Identification of endothelin-1, endothelin-2 and endothelin-3 in human endometrium.

This study determined the presence of specific endothelin isoforms in human endometrium using high performance liquid chromatography (HPLC) combined with radioimmunoassay, and immunocytochemistry to detect the endothelin precursors (proendothelin-1, proendothelin-2 and proendothelin-3). Endothelin-like immunoreactivity was measured in HPLC eluates using antisera raised in rabbits against the carboxy-terminal heptapeptide of endothelin-1, which is common to the three endothelin isoforms. Of eight endometrial samples analysed by HPLC, three were in the proliferative phase of the cycle, and five in the secretory phase. Endothelin-1 was detected in seven samples, whereas endothelin-2 and endothelin-3 were seen in four and five specimens, respectively. No relationship was seen between endothelin isoforms and the stage of the cycle. Immunocytochemistry was performed on five proliferative and three secretory phase tissues. When present, staining for the precursor proendothelins was localized to endometrial glandular and luminal epithelium (proendothelin-1, 5 of 8; proendothelin-2, 5 of 8; proendothelin-3, 6 of 8). In two sections, staining was also seen in vascular endothelium using antibody raised against proendothelin-1 (n = 1) and proendothelin-3 (n = 1). These data provide evidence that the three endothelin isoforms are present in human endometrium, and suggest that these potent vasoactive agents may play a role in the paracrine control of the uterine vascular bed.

Characterization of endothelin isoforms in human heart: endothelin-2 demonstrated.

In humans, three endothelin (ET) isoforms are predicted to exist by analysis of genomic DNA. However, evidence for the presence of all three mature ET peptides and their precursors remains unclear. Our aim was to identify the ET isoforms present in human heart, using radioimmunoassay (RIA) and reverse-phase high-performance liquid chromatography (RP-HPLC). Antisera raised against the ET-1[15-21] terminal sequence were specific for mature ETs, showing no cross-reactivity with their precursor pro-ETs. Antisera raised against the pro-ET-1[31-38] terminal sequence was specific for pro-ET-1, showing no cross-reactivity with other ET peptides. In extracts of human cardiac tissues, the concentrations of immunoreactive (IR) mature ET and pro-ET-1 were found to be as follows: left atrium (n = 3): 282.3 +/- 113.0, 21.9 +/- 11.0, respectively; right atrium (n = 5): 308.3 +/- 95.4, 43.1 +/- 12.8, respectively; left ventricle (n = 6): 218.5 +/- 64.6, 47.9 +/- 11.9, respectively; right ventricle (n = 4): 215.1 +/- 79.8, 53.9 +/- 13.0, respectively (fmol/g wet weight, mean +/- SEM, for total IR mature ET and pro-ET-1, respectively). RP-HPLC showed peaks of immunoreactivity that coeluted with authentic ET-1 in all extracts of human left atria and ventricle tested. In addition, peaks were also present corresponding to ET-2, ET-3, and pro-ET-1. These results suggest that in addition to ET-1 and pro-ET-1, ET-2 and ET-3 are present in the human heart.

Immunomodulation of dog islets using a cocktail of monoclonal antibodies.

Islet allografts are particularly vulnerable to rejection, and current immunosuppressive agents are deleterious to their function. They are, however, highly suitable for 'immunomodulation', i.e., the removal or inactivation of passenger leukocytes to reduce their immunogenicity. For this purpose we have used 3 rat anti-dog monoclonal antibodies (Mabs) which are synergistic for leukocytolysis in the presence of autologous dog serum. Spleen cells or purified islets treated with these Mabs together with autologous serum were tested in mixed leukocyte and islet co-culture assays. The stimulatory properties of the Mab-pretreated splenocytes or islets were markedly reduced; moreover, the Mab cytolytic activity was shown to be confined to the leukocyte target cells and did not affect islet secretory function upon glucose stimulation. We conclude that this method of modifying the immunogenicity of dog islets could lead to successful islet grafting in vivo, allowing the reduction of conventional immunosuppression. Successful in vivo studies in this model, which are currently in progress, could have implications for clinical islet transplantation.