Connective tissue response to fractionated thermo-ablative Erbium: YAG skin laser treatment.

Indications for and prevalence of laser therapies with a fractionated laser beam have risen significantly. However, as of yet, little is known about the underlying molecular changes, especially with respect to dermal extracellular-matrix remodelling, wound healing and inflammation. This study aimed at the investigation of the connective tissue response of sun-damaged skin following fractionated laser treatment. Seven patients received a laser therapy on the lateral side of the neck of wrinkles grade III-IV (Glogau scale) using a fractionated thermo-ablative erbium yttrium aluminium garnet (Er:YAG) laser (2940 nm, BURANE XL; Quantel Derma, Erlangen, Germany). Skin biopsies were taken at baseline from untreated skin, 1 and 6 weeks after laser intervention to investigate hyaluronan (HA), collagen-I (Coll-I) and collagen-III (Coll-III) remodelling as well as alteration of matrix metalloproteinase 1 expression (MMP-1). To address this issue, HA synthesizing (HA synthetases, HAS) and degrading (hyaluronidases, HYAL) enzymes were measured at mRNA-level using a real-time PCR. Furthermore, immunohistochemical staining for HA was performed by using the HA binding protein (HAbP) and for Coll-I, Coll-III and MMP-1 by using monoclonal antibodies. The degree of inflammation was correlated descriptively. Our findings were that at the two examined read out points, HAS and HYAL showed a slight response alluding to HA synthesis under minimal signs of inflammatory reaction. Concordantly, although to a varying degree, an increase in the HA content of the skin after laser treatment could be detected by immunhistochemistry. During remodelling, Coll-I, Coll-III and MMP-1 showed a cyclic course with a peak after 1 week. Conclusively, our results indicate a light alteration of the HA metabolism towards synthesis and a transient collagen neogenesis caused by a single fractionated thermo-ablative laser skin intervention. Clinical improvement might be attributed to synergistic effects between collagen neogenesis and the water binding capacities of HA and its influence on skin contraction and remodelling.

New E-beam-initiated hyaluronan acrylate cryogels support growth and matrix deposition by dermal fibroblasts.

Cryogels made of components of natural extracellular matrix components are potent biomaterials for bioengineering and regenerative medicine. Human dermal fibroblasts are key cells for tissue replacement during wound healing. Thus, any biomaterial for wound healing applications should enable growth, differentiation and matrix synthesis by these cells. Cryogels are highly porous scaffolds consisting of a network of interconnected pores. Here, we used a novel group of cryogels generated from acrylated hyaluronan where the polymerization was initiated by accelerated electrons (E-beam). This novel procedure omits any toxic polymerization initiators and results in sterile, highly elastic scaffolds with adjustable pore size, excellent swelling and low flow resistance properties. We show that these cryogels are effective 3D-substrates for long-term cultures of human dermal fibroblasts in vitro. The cells proliferate for at least 28days throughout the cryogels and deposit their own matrix in the pores. Moreover, key modulators of dermal fibroblasts during wound healing like TGFß and PDGF efficiently stimulated the expression of wound healing-relevant genes. In conclusion, electron beam initiated cryogels of acrylated hyaluronan represent a functional and cell compatible biomaterial that could be adapted for special wound healing applications by further functionalization.

Investigation of arthritic joint destruction by a novel fibroblast-based model.

The key pathologic mechanism in rheumatoid arthritis (RA) is the destruction of cartilage by fibroblasts. In a severe combined immunodeficient (SCID) mouse model, this process can be modulated by gene transfer using invasive LS48 fibroblasts. This study aims to investigate the effect of interleukins (IL) -11 and -12 on cartilage destruction when transferred into LS48, and of IL-15 when transfected into non-invasive 3T3 cells; to compare three transduction systems (a lentiviral vector system, a retroviral vector system, and a particle-mediated gene transfer); and to establish an in vitro cartilage destruction system based on LS48 cells. Transduced fibroblasts were injected into SCID mice knee joints, and disease progression assessed microscopically. Distinctive morphologic pattern revealed invasion of fibroblasts into the articular cartilage by transfected, as well as non-transfected, LS48 cells. IL-12 and IL-15 did not alter swelling or cartilage destruction. Animals treated with IL-11-transfected cells showed reduced cartilage damage but no changes in swelling. Efficacy of gene transfer to establish transfected fibroblasts was shown to be >85% for lentiviral transfer, compared to <10% for retroviral transfer and gene gun. Furthermore, cells were co-incubated with porcine cartilage. Transduction of IL-11 led to a reduction of apoptosis in chondrocytes. These findings suggest that cartilage destruction by invasive fibroblasts can be modulated by gene transfer. Lentiviral vector systems offer the most effective approach for gene transduction. In vitro fibroblast/cartilage co-cultures present a convenient system for the assessment of novel therapeutic strategies toward reduction of articular destruction.

