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Introduction: The mono- and digalactosyldiacylglycerol (MGDG and DGDG) galactolipids have been purified from the thermophilic blue-green alga Phormidium sp. ETS-05 that colonizes the therapeutic thermal mud of Abano Terme and Montegrotto Terme, Italy. Both compounds present a marked composition in polyunsaturated fatty acids, mainly omega-3. The therapeutic thermal mud is applied mainly to osteoarthritic cartilage patients. In the present study the effect of MGDG treatment on proteins and factors expressed by human articular cartilage cells in culture and on pathways activated in inflammatory conditions was studied., Methods: Primary cultures of human articular chondrocytes were used at cell passage number 1 (P1). Cells were treated in serum-free medium with inflammatory cytokines in the presence and in the absence of MGDG. Western blot was performed on collected medium and on cell layers. At least three different experiments were performed on primary cultures. The quantitation of the MGDG effect was performed by densitometric scanning of Western blots. p38 Mitogen Activated Protein Kinase (p38) activation, Nuclear Factor-kappaB (NF-kB) activation and Prostaglandin E(2) (PGE(2)) quantitation were performed by commercially available assays. Results are given as the mean values ± SD. All statistical analyses were performed using GraphPad software. The two-tailed Student's t -test was performed., Results: We report that MGDG: 1) represses the expression of interleukin-6 (IL-6) and interleukin-8 (IL-8) induced by interleukin-1alpha (IL-1α) or IL-1α + tumor necrosis factor α (TNFα) interfering with the p38 and NF-kB pathways; 2) is not toxic for the cells and does not affect the cell phenotype; 3) strongly enhances COX-2 expression induced by IL-1α or IL-1α + TNFα; 4) represses mPGES expression induced by IL-1α and the synthesis of PGE(2) and induces the synthesis of 15-deoxy-Δ 12,14-prostaglandin J(2) (15ΔPGJ(2)). In addition, the COX-2 product 15ΔPGJ(2) added to the cells: 1) strongly represses IL-6 and IL-8 induced by IL-1α; 2) represses mPGES expression induced by IL-1α and the synthesis of PGE(2)., Conclusions: All together these data suggest that MGDG has an anti-inflammatory activity in human articular cartilage and possibly activates an anti-inflammatory loop triggered by COX-2 via 15ΔPGJ(2) production, indicating a possible role of COX-2 in resolution of inflammation. The purified compound is a novel anti-inflammatory agent potentially active for human articular cartilage pathologies related to inflammation.

Objective: The aim of this study is to verify whether stratified epithelium-specific antinuclear antibodies are present in the sera of patients with erosive oral lichen planus and cutaneous lichen planus., Methods: We studied the pre-immune and immune serum of a rabbit immunized with a peptide corresponding to the N-terminus of the 70-kDa antigen chronic ulcerative stomatitis protein; sera from two patients, one with oral erosive lichen planus and one with cutaneous lichen planus who presented stratified epithelium-specific antinuclear antibodies at high titer; and a third serum from a patient with cutaneous lichen planus without stratified epithelium-specific antinuclear antibodies., Results: We demonstrated that the protein bands recognized by the serum of the rabbit immunized with an epitope of chronic ulcerative stomatitis protein co-migrated by SDS-PAGE with the protein bands recognized by the serum of a patient affected by oral erosive lichen planus and by the serum of a patient with cutaneous lichen planus, both containing antibodies directed against a 70-kDa antigen., Conclusions: Our results confirm that antibodies specifically directed against the chronic ulcerative stomatitis protein are not a distinctive marker of chronic ulcerative stomatitis, but may also be detected in oral erosive and cutaneous lichen planus.

The insect Lazarillo and the mammalian apolipoprotein D (ApoD) are orthologous members of the lipocalin protein family. We report the cloning and embryonic expression of chicken ApoD, the first molecularly characterized nonmammalian ApoD. We also report the ApoD expression in mouse during postnatal development and some novel aspects of the expression of the paralogous lipocalin prostaglandin D-synthase (PGDS) and discuss these results in view of the lipocalin family evolution in vertebrates. ApoD is expressed in subsets of central nervous system (CNS) neurons and glia during late chicken embryogenesis. Contrary to mouse ApoD, no expression appears in neural crest-derived cephalic mesenchyme and blood vessel pericytes. Also, ApoD is expressed in developing chicken feathers. These expressions are corroborated by quantitative reverse transcriptase-polymerase chain reaction profiles. ApoD is expressed during mouse postnatal development in a subset of CNS neurons, astrocytes and oligodendrocytes, but also in meninges and pericytes. Chicken PGDS is expressed in brain meninges and perivascular cells. Our results suggest that the amniote last common ancestor expressed ApoD and PGDS in the brain during embryogenesis. ApoD appears restricted to ectodermal derivatives, whereas PGDS is expressed by derivatives of the three germ layers.

