Crystal structure of human peptidylarginine deiminase type VI (PAD6) provides insights into its inactivity.

Human peptidylarginine deiminase isoform VI (PAD6), which is predominantly limited to cytoplasmic lattices in the mammalian oocytes in ovarian tissue, is essential for female fertility. It belongs to the peptidylarginine deiminase (PAD) enzyme family that catalyzes the conversion of arginine residues to citrulline in proteins. In contrast to other members of the family, recombinant PAD6 was previously found to be catalytically inactive. We sought to provide structural insight into the human homologue to shed light on this observation. We report here the first crystal structure of PAD6, determined at 1.7 Šresolution. PAD6 follows the same domain organization as other structurally known PAD isoenzymes. Further structural analysis and size-exclusion chromatography show that PAD6 behaves as a homodimer similar to PAD4. Differential scanning fluorimetry suggests that PAD6 does not coordinate Ca2+ which agrees with acidic residues found to coordinate Ca2+ in other PAD homologs not being conserved in PAD6. The crystal structure of PAD6 shows similarities with the inactive state of apo PAD2, in which the active site conformation is unsuitable for catalytic citrullination. The putative active site of PAD6 adopts a non-productive conformation that would not allow protein-substrate binding due to steric hindrance with rigid secondary structure elements. This observation is further supported by the lack of activity on the histone H3 and cytokeratin 5 substrates. These findings suggest a different mechanism for enzymatic activation compared with other PADs; alternatively, PAD6 may exert a non-enzymatic function in the cytoplasmic lattice of oocytes and early embryos.

The dimensions and characteristics of the subepidermal nerve plexus in human skin--terminal Schwann cells constitute a substantial cell population within the superficial dermis.

BACKGROUND: The skin constitutes the largest sensorial organ. Its nervous system consists of different types of afferent nerve fibers which spread out immediately beneath the skin surface to sense temperature, touch and pain. OBJECTIVE: Our aim was to investigate the dimension and topographic relationship of the different nerve fibers of the subepidermal nerve plexus in human hairy skin and to analyze numbers and marker expression of terminal Schwann cells. METHODS: Nerve fibers and Schwann cells were investigated on dermal sheet preparations and thick sections of skin from various body regions of 10 individuals. RESULTS: The dimension of subepidermal nerve fibers varied between different body sites with highest values in chest skin (100 ± 18 mm/mm(2)) and lowest in posterior forearm skin (53 ± 10 mm/mm(2)). The majority of fibers (85.79%) were unmyelinated, thus representing C-fibers, of which 7.84% were peptidergic. Neurofilament-positive fibers (A-fibers) accounted for 14.21% and fibers positive for both neurofilament and myelin (Aß-fibers) for only 0.18%. The number of Schwann cells varied in accordance with nerve fiber length from 453 ± 108 on chest skin to 184 ± 58/mm(2) in skin of the posterior forearm. Terminal Schwann cells showed a marker profile comparable to Schwann cells in peripheral nerves with the notable exception of expression of NGFr, NCAM, L1CAM and CD146 on myelinating Schwann cells in the dermis but not in peripheral nerves. CONCLUSION: Our data show that terminal Schwann cells constitute a substantial cell population within the papillary dermis and that both nerve fiber length and Schwann cell numbers vary considerably between different body sites.

Fixed drug eruption caused by mefenamic acid: a case series and diagnostic algorithms.

BACKGROUND: Fixed drug eruption is a fairly common drug-induced hypersensitivity reaction of the skin and the mucous membranes, which is characterized by the re-occurrence of the lesion(s) exactly on the previously involved sites after repeated administration. The pathogenetic mechanisms of this site-specificity are not fully elucidated. PATIENTS AND METHODS: We report on three cases of fixed drug eruption, including a non-pigmenting generalized bullous fixed drug eruption, caused by mefenamic acid in its pure form. RESULTS AND CONCLUSION: Provocation tests with the assumed causative drug represent the gold standard for establishing the diagnosis and for identifying the culprit. Advantages and pitfalls of topical and systemic provocation tests as diagnostic approaches are discussed.

Embryonic stem cell factors undifferentiated transcription factor-1 (UFT-1) and reduced expression protein-1 (REX-1) are widely expressed in human skin and may be involved in cutaneous differentiation but not in stem cell fate determination.

