Scale-up of the Internet-based Professional Learning to help teachers promote Activity in Youth (iPLAY) intervention: a hybrid type 3 implementation-effectiveness trial.

BACKGROUND: Whole-of-school programs have demonstrated success in improving student physical activity levels, but few have progressed beyond efficacy testing to implementation at-scale. The purpose of our study was to evaluate the scale-up of the 'Internet-based Professional Learning to help teachers promote Activity in Youth' (iPLAY) intervention in primary schools using the RE-AIM framework. METHODS: We conducted a type 3 hybrid implementation-effectiveness study and collected data between April 2016 and June 2021, in New South Wales (NSW), Australia. RE-AIM was operationalised as: (i) Reach: Number and representativeness of students exposed to iPLAY; (ii) Effectiveness: Impact of iPLAY in a sub-sample of students (n = 5,959); (iii) Adoption: Number and representativeness of schools that received iPLAY; (iv) Implementation: Extent to which the three curricular and three non-curricular components of iPLAY were delivered as intended; (v) Maintenance: Extent to which iPLAY was sustained in schools. We conducted 43 semi-structured interviews with teachers (n = 14), leaders (n = 19), and principals (n = 10) from 18 schools (11 from urban and 7 from rural locations) to determine program maintenance. RESULTS: Reach: iPLAY reached ~ 31,000 students from a variety of socio-economic strata (35% of students were in the bottom quartile, almost half in the middle two quartiles, and 20% in the top quartile). EFFECTIVENESS: We observed small positive intervention effects for enjoyment of PE/sport (0.12 units, 95% CI: 0.05 to 0.20, d = 0.17), perceptions of need support from teachers (0.26 units, 95% CI: 0.16 to 0.53, d = 0.40), physical activity participation (0.28 units, 95% CI: 0.10 to 0.47, d = 0.14), and subjective well-being (0.82 units, 95% CI: 0.32 to 1.32, d = 0.12) at 24-months. Adoption: 115 schools received iPLAY. IMPLEMENTATION: Most schools implemented the curricular (59%) and non-curricular (55%) strategies as intended. Maintenance: Based on our qualitative data, changes in teacher practices and school culture resulting from iPLAY were sustained. CONCLUSIONS: iPLAY had extensive reach and adoption in NSW primary schools. Most of the schools implemented iPLAY as intended and effectiveness data suggest the positive effects observed in our cluster RCT were sustained when the intervention was delivered at-scale. TRIAL REGISTRATION: ACTRN12621001132831.

T-cell tolerance to the developing equine conceptus.

One of the most intriguing and dramatic examples of immunological tolerance is displayed by the mammalian foetal-placental unit, which thrives as a semi-allograft in the mother's uterus during pregnancy. The success of the so-called foetal allograft stands in stark contrast to the failure of most tissue and organ grafts to survive without genetic matching of donor and recipient or drastic immunosuppression of the recipient's immune system. Experiments conducted over the past 60 years have revealed multiple mechanisms that enable the conceptus to avoid immunological detection or destruction. Many of these mechanisms are directed towards evading immune-mediated damage by maternal T lymphocytes, and they can be grouped into three classes: (i) downregulation of major histocompatibility complex (MHC) gene expression in placental trophoblast cells; (ii) local and systemic alterations in maternal immune reactivity; and (iii) innate defence mechanisms of the trophoblast cells that comprise the barrier between foetal and maternal tissues. The redundancy in these protective mechanisms helps ensure the transmission of life from generation to generation and provides a rich field of study of ways in which functional immunological tolerance can be manifest. The variation in placental forms and function among mammalian species present opportunities to discover and understand novel tolerogenic mechanisms that may have broad application in biology, medicine and animal husbandry. This review focuses on the evidence obtained from studies of pregnancy in the mare that support the case for selective T-cell tolerance to the mammalian conceptus.

Identification of equine major histocompatibility complex haplotypes using polymorphic microsatellites.

