Reproductive immunology in viviparous mammals: evolutionary paradox of interactions among immune mechanisms and autologous or allogeneic gametes and semiallogeneic foetuses.

Literally, reproductive immunology was born in bovine on-farm reproduction where seminal experiments intended for developing methods for embryo transfer in cattle were performed. Actually, these experiments led to two of major concepts and fundamental principles of reproductive immunology using the bovine species as a model for biomedical research, namely the concept of acquired immunological tolerance and the paradox of the semiallogeneic bovine foetus whereby such organism can develop within an immunologically competent host. Peter Medawar, a scientist who together with Frank Macfarlande Burnet shared the 1960 Nobel Prize in physiology or medicine for discovery of acquired immunological tolerance, while studying dizygotic cattle twins, thereby giving birth to reproductive immunology. Also, these findings significantly influenced development of organ transplants and showed that using farm animals as models for studying transplantation immunology had general relevance for mammalian biology and health including those of humans. However, the interest for further research of the fascinating maternal immune influences on pregnancy and perinatal outcomes and of the prevention and treatment of immunologically mediated reproductive disorders in viviparous mammals of veterinary relevance by veterinary immunologists and reproductive clinicians have been very scarce regarding the application of nonspecific immunomodulatory agents for prevention and treatment of subfertility and infertility in pigs and cattle, but still broadening knowledge in this area and hold great potential for improving such therapy in the future. The aim of the current overview is to provide up-to-date information and explaining/translating relevant immunology phenomena into veterinary practice for specialists and scientists/clinicians in reproduction of animals.

The effect of thymectomy on the competitive potential of C57BL/6 bone marrow graft.

When a mixture of bone marrow cells derived from mice of the CBA-T6T6 and C57BL/6 strains and Lewis rats is injected into lethally irradiated (C57BL/6 X CBA-T6T6)F1 hybrid recipients, a process of cell interaction takes place, which in less than 2 weeks leads to the complete and permanent take over in hematopoietic and lymphoid organs by the C57BL/6 cell population. This is shown by the chromosome marker technique (T6) analysis. Adult thymectomy of F1 hybrid hosts prior to irradiation and bone marrow transplantation does not alter the competitive potential of the C57BL/6 donor cell population. However, neonatal thymectomy of the prospective C57BL/6 donors significantly impairs their "superiority" as donor cells, and CBA-T6T6 dividing cells persist at all times in all tissues analyzed. The results lend support to the concept of thymus-dependent precursor cells residing in "prethymic" hematopoietic tissue.

Chimaerism in lymph nodes of F1 into irradiated parental recipient chimaeras rejecting skin allografts from the other parent.

Mice of the C57BL strain were irradiated with 800 R over the whole body. The next day they received i.v. a mixture of 50 x 10(6) spleen and bone marrow cells from (C57BL x CBA-T6T6)F1 hybrid mice, and were challenged with CBA-T6T6 skin grafts later on. About 20% of the recipients rejected the CBA-T6T6 skin, whereas the others were completely tolerant for more than 200 days. By using the cytotoxic test, we found that both tolerant and nontolerant recipients were complete chimaeras, i.e., had only (C57BL x CBA-T6T6)F1 cells in their lymph nodes. However, analysis of the same mice by the chromosome marker technique disclosed a proportion of host (C57BL) cells in lymph nodes of both tolerant and nontolerant chimaeras. The percentage of host metaphases in nontolerant chimeras was significantly higher than that in tolerant chimaeras (P less than 0.01). It is possible therefore that the CBA-T6T6 skin grafts were rejected by residual host (C57BL) cells rather than (C57BL x CBA-T6T6)F1 cells reacting against skin-specific transplantation antigen(s) of the parental graft.

An association between serum testosterone level and HLA phenotype.