Undifferentiated transcription factor-1 (UTF-1) and reduced expression protein-1 (REX-1) are used as markers for the undifferentiated state of pluripotent stem cells. Because no highly specific cytochemical marker for epidermal stem cells has yet been identified, we investigated the expression pattern of these markers in human epidermis and skin tumours by immunohistochemistry and in keratinocyte cell cultures. Both presumed stem cell markers were widely expressed in the epidermis and skin appendages. Distinct expression was found in the matrix cells of the hair shaft. Differentiation of human primary keratinocytes (KC) in vitro strongly downregulated UTF-1 and REX-1 expression. In addition, REX-1 was upregulated in squamous cell carcinomas, indicating a possible role of this transcription factor in malignant tumour formation. Our data point to a role for these proteins not only in maintaining KC stem cell populations, but also in proliferation and differentiation of matrix cells of the shaft and also suprabasal KC.

Lymphatic precollectors contain a novel, specialized subpopulation of podoplanin low, CCL27-expressing lymphatic endothelial cells.

Expression of the lymphoendothelial marker membrane mucoprotein podoplanin (podo) distinguishes endothelial cells of both blood and lymphatic lineages. We have previously discovered two distinct subpopulations of lymphatic endothelial cells (LECs) in human skin that were defined by their cell surface densities of podoplanin and were designated LEC podo-low and LEC podo-high. LEC podo-low is restricted to lymphatic precollector vessels that originate from initial LEC podo-high-containing lymphatic capillaries and selectively express several pro-inflammatory factors. In addition to the chemokine receptor protein Duffy blood group antigen receptor for chemokines, these factors include the constitutively expressed chemokine CCL27, which is responsible for the accumulation of pathogenic CCR10+ T lymphocytes in human inflammatory skin diseases. In this study, we report that CCR10+ T cells accumulate preferentially both around and within CCL27+ LEC podo-low precollector vessels in skin biopsies of human inflammatory disease. In transmigration assays, isolated CCR10+ T lymphocytes are chemotactically attracted by LEC podo-low in a CCL27-dependent fashion, but not by LEC podo-high. These observations indicate that LEC podo-low-containing precollector vessels constitute a specialized segment of the initial lymphatic microvasculature, and we hypothesize that these LEC podo-low-containing vessels are involved in the trafficking of CCR10+ T cells during skin inflammation.

Expression of VEGF-A/C, VEGF-R2, PDGF-alpha/beta, c-kit, EGFR, Her-2/Neu, Mcl-1 and Bmi-1 in Merkel cell carcinoma.

Merkel cell carcinoma is a rare but very aggressive tumor of the skin. With current treatment options, Merkel cell carcinoma is associated with a high incidence of recurrence and metastasis. Targeted anticancer therapies such as receptor tyrosine kinase inhibitors and antisense oligonucleotides have been found to be a promising new type of treatment for various types of cancer. To evaluate whether the use of targeted therapies is a possible treatment option in Merkel cell carcinoma, we determined the expression of the target molecules c-kit, Mcl-1, Bmi-1, vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF-receptor 2 (VEGF-R2), platelet-derived growth factor (PDGF)-alpha, PDGF-beta, epidermal growth factor receptor (EGFR) and Her-2/Neu in a tissue microarray of 32 samples of 29 patients with Merkel cell carcinoma. C-kit-positive samples were analyzed for mutations in exons 9 and 11. The tissue microarray was stained immunohistochemically with antibodies directed against the above-mentioned proteins, and an immunoreactivity score was calculated. DNA was extracted from c-kit-positive samples and was analyzed for exon 9 and 11 mutations using direct DNA sequencing. We found that c-kit (7%), Mcl-1 (88%), Bmi-1 (78%), VEGF-A (91%), VEGF-C (75%) VEGF-R2 (88%), PDGF-alpha (72%) and PDGF-beta (13%) were expressed in Merkel cell carcinomas. All samples showed a lack of EGFR and Her-2/Neu expression. Analysis of c-kit revealed no mutations. As VEGF-A, VEGF-C, VEGF-R2, PDGFs and c-kit are targets of new cytostatic agents used in the treatment of other cancers, inhibition by a multitargeted chemotherapy could be a very promising treatment option. High expression of Bmi-1 and Mcl-1 warrants further studies on the use of antisense oligonucleotides in Merkel cell carcinoma.