A system for identifying equine major histocompatibility complex (MHC) haplotypes was developed based on five polymorphic microsatellites located within the MHC region on ECA 20. Molecular signatures for 50 microsatellite haplotypes were recognized from typing 353 horses. Of these, 23 microsatellite haplotypes were associated with 12 established equine leucocyte antigen (ELA) haplotypes in Thoroughbreds and Standardbreds. Five ELA serotypes were associated with multiple microsatellite subhaplotypes, expanding the estimates of diversity in the equine MHC. The strong correlations between serological and microsatellite typing demonstrated a linkage to known MHC class I protein polymorphisms and validated this assay as a useful supplement to ELA serotyping, and in some applications, a feasible alternative method for MHC genotyping in horse families and in population studies.

Equine clinical genomics: A clinician's primer.

The objective of this review is to introduce equine clinicians to the rapidly evolving field of clinical genomics with a vision of improving the health and welfare of the domestic horse. For 15 years a consortium of veterinary geneticists and clinicians has worked together under the umbrella of The Horse Genome Project. This group, encompassing 22 laboratories in 12 countries, has made rapid progress, developing several iterations of linkage, physical and comparative gene maps of the horse with increasing levels of detail. In early 2006, the research was greatly facilitated when the US National Human Genome Research Institute of the National Institutes of Health added the horse to the list of mammalian species scheduled for whole genome sequencing. The genome of the domestic horse has now been sequenced and is available to researchers worldwide in publicly accessible databases. This achievement creates the potential for transformative change within the horse industry, particularly in the fields of internal medicine, sports medicine and reproduction. The genome sequence has enabled the development of new genome-wide tools and resources for studying inherited diseases of the horse. To date, researchers have identified 11 mutations causing 10 clinical syndromes in the horse. Testing is commercially available for all but one of these diseases. Future research will probably identify the genetic bases for other equine diseases, produce new diagnostic tests and generate novel therapeutics for some of these conditions. This will enable equine clinicians to play a critical role in ensuring the thoughtful and appropriate application of this knowledge as they assist clients with breeding and clinical decision-making.

Report of the Second Havemeyer EHV-1 Workshop, Steamboat Springs, Colorado, USA, September 2008.

This report summarises the findings of the Second Havemeyer EHV-1 Workshop, which was held in Steamboat Springs, Colorado, USA in September 2008. A total of 38 delegates, consisting of veterinary clinicians and scientists from academia and industry participated in a series of sessions that focused on equine herpesvirus myeloencephalopathy (EHM). Each session consisted of a review, followed by short presentations on current research topics. The sessions included EHM epidemiology, in vivo and in vitro models for studying EHM, EHV-1 virulence determinants, real-time PCR diagnostics, antiviral medications and new vaccination technologies. The report summarises the key advances identified during and since the meeting. Citations are restricted to selected reviews and papers published since the workshop.

Genome-wide association study of equine herpesvirus type 1-induced myeloencephalopathy identifies a significant single nucleotide polymorphism in a platelet-related gene.

Equine herpesvirus type 1 (EHV-1)-induced myeloencephalopathy (EHM) is a neurologic disease of horses that represents one outcome of infection. The neurologic form of disease occurs in a subset of infected horses when virus-induced endothelial cell damage triggers vasculitis and subsequent ischemic insult to the central nervous system. EHM causes considerable animal suffering and economic loss for the horse industry. Virus polymorphisms have been previously associated with disease outcome but cannot fully explain why only some horses develop EHM. This study investigated the role of host genetics in EHM. DNA samples were collected from 129 horses infected with EHV-1 (61 that developed EHM and 68 in which disease resolved without the development of neurologic signs) during natural outbreaks or experimental infections. A genome-wide association study (GWAS) was performed to investigate host genetic variations associated with EHM. Genotyping was performed using the Illumina SNP50 and SNP70 arrays and a custom Sequenom array. Mixed linear model (MLM) analysis using a recessive model identified one marker that surpassed the threshold for genome-wide significance (P<0.001) after Bonferroni correction. The marker (BIEC2_946397) is in an intron of the tetraspanin 9 (TSPAN9) gene, which is expressed in endothelial cells and platelets. The GWAS identified a region in the horse genome that is associated with EHM in the sample population and thus warrants further exploration. Understanding the contribution of host genetic variation to the development of EHM will enhance our knowledge of disease pathophysiology, and lead to improved strategies for treating individual cases and managing outbreaks.

