Christina Reinisch

Expression of VEGF-A/C, VEGF-R2, PDGF- α / β , 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)-α, PDGF-β, 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-α (72%) and PDGF-β (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.

Lymphatic Precollectors Contain a Novel, Specialized Subpopulation of Podoplaninlow, 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 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).

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 Aβ‐fibers, Aδ‐ 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 ± 0.138mm2 (mean ± standard deviation). Within this area, 756 ± 386 Merkel cells/mm2 (mean ± standard deviation) were present compared with less than 50/mm2 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. Ann Neurol 2005;58:88–95

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.

Inhibitors of Differentiation/DNA Binding Proteins Id1 and Id3 Are Regulated by Statins in Endothelial Cells

Id proteins (inhibitors of differentiation), which are involved in the control of cell cycle progression, can delay cellular differentiation and senescence and have been implicated in angiogenesis. The regulation of Id proteins in endothelial cells (ECs) by proangiogenic statins has not been investigated yet and remains unresolved. In this study, human dermal microvascular ECs (HDMECs) were stimulated with fluvastatin, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and serum in vitro. The regulation of Id1, Id3, p21, p27, and p53 and the phosphorylation of AKT was investigated by Western blotting. Id1 was up-regulated by fluvastatin and serum, but not by VEGF and HGF. Fluvastatin did not regulate p21 and p27, but down-regulated Id3 and p53 slightly. In contrast to VEGF and HGF, fluvastatin did not result in AKT phosphorylation, indicating that this pathway is not involved in the control of endothelial Id1 expression. These experiments demonstrate for the first time that Id1 can be up-regulated and p53 down-regulated by a statin in HDMECs. Regulation of these proteins in ECs may account for the proangiogenic effect of statins.

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.

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 (Aβ-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.

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.

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.