Extracellular Fatty Acid Binding Protein (Ex-FABP) is a 21 kDa lipocalin, expressed during chicken embryo development in hypertrophic cartilage, in muscle fibres and in blood granulocyte. The protein selectively binds with high affinity fatty acids, preferably long chain unsaturated fatty acids in chondrocyte and myoblast cultures Ex-FABP expression is increased by inflammatory-agents and repressed by anti-inflammatory-agents. In adult cartilage, Ex-FABP is expressed only in pathological conditions such as in dyschondroplastic and osteoarthritic chicken cartilage. We propose that lipocalin Ex-FABP represents a stress protein physiologically expressed in tissues where active remodelling is taking place during development and also present in tissues characterized by a stress response due to pathological conditions.

Background: To verify the expression of neutrophil gelatinase associated lipocalin (NGAL), molecule now arising great interest because of its proposed involvement in cell-cycle regulation, acute phase response and immunomodulation, into extracellular fluids of female reproductive tract, in order to provide useful data to understand its biological functions. The data collected are purely qualitative, just meant to reveal the presence of lipocalin into the assayed fluids, and they have to be considered as preliminary for a quantitative study (in progress at the moment) based on a double antibody radioimmunoassay., Methods: Three kinds of extracellular fluid were randomly sampled: amniotic fluid (13 samples), cervical mucus (10 samples), coloster (20 samples). The inclusion criteria concerning the selection of the donor women were: age (fertile period), healthy state and pregnancy state. All the samples underwent protein assay, electrophoresis and western blot., Results: All the samples examined revealed NGAL's presence., Conclusions: The positive results of this study seem to strengthen the hypothesis related to NGAL biological functions, specifically the ones that suggest its role into cell differentiation, embryonic development and inflammation. Therefore the female reproductive tract is suggested as a new promising study object in this research field.

Objective: We have isolated and characterized in our laboratory a lipocalin specifically binding unsaturated long chain fatty acids (Ex-FABP). In developing chicken embryo long bones, Ex-FABP first appears at the boundary of the cone of hypertrophic cartilage. 'In vitro' EX-FABP is highly expressed by differentiating hypertrophic chondrocytes. Ex-FABP is expressed also in the forming myotubes both 'in vivo' and 'in vitro'. In cultured chondrocytes, Ex-FABP expression is strongly induced by treatment with inflammatory agents such as the bacterial liposaccharide LPS or interleukin-6. The possible mechanism for this induction was investigated. Expression of Ex-FABP was studied in other stress conditions., Design: To investigate a possible mechanism for Ex-FABP induction by LPS or interleukin-6, we have cultured the cells in the presence of either hydrogen peroxide or the NO donor SNAP (S-nitrosil-acetil-D, L-penicillamine), two agents known to produce cellular stresses through the activation of specific signalling pathways. To investigate Ex-FABP expression in other stress conditions, chondrocytes were cultured for 3 days in the presence of alpha,alpha-dipyridyl, an agent inhibiting prolyl hydroxylase activity and collagen secretion. Supplement of this agent to the culture medium results in an impairment of collagen secretion and assembly and the consequent altered interaction of the cell with the surrounding extracellular matrix. In addition Ex-FABP expression was studied also in chondrocytes cultured in the absence of serum, a stress condition activating cell defence mechanisms., Results: We have excluded that induction of Ex-FABP expression by inflammatory agents is mediated by oxidative stress or NO production. Ex-FABP expression was induced also by changes in the hypertrophic chondrocyte microenvironment, considered either as extracellular matrix surrounding the cell in culture or as nature and concentration of growth factor in the culture medium., Conclusions: No definitive data are so far available on the possible role of Ex-FABP when induced by cellular stresses. The capacity of the protein to specifically bind and transport unsaturated long chain fatty acids suggests that lipid metabolism and fatty acid utilization by the cells may be involved. Based on literature data the NRL/N-GAL (neu-related lipocalin/neutrophil gelatinase-associated lipocalin) protein was proposed as a possible mammal counterpart of the chick Ex-FABP. We have suggested that Ex-FABP and NRL/NGAL expression in forming bones and muscles is part of a 'physiological' acute phase response. Interestingly the expression of Ex-FABP and NRL/NGAL is also activated in osteoarthritic cartilage and in the case of NRL/N-GAL during neoplastic transformation of chondrogenic lineage cells.

