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 nonreplicating smallpox candidate vaccines defective vaccinia Lister (dVV-L) and modified vaccinia Ankara (MVA) elicit robust long-term protection.

Current smallpox vaccines are live vaccinia viruses that replicate in the vaccinee inducing immunity against the deadly disease smallpox. Replication resulting in virus spread within the host, however, is the major cause of severe postvaccinal adverse events. Therefore, attenuated strains such as modified vaccinia Ankara (MVA) or LC16m8 are candidates as next generation vaccines. These strains are usually grown in primary cells in which mass production is difficult and have an unknown protective potential in humans. Proven vaccine strains of defined origin and modern production techniques are therefore desirable. In this study, defective vaccinia virus (dVV) lacking a gene essential for replication (derived from the Lister vaccine in a complementing cell line) was compared with the Wyeth smallpox vaccine strain and with MVA in mouse animal models using cowpox and ectromelia virus challenge. Similar to MVA, prime-boost immunizations with defective vaccinia induced robust long-term immunity, suggesting it as a promising next generation smallpox vaccine.

Immunogenicity and safety of defective vaccinia virus lister: comparison with modified vaccinia virus Ankara.

Potent and safe vaccinia virus vectors inducing cell-mediated immunity are needed for clinical use. Replicating vaccinia viruses generally induce strong cell-mediated immunity; however, they may have severe adverse effects. As a vector for clinical use, we assessed the defective vaccinia virus system, in which deletion of an essential gene blocks viral replication, resulting in an infectious virus that does not multiply in the host. The vaccinia virus Lister/Elstree strain, used during worldwide smallpox eradication, was chosen as the parental virus. The immunogenicity and safety of the defective vaccinia virus Lister were evaluated without and with the inserted human p53 gene as a model and compared to parallel constructs based on modified vaccinia virus Ankara (MVA), the present "gold standard" of recombinant vaccinia viruses in clinical development. The defective viruses induced an efficient Th1-type immune response. Antibody and cytotoxic-T-cell responses were comparable to those induced by MVA. Safety of the defective Lister constructs could be demonstrated in vitro in cell culture as well as in vivo in immunodeficient SCID mice. Similar to MVA, the defective viruses were tolerated at doses four orders of magnitude higher than those of the wild-type Lister strain. While current nonreplicating vectors are produced mainly in primary chicken cells, defective vaccinia virus is produced in a permanent safety-tested cell line. Vaccines based on this system have the additional advantage of enhanced product safety. Therefore, a vector system was made which promises to be a valuable tool not only for immunotherapy for diseases such as cancer, human immunodeficiency virus infection, or malaria but also as a basis for a safer smallpox vaccine.

Humoral and cell-mediated immunity to vero cell-derived influenza vaccine.

A candidate trivalent influenza whole virus vaccine produced in a continuous mammalian cell line (Vero), and analogous commercially available egg-derived vaccines, were compared for their ability to induce humoral and cell-mediated immunity in Balb/c mice. Substantial haemagglutination-inhibition titre and high levels of influenza virus-specific IgG were found in all groups of immunized mice, irrespective of the vaccine formulation. The IgG responses were predominantly of IgG1 and IgG2a/2b isotypes. Virus-specific secretory IgA antibodies were detected only in mice immunized intranasally with a live virus, derived either from Vero cells or eggs. T-cell proliferative responses and T-helper 1 type cytokine release was significantly higher in mice immunized with Vero cell-derived influenza vaccine compared to egg-derived vaccine formulations. We have demonstrated that the immunogenicity of the trivalent Vero cell-derived whole influenza virus vaccine was comparable to that of the equivalent egg-derived vaccine, with respect to humoral immune response and was superior with respect to cellular response.

Natural IgM antibodies in baby rabbit serum bind high-mannose glycans on HIV type 1 glycoprotein 120/160 and activate classic complement pathway.

