The development and characterization of an enhanced composite skin substitute based on collagen and poly(ε-caprolactone) are reported. Considering the features of excellent biocompatibility, easy-manipulated property and exempt from cross-linking related toxicity observed in the 1:20 biocomposites, skin substitutes were developed by seeding human single-donor keratinocytes and fibroblasts alone on both sides of the 1:20 biocomposite to allow for separation of two cell types and preserving cell signals transmission via micro-pores with a porosity of 28.8 ± 16.1 μm. The bi-layered skin substitute exhibited both differentiated epidermis and fibrous dermis in vitro. Less Keratinocyte Growth Factor production was measured in the co-cultured skin model compared to fibroblast alone condition indicating a favorable microenvironment for epidermal homeostasis. Moreover, fast wound closure, epidermal differentiation, and abundant dermal collagen deposition were observed in composite skin in vivo. In summary, the beneficial characteristics of the new skin substitutes exploited the potential for pharmaceutical screening and clinical application.

There is growing evidence that cholecystokinin (CCK) affects growth and differentiation of anterior pituitary cells, via the CCK-B receptor. The possibility of an autocrine / paracrine role for CCK to modulate hormone secretion in human pituitary tumour cells is demonstrated here by RT-PCR and direct sequencing. In support of this conclusion, a neutralising antibody against the CCK peptide exhibited a dose dependent inhibition of hormone secretion by functionless pituitary adenomas. Total RNA was extracted from human pituitary adenomas, reverse transcribed into cDNA and subjected to PCR using primers specific for the gene for CCK, CCK-A and CCK-B receptors. PCR bands of the predicted length were observed in all tumours using human CCK gene and CCK-B receptor primers. Restriction digestion and direct sequence analysis provided further evidence that they represented both the human CCK peptide along with the CCK-A and/B receptor mRNA. CCK-33 and CCK octapeptide sulphate (CCK-8s) both powerfully stimulated phosphatidylinositol hydrolysis, providing evidence for functional activity of the CCK-A and/B receptors. A direct stimulatory effect of CCK peptides on both LH and FSH secretion is reported for the first time, whereas stimulatory effects on GH were blocked by antagonists to CCK. These results may indicate an autocrine role for CCK in the functioning and perhaps development of human pituitary tumours.

Estradiol 17-beta dehydrogenase (OE2DH) is an enzyme involved in both the activation and the inactivation of estrogen, which plays an important role in breast cancer (BC). Polyunsaturated fatty acids (PUFA's) are also implicated in the development of BC, although the mechanisms are not fully understood. The aim of this investigation was to examine the growth effects of the n-3 PUFA-eicosapentaenoic acid (EPA) on the MCF7 human BC cell line in parallel with its effects upon OE2DH. A bell shaped growth inhibition of MCF7 cells was observed at around 50 muM. A single dose of EPA significantly inhibited cell proliferation at every time point measured over a twelve-day period. In the presence of 17beta-estradiol (E-2) alone, cell proliferation was increased peaking at 1 nM E-2. The effect was completely abolished by co-incubation with 50 muM EPA. OE2DH activity was measured in both the reductive [estrone (E-1) --> E-2] and the oxidative [E2 --> E-1] directions. These results show that whilst 50 muM EPA weakly stimulates OE2DH activity in the reductive direction, it strongly drives the reaction in the oxidative direction. This effect was specific to EPA as the structurally related n-6 fatty acid arachidonate, had no effect. These results suggest that EPA might, in part, exert its effects in BC through driving OE2DH activity towards the production of the less biologically potent E-1 as well as having a direct inhibitory effect on activity.

