Halle Morton

AN EARLY PREGNANCY FACTOR DETECTED IN HUMAN SERUM BY THE ROSETTE INHIBITION TEST

Modification of maternal lymphocyte activity has been demonstrated early in pregnancy by the rosette inhibition test. Normal human lymphocytes showed a similar depression of activity after incubation in serum from pregnant women, indicating that the response was caused by a serum factor. This early pregnancy factor has been differentiated from other substances which appear later in pregnancy and which may also be involved in the suppression of the maternal response. The results of this investigation suggest that the early pregnancy factor may be necessary for the continued viability of the early embryo.

Early pregnancy factor: An extracellular chaperonin 10 homologue

Early pregnancy factor (EPF) has been identified as a homologue of chaperonin 10 (cpn10) with immunosuppressive and growth factor properties. As a homologue of cpn10, it belongs to the heat shock family of proteins (hsp) but, unlike other members of this family, EPF is detected extracellularly. Early pregnancy factor was first discovered in pregnancy serum by the rosette inhibition test, and the novelty of its discovery was that its presence could diagnose pregnancy within 6–24 h of a fertile mating. As well as being a monitor of the presence of a viable embryo, it is necessary for embryonic survival. In this capacity it acts as both an immunosuppressant and growth factor. Early pregnancy factor is also a product of proliferating primary and neoplastic cells and functions as an autocrine growth factor both in vivo and in vitro. It has a modifying effect on the outcome of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Early pregnancy factor is considered to be one of the major factors involved in the modification of multiple sclerosis observed during pregnancy.

Ovum Factor: A First Signal of Pregnancy?

ABSTRACT: Previous studies have shown that, in the mouse, a factor is produced by the fertilized ovum within 24 h of mating. It cooperates with prolactin to stimulate ovarian production of component B of early pregnancy factor (EPF). This paper presents an initial characterization of the substance, termed ovum facor (OF).

Monoclonal antibodies to early pregnancy factor perturb tumour cell growth

The pregnancy‐associated substance early pregnancy factor (EPF) has previously been reported as a product of tumours of germ cell origin. More recently EPF (or an EPF‐related substance, tEPF) has also been detected in the serum of patients bearing tumours of non‐germ cell origin. We report here the production of tEPF by a variety of cultured transformed and tumour cell lines, of both germ and non‐germ cell origin. Antibodies specific for EPF remove all tEPF activity from tumour cell conditioned medium, tEPF production is found to be associated with cell division; tEPF is no longer detected after growth arrest or differentiation. Co‐culture of tumour cells with increasing doses of anti‐EPF monoclonal antibodies resulted in a significant, dose‐dependent decrease in rate of cell growth and viability. Similar anti‐EPF concentrations had no effect on the concanavalin A induced proliferation of mouse spleen cells. These studies suggest, therefore, that tEPF is a growth‐regulated product of cultured tumour and transformed cells. These cells are also dependent upon tEPF for continued growth, i.e. tEPF is acting in the autocrine mode.

Early pregnancy factor has immunosuppressive and growth factor properties

Early pregnancy factor (EPF) was first described as a pregnancy-associated substance, although recent studies suggest a more general link with cell development. It is a product of actively dividing cells and its apparent functional importance to them suggests its potential as a regulator of cell proliferation. The recent discovery of EPF in platelets has provided a comparatively rich and readily available source of EPF. The purification procedures employed to isolate EPF from this source have also been applied to pregnancy serum and urine, medium conditioned by oestrous mouse ovaries (stimulated with prolactin and embryo-conditioned medium), medium conditioned by tumour cells, and serum from rats 24 h after partial hepatectomy (PH). In all instances, biological activity followed the same pattern throughout. Furthermore, the final active reversed-phase high-performance liquid chromatography fraction from all sources was bound specifically by immobilized anti-EPF monoclonal antibodies (MAbs), indicating that the active fractions produced from these diverse sources are very closely related, if not identical. Some differences have been observed in the behaviour of EPF in various conditions. EPF is produced by proliferating tumour cells and by liver cells post-PH, and passive immunization studies with anti-EPF MAbs have shown that these cells need EPF for survival. In contrast, EPF has not been detected as a product of the pre-embryo, and addition of anti-EPF MAbs to embryo cultures does not adversely affect development from the 2-cell to the blastocyst stage. Although the pre-embryo is not dependent on EPF for its development in vitro, neutralization of EPF in vivo by anti-EPF MAbs retards its development. Thus, EPF appears to play an indirect role in maintaining the pre-embryo. By virtue of its ability to suppress the delayed-type hypersensitivity reaction, it has been suggested that EPF might act as an immunological response modifier of the maternal immune system. Alternatively, the effect of EPF on lymphocytes may be to reduce the expression of all or some cytokines and this could inhibit development. Whether or not EPF acts more directly as an autocrine growth factor from around the time of implantation, when the embryo first begins synthesis of EPF, is not known and remains to be investigated.

Ovum Factor and Early-Pregnancy Factor

Publisher Summary This chapter describes experimental evidences, which shows that production of early pregnancy factor (EPF) is initiated by fertilization and that its presence in serum monitors the viability of the conceptus for at least the first half of gestation. Furthermore, EPF is necessary for the continued survival of the conceptus. The tissues involved in the production of EPF are discussed and the role of EPF in the maintenance of the embryo is also considered. The role of EPF in maintaining embryonic viability may involve immunological protection. The characteristics of EPF meet the criteria needed for an effective immunosuppressant capable of inhibiting maternal rejection of the embryo. Immunomodulation begins within hours of fertilization and is reversible within 24 hours of loss of the embryo. The effect of EPF is selective; it binds to a specific lymphocyte population, recruiting suppressor cells that in turn release soluble suppressor factors, genetically restricted in their behavior. The target cells affected by these suppressor factors are of the same T-lymphocyte population as those involved in graft rejection. The discovery of EPF and the ensuing research has depended on the activity of EPF in the rosette inhibition test.

