Thy-1 (CD90) is an interacting partner for CD97 on activated endothelial cells.

Leukocyte recruitment in response to inflammatory signals is governed, in part, by binding to Thy-1 (CD90) on activated endothelial cells (EC). In this study, we characterized the adhesion G-protein coupled receptor CD97, present on peripheral myeloid cells, as a novel interacting partner for Thy-1. CD97 was upregulated on polymorphonuclear cells (PMNC) of patients with psoriasis. In psoriatic skin lesions, CD97(+) myeloid cells colocalized with Thy-1(+) EC of small vessels in microabscesses, suggesting an interaction between CD97 and Thy-1 that was further examined by adhesion and protein-binding assays. PMNC and cell lines stably overexpressing CD97 adhered specifically to Thy-1(+)-activated human dermal EC, Thy-1(+) CHO cells, and immobilized Thy-1 protein. Binding of the CD97(+) CHO clones correlated with their CD97 expression level. Soluble CD97 bound specifically to immobilized Thy-1 protein, as well as Thy-1(+)-activated EC and CHO cells. In all assays, cellular adhesion or protein binding was blocked partially by CD97 and Thy-1-blocking mAb. Our data suggested that CD97 interacts via its stalk with Thy-1 because mAb directed to the stalk of CD97 showed stronger blocking compared with mAb to its epidermal growth factor-like domains, and binding was calcium independent. Moreover, soluble CD97 without the stalk and soluble EMR2, containing highly homologous epidermal growth factor-like domains but a different stalk, failed to bind. In summary, binding of leukocytes to activated endothelium mediated by the interaction of CD97 with Thy-1 is involved in firm adhesion of PMNC during inflammation and may play a role in the regulation of leukocyte trafficking to inflammatory sites.

Suppression of hyaluronan synthase 2 expression reflects the atrophogenic potential of glucocorticoids.

We recently demonstrated that dexamethasone, a strongly atrophogenic glucocorticoid (GC), downregulated hyaluronan (HA) production in human keratinocytes and fibroblasts in vitro and after topical application to human skin in vivo via suppression of hyaluronan synthase 2 (HAS2). To test whether HAS2 activity might discriminate the atrophogenic potential of other substances applied topically to skin, we compared the HA metabolism in cultured human fibroblasts following in vitro treatment with GCs and the non-atrophogenic calcineurin inhibitor tacrolimus. GCs suppressed HAS2 mRNA expression and decreased HA as shown by qRT-PCR or ELISA. Tacrolimus did not affect hyaluronan-metabolizing enzymes nor total HA. Neither mometasone nor tacrolimus affected the expression of collagen mRNAs in human fibroblasts. In conclusion, suppression of HAS2 activity may represent an early process of GC-induced skin atrophy that precedes the suppression of collagens. Analysis of HAS2 regulation may thus be of value to assess the atrophogenic potential of drugs being developed.

Lysophosphatidylcholine-mediated functional inactivation of syndecan-4 results in decreased adhesion and motility of dendritic cells.

Following antigen contact, maturation and migration of DCs into lymphatic tissues are crucial to the developing immune response or maintenance of tolerance. Lysophosphatidylcholine (LysoPC) is generated during apoptosis of cells and acts as a "find-and-eat-me" signal thought to prevent autoimmunity. Moreover, LysoPC can activate PKCδ and initiates a signaling cascade that leads to phosphorylation and inactivation of syndecan-4 (SDC4), a heparansulfate proteoglycan integrin co-receptor. In human monocyte-derived DCs, we recently demonstrated that SDC4 is upregulated during maturation thereby stimulating DC motility. Here, we investigate the effects of LysoPC on DC motility as well as on the involvement of PKCδ phosphorylation-dependent regulation of DC motility by SDC4 and PKCα. Employing a static adhesion assay and videomicroscopy, we show that LysoPC inhibits adhesion of DCs to fibronectin and motility of DCs by decreasing podosome formation. Moreover, DC podosome formation and motility, which both are regulated by SDC4 and subject to control by PKCδ-dependent phosphorylation of SDC4, were inhibited in LysoPC-matured DCs. Thus, these DC are defective in adhesion and migration. Based on our results, we hypothesize that LysoPC released during apoptosis might delay DC migration to lymphoid organs and thus prevent autoimmunity.