Poly(ε-caprolactone) (PCL) fibres were produced by wet spinning from solutions in acetone under low shear (gravity flow) conditions. As-spun PCL fibres exhibited a mean strength and stiffness of 7.9 MPa and 0.1 GPa, respectively and a rough, porous surface morphology. Cold drawing to an extension of 500% resulted in increases in fibre strength (43 MPa) and stiffness (0.3 GPa) and development of an oriented, fibrillar surface texture. The proliferation rate of Swiss 3T3 mouse fibroblasts and C2C12 mouse myoblasts on as-spun, 500% cold-drawn and gelatin-modified PCL fibres was determined in cell culture to provide a basic measure of the biocompatibility of the fibres. Proliferation of both cell types was consistently higher on gelatin-coated fibres relative to as-spun fibres at time points below 7 days. Fibroblast growth rates on cold-drawn PCL fibres exceeded those on as-spun fibres but myoblast proliferation was similar on both substrates. After 1 day in culture, both cell types had spread and coalesced on the fibres to form a cell layer, which conformed closely to the underlying topography. The high fibre compliance combined with a potential for modifying the fibre surface chemistry with cell adhesion molecules and the surface architecture by cold drawing to enhance proliferation of fibroblasts and myoblasts, recommends further investigation of gravity-spun PCL fibres for 3-D scaffold production in soft tissue engineering.

Tissue engineering of skin based on collagen:PCL biocomposites using a designed co-culture system is reported. The collagen:PCL biocomposites having collagen:PCL (w/w) ratios of 1:4, 1:8, and 1:20 have been proven to be biocompatible materials to support both adult normal human epidermal Keratinocyte (NHEK) and mouse 3T3 fibroblast growth in cell culture, respectively, by Dai, Coombes, et al. in 2004. Films of collagen:PCL biocomposites were prepared using non-crosslinking method by impregnation of lyophilized collagen mats with PCL/dichloromethane solutions followed by solvent evaporation. To mimic the dermal/epidermal structure of skin, the 1:20 collagen:PCL biocomposites were selected for a feasibility study of a designed co-culture technique that would subsequently be used for preparing fibroblast/biocomposite/keratinocyte skin models. A 55.3% increase in cell number was measured in the designed co-culture system when fibroblasts were seeded on both sides of a biocomposite film compared with cell culture on one surface of the biocomposite in the feasibility study. The co-culture of human keratinocytes and 3T3 fibroblasts on each side of the membrane was therefore studied using the same co-culture system by growing keratinocytes on the top surface of membrane for 3 days and 3T3 fibroblasts underneath the membrane for 6 days. Scanning electron microscopy (SEM) and immunohistochemistry assay revealed good cell attachment and proliferation of both human keratinocytes and 3T3 fibroblasts with these two types of cells isolated well on each side of the membrane. Using a modified co-culture technique, a co-cultured skin model presenting a confluent epidermal sheet on one side of the biocomposite film and fibroblasts populated on the other side of the film was developed successfully in co-culture system for 28 days under investigations by SEM and immunohistochemistry assay. Thus, the design of a co-culture system based on 1:20 (w/w) collagen:PCL biocomposite membranes for preparation of a bi-layered skin model with differentiated epidermal sheet was proven in principle. The approach to skin modeling reported here may find application in tissue engineering and screening of new pharmaceuticals.

Cholecystokinin (CCK) is a peptide found widely distributed throughout the gut and nervous systems. There is growing evidence that it affects growth and differentiation of several types of cell, including anterior pituitary cells, via two receptor subtypes, A and B. An autocrine/paracrine role for CCK in GH3 rat pituitary tumor cells has been reported. Herein, we demonstrate that functionless human pituitary tumors also possess CCK-B receptors. Total RNA was extracted from 30 human functionless pituitary tumors, reverse transcribed into cDNA, and subjected to PCR using primers specific for CCK-A and CCK-B receptors. The primers were targeted against different exons of the receptor genes, allowing identification of cDNA amplification. PCR bands of the predicted length were observed in all tumors using CCK-B receptor primers. Restriction digestion and direct sequence analysis of the products provided further evidence that they represented CCK-B receptor mRNA. CCK-A receptor mRNA was not detected by this RT-PCR analysis. In culture, CCK and CCK octapeptide sulphate powerfully stimulated phosphatidylinositol hydrolysis, providing evidence for functional activity of the CCK-B receptors. Similar analyses on pituitary-derived fibroblasts yielded negative results, indicating that the CCK-B receptor mRNA was expressed by tumors cells. These results suggest a role for CCK in the development of human functionless pituitary tumors.