Serum from rodents and felines has been found very effective in complement-dependent lysis of HIV-1, even in nonimmunized animals, but the effector molecules in animal serum and target structures on HIV-1 envelope gp120/160 responsible for complement activation were not determined. We have found that the natural anti-carbohydrate-specific IgM antibodies present in baby rabbit serum were able to lyse effectively the CD4+ T cells coated with the whole virus or with a recombinant gp120/160, irrespectively of the virus strain or glycoprotein expression system. When the high mannose-type glycans on gp160 were enzymatically removed by endoglycosidase F or blocked with the specific lectins, the complement activation and subsequent cell lysis were abolished. IgM-depleted baby rabbit serum was not able to lyse the gp120/160- and/or whole virus-coated target cells. These results suggest that the target structures for complement-activating and naturally occurring IgM antibodies in baby rabbit serum are high-mannose residues on HIV-1 envelope glycoprotein.

The role of complement and gp120-specific antibodies in virus lysis and CD4+ T cell depletion in HIV-1-infected patients.

The substantial virus lysis was induced by HIV-1-infected patient serum and normal human complement serum in the presence of purified patient IgG. Non-infected CD4+ T cells coated with the whole virus or with a recombinant HIV-1 envelope gp120 and sensitised with patient IgG were also shown to be susceptible to complement-dependent lysis. The serum level of complement regulatory protein in a fluid phase, the C1-esterase inhibitor, was significantly correlated with serum concentration of C1q-circulating immune complexes (P=0.0062), but inversely with CD4+ T cell count (P < 0.0001). Accordingly, the disease progression in HIV-1-infected patients was significantly correlated with the level of complement activation as determined by serum level of C1-esterase inhibitor (P=0.0001), and inversely correlated with CD4+ cell count (P < 0. 0001) and gp120-specific antibody titre (P=0.0086). These results strongly suggest that the complement activation by gp120-specific antibodies play a very important role in virus clearance, but also in depletion of infected as well as gp120-coated non-infected CD4+ bystander T cells during the course of HIV-1 infection.

Inhibition of classical complement activation by sera from HIV-1-positive patients.

It was shown that gp120/160-coupled CD4+ T cells could be lysed by complement activation, but the target cell lysis was strongly inhibited by the majority of HIV-1-positive sera. Significantly more sera from HIV-1-infected patients with CD4+ T cell count higher than 500 microl (N = 38) as well as from patients with 200-500/microl (N = 32) showed strong inhibition of complement activation as compared to sera from those with less than 200 CD4+ T cells/microl (N = 28) (P = 0.0064 and 0.0012, respectively). Consequently, highly significant correlation between CDL inhibitory activity and CD4+ T cell count in HIV-1-infected patients was found by Spearman's rank order analysis (R = 0.399, P < 0.001). CH50 titer and functional C1-inhibitor level were significantly lower in inhibitory as compared to the noninhibitory sera (P < 0.001) and to controls (P < 0.001). The C3 activation products-C3-circulating immune complexes were not increased in inhibitory sera (P = 0.014) suggesting that inhibition of complement activation occurred at or before C3 activation level. C4d fragments and antigenic C1-INH concentration were significantly increased in both categories of HIV-1-positive sera, P < 0.001. These findings indicate that persistent and massive stimulation of complement system with HIV-1 envelope glycoproteins during all stages of the disease induced impairment of classical pathway activation by an inhibitory factor in a majority of patients, which might contribute to the onset of opportunistic infections and AIDS.

Immunosuppression of in vivo and in vitro lymphocyte responses in swine induced by Trichinella spiralis or excretory-secretory antigens of the parasite.