Rarefaction of the peripheral nerve network in diabetic patients is associated with a pronounced reduction of terminal Schwann cells.

OBJECTIVE: Peripheral neuropathy is the most frequent neurological complication in diabetic patients. The diagnosis is established by both clinical neurological examination and demonstration of reduced epidermal nerve fibers in skin biopsies (1). Whereas the decrease of free nerve endings has been extensively studied in diabetic patients (2,3), no data are available on possible changes of terminal Schwann cells. Besides their role as scaffold for peripheral nerves, they also play an important role in supporting survival and function of peripheral nerves (4). RESEARCH DESIGN AND METHODS: We analyzed the subepidermal nerve plexus in dermal sheet preparations of deceased diabetic and nondiabetic patients by immunostaining for detection of the neural cell adhesion molecule and quantification of the subepidermal nerve plexus. RESULTS AND CONCLUSIONS: The subepidermal nerve plexus, comprising nerve fibers and ensheathing Schwann cells, was significantly reduced in diabetic patients. Whether the reduction in terminal Schwann cells is cause or consequence of the loss of peripheral nerve fibers remains to be investigated.

Expression of BMI-1 in normal skin and inflammatory and neoplastic skin lesions.

BACKGROUND: BMI-1 is involved in the maintenance of stem cells and functions as an oncogene in both lymphomas and solid carcinomas, acting by downregulation of p16ink4a. We have investigated the expression profile of BMI-1 in normal and inflamed skin as well as in basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs). METHODS: BMI-1 expression was determined by immunohistochemistry and immunofluorescence, and evaluated semiquantitatively. RESULTS: BMI-1 was weakly expressed in nuclei of basal and sometimes suprabasal keratinocytes, in basal cells of sebaceous glands, weakly to moderately in the bulge area and the external root sheath of hair follicles, and strongly in sweat glands. Whereas BCCs showed strong and diffuse BMI-1 expression, SCCs expressed BMI-1 heterogeneously. Strong cytoplasmic expression of BMI-1 was found in dividing cells. CONCLUSIONS: BMI-1 expression marks stem cells within the hair follicle. As BMI-1 was also found in suprabasal keratinocytes and a variety of specialized cells, the distribution of BMI-1 only partly reflects the known distribution of stem cell compartments. BMI-1 is strongly overexpressed in BCCs, tumors linked to dysregulation of the sonic hedgehog pathway, which has been shown to upregulate BMI-1, suggesting a contribution of the BMI-1 oncogene in their pathogenesis.

The touch dome in human skin is supplied by different types of nerve fibers.

Receptor end organs and free-nerve endings in the skin are the peripheral sentinels of the sensorial nervous system encoding for touch, temperature, and pain. Using a novel approach to analyze the outermost nerves of the skin, we visualized for the first time the distinct microanatomical structure of the touch dome of human hairy skin. The dermal nerve fibers of this slowly adapting type 1 mechanoreceptor were embedded in dermal protrusions that could be readily discerned by Laminin-5 staining. Concerning the nerves supplying the touch domes, we found, unexpectedly, that besides Abeta-fibers, Adelta- and C-fibers also were regularly present. The epidermis overlying the nerve convolutes showed a distinctive architecture of the rete ridges clearly demarcated from the surroundings and extending over 0.193 +/- 0.138 mm(2) (mean +/- standard deviation). Within this area, 756 +/- 386 Merkel cells/mm(2) (mean +/- standard deviation) were present compared with less than 50/mm(2) outside the touch dome, demonstrating for the first time a highly discontinuous distribution of these cells in nonglabrous skin. Our findings strongly suggest that the receptive qualities of human touch domes exceed mechanosensation, and that they may serve as multifunctional nerve end organs in human skin.

Ultrastructural characterization of an artificial basement membrane produced by cultured keratinocytes.