Modeling trophoblast differentiation using equine chorionic girdle vesicles.

The chorionic girdle of the equine conceptus is comprised of specialized trophoblast cells which, at day 36-38 of equine pregnancy, gain an invasive phenotype and invade the endometrium to form endometrial cups. Studies of equine endometrial cups remain difficult to perform because of the invasive techniques required to obtain cup tissue and because sampling requires termination of the pregnancy. In this study we developed a system to model trophoblast differentiation and trophoblast-immune interactions in vitro and in vivo. We utilized a method of culturing chorionic girdle pieces in serum-free medium to promote spontaneous formation of vesicle structures enriched for terminally differentiated binucleate cells that secreted equine chorionic gonadotrophin (eCG). Immunohistochemical staining and scanning electron microscopy showed that the cells of the vesicles closely resembled the outer layers of chorionic girdle immediately prior to invasion. Chorionic girdle vesicles were harvested after 72h in culture and ectopically transplanted via injection into the vulvar mucosa of recipient mares. At 7, 14, 21 and 28days after transplantation, biopsies of the injection sites were obtained. Immunohistochemical labeling of cryostat sections of the biopsies with a panel of monoclonal antibodies to horse trophoblast molecules demonstrated survival, differentiation, and presence of trophoblast cells for at least 21days. Serial sections of the biopsies labeled with antibodies to the equine lymphocyte surface markers CD4 and CD8, together with lymphocyte microcytotoxicity assays, revealed that the recipients mounted both cellular and humoral antibody immune responses to the transplanted trophoblast cells. This new method for culturing equine chorionic girdle trophoblast cells, and for transplanting trophoblast vesicles to ectopic sites, should allow identification of key aspects of trophoblast differentiation and the interactions that occur between invasive trophoblast and the maternal immune system.

Report of the 3rd Havemeyer workshop on allergic diseases of the Horse, Hólar, Iceland, June 2007.

Allergic diseases occur in most mammals, although some species such as humans, dogs and horses seem to be more prone to develop allergies than others. In horses, insect bite hypersensitivity (IBH), an allergic dermatitis caused by bites of midges, and recurrent airway obstruction (RAO), a hyperreactivity to stable born dust and allergens, are the two most prevalent allergic diseases. Allergic diseases involve the interaction of three major factors: (i) genetic constitution, (ii) exposure to allergens, and (iii) a dysregulation of the immune response determined by (i) and (ii). However, other environmental factors such as infectious diseases, contact with endotoxin and degree of infestation with endoparasites have been shown to influence the prevalence of allergic diseases in humans. How these factors may impact upon allergic disease in the horse is unknown at this time. The 3rd workshop on Allergic Diseases of the Horse, with major sponsorship from the Havemeyer Foundation, was held in Hólar, Iceland, in June 2007 and focussed on immunological and genetic aspects of IBH and RAO. This particular venue was chosen because of the prevalence of IBH in exported Icelandic horses. The incidence of IBH is significantly different between Icelandic horses born in Europe or North America and those born in Iceland and exported as adults. Although the genetic factors and allergens are the same, exported adult horses show a greater incidence of IBH. This suggests that environmental or epigenetic factors may contribute to this response. This report summarizes the present state of knowledge and summarizes important issues discussed at the workshop.

The effect of skin allografting on the equine endometrial cup reaction.