The in vivo and in vitro effects of Trichinella spiralis excretory-secretory (ES) antigens on porcine peripheral blood lymphocyte (PBL) responses induced with mitogens (phytohemagglutinin, PHA; concanavalin A, Con A; pokeweed mitogen, PWM) or unrelated antigen (Protein A) were studied to determine whether ES antigens depress lymphocyte responses in experimental swine trichinosis, and/or if this response was manifested after lymphocytes from infected pigs had been pretreated with ES antigens. Additionally, the range of inhibition of lymphocyte responses was tested in parasite-free pigs using different doses of ES antigens and compared with the responsiveness of control cultures from the same animals. The responses of lymphocytes from pigs inoculated with 4 x 10(3) muscle larvae (ML) were strongly depressed (P < 0.05) at post-inoculation days (PID) 7 (after stimulation with PHA), 14, 35 (Con A or PWM), and 49 (PWM). At PID 56 and 63 the lymphocytes from T. spiralis-infected pigs responded better (P < 0.05) to all three mitogens than those from non-infected controls. After 7 weeks post-inoculation, PBL which were pretreated with 10 or 250 micrograms ml-1 of ES antigens showed significantly weaker (P < 0.05, P < 0.001) responses to PWM or PHA, respectively, than those from non-infected animals. The responsiveness of lymphocytes from both groups of pigs to Protein A was not affected by the pretreatment with ES antigens in vitro. The responses of lymphocytes from the parasite-free pigs induced by PHA, PWM or Protein A were strongly depressed (P < 0.01) after in vitro pretreatment regardless of the dose of ES antigens (5, 10, 15, or 20 micrograms ml-1) applied.

In vitro modulating effects of porcine immunoglobulin G on mitogens-induced lymphocyte response in precolostral, suckling and weaned piglets.

The influence of allogeneic IgG on in vitro reactivity of peripheral blood lymphocytes (PBL) of neonatal colostrum-deprived piglets as well as of suckling and weaned piglets was studied. PBL were preincubated with purified allogeneic IgG for 24 h before their ability to respond to PHA, Con A or PWM was tested. PBL of precolostral piglets pretreated with allogeneic IgG exhibited higher response to PHA (P less than 0.01) than untreated control cells. An increased response of PBL treated with IgG was also observed in suckling piglets as compared to their respective control cells (P less than 0.01). Responsiveness of PBL treated with IgG to PWM was suppressed. No differences in response to Con A regardless of the sources of lymphocytes was observed as compared to IgG untreated controls. The results suggest that pretreatment of lymphocytes of piglets with allogeneic IgG modulates their reactivity to mitogens, suppressing the response to PWM and stimulating the response to PHA, respectively.

Qualitative analyses of cellular immune functions in equine infectious anemia show homology with AIDS.

Equine infectious anemia (EIA) is a disease caused by a lymphocytotropic lentivirus which belongs to the same subfamily as HIV. Because of the very close relationship of their predicted gag and pol gene products and similarities in clinical manifestations of the disease, EIA served as a model to study immunological events involved in the host defence against lymphocytotropic viral infections. The existence of antibody dependent cell-mediated cytotoxicity against autologous EIA virus infected lymphoblasts was demonstrated in vitro at the beginning of an acute attack of the disease. Cytotoxic activity was not found or was very low during chronic infection. Reactivity of peripheral blood lymphocytes to Protein A and allogeneic cells in vitro was significantly suppressed in the presence of an acute serum, while the reactivity to PHA was at the normal level. These results suggest that cellular immune mechanism(s) are also involved in removal of EIA virus infected cells as has been reported recently in HIV-1 infected individuals and that EIAV and HIV immunopathogenesis show similarities in affecting the immune response of the host.

T and B lymphocytes in horses persistently infected with equine infectious anaemia virus.

