Biochemical definition of DLA-A and DLA-B gene products by one-dimensional isoelectric focusing and immunoblotting.

The serologically defined canine MHC gene products (DLA-A and DLA-B) were investigated by one-dimensional isoelectric focusing after detergent phase separation, and immunoblotting (1D-IEF). The animals studied (n = 36) represented three different breeds. Since DLA-A and DLA-B antigens are MHC class I and class II molecules, respectively, cross-reactive polyclonal antibodies specific for human class I or class II molecules were utilized to visualize the protein pattern. The relative positions of the bands were correlated with the DLA-A and DLA-B antigens. In most cases DLA antigens of identical serological type did not differ in their 1D-IEF pattern except for DLA-A9 and the serologically undefined DLA-B gene product DLA-B 'blank'. For DLA-A9, two subtypes (A9.1 and A9.2) were identified in Beagle and associations of A9.1 with the haplotype A9,B4 and of A9.2 with A9,B6 were seen. In contrast, the haplotype A9,B6 in Labradors was associated with A9.1. Whereas all six serologically defined DLA-A antigens possessed distinct isoelectric points (IEP), not all DLA-B antigens could be differentiated by 1D-IEF:B5 as well as B13 coband with one B 'blank' specificity (BE1). Furthermore, a second B 'blank' (BE2) could be identified by 1D-IEF, underlining the advantage of this technique in the immunogenetic analysis of DLA gene products in dogs.

Recognition of monocyte-associated antigens in the dog.

Canine pregnant sera were investigated for monocyte-specific cytotoxic antibodies against a cell panel from 50 unrelated dogs. Contaminating DLA and DLB reactivity was removed by absorption on a random pool of purified lymphocytes. Two monocyte-associated antigens were recognized by two independent clusters of antisera; the existence of further antigens is suggested. In preliminary segregation studies, the inheritance of these determinants in linkage with DLA could not be observed.

Systemic lupus erythematosus in dogs: association to the major histocompatibility complex class I antigen DLA-A7.

The DLA-A,B antigens and the allotypes of the fourth complement component have been determined in German shepherd dogs suffering from systemic lupus erythematosus. We have typed 26 unrelated affected dogs, 11 animals of a three generation family, and 16 dogs of a colony with a high frequency of the disease. The results obtained from the 26 unrelated diseased dogs were compared to those determined in the 23 unaffected German shepherds. The antigen DLA-A7 was found to be predominant in the diseased group with a c2 = 11.02, Pc = 0.02, and a relative risk for the carriers of 11.93. The antigens DLA-A1 and DLA-B5 were negatively associated to the disease (c2 = 14.95, Pc = 0.001, and c2 = 17.16, P = 0.0008 respectively) and thus may be of protective nature. These data were further substantiated by the typing of the three generation family and the colony.

Biochemical evidence that the DLA-B locus codes for a class II determinant expressed on all canine peripheral blood lymphocytes.

We have analyzed the products of the canine DLA-B gene locus by biochemical and serological methods. Due to its ubiquitous expression on lymphocytes this antigen series was thought to be of class I nature. However, by two-dimensional gel electrophoresis and lysostrip experiments we could unequivocally identify the DLA-B antigens as typical class II gene products. The serological polymorphism is caused by the beta-chain, whereas the alpha-chain appears invariant. The presence of these class II gene products on almost all lymphocytes in the dog is in sharp contrast to the numerous reports on their restricted expression in other mammalia such as man and mouse.

[Clinical, psychological and psychosocial factors in spontaneous and recurrent or habitual abortion. Results of a pilot study]. / Medizinische, psychische und psychosoziale Faktoren bei Spontanaborten (SA) und rezidivierenden Spontanaborten (RSA) bzw. habituellen Aborten (HA). Ergebnisse einer Pilotstudie.

In a pilotstudy about medical, psychological and psychosocial factors in spontaneous and recurrent spontaneous abortions 47 women were investigated. 26 of them had one, 14 tow and 7 three or more recurrent spontaneous abortions. Etiologic factors were unknown in about 90% of the cases. Illnesses observed were allergies, thyroid diseases and infections. An influence of social and psychosocial factors could not be proved in our sample. Psychological relevant factors were higher levels of fear and depressive and evasive coping strategies in pregnancy. Certain personality traits (vulnerability, nervousness and tendency to psychosomatic reactions) were found in women with three or more recurrent abortions. All patients showed depressive reactions to pregnancy loss. In a further investigation well defined subgroups of patients shall be compared. The results can serve as guidelines in pregnancy counselling.

Joint report of the Third International Workshop of Canine Immunogenetics. II. Analysis of the serological typing of cells.

Using a standardized microlymphocytotoxicity assay, seven international laboratories evaluated 144 anti-dog lymphocyte antigen (DLA) sera in 319 mixed breed and 152 Beagle dogs. The workshop confirmed the serological definitions for DLA-A2, A3, A9; DLA-B4, B5, B6, B13; DLA-C11(Cwl); and C12(Cw2). Two new specificities were assigned to the DLA-A locus (Aw14 and Aw15) in only the mixed breed dogs. A third specificity (Cw3), was assigned to the DLA-C locus. The antigen and gene frequencies of these alleles differed between the two groups of dogs, but the frequencies of the "blank" were similar in both groups. Future international collaborations will be necessary to definite more completely the polymorphisms of the major histocompatibility complex (MHC) of the dog. Those efforts will benefit from the standard serological test established in this workshop.

Immune transfer studies in canine allogeneic marrow graft donor-recipient pairs.