This research tested the hypothesis that immunological sensitization of mares by skin allografting, followed by the establishment of pregnancy using semen from the skin-graft donor, would give rise to secondary immune responses to the developing horse conceptus, resulting in an earlier demise of the fetally derived endometrial cups. Maiden mares received skin allografts from a stallion homozygous for Major Histocompatibility Complex (MHC) antigens and/or equivalent autografts and were subsequently mated to the skin-graft donor stallion during the next two breeding seasons. Mares that had been immunologically primed to the foreign MHC class I antigens of the skin-graft donor stallion developed strong secondary antibody responses early in their first pregnancies, whereas autografted mares made weak primary antibody responses in their first pregnancies and strong secondary responses in their second pregnancies. In contrast, histological examination of the endometrial cups after surgical pregnancy termination at Day 60 of gestation revealed no discernible differences between allografted and autografted mares, and there were no significant differences in the concentrations and/or duration of secretion of the endometrial cup-specific hormone, equine chorionic gonadotrophin (eCG), between allografted and autografted mares, nor in either group between first and second pregnancies. The vigorous antibody response observed in the pregnant allografted mares supported the first part of our hypothesis, providing evidence of systemic immunological priming. However, there was a lack of an equivalent heightened cellular response to the endometrial cups. These findings provided strong evidence for an asymmetric immune response to the conceptus, characterized by strong humoral immunity and a dampened cellular response.

Report of the equine herpesvirus-1 Havermeyer Workshop, San Gimignano, Tuscany, June 2004.

Amongst the infectious diseases that threaten equine health, herpesviral infections remain a world wide cause of serious morbidity and mortality. Equine herpesvirus-1 infection is the most important pathogen, causing an array of disorders including epidemic respiratory disease abortion, neonatal foal death, myeloencephalopathy and chorioretinopathy. Despite intense scientific investigation, extensive use of vaccination, and established codes of practice for control of disease outbreaks, infection and disease remain common. While equine herpesvirus-1 infection remains a daunting challenge for immunoprophylaxis, many critical advances in equine immunology have resulted in studies of this virus, particularly related to MHC-restricted cytotoxicity in the horse. A workshop was convened in San Gimignano, Tuscany, Italy in June 2004, to bring together clinical and basic researchers in the field of equine herpesvirus-1 study to discuss the latest advances and future prospects for improving our understanding of these diseases, and equine immunity to herpesviral infection. This report highlights the new information that was the focus of this workshop, and is intended to summarize this material and identify the critical questions in the field.

Donor-derived equine mesenchymal stem cells suppress proliferation of mismatched lymphocytes.

REASONS FOR PERFORMING STUDY: Recently, it has been shown that mesenchymal stem cells (MSCs) do not express the major histocompatibility complex (MHC) II antigen and are able to inhibit proliferation of MHC-mismatched stimulated lymphocytes, enabling their use as in vivo allogeneic transplants. However, prior to clinical application of allo-MSCs, in vitro tests are required to confirm the safety of treatment protocols. OBJECTIVES: To evaluate the immunosuppressive capabilities of equine bone-marrow-derived MSCs (BM-MSCs) on MHC-mismatched lymphocytes. STUDY DESIGN: In vitro experiment. METHODS: Phytohaemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) from 3 Thoroughbreds (recipients) were co-cultured with mismatched BM-MSCs from 3 Connemara ponies (donors). Proliferation of lymphocytes was monitored by carboxyfluorescein succinimidyl ester labelling and analysed by flow cytometry. In total, 6 horses were haplotyped using microsatellites to confirm mismatching. Optimisation of the conditions to stimulate Thoroughbred lymphocytes and titration of equine anti-CD4 and anti-CD8 antibodies were performed. Connemara pony and Thoroughbred BM-MSCs were isolated, expanded and characterised by tri-lineage differentiation. Finally, BM-MSCs from both breeds were set up in co-culture at different ratios with stimulated Thoroughbred lymphocytes. Proliferation of CD4(+) and CD8(+) cells was determined by flow cytometry. RESULTS: A high proportion of CD4/CD8 double-positive lymphocytes were found in freshly isolated PBMCs, although this percentage decreased after 4 days of culture. Mismatched BM-MSCs inhibited proliferation of stimulated lymphocytes in a dose-dependent manner, with the greatest suppression occurring at a 1:10 ratio of BM-MSCs to PBMCs. Proliferation of CD4(+) and CD8(+) subpopulations decreased in 1:10 co-culture, with statistical significance in the case of CD8(+) cells, while that of the CD4/CD8 double-positive population was similar to the phytohaemagglutinin control. CONCLUSIONS: The results demonstrate dose-dependent immunosuppression of stimulated lymphocytes by mismatched equine BM-MSCs, supporting their future application in allo-MSC clinical treatments.