The percentage of T and B lymphocytes in the peripheral blood of horses chronically infected with equine infectious anaemia (EIA) virus was determined and the results were compared with the percentage of these cells in healthy uninfected horses. Cells with membrane receptors for sheep erythrocytes (T and active T lymphocytes) were determined by E and A rosette techniques, while cells with receptors for the C3b component of complement and those with receptors for mouse erythrocytes (B lymphocytes), were determined by the EAC rosette method. The percentage of Fe positive cells was assayed by the EA rosette test. The majority of peripheral blood lymphocytes (PBL) from both uninfected and EIA-infected horses formed rosettes of each kind with only three erythrocytes indicating a low density of the corresponding receptors on the cell membrane under the condition of the assays used. The percentage of T lymphocytes in the peripheral blood of diseased horses (52.4 +/- 1.6%), as detected by E rosettes, was significantly (p less than 0.01) higher than in control animals (42.4 +/- 3.5%). In clinically healthy horses 8.9 +/- 1.1% of PBL were identified by A rosettes as active T cells, whereas animals with a chronic form of EIA had a much lower (p less than 0.001) percentage of these cells (4.7 +/- 0.7%). In the B lymphocyte subpopulations the percentages of cells bearing Fc and C3b receptors were markedly elevated (p less than 0.001) in EIA-infected horses (24.7 +/- 0.8% and 42.8 +/- 2.2% respectively) as compared to uninfected animals (15.1 +/- 1.4% and 29.6 +/- 1.2% respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

HLA and red blood group antigens in pregnancy disorders.

Total of 356 women with various types of pregnancy disorders as well as their husbands were classified in four groups regarding the type of the disorder as follows: 1. Recurrent spontaneous abortions (RSA) of unknown etiology (N = 105) and RSA - primary aborters only (N = 84); 2. Blighted ovum (N = 80); 3. Rh immunization in pregnancy (N = 90); 4. ABO immunization in pregnancy (N = 47). Two groups of couples were used as controls: 1. Couples randomly taken from forensic medicine cases of paternity evaluation (N = 104); 2. Couples having two or more children with HLA immunization in pregnancy (N = 78). The couples from all groups were typed for red blood group antigens of ABO, Rhesus, MNSs, Kell, Duffy, Lewis, Kidd and P systems and also for HLA antigens. Significantly higher frequency of antigen HLA-A9 was found in women with RSA (corr. p = 0.0003) and in women with pregnancy disorders caused by Rh immunization (corr. p = 0.0136). In couples with RSA the degree of HLA compatibility was significant (p = 0.0048) and the reactivity of spouses in MLR was significantly decreased (p = 0.0001). Significantly, more low responders in MLR were also found among the women with RSA as compared to the controls (p = 0.0217). Two possible pathologic mechanisms may explain the association between HLA antigens and RSA: 1. immunological defects which are linked to HLA-D/DR region causing malfunction of immunosuppressive mechanisms during pregnancy; 2. endocrinological defect which is linked to HLA region as 21-OH hydroxylase deficiency gene.

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.

Plasma testosterone and dihydrotestosterone in normal and abnormal pregnancy.

The concentration of plasma testosterone (T) and dihydrotestosterone (DHT) was determined in 2 groups of nonpregnant and pregnant women. The 1st group consisted of normal women and the 2nd of women with recurrent pregnancy disorders of unknown etiology. Significantly higher concentration of plasma DHT in nonpregnant women from the 2nd group was found (44.9 +/- 22 ng/100 ml) as compared to nonpregnant normals (24.2 +/- 5.2 ng/100 ml), p less than 0.01. There was no difference in the concentration of plasma T between the groups studied (p = 0.165).

Inhibition of mitogen and specific antigen-induced human lymphocyte proliferation by cadmium.

Cadmium chloride (CdCl2) added to human lymphocyte culture inhibits the proliferative response induced by phytohaemagglutinin (PHA), pokeweed mitogen and allogenic lymphocytes in mixed lymphocyte reaction. Minimally effective concentrations of CdCl2 were 3.3, 1.6 and 1.6 microM, respectively. The inhibition was greatest when CdCl2 was added at initiation of cultures and declined if the addition of CdCl2 was postponed. The presence of CdCl2, regardless of the presence of PHA during the first 24 h of incubation suppressed the proliferative response to subsequent stimulation with PHA, indicating that cadmium affects an early step of blastogenic transformation.

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.