Transfer of immunity occurring with bone marrow grafting was studied using the dog as a preclinical model. Allogeneic bone marrow transplantation (BMT) was performed between DLA-identical beagle litter-mates. The donors were immunized with tetanus toxoid (TT) or sheep red blood cells (SRBC), and their humoral response was monitored by hemagglutination. The recipients of bone marrow from TT-immunized donors showed a marked increase of antibody titer one week posttransplantation, while in the recipients of marrow from SRBC immunized donors the antibody titers were considerably lower. Within the following 60 days the antibody titers in both groups diminished gradually to pregrafting levels. Control experiments in which cell-free plasma from donors immunized with TT and SRBC respectively was transfused indicated that the initial rise of specific antibody titers after marrow grafting is likely to be due to a passive transfer of humoral immunity. A single challenge of these marrow graft recipients with the respective antigen 15-18 weeks posttransplantation led to a secondary type of humoral immune response. In addition, it could be demonstrated that transfer of memory against TT or SRBC was independent from the actual antibody titer and the time of vaccination of the donor. One dog was immunized with TT after serving as marrow donor. When the donor had shown an antibody response, a peripheral blood leukocytes (PBL) transfusion was given to his chimera. Subsequent challenge of the latter resulted in a secondary type of specific antibody response. This indicates that specific cellular-bound immunological memory can be transferred after BMT from the donor to his allogeneic bone marrow chimera by transfusion of peripheral blood leukocytes. The data presented may be of importance in clinical BMT to protect patients during the phase of reduced immune reactivity by transfer of memory cells from histocompatible immunized donors.

Revised classification of the DLA loci by serological studies.

Peripheral blood mononuclear cells from DLA typed dogs were treated with rabbit-anti-dog-beta 2-microglobulin and subsequently with goat-anti-rabbit-immunoglobulin in order to aggregate the DLA class I molecules on the cell membrane (lysostrip). Utilizing a panel of 70 defined DLA-A and DLA-B antisera, lymphocytes treated in this way showed resistance to complement dependent lysis with monospecific DLA-A sera only, whereas reactivity of DLA-B antisera was not blocked; on the contrary, complete lympholysis with each DLA-B antiserum was recognized. Thus, the DLA-B antigens, evidently not associated with beta 2-microglobulin, are designated as candidates for class II gene products. The different reactions of DLA-C antisera after lysostrip did not allow a precise assignment of this antigen series as yet.

Joint report of the Third International Workshop on Canine Immunogenetics. I. Analysis of homozygous typing cells.

The Third International Workshop on Canine Immunogenetics involved 80 potentially DLA-D homozygous typing cells obtained from dogs of various breeds and submitted from five laboratories in Europe and the United States. Mutual reactivity of all cells was studied in mixed leukocyte cultures, and stabilized relative responses were used for analysis. Intralaboratory and interlaboratory comparisons of results suggest that a stabilized relative response of 30% represents an acceptable parameter for "typing responses" indicating phenotypic DLA-D identity of stimulator and responder cells. Using this criterion, 10 clusters of homozygous typing cells were defined and accepted on an international level, and they were assigned the specificities Dw1 to Dw10. At least six additional (provisional) specificities were recognized that were well characterized within individual laboratories but require additional testing before workshop specificities can be assigned. These data show that DLA-D typing is feasible and represents a useful tool in the genetic analysis of the canine major histocompatibility complex. Much work is needed to confirm the present results in family studies, to determine gene frequencies, and to analyze at a molecular level the antigens responsible for mixed leukocyte culture reactivity.

Polymorphism of the fourth complement component in the dog.

The polymorphism of the fourth complement component was studied in 118 unrelated dogs from different breeds and 73 unrelated beagles. In the mixed breed population, five different variants were observed with the following gene frequencies: C4*1 = 3.9%; C4*2 = 13.6%; C4*21 = 6.0%; C4*3 = 9.0% and C4*4 = 51.1%. In beagles, only four variants could be identified with gene frequencies of C4*2 = 24.2%; C4*21 = 1.4%; C4*3 = 20.6% and C4*4 = 46.3%. In both populations analyzed positive linkage disequilibria between certain DLA antigens and C4 variants were recognized. The biochemical analysis of the canine C4 revealed a double-banding pattern for the C4 alpha chain which gives preliminary evidence for two gene loci encoding for the fourth complement component in the dog.

HLA-D and -DR antigens on human amniotic fluid cells. II. Heterogeneous expression of HLA-DR and other cell surface markers.

To evaluate further the feasibility of HLA typing for prenatal diagnosis, we tested human amniotic fluid cells (AFC), known to express HLA-A, -B, and -C antigens, for the presence of HLA-DR antigens using type-specific antisera in the microcytotoxicity assay and a monoclonal antibody directed against the common HLA-DR structure (cDR) in indirect immunofluorescence. Prenatal typing of HLA-DR on AFC in the microcytotoxicity test was possible in only one out of eight families studied. The detected DR2 antigen was confirmed by postnatal typings of cord blood lymphocytes. Thereafter, 23 different AFC cultures were tested with monoclonal antibodies in indirect immunofluorescence. Only six cultures were partially positive (23-35% fluorescent cells) with the monoclonal cDR antibody while all AFC cultures demonstrated strong positive fluorescence (68-100%) with a monoclonal antibody against the common HLA-A, -B, and -C structure (cHLA). These data suggest that only a small subpopulation of AFC expresses class II (HLA-DR) antigens in contrast to the nearly ubiquitous expression of class I (HLA-A, -B, and -C) antigens. Furthermore, the heterogeneous expression of cell surface antigens within the various AFC cultures was substantiated with monoclonal antibodies directed toward cell surface antigens of the OKT, OKM, and Lyt series that have been found to be characteristic for subpopulations of lymphoid and hemapoetic cells. Thus, at present, HLA-DR typing is not reliable for prenatal diagnosis.