[Compliance to compression therapy in patients with existing venous leg ulcers. Results of a cross-sectional study]. / Compliance hinsichtlich der Kompressionstherapie bei Patienten mit floridem Ulcus cruris venosum: Ergebnisse einer Querschnittsuntersuchung.

BACKGROUND AND PURPOSE: Compression therapy is an essential part in the treatment of patients with venous leg ulcers. However, not all patients follow this recommendation. The authors tried to analyze how many patients performed compression therapy at the time of data collection and the reasons for insufficient or missing compression therapy. PATIENTS AND METHODS: Within a cross-sectional study in 73 patients with venous leg ulcers (patients with epifascial varicosis as well as patients with postthrombotic syndrome), conducted between 11/2007 and 01/2009 in the own specialized wound ambulance, it was documented, if the patients performed compression therapy at all. By clinically defined criteria, the attending doctor evaluated the efficiency of the compression therapy and documented who had been responsible for the compression therapy. Patients not wearing any compression were asked for the reasons. 25 patients were registered several times and evaluated separately; for those, the authors tried to find out, if compliance to compression therapy after refreshing of the recommendation had changed. RESULTS: In total, 75% (n = 55) of 73 patients were performing compression therapy at the time of data collection, most of them with short stretch bandages. In 50% (n = 19), compression therapy was sufficient. Most of these patients (n = 24, 63%) put the bandages on by themselves. If the bandages had been put on inadequately, the patients explained this (multiple answers possible) mostly with unfitting shoes or extreme overweight. In the group of patients with compression stockings, compression therapy was optimal in 91%; the patients put the stockings on by themselves in nearly all cases. 25% of the patients (n = 18) did not perform any compression. In 61% (n = 11), they explained this with their own unwillingness. 25 patients were observed more than once. 15 of them showed an unchanged compression standard in the follow-up, in detail with sufficient compression in 73% and an unchanged insufficient compression in 27%. CONCLUSION: In cases of insufficient compression, possible impediments should be identified and eliminated. Patients who do not perform any compression therapy, should be motivated with easy-to-handle compression therapy options like compression stocking systems.

Dermal hyaluronan is rapidly reduced by topical treatment with glucocorticoids.

Skin atrophy is part of the normal ageing process, but is accelerated by topical glucocorticoid (GC) treatments that are widely used in dermatology. Hyaluronan (HA) is one of the most abundant components of the cutaneous extracellular matrix and is involved in tissue homeostasis, hydration, and repair processes, but little is known about the effects of GCs on HA synthesis and stability. Here we examined the regulation of HA metabolism in human skin during GC therapy. Expression of the HA synthesizing enzymes hyaluronan synthase (HAS)-2 and HAS-3 and the HA degrading enzymes HYAL-1, HYAL-2, and HYAL-3 in response to GC treatment was evaluated. HAS-2 expression was markedly suppressed by dexamethasone treatment of cultured fibroblasts and HaCaT keratinocyte cells, and in human skin biopsies taken from volunteers treated with dexamethasone ointment. Consistently, the HA content of cell culture supernatants and in human skin was reduced after dexamethasone treatment. Hyaluronidase expression and activity, on the other hand, was not altered by dexamethasone treatment. These data show that the levels of skin HA rapidly decrease after short-term GC treatment due to a reduction in HA synthesis, while HA degradation is not changed. This may reflect an initiation of skin atrophy in response to topically applied GCs.

New indications for artificial collagen-elastin matrices? Covering exposed tendons.

We present an 80-year-old patient who presented a defect with exposed tendons on the dorsum of the hand after micrographic controlled tumour excision. The defect was closed using a combination of artificial collagen-elastin matrix (Matriderm) covered by an autologous split-skin graft in a 1-step approach resulting in rapid healing and good functional and cosmetic results.

A case of cutaneous Rosai-Dorfman disease refractory to imatinib therapy.

