The bioartificial thyroid: a biotechnological perspective in endocrine organ engineering for transplantation replacement.

A new concept for ex situ endocrine organ bioengineering is presented, focused on the realization of a human bioartificial thyroid gland. It is based on the theoretical assumption and experimental evidence that symmetries in geometrical coordinates of the thyroid tissue remain invariant with respect to developmental, physiological or pathophysiological transformations occuring in the gland architecture. This topological arrangement is dependent upon physical connections established between cells, cell adhesion molecules and extracellular matrix, leading to the view that the thyroid parenchyma behaves like a deformable "putty", moulded onto an elastic stromal/vascular scaffold (SVS) dictating the final morphology of the gland. In particular, we have raised the idea that the geometry of the SVS per se provides pivotal epigenetic information to address the genetically-programmed, thyrocyte and endothelial/vascular proliferation and differentiation towards a functionally mature gland, making organ form a pre-requirementfor organ function. A number of experimental approaches are explored to obtain a reliable replica of a human thyroid SVS, and an informatic simulation is designed based on fractal growth of the thyroid intraparenchymal arterial tree. Various tissue-compatible and degradable synthetic or biomimetic polymers are discussed to act as a template of the thyroid SVS, onto which to co-seed autologous human thyrocyte (TPC) and endothelial/vascular (EVPC) progenitor cells. Harvest and expansion of both TPC and EVPC in primary culture are considered, with specific attention to the selection of normal thyrocytes growing at a satisfactory rate to colonize the synthetic matrix. In addition, both in vitro and in vivo techniques to authenticate TPC and EVPC lineage differentiation are reviewed, including immunocytochemistry, reverse trascriptase-polymerase chain reaction, flow cytomery and proteomics. Finally, analysis of viability of the thyroid construct following implantation in animal hosts is proposed, with the intent to obtain a bioartificial thyroid gland morphologically and functionally adequate for transplantation. We believe that the biotechnological scenario proposed herein may provide a template to construct other, more complex and clinically-relevant bioartificial endocrine organs ex situ, such as human pancreatic islets and the liver, and perhaps a new approach to brain bioengineering.

Solubilization of pituitary receptors for thyrotropin-releasing hormone.

Receptors for thyrotropin-releasing hormone were solubilized by Triton X-100. Membrane fractions from GH3 pituitary tumor cells were incubated with thyrotropin-releasing hormone in order to saturate specific receptor sites before the addition of detergent. The amount of protein-bound hormone solubilized by Triton X-100 was proportional to the fractional saturation of specific membrane receptors. Increasing detergent:protein ratios from 0.5 to 20 led to a progressive loss of hormone . receptor complex from membrane fractions with a concomitant increase in soluble protein-bound hormone. The soluble hormone . receptor complex was not retained by 0.22 micron filters and remained soluble after ultracentrifugation. Following incubation with high (2.5--10%) concentrations of Triton X-100 and other non-ionic detergents, or following repeated detergent extraction, at least 18% of specifically bound thyrotropin-releasing hormone remained associated with particulate material. Unlike the hormone receptor complex, the free hormone receptor was inactivated by Triton X-100. A 50% loss of binding activity was obtained with 0.01% Triton X-100, corresponding to a detergent:protein ratio of 0.033. The hormone . receptor complex was included in Sepharose 6B and exhibited an apparent Stoke radius of 46 A in buffers containing Triton X-100. The complex aggregated in detergent-free buffers. Soluble hormone receptors were separated from excess detergent and thyrotropin-releasing hormone by chromatography on DEAE-cellulose. Thyrotropin-releasing hormone dissociated from soluble receptors with a half-time of 120 min at 0 degrees C, while the membrane hormone . receptor complex was stable for up to 5 at 0 degrees C.

Prolactin cell subpopulations separated on discontinuous Percoll gradient: an immunocytochemical, biochemical, and physiological characterization.