A ligand of human thy-1 is localized on polymorphonuclear leukocytes and monocytes and mediates the binding to activated thy-1-positive microvascular endothelial cells and fibroblasts.

Thy-1 is known to be expressed on fibroblasts, nerve cells, and blood stem cells. Previous studies have shown the induction of Thy-1 on phorbol ester stimulated human dermal microvascular endothelial cells in vitro. In situ Thy-1 expression was found on activated endothelium. In this study we were interested in the localization of a Thy-1 ligand and the characterization of the function of Thy-1 on human dermal microvascular endothelial cells and fibroblasts. Human Thy-1 purified from fibroblast extracts was labeled and used as a probe for the detection of a Thy-1 ligand. In cryostat sections of bullous pemphigoid skin a Thy-1 ligand was found on inflammatory cells, whereas the Thy-1 antigen was expressed on the endothelial cells and fibroblasts. By flow cytometry we could show the expression of a Thy-1 ligand on polymorphonuclear leukocytes and monocytes, whereas lymphocytes did not express this Thy-1 ligand. To study whether Thy-1 is involved in cell-cell adhesion we separated Thy-1-positive and Thy-1-negative cells by magnetic cell separation using the monoclonal antibody AS02. Cell adhesion assays and blocking experiments revealed a direct involvement of the Thy-1/Thy-1 ligand interaction in the binding of monocytes and polymorphonuclear leukocytes to Thy-1-positive activated endothelial cells and fibroblasts.

Functional aspects of the effect of prolactin (PRL) on adrenal steroidogenesis and distribution of the PRL receptor in the human adrenal gland.

Hyperprolactinemia is one of the most common disorders in endocrinology. A role for PRL on the human adrenal gland has been postulated in various clinical studies. We have demonstrated for the first time the expression of the PRL receptor in the human adrenal gland and in human adrenal primary cell cultures using PCR and immunohistochemical methods. Using immunostaining, we could detect the PRL receptor in all three zones of the adrenal cortex. Only weak staining was observed in the adrenal medulla. The influence of PRL on the secretion of cortisol, aldosterone, and androgens in human primary cell cultures was investigated. After stimulation with PRL (10(-7) mol/L), we measured increased concentrations of cortisol (155 +/- 9.8%; P < 0.005%), aldosterone (122 +/- 3.7%; P < 0.005), and dehydroepiandrosterone (121 +/- 8.6%; P < 0.05) in the cell supernatant. PRL did not affect the expression of messenger ribonucleic acid of cytochrome P45017 alpha in human adrenal cell cultures. In conclusion, we found the PRL receptor in the human adrenal gland. We postulate that PRL has a direct effect on adrenal steroidogenesis, thereby regulating adrenal function, which may be of particular relevance in clinical disorders with hyperprolactinemia.

Expression of leptin (Ob) and leptin receptor (Ob-R) in human fibroblasts: regulation of leptin secretion by insulin.