BACKGROUND: Rosai-Dorfman disease is a non-Langerhans cell histiocytosis that recently has been treated successfully with imatinib mesylate in a patient with a systemic variant of the disease. OBSERVATIONS: We describe a 69-year-old man with cutaneous Rosai-Dorfman disease manifesting as progressive, deeply infiltrated skin lesions. Histopathologic examination of the lesions demonstrated dense dermal infiltrate positive for CD68, stabilin-1, and S-100, but not for CD1a. The histiocytes were positive for platelet-derived growth factor receptor alpha, the target molecule for imatinib. During the 5-year course of the disease, multiple therapeutic approaches (tuberculostatic drugs, topical and systemic glucocorticoids, thalidomide, isotretinoin, and methotrexate) did not result in significant improvement. Imatinib mesylate therapy (600 mg/d for 2(1/2) weeks and then 400 mg/d for 10 weeks) had no effect, despite the expression of platelet-derived growth factor receptor alpha on the histiocytes. CONCLUSIONS: Failure of imatinib therapy in our patient may be due to a lack of functioning target molecules, the therapy protocol, or the course of the disease. Cutaneous and systemic variants of Rosai-Dorfman disease may be different clinical entities or at least may respond differently to tyrosine kinase inhibitors.

Changes in quality of life for patients with chronic venous insufficiency, present or healed leg ulcers.

BACKGROUND: Patients with chronic leg ulcers are handicapped in daily life, both by physical complaints and social problems. The aim of our study was not only to assess a possible impairment of quality of life (QOL) of leg ulcer patients but also to evaluate if there is a real improvement of QOL after healing of the ulcer. Patients with chronic venous insufficiency served as the control group. We further analyzed if there were significant differences in the response between patients who were and were not performing compression therapy. PATIENTS AND METHOD: We interviewed three groups of patients (active venous leg ulcer, healed venous leg ulcer and patients with chronic venous insufficiency using the "Freiburger Life Quality Assessment für Venenerkrankungen" (FLQAv). RESULTS: Physical problems, daily handicaps and social problems all increased with age. Contrary to our expectations, healing of a leg ulcer did not lead to a significant increase in QOL. Instead, patients with active ulcers did not regard their QOL as lower than those in the other groups. Compression therapy also did not impair QOL in the three groups. CONCLUSION: Even though ulcer healing is an admirable goal, it does not necessarily lead to an improved QOL, probably because of the numerous comorbidities in this patient group. Nonetheless, it is important to control problems associated directly with the wound to allow ulcer patients to participate actively in everyday life and minimize social problems.

ADAM10 is the constitutive functional sheddase of CD44 in human melanoma cells.

CD44 proteins are cell surface receptors for hyaluronic acid (HA), a component of the extracellular matrix that has multiple effects on cell behavior. CD44 can be shed from the cell surface by proteolytic cleavage. The resulting soluble form can interfere with the interaction between HA and membrane-bound CD44. Soluble CD44 can abolish the cell proliferation-promoting effect of HA on melanoma cell lines, suggesting that a better understanding of the shedding process might identify ways of blocking tumor cell proliferation. ADAM10, ADAM17, and MMP14 have previously been implicated in the shedding of CD44 from various tumor cells. Using immunohistochemistry we demonstrate that ADAM10 and ADAM17 but not MMP14 are significantly expressed on melanoma cells in histological sections. In human melanoma cell lines expression of these proteases could be blocked by transfection with appropriate siRNAs. However, only blocking of ADAM10 expression led to decreased shedding of CD44. In parallel, cell proliferation was promoted. Confocal microscopy demonstrated that ADAM10 and CD44 colocalize on the cell surface. We conclude that ADAM10 is the predominant protease involved in the constitutive shedding of endogenous CD44 from melanoma cells, and that enhancement of ADAM10 activity could be an approach to decrease the proliferation of melanoma cells.

Mathematical model for wound healing following autologous keratinocyte transplantation.