Extracellular fatty acid binding protein (Ex-FABP) is a 21 kDa lipocalin specifically binding fatty acids, expressed during chicken embryo development in hypertrophic cartilage, in muscle fibers and in blood granulocytes. In chondrocyte and myoblast cultures Ex-FABP expression is increased by inflammatory agents and repressed by anti-inflammatory agents. In adult cartilage Ex-FABP is expressed only in pathological conditions such as in dyschondroplastic and osteoarthritic chickens. The possible mammalian counterpart is the Neu-related lipocalin (NRL), a lipocalin overexpressed in rat mammary cancer; NRL is homologous to the human neutrophil gelatinase associated lipocalin (NGAL) expressed in granulocytes and in epithelial cells in inflammation and malignancy and to the Sip24 (super-inducible protein 24), an acute phase lipocalin expressed in mouse after turpentine injection. Immunolocalization and in situ hybridization showed that NRL/NGAL is expressed in hypertrophic cartilage, in forming skeletal muscle fibers and in developing heart. In adult cartilage NRL/NGAL was expressed in articular cartilage from osteoarthritic patients and in chondrosarcoma. Moreover, NRL was induced in chondrocyte and myoblast cultures by an inflammatory agent. We propose that these lipocalins (Ex-FABP, NRL/NGAL, Sip24) represent stress proteins physiologically expressed in tissues where active remodeling is taking place during development and also present in tissues characterized by an acute phase response due to pathological conditions.

We report that Ex-FABP, an extracellular protein belonging to the lipocalin family and involved in the extracellular transport of long-chain fatty acids, is expressed in the forming myotubes both in vivo and in vitro. The presence of the protein and of the mRNA was observed in newly formed myotubes at early stages of chick embryo development by immunohistochemistry and by in situ hybridization. At later stages of development myofibers still expressed both the mRNA and the protein. Ex-FABP expression was observed also in the developing myocardium and the muscular layer of large blood vessels. In agreement with these findings, an initial expression of the mRNA and protein secretion by cultured chicken myoblasts were observed only after the onset of myoblast fusion. Double-immunofluorescence staining of these cultured cells revealed that multinucleate myotubes were stained by antibodies directed against both the Ex-FABP and the sarcomeric myosin, whereas immature myotubes and single myoblasts were not. When added to cultured myoblasts, antibodies against the Ex-FABP induced a strong enhancement of the production of the same protein. In all experiments some cell sufferance and a transient impairment of myotube formation were also observed. The finding that the continuous removal of the Ex-FABP from the culture medium of myoblasts, due to the formation of immune complexes, resulted in an overproduction of the protein suggests a feedback (autocrine) control during myotube differentiation and maturation. We propose that the requirement for increased transport and metabolism of free fatty acid released from the membrane phospholipids and storage lipids, mediated by Ex-FABP, may be essential during differentiation of multinucleated myotubes or that an increased local demand of fatty acids and metabolites may act as a local hormone in tissues differentiating and undergoing morphogenesis., (Copyright 1998 Academic Press.)

Secretion of angiogenesis inhibitors and stimulators is modulated during in vitro differentiation of embryonic chick growth plate chondrocytes. Supernatants from dedifferentiated cells undergoing maturation to hypertrophic chondrocytes in suspension progressively inhibited vascular cell random migration and invasion of basement membrane matrix by endothelial cells. Maximal inhibition was exhibited by conditioned medium from hypertrophic chondrocytes. The same medium also repressed vascular cell migration induced by highly angiogenic Kaposi's sarcoma cell supernatants and prevented formation of an anastomosed network of tube-like structures by endothelial cells plated on matrigel. On the contrary, when the suspension culture of hypertrophic chondrocytes was supplemented with ascorbic acid, a condition leading to the formation of a mineralized tissue similar to calcified cartilage, a dramatic switch to production of angiogenic activity was observed. Medium conditioned by osteoblast-like cells derived from hypertrophic chondrocytes also induced vascular cell migration and invasion of basement membrane matrix. The presence of angiogenic activity in the conditioned medium was assessed also by an in vivo assay in mice using reconstituted basement membrane associated with heparin. Therefore, interactions of chondrocytes with their extracellular matrix are an absolute requirement for the expression of angiogenic activities by hypertrophic chondrocytes at late developmental stages.