Leptin, a hormone of the cytokine family, is mainly synthesized by white adipocytes. As fibroblasts and adipocytes share a common stem cell origin, we hypothesized that connective tissue may be another candidate for leptin synthesis. We demonstrated leptin receptors, inclusive of all isoforms, on cultured fibroblasts (n = 13) by RT-PCR and immunohistochemistry. In contrast to its receptor, basal leptin mRNA expression and protein secretion were found in 8 of 13 cultures, reaching 1.4 ng/350,000 cells.24 h. Incubation with physiological insulin concentrations (1 nmol/liter) increased leptin secretion in fibroblast culture supernatants to 152% of basal levels. A maximal stimulation of the basal level up to 192% was found with 10 nmol/liter insulin after 24 h. Actinomycin D and cycloheximide abolished this effect, providing evidence that active RNA and protein synthesis are involved in insulin's action. Completing these in vitro results, we could show protein expression for leptin and leptin receptors in fibroblasts by immunostaining of human skin biopsies in situ. In conclusion, we provide evidence of leptin synthesis and secretion by human fibroblasts that are regulated by insulin. Leptin produced by fibroblasts may thus exert important local autocrine and paracrine actions and contribute to the total plasma pool. Hence it might in part account for variations in body mass index-dependent reference ranges of leptin as well as disruptions in the relationship between fat content and leptin.

The role of CXCR5 and its ligand CXCL13 in the compartmentalization of lymphocytes in thyroids affected by autoimmune thyroid diseases.

OBJECTIVE: Graves' disease (GD) and Hashimoto's thyroiditis (HT) are characterized by lymphocytic infiltrates partly resembling secondary lymphoid follicles in the thyroid. CXCR5 and its ligand CXCL13 regulate compartmentalization of B- and T-cells in secondary lymphoid organs. The aim of the study was to elucidate the role of this chemokine receptor-ligand pair in thyroid autoimmunity. METHODS: Peripheral blood and thyroid-derived lymphocyte subpopulations were examined by flow cytometry for CXCR5. CXCR5 and CXCL13 cDNA were quantified in thyroid tissues by real-time RT-PCR. RESULTS: We found no differences between the percentages of peripheral blood CXCR5+ T- and B-cells in GD patients (n=10) and healthy controls (n=10). In GD patients, the number of memory CD4+ cells expressing CXCR5 which are functionally characterized as follicular B helper T-cells is higher in thyroid-derived (18+/-3%) compared with peripheral blood T-lymphocytes (8+/-2%). The highest CXCL13 mRNA levels were found in HT (n=2, 86.1+/-1.2 zmol (10(-21) mol) cDNA/PCR) followed by GD tissues (n=16, 9.6+/-3.5). Only low amounts were determined in thyroid autonomy (TA) (n=11) thyroid tissues, irrespective of whether the autonomous nodule (0.5+/-0.1) or the surrounding normal tissue (1.8+/-0.7) had been analyzed. The same differences were found for CXCR5 (HT: 179.1+/-6.8; GD: 17.4+/-10.6; TA(nodule): 0.8+/-0.5; TA(normal): 4.4+/-3.6). In GD, there is a correlation between CXCL13 and CXCR5 mRNA levels and the number of focal lymphocytic infiltrates and germinal centers as well as anti-thyroperoxidase but not anti-TSH receptor autoantibodies. CONCLUSIONS: CXCR5 and CXCL13 play an essential role in maintaining B- and T-cells in lymphocytic infiltrates and ectopic follicles in thyroid tissue from patients affected by autoimmunity.

Chemokine release from activated human dermal microvascular endothelial cells--implications for the pathophysiology of scleroderma?

Long-term exposure to silica (SiO2) may induce silicosis as well as extrapulmonary diseases such as scleroderma. Infiltration of mononuclear cells and release of proinflammatory cytokines from these cells have been suggested to play a role in the development of inflammatory and immunological events typical of scleroderma as well as of silica-induced scleroderma. We showed that silica is able to directly activate cytokine expression in blood monocytes, collagenase expression in cultured dermal fibroblasts and ICAM-1 expression in human dermal microvascular endothelial cells. In the study reported here we found that silica and TNFalpha induce mRNA and protein of the chemokines RANTES and MCP-1 in endothelial cells. In addition, we demonstrated that culture supernatants of silica-treated endothelial cells are chemotactic for mononuclear cells from peripheral blood, suggesting that activation of endothelial cells may contribute to the chemotactic gradient necessary for extravasation of inflammatory blood cells into the surrounding tissue found in early scleroderma. However, a polyclonal anti-RANTES antibody failed to block chemotaxis suggesting that other proteins are involved in this phenomenon. We also studied the expression of RANTES in situ in the skin of systemic sclerosis patients and of healthy individuals. We found abundant RANTES mRNA expression in the skin of SSc patients, whereas in control skin no expression was found. From our data we conclude that RANTES and MCP-1 induction by silica may be an initiating event in inflammatory infiltration, whereas TNFalpha-mediated inflammation may propagate the disease more efficiently.