In times of increasing economical pressure on the health care systems, it is important to optimise the outpatient treatment of chronic wounds. Another aim of wound healing research is to discover agents to accelerate healing. Wound healing trajectories or healing velocities can provide information to demonstrate the endpoints for wound healing. A great problem in clinical trials is to specify these parameters. Therefore, we developed a mathematical model for more transparency. In this initial project, we observed 19 wounds to construct the wound healing trajectories after transplantation of autologous keratinocytes, and the results are so encouraging that investigation in this area will continue. The developed mathematical model describes the clinical observed healing process. It was possible to find parameters to distinguish between old and young patients, retrospectively or prospectively calculate the healing rates and to determine exactly the endpoint of healing. Therefore, our model might be very useful in practices or for studies.

Hyaluronan fragments induce cytokine and metalloprotease upregulation in human melanoma cells in part by signalling via TLR4.

Small fragments of the extracellular matrix component hyaluronic acid (sHA) are typically produced at sites of inflammation and tissue injury and have been shown to be associated with tumor invasiveness and metastasis. Here we report that exposure of human melanoma cells to sHA leads to nuclear factor kB (NFk-B) activation followed by enhanced expression of matrix metalloprotease (MMP) 2 and interleukin (IL)-8, factors that can contribute to melanoma progression. At the receptor level, we found that Toll-like receptor (TLR) 4 is involved in this signalling pathway, similar to the case in dendritic and endothelial cells. Specifically, we found that melanoma cells expressed TLR4 on their surface in vivo and in vitro, and using specific siRNA, we could clearly demonstrate the functional importance of TLR4 in sHA-triggered activation of IL-8 expression in melanoma cells. Furthermore, we also found that sHA treatment enhanced the motility of melanoma cells, an effect that could again be blocked by TLR4-specific siRNA. Together, our results suggest that sHA in melanoma might promote tumor invasiveness by inducing MMP- and cytokine-expression, in part in a TLR4-dependent manner, providing new insights into the relationship between cancer and innate immunity.

Immunologic principles of allergic disease.

Allergy either results from a pathological excessive immune reaction, or from the defective induction of tolerance to otherwise harmless antigens. Allergic reactions are mounted by mechanisms of innate and adaptive immunity. The development of an allergic response can be divided in sensitization and elicitation phases. Immediate type allergic reactions (e.g. anaphylaxis, urticaria, rhinoconjunctivitis allergica, allergic asthma) are mediated by IgE antibodies which are produced by B cells stimulated by allergen-specific Th2 cells. Crosslinking of allergen-specific IgE on membrane surfaces of mast cells and basophilic granulocytes leads to release of soluble mediators which may cause systemic symptoms within minutes to hours. The following infiltration of eosinophilic granulocytes and Th2 cells directs chronic inflammation. Humoral cytotoxic immune reactions (e.g. drug induced cytopenia) are mediated by IgG and IgM antibodies which are directed against membrane associated antigens. IgG and IgM antibodies directed against soluble antigens elicit immune complex mediated cytotoxicity (e.g.drug induced vasculitis). Delayed type immune reactions (e.g.contact dermatitis) are based on the activation of antigen specific CD4(+) and CD8(+) T cells and need 24 h to 48 h to develop. Upon recurrent contact with identical antigens, recruitment of CD4(+) and CD8(+) T cells cause inflammation and cytotoxic induced apoptosis in target cells as well as cytokine mediated leukocyte infiltration. Subsequent immigration of CD4(+) Th2 cells provides anti-inflammatory mechanisms leading to resolution of the inflammatory response and tissue repair.

Ultraviolet-B irradiation enhances melanoma cell motility via induction of autocrine interleukin 8 secretion.