In this study, the central technique of in vitro culture has been used to further investigate whether LH/FSH-expressing, but clinically "functionless" pituitary adenomas are gonadotropinomas or whether their hormone secretion is due to transdifferentiation events. 664 "functionless" pituitary adenomas were examined for hormone secretion by in vitro culture and for hormone content by immunostaining. The results were correlated with the clinical findings. 40% of the tumours (n = 263) secreted at least one of the gonadotropins alone, 8% (n = 53) exhibited various patterns of anterior pituitary hormones, whilst the remaining 52% of tumours were not associated with any hormone. In the secretory tumours, immunostaining revealed only a few scattered hormone-containing cells (5 to 15%). Mild hyperprolactinaemia was observed in some cases, presumably because of pressure effects of the tumours. The majority of the patients suffered clear cut hypopituitarism (p < 0.05). Pre-operatively, gonadotropin hypersecretion was observed in 3 cases, but only one of these secreted hormones in culture. Interestingly, a higher proportion of tumours removed from patients with hypopituitarism showed secretory activity in vitro than those tumours removed from patients showing no hormonal dysfunction or hyperprolactinaemia. We conclude that the term "gonadotropinoma" to describe functionless pituitary tumours associated with LH and/or FSH secretion is a misnomer, because the presence of LH and/or FSH confirmed by in vitro methods in the present series is a result of only a few scattered cells. We suggest that primary pituitary tumour cells differentiate into a secretory type (transdifferentiation), possibly in response to altered serum hormone levels such as decreased steroids. Further work is required to identify the factors which trigger the altered cells' characteristics.

The preparation and characterisation of collagen:PCL composites for manufacture of tissue engineered skin substitutes and models are reported. Films having collagen:PCL (w/w) ratios of 1:4, 1:8 and 1:20 were prepared by impregnation of lyophilised collagen mats by PCL solutions followed by solvent evaporation. In vitro assays of collagen release and residual collagen content revealed an expected inverse relationship between the collagen release rate and the content of synthetic polymer in the composite that may be exploited for controlled presentation and release of biopharmaceuticals such as growth factors. DSC analysis revealed the characteristic melting point of PCL at around 60°C and a tendency for the collagen component, at high loading, to impede crystallinity development within the PCL phase. The preparation of fibroblast/composite constructs was investigated using cell culture as a first stage in mimicking the dermal/epidermal structure of skin. Fibroblasts were found to attach and proliferate on all the composites investigated reaching a maximum of 2×105/cm2 on 1:20 collagen:PCL materials at day 8 with cell numbers declining thereafter. Keratinocyte growth rates were similar on all types of collagen:PCL materials investigated reaching a maximum of 6.6×104/cm2 at day 6. The results revealed that composite films of collagen and PCL are favourable substrates for growth of fibroblasts and keratinocytes and may find utility for skin repair.

Two forms of the growth hormone-releasing hormone (GHRH) receptor (GHRH-R) exist in terms of a polymorphism at codon 57. The most common allele possesses GCG, coding for Ala. This codon can also be ACG, replacing the Ala with Thr. The present study demonstrates that the latter occurs in about 20% of pituitary somatotrophinomas, removed from patients with acromegaly. Somatotrophinomas possessing the alternative allele respond, on average, more strongly to GHRH in terms of GH secretion in vitro than tumors which are homozygous for the more common allele. The distribution of the two allelic forms of the GHRH-R did not significantly differ between acromegalic and non-acromegalic subjects. Thus, while the alternative allelic forms may, at least partially, contribute to the variable response of serum GH levels to i.v. GHRH observed in acromegalic and normal subjects, it is unlikely that subjects possessing the rarer form containing Thr in place of Ala at residue 57 are at increased risk of developing acromegaly.

Craniopharyngioma is the most common childhood tumor and thought to arise from embryonic remnants of Rathke's pouch. The paucity of published data on the molecular basis of these tumors prompted us to examine 22 adamantinomatous craniopharyngiomas looking for genetic abnormalities. Using the X-linked polymorphic androgen receptor gene as a tool for X-chromosome inactivating analysis, we found that a subset of craniopharyngiomas are monoclonal and therefore are probably due to acquired somatic genetic defects. Thus, we investigated these tumours for mutations within three candidate genes, Gsα, Gi2α and patched (PTCH). Using single stranded conformational polymorphism (SSCP), denaturing gradient gel electrophoresis and direct sequencing, the presence of somatic mutations in these genes could not be demonstrated in any tumor. Our data indicate that a subset of craniopharyngiomas are monoclonal and the mutations in the PTCH, Gsα, and Gi2α contribute little if any to cranipharyngioma development.