The monoclonal antibody AS02 recognizes a protein on human fibroblasts being highly homologous to Thy-1.

Recently, we described a novel fibroblast-restricted monoclonal antibody (mAb AS02) that recognizes a membrane-bound antigen. Characterization and isolation of the corresponding antigen showed that mAb AS02 recognized a protein on human fibroblasts that is highly homologous or identical to human Thy-1 antigen (CD90). Partial amino acid sequencing of the corresponding mAb AS02 antigen and comparison with known proteins revealed a 100% homology of the sequenced peptides to the human Thy-1 antigen. Cross-immunodepletion studies with mAb AS02 and an anti-Thy-1 antibody confirmed these results. Utilizing two-dimensional (2D) gel electrophoresis of fibroblast cell extracts and purified antigen, mAb AS02 and the anti-Thy-1-antibody recognized identical protein spots. Furthermore, we demonstrated many identical biochemical properties of the corresponding AS02 antigen and Thy-1 antigen, such as the molecular weight of the core protein and deglycosylation products and the detection of a GPI anchor. In functional assays, the attachment of fibroblasts to collagen I and fibronectin was increased after incubation of fibroblasts with mAb AS02. Therefore, the Thy-1 antigen appears to be involved in the regulation of the adherence of human dermal fibroblasts.

Fibroblasts enhance the invasive capacity of melanoma cells in vitro.

In previous experiments we have shown an enhanced expression of matrix metalloproteinase-1 (MMP-1) in fibroblasts obtained from the border of invasive melanoma in comparison to fibroblasts more distant from the tumour. In the study reported here we sought to determine whether melanoma-derived soluble factors are responsible for the stimulation of MMP-1 expression in fibroblasts. By real-time PCR and enzyme-linked immunosorbent assays, we demonstrated that the stimulation of fibroblasts with melanoma cell conditioned medium led to an increased expression of MMP-1 mRNA as well as MMP-1 protein, whereas melanoma cells themselves did not produce detectable amounts of MMP-1 protein. Basic fibroblast growth factor (bFGF) was detected as an important factor responsible for the enhanced expression of MMP-1 by fibroblasts after stimulation with melanoma cell conditioned medium. In a three-dimensional in vitro invasion assay, we demonstrated that fibroblasts are essential for melanoma cell invasion into a collagen I matrix. These findings support the hypothesis that stromal fibroblasts assist the invasion of melanoma cells through the extracellular matrix by producing elevated amounts of proteolytic enzymes after interaction with soluble factors (e.g. bFGF).

Mature human spermatozoa do not transcribe novel RNA.

Mature spermatozoa contain subsets of mRNA that have been found in human testes before. Based on this finding it was hypothesized that the mRNAs of spermatozoa are transcribed during spermatogenesis. However, up to now there is no proof of the transcriptional inactivity of human sperm. To address this issue we performed in vitro labelling experiments with radio-labelled uridine triphosphate followed by analysis of cellular RNA. There was virtually no radioactive RNA detectable in the RNA purified from human spermatozoa proving the transcriptional inactivity of mature spermatozoa. The spermatozoal RNA obviously results from transcription during spermatogenesis and can be used for diagnostic purposes. These findings might have diagnostic and - possibly - therapeutic value in infertility patients as spermatozoal RNAs might complement the RNA pool of the oocyte after fertilization.

Silica induced scleroderma--clinical and experimental aspects.