Ultraviolet radiation (UVR) is known to be involved in the initiation and progression of malignant melanoma. Many studies have focused on the initiation of melanoma, but less is known about the effect of UVR on established tumor cells. Here, we show that after ultraviolet-B (UVB) irradiation, melanoma cells (MM) are able to secrete autocrine factors that enhance their motility. Time-lapse videomicroscopy of UVB irradiated (15 or 30 mJ/cm(2)) MM showed an initial decrease in MM cell motility one hour after irradiation, with subsequent increase 24 h after UV-B treatment. Conditioned media harvested from MM 24 h following UV-B irradiation specifically enhanced the motility of un-irradiated MM, suggesting that a newly synthesized soluble factor released by UVB MM is involved. As interleukin 8 (IL-8) is known to be up-regulated by different cell types after UV-B irradiation, we investigated IL-8 expression after UVB exposure. Quantitative RT-PCR and ELISA demonstrated an induction of IL-8 in MM by UVB (15 or 30 mJ/cm(2)), and addition of recombinant IL-8 to cell cultures enhanced cell motility to a similar degree than UVB. Importantly, blocking IL-8 activity by a neutralizing anti IL-8 antibody inhibited the up-regulation of MM motility after UVB treatment. We conclude that UVB enhances MM motility and that this effect is mediated at least in part by IL-8 released by MM in an autocrine fashion. Our findings are consistent with the hypothesis that UVB is not only involved in the initiation of melanoma, but may also be important for some aspects of tumor progression.

Switch in syndecan-1 and syndecan-4 expression controls maturation associated dendritic cell motility.

Dendritic cells (DCs) need to mobilize within the extracellular matrix (ECM) during their maturation and concomitant migration from peripheral sites to lymphoid organs. Syndecans are cell surface proteoglycans that mediate the interaction of DCs with the ECM. Here we investigated the influence of syndecans on dendritic cell motility and morphology. Langerhans cells of the epidermis and monocyte-derived DCs were found to undergo a switch in syndecan expression during maturation. Syndecan-1 was downregulated and syndecan-4 was strongly upregulated within the first hours of lipopolysaccharide-induced dendritic cell maturation and during Langerhans cell emigration from human skin, as shown by flow cytometry and qRT-PCR. Syndecan-1 downregulation was inhibited by syndecan-4 siRNA knock-down, indicating a functional interconnection between enhanced syndecan-4 expression and syndecan-1 downregulation. Syndecan-4 upregulation is functionally involved in dendritic cell motility, as inhibition of syndecan-4 function by means of blocking antibodies or through siRNA knock-down decreased dendritic cell motility. In other experiments, the cytoskeletal component a-actinin was observed to be upregulated in DCs as a consequence of the induction of maturation, and was found to colocalize with syndecan-4. Furthermore, lammellopodial spreading by DCs on fibronectin (FN)-coated surfaces was dependent on syndecan-4. This binding of syndecan-4 to FN and its association with the cytoskeleton may be relevant for syndecan-4-dependent dendritic cell motility. We conclude that the switch in syndecan expression during dendritic cell maturation controls the motility of DCs in a way that appears to be crucial for their mobilization from peripheral sites and subsequent migration to lymphoid tissues.

Dermal fibroblasts induce maturation of dendritic cells.

To trigger an effective T cell-mediated immune response in the skin, cutaneous dendritic cells (DC) migrate into locally draining lymph nodes, where they present Ag to naive T cells. Little is known about the interaction of DC with the various cellular microenvironments they encounter during their migration from the skin to lymphoid tissues. In this study, we show that human DC generated from peripheral blood monocytes specifically interact with human dermal fibroblasts via the interaction of beta(2) integrins on DC with Thy-1 (CD90) and ICAM-1 on fibroblasts. This induced the phenotypic maturation of DC reflected by expression of CD83, CD86, CD80, and HLA-DR in a TNF-alpha- and ICAM-1-dependent manner. Moreover, fibroblast-matured DC potently induced T cell activation reflected by CD25 expression and enhanced T cell proliferation. Together these data demonstrate that dermal fibroblasts that DC can encounter during their trafficking from skin to lymph node can act as potent regulators of DC differentiation and function, and thus may actively participate in the regulation and outcome of DC-driven cutaneous immune responses.

Differential regulation of hyaluronan metabolism in the epidermal and dermal compartments of human skin by UVB irradiation.