Data obtained while investigating growth plate chondrocyte differentiation during endochondral bone formation both in vivo and in vitro indicate that initial chondrogenesis depends on positional signaling mediated by selected homeobox-containing genes and soluble mediators. Continuation of the process strongly relies on interactions of the differentiating cells with the microenvironment, that is, other cells and extracellular matrix. Production of and response to different hormones and growth factors are observed at all times and autocrine and paracrine cell stimulations are key elements of the process. Particularly relevant is the role of the TGF-beta superfamily, and more specifically of the BMP subfamily. Other factors include retinoids, FGFs, GH, and IGFs, and perhaps transferrin. The influence of local microenvironment might also offer an acceptable settlement to the debate about whether hypertrophic chondrocytes convert to bone cells and live, or remain chondrocytes and die. We suggest that the ultimate fate of hypertrophic chondrocytes may be different at different microanatomical sites.

We investigated the induction of specific protein expression by heat shock in dedifferentiated and hypertrophic chick embryo chondrocytes in a culture system that allows 'in vitro' differentiation of cartilage cells [Castagnola, P., Moro, G., Descalzi-Cancedda, F. and Cancedda, R. (1986) J. Cell. Biol. 102, 2310-2317]. As control, we used cultures of embryonic fibroblasts from the whole body and from the skin. In the cell lysates of all cultures we identified four major heat-shock proteins (HSP), with a molecular size corresponding to HSP families previously described (HSP 90, HSP 70, HSP 47 and HSP 26). Some of these proteins were constantly induced when the temperature was raised, others were expressed in a more variable manner. Differences also existed in the relative amount of the HSP synthesized by the four cultures. When we specifically investigated HSP species released into the culture medium, we observed a 43-45 kDa protein constantly expressed and secreted in large amount by the cells. On the basis of its biochemical characteristic and its precipitation by specific antibodies, this protein has been identified as osteonectin (SPARC, BM-40).

Expression of specific differentiation markers was investigated by histochemistry, immunofluorescence, and biosynthetic studies in osteoblasts outgrown from chips derived from tibia diaphyses of 18-day-old chick embryos. The starting osteoblast population expressed type I collagen and alkaline phosphatase in addition to other bone and cartilage markers as the lipocalin Ch21; the extracellular matrix deposited by these cells was not stainable for cartilage proteoglycans, and mineralization was observed when the culture was maintained in the presence of ascorbic acid, calcium and beta-glycerophosphate. During culture, clones of cells presenting a polygonal chondrocyte morphology and surrounded by an Alcian-positive matrix appeared in the cell population. Type II collagen and type X collagen were synthesized in these areas of chondrogenesis. In addition, chondrocytes isolated from these cultures expressed Ch21 and alkaline phosphatase. Chondrocytes were generated also from homogeneous osteoblast populations derived from a single cloned cell. The coexistence of chondrocytes and osteoblasts was observed during amplification of primary clones as well as in subclones. The data show the existence, within embryonic bone, of cells capable in vitro of both osteogenic and chondrogenic differentiation.

Ch21 protein, a developmentally regulated chick embryo protein of 21,000 apparent molecular weight, was purified from culture medium of hypertrophic chondrocytes. The purification method included a DEAE cellulose chromatography column, a CM cellulose chromatography column and a HPLC molecular sieve column. The amino acid sequence of the amino terminal end of the protein was determined. Computer assisted analysis showed significant homology between this sequence and the amino terminal sequences of proteins that belong to the superfamily of the low molecular weight binding proteins sharing a basic framework for the binding and transport of small hydrophobic molecules. Determination of the amino terminal sequence of the chicken retinol binding protein excluded identity between this protein and the Ch21.