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Extracellular matrix (ECM) molecules and growth factors, such as fibroblast growth factor (FGF), play a crucial role in Alzheimer's disease (AD). The purpose of this investigation was to determine whether phenotypic alterations in ECM production are present in non-neuronal AD cells associated with different FGF expression and response. Synthesis of glycosaminoglycans (GAG) and collagen were measured in skin fibroblasts from patients with familial, sporadic AD (FAD and SAD respectively), and from age-matched controls by radiolabeled precursors. Proteoglycans (PG), metalloprotease (MMP)-1, and FGF gene expressions were measured by reverse transcription-polymerase chain reaction. The results showed different ECM neosynthesis and mRNA levels in the two AD fibroblast populations. FAD accumulated more collagen and secreted less GAG than SAD. Biglycan PG was upregulated in FAD while betaglycan, syndecan, and decorin were markedly downregulated in SAD fibroblasts. We found a significant decrease of MMP1, more marked in FAD than in SAD fibroblasts. Constitutive FGF expression was greatly reduced in both pathological conditions (SAD>FAD). Moreover, an inverse high affinity/low affinity FGF receptor ratio between SAD and FAD fibroblasts was observed. FGF treatment differently modulated ECM molecule production and gene expression in the two cell populations. These observations in association with the changes in FGF gene expression and in the FGF receptor number, suggest that cellular mechanisms downstream from FGF receptor binding are involved in the two different forms of AD.

We have investigated the action of sulphated glycosaminoglycans (GAGs) upon primary 11-day chick embryo skin fibroblasts cultured for various periods of time in the presence or absence of fetal calf serum (FCS). Chondroitin 4- and 6-sulphate (CS) added to serum supplemented medium provoked a decrease in DNA synthesis both in sparse and crowded cultures only at high concentrations (250 micrograms/ml). Synthesis of endocellular and secreted proteins was not affected. In contrast, CS administered to fibroblasts for 24 h in serum-free medium stimulated DNA synthesis. We postulate that the CS stimulating effect seen in serum-free cultures might be antagonized by peptide regulators of cell growth normally present in serum.

Age-related differences in the composition and the post-translational modifications of human skin collagens were examined in the present study. The data were compared with results of collagen synthesis from in vivo-aged fibroblasts in culture. Skin extracts and newly synthesized collagen from fibroblast cultures derived from both old and young donor groups showed the same ratio of collagen III to collagen I. Furthermore, no difference was noted in the degree of prolyl and lysyl hydroxylation of collagen I and collagen III Young and old fibroblasts synthesized a similar quantity of collagen in vitro. The data suggest that fibroblasts maintain a uniform level of collagen production, composition and modification independent of the age of the donor.

Pepsin-solubilized collagen I from skin and bone was analyzed with regard to its thermal stability as a triple helical molecule in solution and after in vitro fibril formation. Collagen I from human control bone was compared with samples showing deficiencies or surplus in the degree of hydroxylation of lysine. The helix to coil transitions were studied by circular-dichroism measurements and limited trypsin digestion. Melting of fibrils from standardized in vitro self-assembly was investigated turbidimetrically. Human control bone collagen I has a maximum transition rate (Tm) at 43.3 degrees C in 0.05% acetic acid. This is 1.9 degrees C above control skin (Tm = 41.4 degrees C), most likely, due to a higher degree of prolyl hydroxylation--0.48 in bone vs. 0.41 in skin collagen I. Lysyl overhydroxylation of human and mouse bone collagen I appears to reduce the Tm slightly (approximately 1 degree C). Underhydroxylated bone collagen has a Tm which is 2 degrees C below control. Melting temperatures of in vitro formed fibrils are an indication for higher thermostability in parallel with an increase of lysyl hydroxylation. Accordingly, the melting temperature of such fibrils from human control skin, 49.3 degrees C, exceeds control bone by 1.4 degrees C. The degree of lysyl hydroxylation in these samples is 0.14 and 0.10, respectively. Further underhydroxylation (0.06) reduced it down to 45.4 degrees C, while extensive overhydroxylation did not continue to increase the thermal stability of fibrils.