Inhibition of lymphocyte proliferation and activation: a mechanism used by equine invasive trophoblast to escape the maternal immune response.

At days 36-38 of gestation, the equine invasive trophoblast cells migrate into the endometrium of the pregnant mare to form the endometrial cups. During their migration, they become surrounded by maternal CD4+ and CD8+ T lymphocytes, and stimulate a cytotoxic antibody response to the paternal major histocompatibility complex class I antigens that they express. Nevertheless, endometrial cup cells remain viable at the site of uterine invasion up to days 80-100 of gestation, suggesting the participation of immunomodulatory mechanisms to the maternal cellular immune response. To determine the effects of the invasive trophoblast cells on lymphocyte proliferation, an in vitro co-culture system was developed using isolated equine invasive trophoblast cells and peripheral blood lymphocytes. Fetal fibroblast cells from the same conceptuses were used as controls. The presence of invasive trophoblast cells or their pre-conditioned medium inhibited 50% or more of lymphocyte proliferation, while fetal fibroblasts had no effect. The invasive trophoblast cell inhibitory factor needed to be present constantly to affect lymphocyte proliferation, and it was ineffective if lymphocytes had been previously stimulated to proliferate. The lymphoproliferative inhibitory mechanism affected lymphocyte subpopulations similarly. In addition, lymphocyte expression of cytokine mRNA including IFNgamma, IL-2, IL-4, and IL-10 was affected compared to controls. The implication of these observations in vivo may explain, in part, the apparent equine maternal immune acceptance of the presence and development of endometrial cup cells.

The complete cDNA and deduced amino acid sequence of equine IgE.

The cDNA from a transcript encoding the complete heavy chain of the equine immunoglobulin IgE has been cloned and sequenced. A fragment of the equine epsilon gene was amplified from cDNA using PCR and this fragment was then used to probe a horse cDNA library prepared from peripheral blood lymphocytes. A recombinant clone containing the cDNA encoding the complete horse epsilon chain and its associated V-D-J and leader, was subsequently isolated and sequenced. Comparison of the deduced amino acid sequence of equine IgE with the C epsilon heavy chains of other species indicates it to be most similar to human C epsilon (54%), followed by sheep C epsilon (52%); the greatest sequence similarity was found in the C epsilon 3 and C epsilon 4 domains among the species compared. The associated V-region had greatest similarity in FR1 to those reported for sheep and the cow (> 80%) but less than 60% to the VH sequences of other species in FR3.

An ordered BAC contig map of the equine major histocompatibility complex.

A physical map of ordered bacterial artificial chromosome (BAC) clones was constructed to determine the genetic organization of the horse major histocompatibility complex. Human, cattle, pig, mouse, and rat MHC gene sequences were compared to identify highly conserved regions which served as source templates for the design of overgo primers. Thirty-five overgo probes were designed from 24 genes and used for hybridization screening of the equine USDA CHORI 241 BAC library. Two hundred thirty-eight BAC clones were assembled into two contigs spanning the horse MHC region. The first contig contains the MHC class II region and was reduced to a minimum tiling path of nine BAC clones that span approximately 800 kb and contain at least 20 genes. A minimum tiling path of a second contig containing the class III/I region is comprised of 14 BAC clones that span approximately 1.6 Mb and contain at least 34 genes. Fluorescence in situ hybridization (FISH) using representative clones from each of the three regions of the MHC localized the contigs onto ECA20q21 and oriented the regions relative to one another and the centromere. Dual-colored FISH revealed that the class I region is proximal to the centromere, the class II region is distal, and the class III region is located between class I and II. These data indicate that the equine MHC is a single gene-dense region similar in structure and organization to the human MHC and is not disrupted as in ruminants and pigs.

Characterization of equine natural killer and IL-2 stimulated lymphokine activated killer cell populations.