Ghrelin is a recently identified endogenous ligand of the GH secretagogue (GHS) receptor. It was originally isolated from the stomach, but has also been shown to be present in the rat hypothalamus. It is a 28-amino acid peptide with an unusual octanoylated serine 3 at the N-terminal end of the molecule, which is crucial for its biological activity. Synthetic GHSs stimulate GH release via both the hypothalamus and the pituitary, and the GHS receptor (GHS-R) has been shown by us and others to be present in the pituitary. We investigated whether ghrelin messenger ribonucleic acid (mRNA) and peptide are present in the normal human hypothalamus and in normal and adenomatous human pituitary. RNA was extracted from pituitary tissue removed at autopsy and transsphenoidal surgery (n = 62), and ghrelin and GHS-R type 1a and 1b mRNA levels were investigated using real-time RT-PCR. Both ghrelin and GHS-R mRNA were detected in all samples. Corticotroph tumors showed significantly less expression of ghrelin mRNA, whereas GHS-R mRNA levels were similar to those in normal pituitary tissue. Gonadotroph tumors showed a particularly low level of expression of GHS-R mRNA. Immunohistochemistry, using a polyclonal antibody against the C-terminal end of the ghrelin molecule, revealed positive staining in the homolog of the arcuate nucleus in the human hypothalamus and in both normal and abnormal human pituitary. Pituitary tumor ghrelin peptide content was demonstrated using two separate RIA reactions for the N-terminal and C-terminal ends of the molecule. Both forms were present in normal and abnormal pituitaries, with 5 +/- 2.5% octanoylated (active) ghrelin (mean +/- SD) present as a percentage of the total. We suggest that the presence of ghrelin mRNA and peptide in the pituitary implies that the locally synthesized hormone may have an autocrine/paracrine modulatory effect on pituitary hormone release.

I. Ozaki, Y. Yaegashi, T. Kimura, M. Baba, M. Matsunaga, I. Hashimoto (Japan) 41 Lysyl oxidase, the extracellular matrix-forming enzyme, in rat brain injury sites GM Gilad, HM Kagan, VH Gilad (Israel, USA) 45 Bisphenol-A differently affects estrogen receptors-a in estrous-cycling and lactating female rats AM Aloisi, D. Della Seta, I. Ceccarelli, F. Farabollini (Italy) 49 Peripheral capsaicin receptors increase in the inflamed rat hindpaw: a possible mechanism for peripheral sensitization SM Carlton, RE Coggeshall (USA) 53

A previous study has suggested that a G to A base change at position 169 of the GHRH-receptor gene in human somatotrophinomas is a mutation and confers hypersensitivity to GHRH. The alternative base converts codon 57 from GCG to AGC, resulting in replacement of alanine (Ala) with threonine (Thr). In the present study, two of five human GH-secreting somatotrophinomas were found to possess the codon 57 AGC sequence. The GCG allele was also detected, indicating heterozygosity. However, the patients' normal blood-derived DNA also yielded the same sequence pattern, indicating that the Ala --> Thr amino acid change is a normal polymorphism, and not a somatic mutation. Nevertheless, in vitro, the tumors possessing the Ala --> Thr amino acid change responded very strongly to GHRH in terms of cAMP formation, being increased 40- and 200-fold, in comparison to the 2-fold increases by tumors without the alternative GHRH-receptor sequence. Likewise, the in vitro response of GH secretion to GHRH was elevated. One of the two tumors with the alternative Thr residue, and the highest responder to GHRH, possessed a gsp mutation, despite the fact that these defects are thought to reduce responsiveness to GHRH. These results fail to confirm that the GCG --> AGC at codon 57 of the GHRH-receptor gene is a mutation, but do support the concept that the alternative form with Thr confers increased sensitivity to GHRH.