Early pregnancy factor treatment suppresses the inflammatory response and adhesion molecule expression in the spinal cord of SJL/J mice with experimental autoimmune encephalomyelitis and the delayed-type hypersensitivity reaction to trinitrochlorobenzene in normal BALB/c mice

Early pregnancy factor (EPF) is a secreted protein, present in serum during early pregnancy and essential for maintaining viability of the embryo. It is a homologue of chaperonin 10 (Cpn10) but, unlike Cpn10, it has an extracellular role. EPF has immunosuppressive and growth regulatory properties. Previously we have reported the preparation of recombinant EPF (rEPF) and shown that treatment with rEPF will suppress clinical signs of MBP-EAE in Lewis rats and PLP-EAE in SJL/J mice. In the present study, these findings have been extended to investigate possible mechanisms involved in the action of EPF. Following treatment of mice with rEPF from the day of inoculation, there were fewer infiltrating CD3+ and CD4+ cells in the parenchyma of the spinal cord during the onset of disease and after the initial episode, compared with mice treated with vehicle. Expression of the integrins LFA-1, VLA-4 and Mac-1 and of members of the immunoglobulin superfamily of adhesion molecules ICAM-1 and VCAM-1 was suppressed in the central nervous system (CNS) following rEPF treatment. The expression of PECAM-1 was not affected. To determine if rEPF suppressed T cell activation in the periphery, the delayed-type hypersensitivity (DTH) reaction of normal BALB/c mice to trinitrochlorobenzene (TNCB) following treatment with rEPF was studied. The results showed that treatment with rEPF suppressed the DTH reaction, demonstrating the ability of EPF to downregulate the cell-mediated immune response. These results indicate that suppression of immunological mechanisms by rEPF plays a major role in the reduction of clinical signs of disease in experimental autoimmune encephalomyelitis (EAE).

Early pregnancy factor suppresses experimental autoimmune encephalomyelitis induced in Lewis rats with myelin basic protein and in SJL/J mice with myelin proteolipid protein peptide 139-151

Early pregnancy factor (EPF) is a secreted protein with immunosuppressive and growth factor properties. During pregnancy, it appears in maternal serum within 6-24 h of fertilization, is present for at least the first two-thirds of pregnancy in all species studied and is essential for embryonic survival. It is a homologue of chaperonin 10, a heat shock protein, but, unlike other members of this family, EPF has an extracellular role. As it has the ability to modulate CD4+ T cell-dependent immune responses, its role in treatment of experimental autoimmune encephalomyelitis (EAE) was investigated. EAE is a CD4+ T cell-mediated disease, the best available animal model of multiple sclerosis (MS). Two models of EAE were investigated, acute EAE induced in Lewis rats by inoculation with myelin basic protein (MBP-EAE) and chronic relapsing EAE induced in SJL/J mice by inoculation with myelin proteolipid protein peptide (residues 139-151) (PLP-EAE). EPF, delivered intraperitoneally or orally to rats or intraperitoneally to mice, suppressed clinical signs of disease. Mice with PLP-EAE were also treated with interferon-beta, with and without EPF. Both EPF and IFN-beta suppressed clinical signs of EAE and, when administered together, gave greater suppression than when given separately. These findings suggest that EPF may be a potential candidate for use in treatment of MS and may be of use in combined therapy with IFN-beta.

Rosette inhibition test: A multicentre investigation of early pregnancy factor in humans

The rosette inhibition test for the detection of early pregnancy factor is described in detail. The extended methodology presented here represents the cumulative experience of three independent laboratories. Special reference is made to the effect on the assay of varying the conditions of rosette formation between lymphocytes and sheep red blood cells. Antilymphocyte sera prepared for use in the rosette inhibition test fell into three categories: (i) with no rosette inhibiting activity, (ii) with rosette inhibiting activity which is not affected by the presence of EPF, and (iii) rosette inhibiting activity which is significantly increased in the presence of EPF. To date, this third reaction has been found to be a specific indication of the presence in serum of early pregnancy factor.

A protective effect of early pregnancy factor on experimental autoimmune encephalomyelitis induced in Lewis rats by inoculation with myelin basic protein

Experimental autoimmune encephalomyelitis (EAE) is an organ-specific autoimmune disease characterised by inflammation and demyelination of the central nervous system and is the best available animal model of multiple sclerosis (MS). Since previous studies have shown that EAE is less severe or is delayed in onset during pregnancy and that administration of the pregnancy hormone early pregnancy factor (EPF) down-regulates EAE, experiments in the present study were designed to explore further the role of EPF in EAE. By using the rosette inhibition test, the standard bioassay for EPF and, by semi-quantitative RT-PCR techniques, we have now shown that inflammatory cells from the spinal cord of rats with EAE can produce and secrete EPF, with production being greatest during recovery from disease. Administration of EPF to rats with EAE resulted in a significant increase in the expression of IL-4 and IL-10 mRNA and a significant decrease in IFN-gamma mRNA expression in spinal cord inflammatory cells. Encephalitogenic MBP-specific T cell lines were prepared from popliteal lymph nodes of rats with EAE. Proliferation assays using these cells demonstrated the ability of exogenous EPF to down-regulate the responses of T lymphocytes to MBP.