A recent study in our laboratories on the growth of keratinocytes at the culture medium/air interface has led to the identification of a novel thin sheet-like matrix that supports adherent cells. This novel matrix consists of components secreted by keratinocytes, including type IV collagen, and laminins 1 and 5, that self-assembled to a membrane structure. In the present study, a detailed ultrastructural characterization of this membrane was done with high-resolution electron microscopy after negative staining. The basic organization of the membrane was found to be a dense network of 8- to 10-nm-wide irregular rod-like elements. High-resolution examination and immunolabeling showed that type IV collagen filaments form the core of these elements, and other components including heparan sulfate proteoglycan in the form of 4.5- to 5-nm-wide ribbon-like "double tracks" are aggregated around it. These detailed features of the membrane strikingly resembled those of the basement membrane in vivo. These ultrastructural similarities indicate that the membrane may also have basement membrane-like functional properties, and suggest that it should be considered for testing in future medical applications.

Ultrastructural localization of caspase-14 in human epidermis.

Caspase-14 has been implicated in the formation of stratum corneum because of its specific expression and activation in terminally differentiating keratinocytes. However, its precise physiological role and its protein substrate are elusive. We studied the ultrastructural localization of caspase-14 in human epidermis to compare its distribution pattern with that of well-characterized differentiation markers. Immunogold cytochemistry confirmed that caspase-14 is nearly absent in basal and spinous layers. In the granular, layer nuclei and keratohyalin granules were labeled with increasing intensity towards the transitional layer. Particularly strong caspase-14 labeling was associated with areas known to be occupied by involucrin and loricrin, whereas F-granules, occupied by profilaggrin/filaggrin, were much less labeled. A high density of gold particles was also present at the forming cornified cell envelope, including desmosomes. In corneocytes, intense labeling was both cytoplasmic and associated with nuclear remnants and corneodesmosomes. These observations will allow focusing efforts of biochemical substrate screening on a subset of proteins localizing to distinct compartments of terminally differentiated keratinocytes.

Sheet preparations expose the dermal nerve plexus of human skin and render the dermal nerve end organ accessible to extensive analysis.

Since vertical tissue sections used for the study of the human cutaneous nervous system inherently allow visualization of only a small part of the mainly horizontally oriented cutaneous nerves, we searched for possibilities to extend this view. We now propose a method based on the immuno-staining of dermal sheet preparations for subsequent analysis by electron-, light- or laser scanning microscopy. Dermal sheet preparations for the first time allowed the imaging of the complex structure of the nerve end organ over several cm2, and facilitated viewing of its topological relationship to other tissue components. We could visualize that the bulk of free ending nerve fibers ramified within 25 microm of the dermo-epidermal junction, whereas below that only larger nerve bundles were present. This method further allowed the detection and quantification of NCAM/CD56+ non-myelinating Schwann cells which envelope terminal axons within the dermis. Depending on the body region, we detected between 140 to over 300 individual terminal Schwann cells per mm2 skin surface. Our method should allow the acquisition of new insights into the highly organized architecture of the skin nerve end organ. Its further application will give new impetus in the investigation of alterations of this skin compartment under pathological conditions.

A basement membrane-like matrix formed by cell-released proteins at the medium/air interface supports growth of keratinocytes.

Epithelial cells require adherence to a matrix for regular growth. During standard keratinocyte cell culture in serum-free medium, we observed that cell colonies formed not only on the bottom of the culture vessels but also at the medium/air interface. Coomassie blue staining detected a protein membrane that extended up to several centimeters between the colonies of floating cells. Ultrastructural investigation of this membrane revealed structures closely resembling those of basement membranes, and immunochemical staining confirmed the presence of laminins-1 and -5 as well as collagen IV, representative components of basement membranes. Cells attached to the floating membrane proliferated and could be cultivated for up to six months. When keratinocyte-conditioned medium was filtered and transferred to a culture vessel without cells, the protein membrane at the liquid/air interface formed within one week suggesting self-assembly of cell-released proteins. Our findings provide a basis for the production of epidermal basement membranes for potential medical uses.

Inhibition of human neutrophil chemotaxis toward interleukin 8 with six clinical antithrombin concentrates in vitro.