Natural killer (NK) cells are an important component of the innate immune system. Though intensively studied in humans and rodents. NK cells remain less well characterized in other species. Studies are often limited by the lack of specific cell markers; however, the mAb NK-5C6 has been suggested to recognize an evolutionarily conserved molecule on NK cells and reacts with cells from several species. This mAb was used in the current investigation to identify and characterize equine NK cells, and was found to label approximately 10% of peripheral blood lymphocytes (PBL). Two-color flow cytometry analysis identified the NK-5C6+ cell population as being CD3-CD4- and CD8-, but positive for MHC class I and LFA-1 expression. Depletion of CD3+ T cells increased the percent NK-5C6+ cells in PBL; this enriched population demonstrated a specific cytotoxic response against a major histocompatibility complex (MHC) deficient NK target cell line (K-562), but not MHC+ target cells (EqT8888). These results provide evidence for an equine NK cell population, which exhibits endogenous lytic activity and a phenotype similar to that of human and mouse NK cells. Stimulation of peripheral blood mononuclear cells (PBMC) with IL-2 promoted the development of LAK cells. These cells were predominantly CD3+ T cells, demonstrated intracellular perforin expression, and effectively lysed both K-562 and EqT8888 target cells. Hence, equine NK cells can be identified by the NK-5C6 mAb and distinguished from IL-2 stimulated LAK cells by their cytotoxic response to specific target cell lines.

Immortalization of equine trophoblast cell lines of chorionic girdle cell lineage by simian virus-40 large T antigen.

Immortalized cell lines have many potential experimental applications including the analysis of molecular mechanisms underlying cell-specific gene expression. We have utilized a recombinant retrovirus encoding the simian virus-40 (SV-40) large T antigen to construct several immortalized cell lines of equine chorionic girdle cell lineage - the progenitor cells that differentiate into the equine chorionic gonadotropin (eCG) producing endometrial cups. Morphologically, the immortalized cell lines appear similar to normal chorionic girdle cells. Derivation of the immortalized cell lines from a chorionic girdle cell lineage was verified by immunological detection of cell-surface antigens specific to equine invasive trophoblasts. The cell lines differed, however, from mature chorionic girdle cells or endometrial cup cells in that they did not produce eCG and did express MHC class I molecules. Thus, these cell lines appear to have been arrested at a stage of development prior to final differentiation into endometrial cup cells. It was also determined that some of these cell lines as well as endometrial cups express the estrogen receptor-related receptor beta gene, but not the glial cell missing gene (GCMa) both of which are expressed in the murine and human placenta. Among these cell lines, three (eCG 50.5, 100.6 and 500.1) express eCG alpha mRNA. Since regulation of eCG alpha subunit gene is largely unknown, we investigated the signal transduction pathways regulating the eCG alpha subunit gene. Both activators of protein kinase A (PKA) and protein kinase C (PKC) induced the expression of eCG alpha subunit expression 3.2 (P<0.05)- and 1.9 (P<0.05)-fold respectively, in the eCG 500.1 cell line. However, activation of these pathways failed to induce eCG beta subunit expression. In conclusion, lines of equine trophoblast cells have been immortalized that display markers characteristic of those with the equine chorionic girdle and endometrial cup cell lineage. A subset of these cells expresses the eCG alpha subunit gene which is responsive to activators of the PKA and PKC signal transduction pathways.

Paternal and maternal major histocompatibility complex class I antigens are expressed co-dominantly by equine trophoblast.

Invasive equine trophoblast cells of the chorionic girdle express high levels of paternally inherited Major Histocompatibility Complex (MHC) class I antigens prior to migration into the endometrium to form the so-called endometrial cups. Three groups of experiments were performed to determine if maternally inherited MHC class I antigens are expressed on chorionic girdle cells. Results indicated that maternally and paternally inherited MHC class I antigens are co-dominantly expressed by cells of the invasive equine trophoblast, and therefore, that the expression of polymorphic equine MHC class I genes does not appear to be affected by genomic imprinting in this tissue. The demonstration that cells of the chorionic girdle were immunogenic supports the hypothesis that invasion of the maternal endometrium by chorionic girdle cells stimulates the production of anti-paternal alloantibodies normally observed in early horse pregnancy. The co-dominant expression of MHC class I antigens by invasive chorionic girdle cells has important implications for the mechanism of recognition of allogeneic fetal MHC class I antigens by the maternal immune system.