The concentration of testosterone was determined in sera of 122 HLA-typed women. Subsequently the women were classified in the category below or above the mean serum testosterone concentration. When the frequencies of HLA antigens were compared in these two categories of women, it was found that HLA-B5 and HLA-B12 antigens were significantly increased in the category of women with serum testosterone level above the mean concentration (P less than 0.01 and P less than 0.05, respectively). The frequency of HLA-B8 antigen was significantly decreased in the same category of women (P less than 0.05). The comparisons of the mean testosterone values of each HLA antigen and the variance analysis have also shown significant differences between the mean of HLA-B8 antigen and the means of other HLA antigens--HLA-A2, A3, A9, B5, B12 and Bw35. These results gave further conclusive evidence that gene(s) inside HLA region influence either the androgen hormone metabolism itself or cellular sensitivity to hormonal action as it has been presented for congenital adrenal hyperplasia.

Prenatal diagnosis of congenital adrenal hyperplasia (21-hydroxylase deficiency) in Croatia.

We report on the prenatal diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase in 20 at-risk pregnancies (16 salt-wasting and 4 simple virilizing families). We have diagnosed 3 affected fetuses (2 males and 1 female), 3 healthy homozygotes (2 males and 1 female), and 14 healthy heterozygotes (7 females and 7 males). These data were collected over 4 years. In 16 fetuses, the diagnosis was made with measurements of 17-hydroxyprogesterone (17-OHP) and delta-4-androstenedione (delta) in amniotic fluid (AF), human leukocyte antigen (HLA) typing of amniotic cells, as well as karyotypes between the 16th and 18th weeks of gestation. In 4 fetuses, DNA analysis of amniotic cells was also performed. In 3 pregnancies in which affected fetuses were suspected (on the basis of HLA typing and measurements of 17-OHP and delta concentrations in AF), the fetuses were electively aborted between the 17th to 19th weeks of gestation by parental decision. In all aborted fetuses, diagnosis was confirmed with HLA typing, autopsy findings of hyperplastic adrenal glands, and ambiguous genitalia in female fetuses. Postnatal diagnosis was confirmed in healthy fetuses with HLA typing and serum measurements of 17-OHP concentrations, and in 4 of them with DNA analysis. In 3 of the 4 families, DNA analyses revealed the following mutations: in Family 1, the index case mutation was Intron 2, Exon 3/Exon 6, and the fetus was Normal/Exon 6; in Family 2, the index case mutation was Ex1 Int2 Ex3/ Int2, and the fetus was Ex1 Int2 Ex3/Normal; and in Family 3, the index case mutation was Ex8(356)/Ex8(356), and the fetus was Ex8(356)/ Normal. We also report one case of prenatal diagnosis and treatment. Dexamethasone 0.5 mg BID (20 micrograms/kg/d) was given starting at 6th week of gestation. Prenatal diagnosis suggested, but did not prove, that the female fetus was a heterozygote as the fetus lacked the paternal mutation Ex8(318). No mutation was found in the mother. The fetus, the mother, and the affected sib shared a haplotype, further suggesting heterozygosity. The unaffected status was confirmed postnatally.

Implication of molecular analysis of HLA-A*02 subtyping for unrelated bone-marrow donor selection.

The HLA-A*02 allele is the most heterogeneous allele at HLA-A locus with 22 different subtypes so far identified. All of these subtype polymorphisms are located in alpha 1 and alpha 2 domains which are responsible for peptide biding and HLA restricted recognition by T-cell receptor. The aim of the present study was to determine the frequency of different HLA-A*02 alleles in 33 healthy unrelated Croatians. HLA-A*02 subtyping has also been retrospectively performed in 2 recipient-unrelated donor pairs and in 4 recipient-HLA phenotypically identical parent pairs. All subjects, previously typed as HLA-A2 by serology were tested using HLA-A*02 ARMS-PCR kit which discriminates 17 different A*02 alleles. Among 17 A*02 alleles we have found 4 different A*02 subtypes in healthy unrelated Croatians. The most frequent A*02 allele was A*0201 (84%). The frequency of the remaining A*02 alleles were as follows: A*0205 (3%), A*0207 (6%) and A*0213 (6%). Among 6 tested bone-marrow transplantation (BMT) pairs, only one has been found to be A*02 subtype incompatible (A*0201/A*0205). Four different A*02 alleles are found in Croatian population with the predominance of A*0201. However these results suggest that A*02 subtyping is also necessary for optimal matching of HLA-A*02 positive donor-recipient pairs in HLA incompatible BMT.