OBJECTIVE: Antithrombin exerts direct effects on neutrophils by inhibiting chemokine-induced migration. This study examined the potency of different pharmaceutical antithrombin preparations in inhibiting neutrophil chemotaxis toward interleukin 8. METHODS: Cell migration was tested by the leading front assay in modified Boyden microchemotaxis chambers bearing nitrocellulose filters. Human neutrophils were incubated with six different antithrombin concentrates or an immunopurified antithrombin preparation at concentrations of 1 micro IUeth-5 IU/ml. RESULTS: All antithrombin concentrates irrespective of the pharmaceutical source deactivated neutrophil chemotaxis. At concentrations below 100 mIU/ml neutrophil chemotaxis toward interleukin 8 was decreased by the antithrombin preparations with varying potency, but at 1 mIU/ml no significant differences were observed. CONCLUSIONS: As the ability of antithrombin to deactivate neutrophil chemotaxis toward interleukin 8 shows differences depending on the source of commercial antithrombin, these results suggest that at equivalent WHO standard concentrations clinical antithrombin concentrates may differ in anti-inflammatory potential.

Inhibition of plasma-dependent monocyte chemokinesis and cytokine-triggered endothelial activation for neutrophil transmigration by administration of clopidogrel in man.

Mediators released by spontaneously activated platelets may contribute to alterations in endothelial and leukocyte dysfunctions. We investigated the roles of clopidogrel and aspirin in ex vivo endothelial activation for interactions with leukocytes. Eight healthy volunteers received clopidogrel or aspirin for 8 days. Blood samples were taken before, during, and after treatment. Levels of adhesion molecules and platelet-derived mediators in these samples were measured using commercially available test kits, and effects of plasma on endothelial cells and leukocytes were investigated in neutrophil transendothelial migration, monocyte-endothelial adhesion and leukocyte migration assays. Plasma samples from clopidogrel-treated persons induced diminished chemokinesis of monocytes. Tumour necrosis factor-induced priming of endothelial cells for enhanced neutrophil transmigration was also diminished by pretreatment of endothelial cells, but not of neutrophils, with plasma derived from subjects during clopidogrel treatment. Plasma from the aspirin group had no such effects. Administration of clopidogrel but not aspirin significantly decreased serum levels of soluble intercellular adhesion molecule-1, whereas no changes in levels of soluble vascular cell adhesion molecule-1, P-selectin, L-selectin, von Willebrand factor, platelet-derived growth factor, vascular-endothelial growth factor, and transforming growth factor-beta were observed. Inhibition of plasma-promoted endothelial activation by clopidogrel may indicate a novel role in the prevention of atherosclerosis.

Syndecan-4 mediates antithrombin-induced chemotaxis of human peripheral blood lymphocytes and monocytes.

Antithrombin inhibits chemokine-induced migration of neutrophils by activating heparan sulfate proteoglycan-dependent signaling. Whether antithrombin affects migration of other types of leukocytes is not known. We investigated the effects of antithrombin on spontaneous and chemokine-triggered migration of lymphocytes and monocytes from human peripheral blood in modified Boyden chamber micropore filter assays. Lymphocyte and monocyte populations from human peripheral blood were purified using magnetic antibody cell sorting. The signaling mechanisms required for antithrombin-dependent migration were studied using signaling enzyme blockers. Expression of heparan sulfate proteoglycan core protein was studied by RT-PCR and flow cytometry. The antithrombins used were Kybernin P from human plasma and a monoclonal-antibody-purified preparation from this plasma. Pretreatment of lymphocytes and monocytes with antithrombin inhibited chemotaxis toward optimal concentrations of interleukin-8 or Rantes (regulated upon activation normal T-cell expressed and activated) at concentrations of antithrombin as low as 10 nU/ml. In the absence of the chemokines, direct exposure of cells to gradients of antithrombin stimulated migration. Effects of antithrombin were abolished by pretreating cells with heparinase-1, chondroitinase, sodium chlorate and anti-syndecan-4 antibodies. Expression of syndecan-4 mRNA and protein in monocytes and lymphocytes was demonstrated in RT-PCR and anti-syndecan-4 immunoreactivity assays, respectively. In the presence of pentasaccharide, antithrombin lost its effect on cells. Data indicate that antithrombin directly inhibits chemokine-stimulated migration of monocytes and lymphocytes via the effects of its heparin-binding site on cell surface syndecan-4 by activation of protein kinase C and Rho signaling.