. In such cases, which have a poor prognosis, the postoperative success rate drops to below 50% due to graft rejection. The origin and point of attack of the immune reaction to the graft are the MHC antigens of the donor cornea, which occur on almost all nucleated cells. Since they are capable of triggering a T cell activation in the recipient they set immunological effector mechanisms in motion which lead to elimination of the cells carrying the alloantigen. Corresponding to the MHC antigen expression (both Class I as well as Class II) on all cells in all layers of the cornea, rejection reactions of all the various cell types (epithelial, stromal, endothelial) are also seen. By using histocompatible donor corneas it is possible to influence the afferent arm of the immune response preoperatively and thus to diminish its effect on the postoperative course.

HLA antigens and humoral antithyroid immunity have been studied in 97 thyrotoxic patients [48 without and 49 with endocrine ophthalmopathy (EOP)]. A statistical significant increase of HLA-B8 frequency could be observed only in the thyrotoxic patients with EOP, whereas in patients without EOP B8 frequency was similar as to the one in the controls. The C locus antigen Cw3 was significantly different between both groups of patients: increased in those with EOP and statistically significantly decreased in those without EOP. Thyroid microsomal antibodies were found to be associated with HLA-B8 positivity and were noted with highest incidence in B8 positive EOP patients. No such association was observed for thyroglobulin antibodies. These data show a genetic heterogeneity in thyrotoxicosis patients with and without EOP. HLA-Cw3 positivity represents and increased risk to develop EOP in thyrotoxicosis, as frequency of this antigen was significantly decreased in thyrotoxicosis patients without any signs of EOP. The increased frequency for microsomal antibodies in thyrotoxic patients with EOP, especially in B8 positives underlines the genetically influenced autoimmune reactivity in that disorder.

Twenty-one patients with dermatitis herpetiformis were typed for HLA-ABC and -DR determinants. The incidence of HLA-AI, -B8 and -DR3 antigens was found to be significantly higher (P: = 10(-3), less than 10(-6) and less than 10(-6), respectively) among patients with dermatitis herpetiformis than among the normal population. HLA-DR3 was found in 85.7% of patients, HLA-B8 in 66.7% and HLA-AI in 61.9% only. These results indicate that HLA-DR3 is the antigen primarily associated in dermatitis herpetiformis and the latter antigens (HLA-AI and -B8) are present in increased incidence, probably due to the known linkage disequilibrium of these antigens with HLA-DR3.

Histocompatibility antigens play a certain role in the development of testicular tumours. 151 patients with testicular cancer (86 non-seminomatous germ cell tumours--NSGCT--and 65 pure seminoma) were typed for the HLA-antigens of the A, B, C and DR locus. 24 patients of the pure seminoma group and 50 patients of the NSGCT group had an elevated serum HCG level preoperatively. The antigen DR-5 was elevated in the seminoma group whereas the incidence of B-13 was increased in the NSGCT group. In terms of antigen distribution HCG-positive seminoma resembles seminomatous tumours rather than NSGCT.

A further case of blood chimerism in male twins concerning red and white cells is described. The erythrocyte chimerism could be detected by 5 out of 16 genetic marker systems and the lymphocyte chimerism by the analysis of autosomal polymorphisms of peripheral blood lymphocytes.

Sixteen patients with meningopolyneuritis of Garin-Bujadoux-Bannwarth (MPN-GBB) were examined for HLA-DR antigens. In contrast to data in Lyme disease (LD), which is caused by an identical or closely related spirochete, no significant association was found between the neurological disease and HLA-DR. The reported association between neurological disease and HLA-DR2 in LD may be due to the inclusion of cases with neurological disease and arthritis, since chronic arthritis in LD seems to be well correlated with HLA-DR2.

A method for the calculation of the probability of paternity for the HLA system using only the typing results from the child and the putative father, without taking into account the data of the mother, is presented. Furthermore, the usability of the formulas by Mayr and Pausch (Z. Immun.-Forsch. 150, 447 (1975)) for the computation of the chance of paternity exclusion in such cases is demonstrated.

In 19 patients with idiopathic adrenal insufficiency and in 19 controls, cellular (as measured by the migration inhibition test) and humoral (by means of immunoflourescence) immune reaction to adrenal antigens were investigated. Significant inhibition of migration was observed in 12 patients with adrenal microsomes; migration of mononuclear cells was however within normal range in 18 out of 19 controls. Adrenal antibodies could be detected in 11 (53%) of the patients. In a further series of investigations adrenal microsomes have been preincubated with specific antibody and used in migration inhibition tests. The migration indices observed with microsomes were compared to those obtained with microsomes preincubated with adrenal antibody. Migration inhibition with microsomes could be blocked by antibody in almost all antibody-positive patients. No uniform trend on the migration of mononuclear cells was found in the antibody-negative patients. A blocking of the cellular immune reaction by specific antibody seems likely. Possible mechanisms which could lead to such phenomena as well as the biologic significance of adrenal antibodies are discussed.

Using the data obtained by typing 450 unrelated people of the Viennese population, the phenotype and gene frequencies of 37 HL-A factors governed by the 3 SD loci were determined. The haplotype frequencies involving SD-1 und SD-2 genes were calculated and the associations between the 3 SD loci have been analysed.

In 17 cases positive cytotoxic anti-HL-A antibodies were gained from 26 immunizated pairs. On the whole this corresponds to the data to be found in literature [10, 17]. The antibodies against the antigens of the I. locus will appear more rapidly, younger persons will form antibodies more easily. The difference in one single antigen results in favourable findings when the pairs are composed; in spite of that, however, cross-reacting antibodies may frequently be obtained, particularly when boostered to produce sera with a high titre. No side effects were detected in the immunizated group; yet there is a certain reason to suppose that the production of anti-HL-A sera by artificial immunization cannot be compared with the usual production of AB0 sera. The immunopathological significance of the HL-A-system is still elucidated insufficiently; by no means it is confined to histocompatibility. Only those institutes should be concerned with artificial immunization which are able to perform the necessary immunological controls exactly in each case.

Forty Caucasian patients with classical or definite rheumatoid arthritis were tested for 7 HLA-DR alloantigens and 37 HLA-A, B, and C antigens and compared with 125 and 450 healthy controls, respectively. HLA-DRw4 was significantly (corrected p = 10(-3) increased in rheumatoid arthritis (55%) compared with controls (23%). There was no significant correlation between clinical, radiological or serological disease parameters and HLA-DRw4. The frequency of HLA-B8 was increased in the patient group; however, the corrected p value was not significant.