Monika Schmelz

Proliferative merkel cells were not detected in human skin

The fetal development of Merkel cells-neuroendocrine cells of the skin—has been a matter of debate for a long time. Recent results have helped to confirm their intraepidermal development in humans. Simple epithelial cytokeratins (CK) nos. 8, 18, 19 and 20 are well established markers at the light microscopic level. These cells could be detected from fetal week 8 within the epidermis with an enormous increase during the following weeks. This gives rise to the question as to whether Merkel cells are undergoing mitoses or whether they are derived from basal keratinocytes. We studied fetal and adult skin using antibodies to simple epithelial CK and to Ki67, a human nuclear cell proliferation-associated antigen in an attempt to answer these questions. In human adult and fetal skin of various stages we could not detect any Merkel cells undergoing cell division. These results suggest that Merkel cells are postmitotic cells to be renewed from undifferentiated keratinocytes with stem cell characteristics.

Drebrin is a widespread actin-associating protein enriched at junctional plaques, defining a specific microfilament anchorage system in polar epithelial cells

Using immunoblotting, immunprecipitation with subsequent fragment mass spectrometry, and immunolocalization techniques, we have detected the actin-binding ca. 120-kDa protein drebrin, originally identified in - and thought to be specific for - neuronal cells, in diverse kinds of human and bovine non-neuronal cells. Drebrin has been found in numerous cell culture lines and in many tissues of epithelial, endothelial, smooth muscle and neural origin but not in, for example, cardiac, skeletal and certain types of smooth muscle cells, in hepatocytes and in the human epithelium-derived cell culture line A-431. By double-label fluorescence microscopy we have found drebrin enriched in actin microfilament bundles associated with plaques of cell-cell contact sites representing adhering junctions. These drebrin-positive, adhering junction-associated bundles, however, are not identical with the vinculin-containing, junction-attached bundles, and in the same cell both subtypes of microfilament-anchoring plaques are readily distinguished by immunolocalization comparing drebrin and vinculin. The intracellular distribution of the drebrin- and the vinculin-based microfilament systems has been studied in detail by confocal fluorescence laser scanning microscopy in monolayers of the polar epithelial cell lines, MCF-7 and PLC, and drebrin has been found to be totally and selectively absent in the notoriously vinculin-rich focal adhesions. The occurrence and the possible functions of drebrin in non-neuronal cells, notably epithelial cells, and the significance of the existence of two different actin-anchoring junctional plaques is discussed.

Prognostic Criteria for Squamous Cell Cancer of the Skin

BACKGROUND Non-well-differentiated cutaneous squamous cell carcinomas may display a more aggressive behavior. It is important to better define prognostic criteria for these tumors. METHODS This was a retrospective case-control analysis of a squamous cell carcinoma database. Patients with non-well-differentiated and well-differentiated tumors were matched based on site of tumor, age, and immunocompromised status. Comparisons included demographics, histology, immunohistochemical protein expressions (Ki-67, p53, E-cadherin, cyclin D1), and clinical outcomes. RESULTS Demographic features were similar between cases (n=30) and controls (n=30). Non-well-differentiated tumors were larger (1.8 cm versus 1.3 cm, P=0.08), deeper (0.81 cm versus 0.32 cm, P<0.0001), and had greater recurrence (P=0.003). Non-well-differentiated tumors showed increased proliferation rate, Ki-67 index (77% versus 61%, P=0.001); no significant difference in activity of p53, E-cadherin, and cyclin D1 between the two groups. CONCLUSIONS Tumor differentiation and depth are important pathologic and prognostic criteria for cutaneous squamous cell carcinoma. Immunohistochemistry helps describe patterns of biomarker protein expression and may exemplify aggressive subtypes.

Implantation of a three-dimensional fibroblast matrix improves left ventricular function and blood flow after acute myocardial infarction.

This study was designed to determine if a viable biodegradable three-dimensional fibroblast construct (3DFC) patch implanted on the left ventricle after myocardial infarction (MI) improves left ventricular (LV) function and blood flow. We ligated the left coronary artery of adult male Sprague-Dawley rats and implanted the 3DFC at the time of the infarct. Three weeks after MI, the 3DFC improved LV systolic function by increasing (p < 0.05) ejection fraction (37 ± 3% to 62 ± 5%), increasing regional systolic displacement of the infarcted wall (0.04 ± 0.02 to 0.11 ± 0.03 cm), and shifting the passive LV diastolic pressure volume relationship toward the pressure axis. The 3FDC improved LV remodeling by decreasing (p < 0.05) LV end-systolic and end-diastolic diameters with no change in LV systolic pressure. The 3DFC did not change LV end-diastolic pressure (LV EDP; 25 ± 2 vs. 23 ± 2 mmHg) but the addition of captopril (2mg/L drinking water) lowered (p < 0.05) LV EDP to 12.9 ± 2.5 mmHg and shifted the pressure–volume relationship toward the pressure axis and decreased (p < 0.05) the LV operating end-diastolic volume from 0.49 ± 0.02 to 0.34 ± 0.03 ml. The 3DFC increased myocardial blood flow to the infarcted anterior wall after MI over threefold (p < 0.05). This biodegradable 3DFC patch improves LV function and myocardial blood flow 3 weeks after MI. This is a potentially new approach to cell-based therapy for heart failure after MI.