The molecular mechanisms underlying pituitary tumor development are largely unknown, but presumably involve inactivation of the MENl gene, and in prolactinomas, uncoupling of the dopamine receptor (DRD2) from intracellular effectors. To test this notion, 50 sporadic pituitary tumors (28 non-functioning, 22 prolactinomas) were analyzed for somatic MENl mutations, and 14 prolactinomas for DRD2 mutations. PCR amplification of all coding exons of the DRD2 and the MENl genes was followed by denaturing gradient gel electrophoresis (DGGE) analysis and DNA sequencing. No abnormal sequences were detected in the prolactinomas within the DRD2 gene. Two tumors displayed novel missense mutations in the MENl gene: C->T alteration at g4795, altering proline 245 to leucine in a prolactinoma, and C->A change at position g2677, altering arginine 130 to serine in a non-functioning tumor. There is no evidence of inactivating mutations at the DRD2 gene level in prolactinomas, and somatic mutations of the MENl gene in pituitary tumors are infrequent. Reprint address: Eitan Friedman, M.D., Ph.D. The Susanne-Levy Gertner Oncogenetics Unit Institute of Genetics Chaim Sheba Medical Center Tel Hashomer 52621, Israel e-mail: eitan21 l@netvision.net. i l

We herein review published findings on the clinical characteristics of acromegalic patients harboring pituitary somatotrophinomas expressing adenylyl cyclase activating gsp mutations and present an update of our own data on a large series of 176 patients with and without these oncogenes. Gsp oncogenes are the result of point mutations in either codon 201 or 227 of the Gs-alpha subunit of the Gs-protein which controls adenylyl cyclase. They result ultimately in increased intracellular cAMP levels and thus in excessive growth hormone (GH) secretion. Our large series has allowed us to characterise patients with mutations in codon 201 and the far rarer group possessing codon 227 defects. Both groups were compared with patients without gsp oncogenes. In accordance with previous findings, there was no statistically significant difference in age of the patients belonging to each group, the overall average tumor diameter nor in pre-operative serum GH levels, although the latter showed a tendency to be lower in patients with gsp oncogenes. The distribution of different types of response during an oral glucose tolerance test (no change, paradoxical rise or greater than 50% decrease in serum GH levels) did not differ between the 3 groups. However, the incidence of microadenomas was higher in acromegalics expressing gsp oncogenes in patients possessing mutations in codon 227. Additionally, the incidence of invasiveness was much lower (10% v. 33%) in those tumors with mutations in codon 227. Finally, previous in-vitro data indicating that gsp oncogene-expressing tumors may respond more efficiently to the somatostatin analogue, octreotide, have been confirmed by subsequent in-vivo studies showing a better reduction in serum GH levels in patients with gsp oncogenes. These latter findings suggest that presence of gsp oncogenes may be a marker for good reponsiveness to octreotide. Assessment of gsp oncoge status of surgically removed pituitary somatotrophinomas may thus be helpful in designing optimal medical therapies in those acromegalics requiring further post-operative management of the disease.

In this review, the value of assessing proliferation parameters in surgically resected pituitary tumour tissue is analyzed. Histological examination of basal dura biopsies identifies invasive growth even when intraoperatively not apparent to the surgeon. Determination of DNA-polymerase activity, Ki-67 immunohistochemistry and DNA-flow-cytometry shows a clear difference in the proliferative potential of enclosed and invasive pituitary adenomas. Among the various endocrinologically differentiated groups ACTH-secreting adenomas associated with Nelson's syndrome and thyrotropinomas were the most rapidly proliferating. At present, however, our results reveal that the prognosis of an individual patient cannot be reliably predicted on the basis of such studies.

Growth hormone releasing peptide (GHRP-6) is a synthetic hexapeptide which specifically stimulates secretion of growth hormone (GH) by pituitary somatotrophs. The precise intracellular mechanism by which this is achieved has not been deciphered although it is known to involve protein kinase C (PKC) and Ca2+ but to be cAMP-independent. We have used cell cultures of human pituitary somatotrophinomas to demonstrate powerful effects of GHRP-6 on membrane phosphatidylinositol (PI) turnover, a second messenger system which leads to activation of PKC and mobilisation of intracellular Ca2+ reserves. Incubation of somatotrophinoma cells with GHRP-6 led to a dose-dependent stimulation of rate of PI turnover. GH secretion was increased in parallel. Effects were discernable after only 15 minutes incubation and rose to a maximum at 2 hours. PI turnover was stimulated by GHRP-6 in 8 of 8 tumours examined, effects ranging from 2.1 - 7.9 fold increases. Stimulation of GH secretion by GHRP-6 was independent of presence of gsp oncogenes, emphasising the cAMP-independent nature of its effects. These results provide evidence that the GH-stimulatory effects of GHRP-6 are achieved through activation of the PI second messenger system and thus support earlier findings that PKC and Ca2+ play central roles in mediating the effects of GHRP-6.