A single-step procedure was devised to separate PRL cells from the rat anterior pituitary gland. After dissociation, cells were centrifuged on a Percoll gradient. Three layers were recovered. The composition of the different layers was evaluated using immunocytochemistry (with antisera to the six pituitary hormones), and in situ hybridization [with DNA complementary to PRL or to GH messenger RNA (mRNA)]. Both methods yielded identical values. PRL cells were recovered in the lower density layer (layer 1) with a good yield (that is 81% of the total PRL cells of the initial cell suspension) and in addition, markedly enriched (indeed 85% of the cells in layer 1 stained for PRL). A second layer (layer 2: intermediate density) contained most of the remaining PRL cells which were, however, heavily contaminated mainly by GH cells and cells that did not stain for any of the known pituitary hormones. A third layer (layer 3: higher density) was enriched in GH cells to 93% (representing, however, only 10% of the initial pituitary GH cells). In addition, PRL and GH were measured by RIA in culture medium and in cell lysates. Hormone biosynthesis was monitored by polyacrylamide gel electrophoresis and autoradiography after culture in the presence of [35S]methionine. These experiments confirmed that layer 1 was enriched in cells containing, and producing, PRL and depleted from GH cells. Cells in layer 2 contained and produced more GH than PRL. PRL cells from layer 1 responded to dopamine and to vasoactive intestinal polypeptide in the same way as PRL cells in the unseparated pituitary cell population. In contrast PRL cells in layer 2 had a lower basal secretion rate but a higher response to vasoactive intestinal polypeptide. Unless this represents a paracrine effect of non-PRL cells, PRL cells in layer 2 exhibit different properties and may therefore form a distinct subpopulation of PRL cells.

An ultrastructural and functional characterization of rat somatotrophs highly enriched on a continuous Percoll density gradient.

We here describe the use of a continuous Percoll density gradient in preparing a fraction of consistently more than 95% rat somatotrophs. Recovery was about 50% and cell viability 98-99.5%. Two groups of somatotrophs were identified, one (74-82%, type II) heavily and another (18-26%, type I) sparsely granulated. Freshly prepared somatotrophs responded to growth hormone-releasing factor but the responsiveness was greatly enhanced after 3 days' culture. Electron microscopy revealed a well-preserved morphological integrity in both freshly prepared and cultured somatotrophs. culture reduced the average granule profile area and granule volume density of type II cells. This population of pure and well-functioning somatotrophs is suited for studies on the cellular mechanisms involved in growth hormone secretion.

The effect of angiotensin II receptor antagonists on diethylstilbestrol-induced vascular changes in the rat anterior pituitary gland: a quantitative evaluation.

The effects if diethylstilbestrol (DES) and of angiotensin II (Ang II) receptor antagonists, such as losartan (selective AT1 receptor antagonist) or PD 123319 (selective AT2 receptor antagonist) on the anterior pituitary microvasculature were studied by means of computer-assisted image analysis. The vascularization was visualized using Selye's method modified by Poely et al. (1964). It was found that DES induced a sharp increase in vessel area, mean vessel diameter and perimeter, whereas mean vessel number was reduced. These DES-induced changes were inhibited by simultaneous administration of losartan. On the other hand, PD 123319 was less effective. These findings suggest an involvement of Ang II, acting mainly via AT1 receptors, in the mechanism of estrogen-induced vascular changes in the rat anterior pituitary gland.

An easy method for the removal of Epon resin from semi-thin sections. Application of the avidin-biotin technique.

A simple procedure is described for removing Epon resin from semi-thin 1 micron sections, which permits excellent postembedding immunohistochemical staining (avidin-biotin complex technique). The procedure was developed for the detection of growth hormone and prolactin in bovine adenohypophysis fixed with 2% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4-7.6. The results indicate that the removal of the epoxy embedding medium prior to the application of the immunohistochemical reagents was essential for the successful localization of the antigenic determinants of the two hormones. The immunocytochemical reactivity was obtained only after treating the sections with a solution of potassium hydroxide in a mixture of absolute methyl alcohol and propylene oxide (Maxwell's solution). An enhanced immunoreactivity was obtained when this treatment was followed by an additional treatment with either 4% hydrogen peroxide or a saturated aqueous solution of sodium metaperiodate. Because of the easy preparation of the Epon removal solution and the good structural preservation without damage to the antigenic determinants, Maxwell's solution is suggested as a good etching agent which can be used in immunohistochemical studies on semi-thin sections with excellent results.