Cellular immune suppressor activity resides in lymphocyte cell clusters adjacent to granulomata in human coccidioidomycosis

ABSTRACT The in situ immunologic response in human coccidioidomycosis remains undefined. To explore this further, pulmonary necrotizing coccidioidal granulomata were examined using immunohistochemical staining for lymphocyte subsets and for the cytokines interleukin-10 (IL-10) and gamma interferon (IFN-γ). Discrete perigranulomatous lymphocytic clusters were seen in eight of nine tissues examined. In these tissues, T lymphocytes (CD3+) significantly outnumbered B lymphocytes (CD20+) in the mantle area of the granulomata (P = 0.028), whereas the clusters were composed of roughly equal numbers of T and B lymphocytes. While the number of cells in the mantle expressing IL-10 was similar to those in the perigranulomatous clusters, there were significantly more cells expressing IFN-γ in the mantle than in the clusters (P = 0.037). Confocal microscopy revealed that CD4+ T lymphocytes and B lymphocytes are associated with IL-10 production. CD4+CD25+ T lymphocytes were identified in the perigranulomatous clusters but were not associated with IL-10 production. This is the first report noting perigranulomatous lymphocyte clusters and IL-10 in association with human coccidioidal granulomata and suggests that down-regulation of the cellular immune response is occurring within coccidioidal granulomata.

Laminin-5-mediated gene expression in human prostate carcinoma cells.

Interactions between extracellular matrix (ECM) proteins and prostate carcinoma cells provide a dynamic model of prostate tumor progression. Previous work in our laboratory showed that laminin‐5, an important member of a family of ECM glycoproteins expressed in the basal lamina, is lost in prostate carcinoma. Moreover, we showed that the receptor for laminin‐5, the α6β4 integrin, is altered in prostate tumors. However, the genes that laminin‐5 potentially regulates and the significance of its loss of expression in prostate cancer are not known. We selected cDNA microarray as a comprehensive and systematic method for surveying and examining gene expression induced by laminin‐5. To establish a definitive role for laminin‐5 in prostate tumor progression and understand the significance of its loss of expression, we used a cDNA microarray containing 5289 human genes to detect perturbations of gene expression when DU145 prostate carcinoma cells interacted with purified laminin‐5 after 0.5, 6, and 24 h. Triplicate experiments showed modulations of four, 61, and 14 genes at 0.5, 6, and 24 h, respectively. Genes associated with signal transduction, cell adhesion, the cell cycle, and cell structure were identified and validated by northern blot analysis. Protein expression was further assessed by immunohistochemistry. Mol. Carcinog. 30:119–129, 2001.

Differences of bcl-2 protein expression between Merkel cells and Merkel cell carcinomas

The bcl‐2 gene, originally identified in B‐cell lymphomas, encodes for proteins which may assume oncogenic functions by blocking apoptosis. Bcl‐2 proteins are broadly distributed among various tissues, including epithelial ones. Within the skin, bcl‐2 is strongly expressed in melanocytes, but its further distribution is yet unclear. The Merkel cells, neuroendocrine‐epithelial cells of the skin, are present within the epidermis and hair follicles, mostly nerve‐associated, and are believed to be postmitotic and long lived. Possibly they give rise to the malignant Merkel cell carcinomas. In the present study we investigated the bcl‐2 expression on the protein level by means of immunohistochemical techniques including double confocal laser scanning microscopy, as well as on the RNA level by RT‐PCR techniques, in Merkel cells, Merkel cell carcinomas, and cell lines. Merkel cells were identified by double staining for cytokeratins 20 or 8/18. We demonstrate that fetal epidermal and dermal Merkel cells are immunostained for bcl‐2 protein, most of them clearly weaker than melanocytes. Adult Merkel cells also express bcl‐2 protein very heterogeneously, mostly weak. In contrast, Merkel cell carcinomas are visually strongly positive for bcl‐2 protein with some degree of heterogeneity. This is different from malignant melanomas in which bcl‐2 expression is reduced as compared to normal melanocytes. Bcl‐2 gene expression was also shown for Merkel cell carcinoma cell lines on both the mRNA and the protein level. Possibly bcl‐2 protein expression is downregulated during the life span of Merkel cells, arguing that they may succumb to a certain cell turnover. The comparably high bcl‐2 protein level in Merkel cell carcinomas may reflect peculiar biological and clinical characteristics.