Combination of cyclosporin and methotrexate for prophylaxis of acute graft-versus-host disease after allogeneic bone marrow transplantation for leukemia.

From May 1985 to July 1989, 76 patients with leukemia (30 acute myelogenous leukemia, 24 acute lymphoblastic leukemia and 22 chronic myeloid leukemia) were randomized to receive either cyclosporin (CSP) alone (n = 39) or CSP combined with methotrexate (CSP + MTX, n = 37) for graft-versus-host disease (GVHD) prophylaxis. Patients were conditioned with total body radiation and cyclophosphamide followed by bone marrow infusion from an HLA-identical sibling. Engraftment of the transplanted bone marrow was similar in both groups. The incidence of moderate to severe acute GVHD was significantly higher in the CSP group compared with the CSP + MTX group (20 (51%) versus 9 (25%), chi 2 = 4.76, p less than 0.02). There was no significant difference in the incidence of chronic GVHD. Survival was significantly better for the CSP + MTX group (63 +/- 16%) compared to CSP alone (42 +/- 18%). Leukemia-free survival tended to be better for the CSP + MTX group (55 +/- 17% versus 32 +/- 16%).

HLA antigen studies in women with recurrent gestational disorders.

The frequencies of HLA antigens were determined in an experimental group of women with recurrent gestational disorders mainly expressed as spontaneous abortions of unknown etiology. The estimated antigen frequencies in this group were compared with the antigen frequencies in 158 unrelated persons (79 couples with no record of secondary infertility). A significantly higher frequency of antigen HLA-A9 was found in the experimental group as compared with the controls (corrected P = 0.0015). A greater degree of HLA compatibility was found between each woman from this group and her husband, considering 45 couples, as compared with 79 control couples. These results indicate that gene(s) close to the HLA region may have an influence on the reproductive performance in humans.

Experience with bone marrow transplantation for severe aplastic anemia and leukemia. Two years experience with 31 patients in Zagreb (Yugoslavia).

Since 1983, twelve patients with severe aplastic anemia and nineteen patients with leukemia were treated with bone marrow transplantation. Six patients with severe aplastic anemia are still alive at 300 to 1,210 days after transplantation, and twelve patients with leukemia are alive at 199 to 671 days. The incidence of GvHD was relatively high (60%). Bacterial infections were the main causes of death.

Cyclosporin neurotoxicity in patients treated with allogeneic bone marrow transplantation.

Toxicity to the central nervous system was observed in 3 out of 15 patients receiving cyclosporin prophylaxis for graft-versus-host-disease after allogeneic bone marrow transplantation. Neurological clinical features included grand mal seizures, dysarthria and vestibular and cochlear toxicity. In all three patients, cyclosporin plasma levels were increased at the time of overt clinical neurological signs. The symptoms resolved quickly with the reduction of the cyclosporin dose.

The preexcitation syndrome: epidemiological and genetic study.

A sample of 4210 subjects of both sexes aged 35-54 years was examined, chosen at random from six regions of Croatia. An electrocardiogram at rest was performed in all subjects and changes analyzed by the Minnesota code. A short P-R interval together with a widening QRS complex and a delta wave was found in 0.05%, while 42 (1.0%) of the examinees had a short P-R interval, but only 0.21% were symptomatic. Three years after the first examination 0.06% of the subjects had preexcitation with a delta wave, and in one subject it appeared after three years. 0.35% of the subjects had a short P-R interval after three years but only 0.18% were symptomatic and in 22 (0.65%) it had disappeared in three years. After 13 years these subjects did not appear for an examination, and the short P-R interval did not appear in any of other subjects during this period. There were more short P-R intervals: 3.22% in females and 1.96% in males, but 0.33% only were symptomatic. Antigens of the human leukocyte group A (HLA) system were analyzed in 46 patients: the Wolff-Parkinson-White syndrome was found in 35, while 11 had the Lown-Ganong-Levine syndrome. Antigens of the HLA-A, HLA-B and HLA-DR locuses were determined by the microlymphocytotoxicity method. The results of the frequency of HLA system antigens were compared to the results of the control group of a Croatian population consisting of 175 people. There was an increased frequency of HLA-A9 and HLA-B5 (P = 0.026 and 0.0092) in the investigated population as a whole. The participation of HLA-A3 antigen was significantly less among patients (P = 0.03), while HLA-B14 antigen was not found in patients with preexcitation. Within 10 HLA-DR locuses, HLA-DR7 antigen was rather more frequently present, although this was not statistically significant (P = 0.173).