The maternal leucocyte response to the endometrial cups in horses is correlated with the developmental stages of the invasive trophoblast cells.

Invading trophoblasts form endometrial cups in the endometrium of the pregnant mare. In the present study we characterized the maternal leucocyte response to endometrial cups from their formation to their regression. The maternal leucocyte response was correlated with the stages of trophoblast development. (1) Aggregates of CD4+ and CD8+ cells were present between the migrating and differentiating endometrial cup trophoblasts and surrounding the forming endometrial cups. (2) Numbers of CD4+ cells within the mature endometrial cups were much reduced. At the periphery of the endometrial cups CD4+ and CD8+ cells were found in patchy accumulations around endometrial glands; small clusters of CD79+ B lymphocytes were present as well. (3) Scattered CD4+ and CD8+ cells were found within dying endometrial cups; areas of cell death were infiltrated with neutrophils. Large aggregates of CD4+ cells and CD8+ cells, and small but numerous clusters of CD79+ cells and eosinophils, were found outside of the dying endometrial cups. The CD4+ or CD8+ cells were mostly CD3+ T cells; some were probably macrophages which can express both of these markers in horses. The correlation between the developmental stages of the endometrial cup trophoblast and the maternal leucocyte response suggests a complicated cytokine-mediated regulatory network.

Analysis of MHC class I expression in equine trophoblast cells using in situ hybridization.

Down-regulation of major histocompatibility complex (MHC) genes by trophoblast cells is considered to be a primary mechanism preventing maternal immune rejection of the fetal-placental unit in mammalian pregnancy by rendering these cells, which form the primary barrier between mother and fetus, relatively non-antigenic. In situ hybridization with probes encoding human and horse MHC class I genes was used to characterize the pattern of MHC class I mRNA expression in the various forms of horse trophoblast. Strong hybridization signals were observed in the invasive trophoblast cells of chorionic girdle tissue. In contrast, no hybridization signal specific for MHC class I mRNA transcripts was observed in the descendent endometrial cup trophoblast cells. In the non-invasive trophoblast cells of the allantochorion, no hybridization signals specific for horse MHC class I mRNA transcripts were consistently detected. In parallel to the in vivo results, strong hybridization signals were observed in the small, mononuclear cells present in chorionic girdle cell explant cultures, but not in the population of large binucleate cells corresponding to endometrial cup cells. The results obtained using in situ hybridization are consistent with the hypothesis that expression of MHC class I genes may be controlled at the transcriptional level in horse invasive and non-invasive trophoblast cells, and suggest that down-regulation of MHC class I antigen expression in endometrial cup cells may be accomplished by the same mechanisms in vivo and in vitro.

Lymphocyte alloantigens of the horse. II. Antibodies to ELA antigens produced during equine pregnancy.

Evidence is presented for a reproducible maternal immune response to histocompatibility antigens during equine pregnancy. Mares were stimulated as a result of pregnancy to produce cytotoxic antibodies to paternal lymphocyte alloantigens. The majority of these antibodies were directed against antigens of the equine lymphocyte antigen (ELA) system, which is the major histocompatibility complex (MHC) of the horse. In 16 experimental pregnancies produced using 12 mares and 4 stallions which had been typed for ELA antigens, there was correlation between ELA incompatibility between sire and dam and the appearance of antibody in the serum of the dam as a result of pregnancy. In ELA incompatible pregnancies, antibody was first detected very early in pregnancy, usually by day 60 in a 336-day gestation. The appearance of antibody following shortly the development of the endometrial cups, which can be seen macroscopically for the first time between days 38 and 40. The strength (titre) of the cytotoxic antibody response appeared to vary with the paternal ELA antigens expressed by the fetus. Circulating antibody to 'third party' alloantigens generated during prior pregnancies did not alter the kinetics of the appearance of antibody in a current pregnancy. Evidence for an anamnestic response was obtained in second pregnancies in which the histocompatibility difference between mare and fetus was the same as in the first pregnancy. However, this accelerated response did not occur before the time of formation of the endometrial cups. Serum samples from approximately 90% of parturient mares on horse farms in New York State contained antibody reactive with paternal lymphocyte alloantigens.