Six-day limb skin from a chick embryo was cultured in vitro for seven days in a complete medium either supplemented or not with histones. At the end of the incubation period, the chick embryo skin cultured in the absence of histones was found to undergo keratinization, the converse being true for the limbs cultured in the presence of histones. In the latter, when H3-leucine and C14-cystine were added to the medium, a sharp decrease in the labeled amino acid incorporation was found.

Epithelial-mesodermal tissue interactions have been shown to be required for normal cytodifferentiation of chick embryo skin. Six-day limb skin does not develop in a protein free chemically defined medium, but keratinization has been observed in medium containing chicken serum. In the present study the authors show that the addition of human serum may stimulate the in vitro differentiation of explants of six-day chick embryo skin. Human serum is able to support skin keratinization and this finding has been confirmed by histological and histochemical criteria. Synthesis of proteins in tissue cultures supplemented with human serum has been studied by use of labeled amino acids such as H3-Leucine and C14-Cystine. These incorporation studies show the existence of macromolecular factors in human serum which may be responsible for the observed skin differentiation.

In the present study organ cultures of 6 day-old chick embryo matatarsal skin were used to determine the effect of hepatectomized rat serum in modulating epithelial differentiation. Six day-old chick embryo skin, maintained in organ culture for 6-7 days with a medium containing adult rat serum, was seem to undergo differentiation and keratinization faster then in vivo. Epidermis cultured for 6-7 days with a medium containing partially hepatectomized rat serum does not differentiate and the squamous layer is absent. Studies with 3H-cystine show a sharp decrease in the labelled amino-acids incorporation in the cultures added with serum obtained after partial hepatectomy.

In the present work the Authors have studied the differentiation and the cellular multiplication of chick embryo skin incubated in vitro with normal human sera or with pathological human sera from patients affected by Kerion Celsi. In the following research the Authors have studied the skin structure by an optical and electron microscope and the thymidine 3H incorporation. The result demonstrates that the pathological human serum does not interfere with the multiplication and the differentiation of the skin in vitro, but displays a cytoaggressive action that induces a noticeable cytoplasmic vacuolation.

Skin explants obtained from 12-day old chick embryos, maintained in organ culture containing adult rat serum, show a lower epidermal growth in comparison with skin explants cultured with other adult animal serum; nevertheless the keratogeneous differentiation of the epidermal cell is evident. When the cultures are incubated with medium containing partially hepatectomized rat serum epidermal-cell differentiation and proliferation are absent.

1. Two forms (I and II) with acidic pH optima and a neutral form of beta-hexosaminidase has been separated by DEAE-cellulose chromatography and characterized in skin and lung of 7, 9, 11, 14 day chick embryos and 1 day old chicken. 2. Forms I and II are similar to hexosaminidase A and B for their behaviour on DEAE-cellulose chromatography, Concanavalin A-Sepharose column and thermal stability. 3. Neutral form has a neutral pH optimum and higher molecular weight and a more acidic I. P. than forms I and II, a low beta-N-acetylgalactosaminidase activity and it is not bound by a Concanavalin A-Sepharose column and in that resemble hexosaminidase C and/or other neutral hexosaminidases. 4. We have found differences in the percentage of neutral form and in the specific activities of the extracts in the skin in different stages of development. 5. No significant differences were observed in the lung.

beta-N-acetyl-D-glucosaminidase, beta-N-acetyl-galactosaminidase and a beta-D-galactosidase activity was determined in untreated and lectins (ConA, PNA, SBA and WGA) treated chick embryonic skin fibroblasts at two incubation stages. Activity of all three glycosidases increased between 7 and 14 incubation days. ConA and WGA affected the levels of enzymatic activity; while SBA and PNA were uneffective. We discuss these findings in relation to a possible role of glycosidases in controlling mesenchymal GAG turnover.

In the present study the authors have carried out further researches on the differentiation of six day limb skin from chick embryo cultured "in vitro" in a complete medium supplemented with histones. When histones were added to the medium in the first two days "in vitro", epidermal keratinization was not observed. However the addition of histones after four days "in vitro" did not interfere with epidermal differentiation.