A prospective study on the etiology of uveitis.

We prospectively studied and classified 152 cases of endogenous uveitis treated at the Ophthalmological Clinic in Split, Yugoslavia, according to clinical presentation, several immunologic parameters including HLA subtyping, and the presence of an associated systemic disease. Using our diagnostic criteria we successfully classified 91.4% of patients. Anterior uveitis was the most common form of clinical presentation (94 patients, 61.8%), whereas an intermediate uveitis was present in only 5.3% of patients (8 patients). By HLA typing we found that 115 patients (75.7%), were positive for HLA-B27 (57 patients, 50%), B5 (30 patients, 26%) and B35 (28 patients, 24%) risk antigens. Of these patients 96 (83%) had an associated systemic illness. In the 8.6% of cases (13 cases) in which the etiology of an endogenous uveitis was not determined, none were associated with an HLA-B risk antigen. Our study emphasizes the association of HLA-B risk antigens and concurrent systemic disease in patients with endogenous uveitis.

Etiology and clinical features of anterior uveitis in southern croatia (dalmatia).

Patients with endogenous uveitis represent 6.5+ of patients in University Hospital Split, which serves most of South Croatia. Within a four-year period 208 patients were treated for endogenous uveitis. Results of clinical-laboratory examinations and treatment of 112 subjects suffering from anterior uveitis are presented and compared. Acute anterior uveitis (AAU) was the commonest form of uveal inflammation. It was present in 49+ of all uveitis cases and in 91.1+ of all anterior uveitis cases (AU). 67.6+ of the subjects with AAU had and 32.4+ did not have the HLA B(27) antigen. The inflammatory pattern in B(27)(+) patients was typical of B(27)(+) AAU. Patients with B(27)(+) AAU exhibited the same inflammatory pattern as those with B(7)(+) AAU. B(27)(+) AAU patients had significantly more systemic/rheumatic diseases (p>0.05), while patients with B(27)(-) AAU had significantly more infectious diseases (p>0.05). Forty percent of the patients with chronic anterior uveitis suffered from juvenile rheumatoid arthritis. The authors observed the rise in peripheral blood IgG, IgA, IgM, CD(2)(+), CD(4)(+) and B cells during the acute phase of AAU. Normalization of B cells (CD(20)(+)) was observed in early remission of anterior uveitis, about eight weeks after the onset of the disease.

Activation of pleural macrophages by intrapleural application of Corynebacterium parvum.

A single ipl injection of 0.25 mg CP into CBA mice led to accumulation of macrophages in the pleural cavity, but it did not influence RES as an injection given iv ipl CP caused a three-to-five-fold increase in the number of nucleated cells in the pleural cavity which persisted at least 14 days. Of these cells 86% were macrophages as shown by their esterase activity. Less than 30% of cells from the pleural cavity of normal mice were esterase positive. Macrophages from the pleural cavity of CP-treated mice were capable of destroying in vitro cultures of a syngeneic mammary carcinoma, while normal pleural macrophages exerted no effect; the former were not cytotoxic for either syngeneic or allogeneic embryo fibroblasts. Ipl CP protected mice against iv injected mammary carcinoma cells; given to mice 7 days after iv inoculation of tumor cells it significantly reduced the number of tumor nodules in their lungs.

Analysis of HLA antigens in Croatian patients with psoriasis.