Functional and Anatomic Aspects of Central Nervous Cardiovascular Regulation

The behavior of animals and men can be defined as adaptation toward changes in the individual’s internal or external milieu. It comprises emotional, motoric, cognitive, and autonomic reaction patterns. In particular, autonomic cardiovascular reactions are initiated to meet the metabolic demands of the organism during different forms of behavior in order to maintain or reestablish homeostasis. This definition can apply to most of the cardiovascular reaction patterns seen during, for example, adaptation to exercise or emotional behavior, when an increase in cardiac output and a redistribution of regional blood flow is observed. With these circulatory changes, blood flow is directed toward those vascular beds that supply the organs involved in the adaptive responses of the organism. It might, however, be difficult to accept this definition if one considers the cardiovascular changes observed during sleep (Fig. 1). There is no simple explanation why there should be a decrease in muscle blood flow and an increase in mesenteric and renal conductance (see also Mancia et al. 1970), resulting in a decrease in peripheral vascular resistance and a decrease in arterial blood pressure with episodal increases, during rapid eye movement (REM) sleep.

A phase II study of belinostat (PXD101) in relapsed and refractory aggressive B-cell lymphomas: SWOG S0520.

Abstract Recent advances in diffuse large B-cell lymphomas (DLBCL) have underscored the importance of tumor microenvironment in escaping host anti-tumor responses. One mechanism is loss of major histocompatibility Class II antigens (MHCII) associated with decreased tumor infiltrating T lymphocytes (TIL) and poor survival. Transcription of MHCII is controlled by CIITA which in turn is regulated by histone acetylation. In this study, we hypothesized that HDAC inhibition with belinostat increases MHCII, CIITA expression, TIL and improves patient outcomes. Primary objective was evaluation of toxicity and response. Twenty-two patients were enrolled for the study. Belinostat was well tolerated with mild toxicity. Two partial responses were observed at 5, 13 months after registration for an overall response rate (ORR) (95% CI) of 10.5% (1.3–33.1%), and three patients had stable disease for 4.7, 42.3+, and 68.4 + months with minimum 3-year follow-up. Included correlative studies support the hypothesis and serve as the basis for SWOG S0806 combining vorinostat with R-CHOP.

A NOVEL TYPE OF ADHERING JUNCTION IN AN EPITHELIOID TUMORIGENIC RAT CELL CULTURE LINE

Abstract Two major types of plaque-bearing adhering junctions are commonly distinguished: the actin microfilament-anchoring adhaerens junctions (AJs) and the desmosomes anchoring intermediate-sized filaments (IFs). Both types of junction usually possess the common plaque protein, plakoglobin, whereas the other plaque proteins and the transmembrane cadherins are mutually exclusive. For example, AJs contain E-, N-, or P-cadherin in combination with α- and β-catenin, vinculin and α-actinin, whereas in desmosomes, desmogleins and desmocollins are associated with desmoplakin and one or several of the plakophilins (PP1–3). Here we describe a novel type of adhering junction comprising proteins of both AJs and desmosomes and the tight junction (TJ) plaque protein, ZO-1, in a newly established, liver-derived tumorigenic rat cell line (RMEC-1). By immunofluorescence microscopy, cell-cell contacts are characterized by mostly continuous-appearing lines which are usually resolved by electron microscopy as extended arrays of closely spaced small plaque subunits. These plaque-covered regions are positive for plakoglobin, α- and β-catenin, the arm-repeat protein p120, vinculin, desmoplakin and protein ZO-1. They are positive for E-cadherin in cultures early on in passaging, but tend to turn negative for all known cadherins in densely grown cultures. On immunoblotting SDS-PAGE-separated proteins from dense-grown cell monolayers, “pan-cadherin” antibodies have reacted with a band at ∼140 kDa, identified as N-cadherin by peptide fingerprinting of the immunoprecipitated protein, which for reasons not yet clear is modified or masked in immunolocalization experiments. The exact histological derivation of RMEC-1 cells is not known. However, the observations of several endothelial markers and the fact that all cells are rich in IFs containing vimentin and/or desmin, while only subpopulations also reveal IFs containing CKs 8 and 18, is suggestive of a mesenchymal, probably endothelial origin. We discuss the molecular relationship of this novel type of extended junction with other types of adhering junctions.