Preparation and characterization of polyclonal antibodies against human chaperonin 10.

Early pregnancy factor (EPF) has been identified as an extracellular homologue of chaperonin 10 (Cpn10), a heat shock protein that functions within the cell as a molecular chaperone. Here, we report the production of polyclonal antibodies directed against several different regions of the human Cpn10 molecule and their application to specific protein quantitation and localization techniques. These antibodies will be valuable tools in further studies to elucidate the mechanisms underlying the differential spatial and temporal localization of EPF and Cpn10 and in studies to elucidate structure and function.

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.

Oestrogen receptor assay in primary breast cancer: clinical validation and prognostic value.

The results of a cytoplasmic oestrogen receptor assay in 134 cases of primary breast cancer are reported. Overall, 57% of primary cancers tested were oestrogen-receptor 'positive' (ER+) and attention is drawn to the need for careful initial handling of tumour tissue, to ensure an accurate assay result. ER+ tumours occurred more often and showed higher assay values in postmenopausal compared with premenopausal women and nine of 12 ER+ tumours responded to hormone manipulation when disease recurred. Follow-up of primary disease confirmed that women with ER+ tumours have a better prognosis with a lower risk of early recurrence than those with ER-tumours. It is suggested that clinical validation of the oestrogen receptor assay should precede routine clinical use.

Early pregnancy factor in liver regeneration after partial hepatectomy in rats: relationship with chaperonin 10.

Early pregnancy factor is not only a product of dividing embryonic and neoplastic cells, as demonstrated previously, but also of normal proliferating cells. Eight hours after partial hepatectomy in rats, early pregnancy factor was detected in serum. It rose to a peak by 48 hr. Neutralization of early pregnancy factor in vivo by passive immunization with specific antibodies, 18 hr after partial hepatectomy, resulted in a significant decrease in the uptake of [3H]thymidine by the liver remnant, measured 4 to 6 hr later. These results suggest that during liver regeneration, early pregnancy factor is essential to the sequence of events that culminates in DNA synthesis and cell division. Recently we purified early pregnancy factor from human platelets and determined by mass spectrometry a precise molecular mass of 10,843 Da. Amino acid sequencing (approximately 72% of the molecule) demonstrated that early pregnancy factor is highly homologous with chaperonin 10, a stress-inducible mitochondrial protein, and that platelet-derived early pregnancy factor and rat chaperonin 10 share similar biochemical and immunological properties. In this study we show that early pregnancy factor, purified from regenerating rat liver and from serum taken 24 hr after hepatectomy, shares these properties. In addition, antibodies to early pregnancy factor, effective in passive immunization studies, recognize chaperonin 10, whereas chaperonin 10 antibodies bind to early pregnancy factor from regenerating liver and posthepatectomy serum. We propose that early pregnancy factor/chaperonin 10 is selectively released from proliferating cells and, in an autocrine or paracrine mode (or both) is involved in DNA synthesis.