Linear Percoll gradient centrifugation of rat anterior pituitary cells. A simple method for prolactin cell enrichment.

Prolactin (PRL) cells were purified from nulliparous normal female adult Wistar rat pituitary cell suspensions by linear Percoll density gradient centrifugation, a procedure yielding single cells. Lactotrophs were found in two different layers, the first containing 70% PRL cells in the density range 1.055 to 1.065 g/ml, the second with 28% PRL cells in the range 1.070 to 1.080 g/ml. Both cell fractions contained more than 90% viable cells with an intact ultrastructure. The physiological integrity of the 70% enriched PRL cells was assessed by their basal PRL secretion, their secretory response to TRH and dopamine, and their cAMP production in a basal situation and after incubation with dopamine.

Staining of different endocrine cells with hydrochloric acid-toluidine blue in Epon embedded rat tissue.

The usual HCl-toluidine blue staining of different endocrine cells is applicable to paraffin embedded material. A modification for Epon embedded tissue suitable for consecutive light and electron microscopic studies is described which makes it possible to find the same stained cell, both in a semithin section and in subsequent ultrathin sections. This method facilitates the search for scattered specific endocrine cells. Without removing the resin, sections of Epon embedded tissues were hydrolyzed for 17 hr in 1% HCl at 65 C and stained for 2 hr in 0.1% toluidine blue in McIlvaine buffer, pH 5.8. The following cells were stained: C cells in thyroid glands; A and D cells in pancreatic islets; B cells in anterior pituitary; G, D and Ec cells in the gastrointestinal tract; Ad cells of the adrenal medulla.

Pegylated peptides. V. Carboxy-terminal PEGylated analogs of growth hormone-releasing factor (GRF) display enhanced duration of biological activity in vivo.

In the present study, human growth hormone-releasing factor (hGRF) and analogs were successfully pegylated at the carboxy-terminus using a novel solid- and solution-phase strategy. Following synthesis, these pegylated hGRF analogs were evaluated for in vitro and in vivo biological activity. Specifically, hGRF (1-29)-NH2, [Ala15]-hGRF (1-29)-NH2, [desNH2Tyr1, D-Ala2, Ala15]-hGRF(1-29)-NH2 and [His1, Val2, Gln8, Ala15, Leu27]-hGRF(1-32)-OH were each C-terminally extended using a Gly-Gly-Cys-NH2 spacer (previously demonstrated not to alter intrinsic biological activity), and then monopegylated via coupling to an activated dithiopyridyl-PEG reagent. PEG moieties of 750, 2000, 5000 or 10,000 molecular weight (MW) were examined to determine the effect of polymer weight on activity. Initial biological evaluations in vitro revealed that all C-terminally pegylated hGRF analogs retained high growth hormone (GH)-releasing potencies, regardless of the MW of PEG polymer employed. Two of these pegylated hGRF analogs, [desNH2Tyr1, D-Ala2, Ala15]-hGRF (1-29)-Gly-Gly-Cys(NH2)-S-Nle-PEG5000 and [His1, Val2, Gln8, Ala15, Leu27]-hGRF(1-32)-Gly-Cys(NH2)-S-Nle-PEG5000, were subsequently evaluated in both pig and mouse models and found to be highly potent (in vivo potency range = 12-55-fold that of native hGRF). Relative to their non-pegylated counterparts, these two pegylated hGRF analogs exhibited enhanced duration of activity.

Selective alkylation and acylation of alpha and epsilon amino groups with PEG in a somatostatin analogue: tailored chemistry for optimized bioconjugates.