In common with most autoimmune diseases, psoriasis is associated with some HLA antigens. We studied the distribution of HLA antigens in Croatian patients with psoriasis: 108 patients were divided into groups according to family history and age of disease onset. HLA antigens were analyzed serologically and HLA-C alleles were analyzed using polymerase chain reaction. We found significant increases in HLA-A2, -B17, -B37 and -B13 antigens and highly significant increases in HLA-Cw*0602 and DR7 antigens in psoriatic patients compared with controls. Patients with type I psoriasis (early onset, positive family history) showed highly significant associations with Cw*0602 [p < 0.00001; relative risk (RR) = 14.45] and DR7 (p < 0.00001; RR = 15.09) antigens. Patients with type II psoriasis (late onset, no family history) had a significant association with Cw*03 antigen (p = 0.008; RR = 0.17). In conclusion, HLA-B13, -B17, Cw*0602 and -DR7 antigens are associated with a significant risk of psoriasis in the Croatian population and the Cw*0602 allele has the strongest association, especially for type I psoriasis.

Depression and torture.

Torture represents an exceptionally traumatic experience in which horror, helplessness, and hopelessness are extreme. Therefore, it can be expected that depression, along with other trauma-related disorders is present in torture victims at higher rates than in other psychotraumatized individuals. To demonstrate this, we examined two groups of refugees, all suffering the post-traumatic stress disorder. The first group (N = 50) had combat experience but were imprisoned and tortured as well. Members of the second group (N = 29) had combat experience. A third group (N = 30) consisted of local people with no traumatic experience. Using the Hamilton scale, the Beck Depression Inventory and structured dedicated interviews, we tried to determine whether those groups differed in level of depression based on their different levels of traumatic experience. The results of our study indicate that torture victims showed a significantly higher level of depression that is clinically relevant.

HLA class I polymorphism in the Albanian population.

The HLA class I polymorphism was studied in a sample of the Albanian population. Ninety-three unrelated healthy Albanians were typed for HLA-A, -B and -Cw antigens by standard microlyphocytotoxicity test. The antigens with the highest frequencies were: HLA-A2 (34.4%), A3 (14.5%) and A1 (12.4%); B51 (19.3%), B35 (12.4%) and B18 (10.2%); Cw4 (16.2%), Cw7 (16.2%) and Cw6 (10.8%). The HLA haplotypes with high frequency in Albanians included A2-B51 (4.3%), A2-B18 (2.4%), A2-B35 (2.4%), Cw4-B35 (7.6%), and Cw7-B18 (6.5%), which are not significantly different from the other neighboring populations. Low frequency of HLA-A1-B8 haplotype (1.1%) is noted in the Albanian population. The frequency of HLA-B27 antigen (1.1%) is one of the lowest frequencies observed in Caucasians. Such results are important in studies of HLA-A1-B8, HLA-B27 and disease associations. These findings should be also useful in understanding the origin of Albanians, representing a base for future studies about HLA polymorphism in the Albanian population.

Distribution of alleles at two microsatellite loci (D6S273 and TNFa) in Croatian population.

Polymorphism at the level of two microsatellite loci (D6S273 and TNFa) was studied in Croatian population. The most frequent alleles at D6S273 locus are D6S273 134 bp and 136 bp, while at TNFa locus two most frequent alleles are TNFa 117 bp and 99 bp. This study confirms the irregularity in distribution of microsatellite alleles in different populations with the predominance of two or three alleles on these two investigated microsatellite loci.

High resolution molecular typing of HLA class II region in the population of the island of Krk, Croatia.

The HLA class II alleles (DRB1, DRB3, DRB5, DQA1, and DQB1) and haplotypic associations were studied in the population of the island of Krk using the PCR-SSOP method and the 12th International Histocompatibility Workshop primers and probes. Allele and haplotypic frequencies were compared with the general Croatian population. Significant differences were observed between the population of the island of Krk and Croatians for: a) three broad specificities at DRB1 locus (DRB1*01, *15, and *07), b) one allele at DRB3 locus (DRB3*0301), c) one allele at DQA1 locus (DQA1*0201), d) one allele at DQB1 locus (DQB1*0303). Four unusual haplotypic associations, which have not yet been described in the Croatian population, DRB1*1301-DQA1*0103-DQB1*0607, DRB1*1302-DQA1*0102-DQB1*0605, DRB1*1305-DQA1*0102-DQB1*0605 and DRB1*1305-DQA1*0103-DQB1*0603 were observed in the population from the island of Krk.