Clinical and experimental data concerning silica induced systemic sclerosis (SSc) are discussed in comparison to current knowledge of the pathophysiology of idiopathic SSc. About 280 patients with SSc after longterm silica dust exposure, some with associated silicosis, have been reported; 111 of them were analyzed as the largest cohort in our department. Based on clinical and laboratory data, silica induced and idiopathic SSc show similar pathophysiology and similar markers of the disease including vascular involvement, immunological abnormalities, and dysregulation of extracellular matrix metabolism. Experimental studies show that silica dust is able to activate microvascular endothelial cells, mononuclear cells from peripheral blood, and dermal fibroblasts in vitro in a fashion in common with pathophysiological events known from idiopathic SSc.

Pathophysiology of scleroderma: an update.

OBJECTIVES: To review the pathophysiological background of systemic sclerosis in relation to the main components involved: microvascular system, immunological system and fibroblasts of the connective tissue. BACKGROUND: Although many particular aspects of the pathophysiology of systemic sclerosis have been investigated in recent years, the complexity of the pathogenesis and the important links between the components involved remain unclear. METHODS: Literature review. RESULTS AND CONCLUSION: Scleroderma is a connective tissue disorder resulting in a progressive fibrosis of skin and internal organs. The genetic background is not clear. The microvascular system is one of the first targets involved (damage of capillaries, enhanced expression of adhesion molecules interacting with lymphocytes, perivascular infiltrates as starting points for tissue fibrosis). The immune system is unbalanced (selection of T-cell subpopulations, elevated serum levels of several cytokines, occurrence of autoantigens to topoisomerase I, centromeric proteins and RNA polymerases). As far as autoimmunity is concerned the triggering autoantigen is still unknown. Development of connective tissue fibrosis is prominent (subpopulations of fibroblasts with disturbed regulation of collagen turnover by TGF-beta, CTGF and collagen receptors (alpha1beta1, alpha2beta1)). Investigation of pathophysiology of scleroderma is effected by monitoring the serum levels for soluble mediators, by cell culture studies of affected and non-affected fibroblasts and EC, by studying environmentally induced forms of scleroderma and by studies using animal models.

Penicillin G does not alter collagen type I metabolism of dermal fibroblasts in culture.

BACKGROUND: Penicillin G (PenG) has successfully been used for therapy of dermal fibrosis in patients suffering from systemic sclerosis (SSc). However, there is little knowledge on the mechanism of the antifibrotic action of PenG. OBJECTIVE: We studied the effects of PenG on dermal fibroblasts by analysing the influence of various amounts of PenG on the proliferation, synthesis and degradation of collagen by human dermal fibroblasts from healthy donors and 1 SSc patient (collagen high producer). METHODS: Collagen metabolism of cultured dermal fibroblasts was studied by Northern hybridisation for mRNA of collagen I, proline-4-hydroxylase, lysyl hydroxylase, matrix metalloproteinase I and determination of collagen content in culture supernatants. RESULTS: PenG did not alter the expression of the investigated mRNA, independently of the dosage and the incubation times used. The amount of collagen I protein was not influenced. CONCLUSION: There is no evidence of a direct antifibrotic effect of PenG on dermal fibroblasts; therefore, other mechanisms might be responsible for its effect in the treatment of SSc patients.

Detection of human soluble Thy-1 in serum by ELISA. Fibroblasts and activated endothelial cells are a possible source of soluble Thy-1 in serum.

