Model for experimental revascularized laryngeal allotransplantation.

BACKGROUND: Although a human laryngeal transplant has been undertaken successfully, important questions remain that require a suitable animal model. METHODS: A pig model for allotransplantation has been developed. Organ perfusion was studied in nine animals before four transplants were performed in congenic (unrecovered) animals and eight in unmatched (recovered) animals. Larynges were regularly examined endoscopically until death at 14 days. Immunosuppression included the use of tacrolimus. Revascularization was achieved by anastomosing the donor right cervical vascular tree to the recipient common carotid. In recovered animals, four allografts were placed orthotopically and four heterotopically. RESULTS: The pig larynx was perfused adequately via the right cervical vascular tree and congenic grafts were well tolerated. Of eight allografts, seven were well tolerated and remained healthy for the duration of the study (14 days). One allograft became infected between days 4 and 7 after operation. Median operating time was 6 h, with a median cold ischaemia time of 3 h. CONCLUSION: Revascularized allotransplants of the larynx can be undertaken reliably in pigs and this provides a preclinical model for studies of laryngeal transplantation.

Differential expression and immunohistochemical localisation of the phenol and hydroxysteroid sulphotransferase enzyme families in the developing lung.

Reversible sulphation, catalysed by sulphotransferases and sulphatases, of biologically active compounds such as androgens and oestrogens is a sensitive mechanism for regulating their bioavailability, and we have previously hypothesised that this process plays a significant role in the regulation of human fetal lung development. Sulphation is also a major detoxification reaction, contributing significantly to the body's chemical defence mechanism. We have used qualitative and semiquantitative immunological studies to determine the temporal expression and localisation of phenol and hydroxysteroid sulphotransferases during human lung development. Our results show that in the early fetal lung, phenol sulphotransferase expression is at its highest, and is most widely distributed throughout the developing respiratory epithelium. With later development, expression levels decrease and become predominantly restricted to the more proximal airways. In contrast, hydroxysteroid sulphotransferase is present only at very low levels in the early-gestation lung but expression increases rapidly through gestation to reach an apparent peak by 1 year postnatal age. The proximal-to-distal gradients of phenol and hydroxysteroid sulphotransferase expression were similar in mature respiratory epithelium, with immunoreactivity in ciliated cells, non-ciliated secretory cells and basal cells, but with no apparent expression in mucus-secreting cells. These studies provide supporting evidence for the hypothesis that hydroxysteroid sulphotransferase, an androgen-inactivating enzyme, contributes to the role of androgens in retarding the maturation of human lung in utero.

Sulfation of endogenous compounds and xenobiotics--interactions and function in health and disease.

Sulfation is a major detoxication mechanism for endogenous compounds and xenobiotics performed by a family of sulfotransferase isoenzymes. Understanding the normal cellular functions of these different sulfotransferases and the way in which endogenous and exogenous factors are able to influence their activity and expression will provide us with the information necessary to develop novel therapeutic strategies for conditions where sulfation may be implicated. This concept is discussed and is illustrated by examples including adverse drug reactions, fetal development and cancer.

Dehydroepiandrosterone sulfotransferase in the developing human fetus: quantitative biochemical and immunological characterization of the hepatic, renal, and adrenal enzymes.

The sulfation of the adrenal steroid dehydroepiandrosterone (DHEA) is a critical step in the provision of substrates for estrogen biosynthesis by the placenta during pregnancy. This enzyme reaction is catalyzed by a cytosolic sulfotransferase (ST) found in many key body tissues, and we have examined the ontogeny and localization of expression of this important enzyme in three tissues: the liver, adrenal, and kidney. Hepatic DHEA ST expression increased with advancing gestational age before reaching near-adult levels in the early postnatal period, suggesting an increased requirement for this enzyme in the liver as development progresses, whereas in the adrenal and kidney there was no obvious ontogenic pattern. The enzyme was expressed at a 5-fold higher level in the adrenal than in the liver and some 40-fold higher than in the kidney. Comparison of enzyme activity measurements and quantitation of the expression of DHEA ST by immunodot blot analysis with an anti-DHEA ST antibody preparation demonstrated the fragility of the enzyme activity and suggested that immunoquantitation was a superior method for assessment of levels of expression of this enzyme in widely different tissue sources. Examination of the localization of DHEA ST in these tissues by immunohistochemistry showed that in liver, DHEA ST was expressed in embryonic hepatocytes and continued to be expressed in these cells into adulthood, when there was some concentration of immunostaining around central veins. In the fetus, the adrenal enzyme was expressed in the fetal zone, whereas in adult tissue, staining was localized principally to the zona reticularis. Renal DHEA ST was present in the proximal and distal tubules, loops of Henle, collecting ducts, and their progenitors, but was at no time expressed in the vascular glomerulus. In light of the broad substrate specificity of this enzyme toward other steroids, in particular bile acids and cholesterol, the information presented forms a strong basis for further studies into the role of DHEA ST in modulating the activity of a number of biologically active and potentially toxic steroids in the developing human.

Immunochemical characterisation of a dehydroepiandrosterone sulfotransferase in rats and humans.

A member of the rat liver hydroxysteroid sulfotransferase (ST) enzyme family metabolising dehydroepiandrosterone (DHEA) was purified from female rats and used to raise rabbit polyclonal antibodies. Characterisation of this antibody preparation demonstrated that it was specific for DHEA ST, and recognised a single 30-kDa protein on immunoblot analysis of rat liver cytosol which was expressed preferentially in female rat liver, and immunohistochemical localisation of the protein in female rat liver determined that DHEA ST was distributed homogeneously in the cytoplasm of hepatocytes. Examination of the extrahepatic expression of this protein showed it to be located predominantly in the liver, although a small amount of enzyme activity was found in the kidney which was not apparently subject to the same sex difference as the hepatic activity. Immunological analysis suggested that this activity was not due to the action of DHEA ST, but to another, unidentified ST isozyme. The antibody cross-reacted strongly with adult human liver DHEA ST, recognising a protein of 35 kDa on immunoblotting. Using this antibody preparation, the distribution of DHEA ST in mid-trimester human fetal tissues was examined, and it was shown that the enzyme is expressed in the adrenal and liver, but not to any significant extent in the kidney or lung. This antibody therefore provides a powerful tool for investigating the function of DHEA ST.