Hyaluronan (HA), a major component of the cutaneous extracellular-matrix, is involved in tissue repair. Human skin is exposed to and damaged by UVB-irradiation. Here, we investigate the regulation of HA metabolism in human skin during acute UVB-induced inflammation. Expression of HA synthesizing (HAS) and degrading enzymes hyaluronidase (HYAL) as evaluated by quantitative reverse transcribed PCR in response to UVB differed when fibroblasts and HaCaT-keratinocytes, representative cell types in dermis and epidermis, respectively, were compared. Both demonstrated temporally different expression patterns of these genes 3- and 24-hours post-irradiation. This resulted 24-hours post-irradiation in an increase in HAS gene expression in both fibroblasts and HaCaT-keratinocytes, and an increase in HYAL expression only in fibroblasts. HA-production as analyzed by the HA content of conditioned medium was reduced in HaCaT and fibroblast cultures 3-hours post-irradiation, whereas HA increased in HaCaT-cultures 24-hours post-irradiation but remained suppressed in fibroblasts-cultures. Consistently, immunohistochemical staining for HA in human skin 24-hours post-irradiation demonstrated an increased epidermal HA, but a decrease in the dermal compartment. Moreover, analysis of the HA content of dermal microdialysis-fluid revealed increased accumulation of HA degradation products 24-hours post-irradiation. These data demonstrate that there is a complex temporal and spatial regulation of HA-metabolism in skin in response to UVB irradiation.

In situ profiling and quantification of cytokines released during ultraviolet B-induced inflammation by combining dermal microdialysis and protein microarrays.

In skin, an evolving inflammatory or immune response is triggered by early release of a cytokine cascade into the extracellular space. Investigation of extracellular cytokine secretion in situ has been limited by low cut-off filtering membranes and sample volume size and the inability to monitor changes in cytokine protein levels in real-time in situ. Here, we combine for the first time the methods of intradermal microdialysis and antibody protein arraying to profile the early cascade of multiple cytokines in a complex inflammatory response exemplified by ultraviolet B (UVB)-induced inflammation. We observed significant differences of the cytokine and growth factor responses after tissue injury by catheter placement and UVB-induced inflammation. UVB irradiation initiates a rapid proinflammatory response followed by a mixed TH1/TH2 response in which ultimately TH2 cytokines IL-4 and IL10 predominated after 24 h. This most likely indicates the termination and self limitation of the inflammatory response. We conclude that the combination of dermal microdialysis and protein microarray offers a powerful tool to analyze in real-time the complex and rapidly changing interstitial protein milieu during cutaneous inflammatory responses.

Laminar organization of the mouse dentate gyrus: insights from BETA2/Neuro D mutant mice.

The dentate gyrus of rodents is characterized by a highly laminar organization: above a compact granule cell layer, commissural/associational (C/A) fibers terminate on proximal granule cell dendrites and entorhinal fibers terminate on distal granule cell dendrites in a nonoverlapping manner. To gain insights into mechanisms that underlie the formation of this laminar structure, we studied mice deficient for BETA2/NeuroD, a basic helix-loop-helix transcription factor essential for granule cell differentiation. Anterograde tracing was used to label C/A and entorhinal fibers and combined with confocal double immunofluorescence for calbindin, calretinin, parvalbumin, and reelin to visualize putative target cells. The dentate gyrus of mutant mice contained only few granule cells, which formed a cap-like structure adjacent to area CA3. Despite the severe hypoplasia of the dentate gyrus, the remaining BETA2/NeuroD-deficient granule cells expressed mature markers, extended dendrites into the molecular layer, and extended mossy fibers into area CA3. Entorhinal and C/A fibers terminated in a nonoverlapping manner in the dendritic field overlying the rudiment. Entorhinal fibers terminated in the outermost portion of the dentate gyrus where they surrounded reelin-positive Cajal-Retzius cells, and C/A fibers terminated above and within the dentate rudiment. The laminar termination of C/A fibers was closest to normal in zones of the rudiment in which granule cells were densely packed. These data indicate that granule cells are able to differentiate in the absence of BETA2/NeuroD and suggest that the signals underlying the laminar anatomy of the dentate gyrus are present in the absence of most target cells.