The effects of the type and location of polymer grafting on the biological activity of different mono-PEG derivatives of the somatostatin analogue RC160 were evaluated. A chemical strategy to obtain mono-PEG alkylation or acylation of the peptide's alpha-terminal or lysil-epsilon primary amines was devised. Selective BOC protection of the two available primary amines, followed by reaction with two different PEG reagents and removal of the protecting group, was carried out. Chemical characterization, structural studies, and the evaluation of the biological activity of the bioconjugates synthesized allowed the identification of the one having characteristics more suitable for therapeutic application. This corresponds to the mono-epsilon-lysil-pegylated form, obtained by reductive alkylation, where the amine's positive charge is preserved. The results obtained suggest the importance of preliminary studies in the development of new polymer-peptide conjugates with improved pharmacological properties.

Distribution of the titf2/foxe1 gene product is consistent with an important role in the development of foregut endoderm, palate, and hair.

Titf2/foxe1 is a forkhead domain-containing gene expressed in the foregut, in the thyroid, and in the cranial ectoderm of the developing mouse. Titf2 null mice exhibit cleft palate and either a sublingual or completely absent thyroid gland. In humans, mutations of the gene encoding for thyroid transcription factor-2 (TTF-2) result in the Bamforth syndrome, characterized by thyroid agenesis, cleft palate, spiky hair, and choanal atresia. Here, we report a detailed expression pattern of TTF-2 protein during mouse embryogenesis and show its presence in structures where it has not been described yet. At embryonic day (E) 10.5, TTF-2 is expressed in Rathke's pouch, in thyroid, and in the epithelium of the pharyngeal wall and arches, whereas it is absent in the epithelium of the pharyngeal pouches. According to this expression, at E13.5, TTF-2 is present in endoderm derivatives, such as tongue, palate, epiglottis, pharynx, and oesophagus. Later in embryogenesis, we detect TTF-2 in the choanae and whiskers. This pattern of expression helps to define the complex phenotype displayed by human patients. Finally, we show that TTF-2 is a phosphorylated protein. These results help to characterize the domains of TTF-2 expression, from early embryogenesis throughout organogenesis, providing more detail on the potential role of TTF-2 in the development of endoderm and ectoderm derived structures.

A possible differentiation of anterior pituitary cells in collagen gels into neurons.

Dispersed cells from anterior pituitary glands of male rats were cultured embedded in collagen gels or on a plastic surface. After 6 or more days in culture, cells in collagen produced more prolactin than those on plastic. The cultures in collagen had fewer fibroblasts than those on plastic. Many cells cultured in collagen developed processes, and a few cells in every culture had long extended processes that sometimes branched and resembled those of neurons. About 60% of the cells in culture including cells with well developed processes bound the monoclonal antibody A2B5, an antibody that binds to neuronal cells in culture. Fibroblasts did not have detectable binding of A2B5. Some cells with short processes reacted with antiserum to prolactin or ACTH, but the cells with neuronlike processes did not. Collagen gels may provide a matrix in which cells from the anterior pituitary gland can differentiate into neuronlike cells.

Responsiveness of irradiated rat anterior pituitary cells to hypothalamic releasing hormones is restored by treatment with growth hormone.

Hypopituitarism is a common sequela of irradiation in cancer patients. Here we report that recombinant human growth hormone (r-hGH) prevents cell death and restores secretory capacity of irradiated rat pituitary cells in vitro. Dispersed rat pituitary cells from male Sprague-Dawley rats, irradiated with a 9-Gy sublethal dose, were incubated with r-hGH before, after, or before and after irradiation. Treatment with GH resulted in increased cell survival, which reached its maximum at the concentration of 5 nM, with an EC(50) of 3.5 nM. Protective effects of GH on pituitary cells were more pronounced in cultures treated before and after irradiation. Similarly, beneficial effects of GH were observed on the secretory capacity of surviving cells. In fact, irradiated pituitary cells treated with GH secreted substantial amounts of GH, luteinizing hormone, follicle-stimulating hormone, prolactin, thyroid-stimulating hormone and adrenocorticotropic hormone in response to specific releasing hormones. Such effects of GH were prevented in the presence of the specific GH receptor antagonists B2036 and G120K. Our results show that r-hGH exerts a specific protective effect on irradiated rat pituitary cells and suggest possible use of GH as an adjuvant agent for prevention of postirradiation hypopituitarism.