The growth of human cerebral meningiomas depends on various growth factors, including epidermal growth factor (EGF), transforming growth factor (TGF)-alpha and TGF-beta, platelet-derived growth factor (PDGF)-BB, insulin-like growth factor (IGF)-I and IGF-II, and acidic and basic fibroblast growth factors. The latter three have been shown to form autocrine loops that are thought to be a major component of uncontrolled growth in meningioma tissue. Suramin is known to prevent binding of a variety of growth factors to their receptors in mammalian tissue, thus abolishing para- and/or autocrine-mediated cell growth. The authors therefore tested the effect of suramin on the proliferation of cultured human meningioma cells. Suramin (10(-5) to 10(-4) M) significantly inhibited the growth of meningioma cells in culture. The maximum effect observed was with the higher dose (10(-4) M), which resulted in a 40% to 70% reduction in cellular proliferation. This effect was observed in all 15 tumor samples studied and was confirmed by [3H]thymidine uptake. In studies using DNA flow cytometry, suramin inhibited meningioma cell proliferation in five tumor samples by arresting cells in the S and G2/M phases of the cell cycle. Growth factor (EGF, IGF-I, and PDGF-BB)-induced cell proliferation was completely abolished in five tumor samples when 10(-4) M suramin was applied to meningioma cells. Western blot analysis of three tumor samples showed that the intracellular PDGF-BB content of meningioma cells was significantly reduced after treating the cells with 10(-4) M suramin. Binding of iodinated growth factors (that is, [125I]EGF, [125I]IGF-I, and [125I]PDGF-BB) to their receptor sites was prevented by suramin in a dose-dependent manner in 10 meningioma membrane fractions. Lowering of the intracellular PDGF content and prevention of extracellular growth factor receptor binding demonstrates that suramin disrupts autocrine loops and paracrine growth stimulation in meningioma tissue. These data provide evidence that growth of cerebral meningiomas in culture is strongly inhibited by suramin at a concentration of 10(-4) M. Suramin acts as a scavenger neutralizing exogenous growth factors; thus it can interrupt autocrine loops and paracrine stimulation of human meningioma cell growth. The evidence favors suramin as a therapeutic option for controlling meningioma proliferation in patients with inoperable and recurrent high-grade meningiomas.

Dopamine, acting via its specific receptor (DRD2) in the anterior pituitary, tonically inhibits pituitary prolactin secretion and lactotroph proliferation. In addition, dopamine agonist therapy for pituitary prolactinomas results in reduction of prolactin secretion and tumor regression. These observations lead to the speculation that functional dopamine uncoupling may release lactotrophs from the inhibitory effects of dopamine and contribute to the development of prolactin (PRL)-secreting pituitary tumors. We hypothesized that such an uncoupling may occur by inactivating mutation(s) of the DRD2. To test our hypothesis, we examined 79 pituitary tumors, mostly prolactinomas and mixed GH/PRL-secreting, for mutations in the coding exons of the DRD2 gene. We used the polymerase chain reaction and analyzed the fragments for migration abnormalities on denaturing gradient gel electrophoresis, complemented by direct DNA sequencing. No mutations were demonstrated, and all migration abnormalities detected by denaturing gradient gel electrophoresis were due to polymorphisms within the DRD2 gene. In addition, allelic losses in the multiple endocrine neoplasia type 1 region in 11q13 could not be demonstrated in all five informative prolactinomas. We conclude that mutations in the DRD2 gene do not occur in PRL or GH/PRL-secreting pituitary tumors and that allelic loss of 11q13 is uncommon in prolactinomas.