The functions of Thy-1, a 35-kDa cell-surface glycoprotein, and its natural ligand are still unknown. Anchoring to the membrane via linkage to phosphatidyl-inositol (PI) raises the possibility of cleavage off the membrane by PI-specific phospholipases. Soluble Thy-1 (sThy-1) could interfere with the binding of the unknown natural ligand followed by regulation of different cell functions. In this study we established an enzyme-linked immunosorbent assay (ELISA) to measure and quantify sThy-1 in serum and wound fluid. Recombinant human Thy-1 (rhThy-1) was expressed in Drosophila S2 cells, purified from culture supernatant and used as standard for quantitation of sThy- by the ELISA technique. There were no differences in sThy-1 levels in serum of healthy donors and patients with systemic sclerosis, leg ulcers, or rheumatoid arthritis, respectively, detected by ELISA. In contrast, at the local site of inflammation, in wound fluid of venous leg ulcers and in synovial fluid from joint puncture, we found strongly elevated levels of sThy-1 compared with sThy-1 in the serum of the same patient. Thy-1 is expressed in humans on brain cells, fibroblasts, a subpopulation of CD34+ blood stem cells, and possibly activated human dermal microvascular endothelial cells. In this study, we never found Thy-1 mRNA or protein expression in resting endothelial cells as shown by reverse transcriptase polymerase chain reaction (RT-PCR) and flow-cytometry. Thy- expression could be induced on endothelial cells by phorbol myristate acetate and to a lesser extent by tumor necrosis factor-alpha (TNF-alpha). In situ, monoclonal antibodies to Thy-1 did not stain endothelial cells in normal skin, whereas endothelial cells in the synovial membrane of rheumatoid arthritis patients and endothelial cells surrounding melanoma express Thy-1. In summary, our data indicate that Thy-1 is present in soluble form in serum. Furthermore, Thy-1 seems to be a marker for endothelial cell activation. Therefore, activated endothelial cells as well as fibroblasts might be a possible source of sThy-1.

The Thy-1/Thy-1 ligand interaction is involved in binding of melanoma cells to activated Thy-1- positive microvascular endothelial cells.

The expression of adhesion molecules on endothelial cells as well on tumor cells regulates and directs adhesion and transmigration of tumor cells through the endothelial cell barrier as one prerequisite to the formation of metastasis. Thy-1 is an inducible activation-associated cell-adhesion molecule on human dermal microvascular endothelial cells (HDMECs). In this study we investigated whether the Thy-1/Thy-1 ligand interaction may also play a role in adhesion of melanoma cells to endothelial cells. In situ, a strong Thy-1 expression on endothelial cells in melanoma and melanoma metastases was observed. In vitro, Thy-1 expression was stimulated by melanoma-cell-derived soluble factors, reflecting that Thy-1 expression in melanoma is not only due to a nonspecific inflammatory response. TNFalpha and bFGF were not responsible for this effect. In vitro and in situ a Thy-1 ligand was detected on melanoma cells. In cell-adhesion assays we showed the involvement of the Thy-1/Thy-1 ligand interaction in adhesion of melanoma cells to HDMECs. In summary, the data support that the study of the Thy-1/Thy-1 ligand interaction might give a more detailed insight into the regulation and direction of adhesion of melanoma cells to endothelial cells as one critical step in the formation of tumor metastasis.

The fibroblast-specific MAb AS02: a novel tool for detection and elimination of human fibroblasts.

The unwelcome presence of fibroblasts in many cell cultures prevents the long term cultivation of various cell types and work with pure populations. Recently, we described a novel fibroblast-specific monoclonal antibody (MAb AS02) that recognises a membrane-bound antigen. We have now developed a method using the fibroblast-specific MAb AS02 immobilised on goat-anti-mouse-magnetic beads to separate contaminating fibroblasts. An endothelial cell line experimentally contaminated with 5%-50% fibroblasts was successfully purified. Additionally, an endothelial cell line with an initial fibroblast contamination of 1.5% was prepared. A proportion of each preparation was cultured with no separation step being performed, whereas the remainder was cultured after purification with MAb AS02 to exclude the presence of a minor number of fibroblasts (<0.1%). The proportion of fibroblasts increased up to 38% in the fifth passage of culture without elimination of the low initial fibroblast contamination, whereas in the fraction with the separation step, no fibroblasts were detectable by flow cytometry, even after the fifth passage. We also used the antibody to detect the presence of naturally contaminating fibroblasts in thyrocyte cultures. After cultivation of thyrocyte cultures over five passages, the number of fibroblasts increased dramatically up to 50%-80% of the whole population. Subsequently, we successfully applied the method for complete elimination of naturally contaminating fibroblasts from freshly isolated thyrocyte cultures from enzymatically digested thyroid glands. Thus, MAb AS02 is a fibroblast-specific marker that is a useful tool for the detection and elimination of contaminating fibroblasts. The specificity of MAb AS02 permits the universal application of this antibody for human cell cultures of interest.