First identification of Ewing's sarcoma-derived extracellular vesicles and exploration of their biological and potential diagnostic implications.

BACKGROUND INFORMATION: Exosomes are small RNA- and protein-containing extracellular vesicles (EVs) that are thought to mediate hetero- and homotypic intercellular communication between normal and malignant cells.Tumour-derived exosomes are believed to promote re-programming of the tumour-associated stroma to favour tumour growth and metastasis. Currently, exosomes have been intensively studied in carcinomas. However, little is known about their existence and possible role in sarcomas. RESULTS: Here, we report on the identification of vesicles with exosomal features derived from Ewing's sarcoma(ES), the second most common soft-tissue or bone cancer in children and adolescents. ES cell line-derived EV shave been isolated by ultracentrifugation and analysed by flow-cytometric assessment of the exosome-associated proteins CD63 and CD81 as well as by electron microscopy. They proved to contain ES-specific transcripts including EWS-FLI1, which were suitable for the sensitive detection of ES cell line-derived exosomes by qRT-PCRin a pre-clinical model for patient plasma. Microarray analysis of ES cell line-derived exosomes revealed that they share a common transcriptional signature potentially involved in G-protein-coupled signalling, neurotransmitter signalling and stemness. CONCLUSIONS: In summary, our results imply that ES-derived exosomes could eventually serve as biomarkers for minimal residual disease diagnostics in peripheral blood and prompt further investigation of their potential biological role in modification of the ES-associated microenvironment

Tumour exosomes inhibit binding of tumour-reactive antibodies to tumour cells and reduce ADCC.

In order to grow within an immunocompetent host, tumour cells have evolved various strategies to cope with the host's immune system. These strategies include the downregulation of surface molecules and the secretion of immunosuppressive factors like IL-10 and PGE2 that impair the maturation of immune effector cells, among other mechanisms. Recently, tumour exosomes (TEX) have also been implicated in tumour-induced immune suppression as it has been shown that TEX can induce apoptosis in T lymphocytes. In this study, we extend our knowledge about immunosuppressive features of these microvesicles in that we show that TEX efficiently bind and sequester tumour-reactive antibodies and dramatically reduce their binding to tumour cells. Moreover, we demonstrate that this antibody sequestration reduces the antibody-dependent cytotoxicity by immune effector cells, which is among the most important anti-tumour reactions of the immune system and a significant activity of therapeutic antibodies. Taken together, these data point to the fact that tumour-derived exosomes interfere with the tumour-specific function of immune cells and constitute an additional mechanism how tumours escape from immune surveillance.

First report of ectopic ACTH syndrome and PTHrP-induced hypercalcemia due to a hepatoblastoma in a child.

CONTEXT: Only occasionally, endocrine-active tumors develop directly from hepatic tissue, and may lead to paraneoplastic syndromes (PNS). PNS mostly accompany malignancy of adulthood and are exceedingly rare in children. PATIENT: A girl aged 6 years and 9 months presented with a 2-month history of rapidly progressive weight gain, abdominal distension, and polyuria/pollakiuria accompanied by short episodes of abdominal pain. She showed the typical clinical features of Cushing's syndrome and a huge hepatic mass. An abdominal computed tomography (CT) scan revealed a large liver tumor. Blood glucose and serum calcium were greatly elevated. DESIGN AND OBJECTIVE: Case report describing the causative relationship of the clinical findings. METHODS: Physical examination; ultrasound of the abdomen; CT scan of the abdomen and the chest; conventional X-rays; routine hematology; blood chemistry and multiple parameters of calcium and phosphorus metabolism; multisteroid analysis in serum and urine; adrenocortical stimulation and suppression tests; histopathological assessment of the resected tumor; immunohistochemistry for ACTH, beta-endorphin, corticotrophin-releasing hormone (CRH), and PTH-related peptide (PTHrP); electron microscopy of tumor cells; ACTH and CRH extraction from the tumor tissue; and clinical follow-up for more than 20 years. RESULTS: Giant hepatoblastoma (HB; approximately 1000 ml volume) of the right lobe of the liver with combined ectopic ACTH syndrome and PTHrP-induced tumor-associated hypercalcemia. Wide local excision and polychemotherapy led to complete reversal of the paraneoplastic phenotype. CONCLUSIONS: This is the first report of an endocrine-active HB causing both Cushing's syndrome and PTHrP-related 'humoral hypercalcemia of malignancy'. This information should be added to the well-known beta-human chorionic gonadotropin-related paraneoplastic effects of HB in children.

Antithrombin reduces shedding of the endothelial glycocalyx following ischaemia/reperfusion.

AIMS: Antithrombin is an important inhibitor of the coagulation system, additionally exerting specific anti-inflammatory effects on endothelial cells. Healthy vascular endothelium is coated by the endothelial glycocalyx, diminution of which increases capillary permeability, e.g. after ischaemia. Antithrombin is known to infiltrate the glycocalyx, binding to glycosaminoglycans, and to preserve the glycocalyx after application tumour necrosis factor-alpha. We investigated the influence of antithrombin on glycocalyx subjected to ischaemia/reperfusion. METHODS AND RESULTS: Isolated guinea pig hearts were perfused with Krebs-Henseleit buffer (KHB). Antithrombin was applied to achieve physiological levels (1 U/mL) before inducing 20 min of ischaemia (37 degrees C). Hearts were reperfused for 20 min at constant flow (baseline perfusion pressure 70 cmH(2)O) with KHB or KHB plus 2 g% hydroxyethyl starch (130 kDa). Coronary net fluid filtration was assessed directly by measuring transudate formation on the epicardial surface. Post-ischaemic coronary release of syndecan-1 and heparan sulfate was quantified by ELISA. Hearts were perfusion-fixed to visualize the glycocalyx by electron microscopy. Ischaemia/reperfusion caused degradation of the glycocalyx, enhanced coronary perfusion pressure, and increased vascular permeability. Antithrombin significantly reduced post-ischaemic glycocalyx shedding, coronary perfusion pressure, coronary leak, and tissue oedema formation compared to untreated hearts. Additional application of colloid augmented these actions of antithrombin. Electron microscopy revealed a mostly intact glycocalyx after antithrombin treatment. CONCLUSION: Antithrombin preserves the endothelial glycocalyx, sustaining the vascular barrier function and reducing interstitial oedema. The potentiated effect of colloid in these hearts suggests that the prevention of shedding should be of functional benefit also in vivo.

TNF-alpha induced shedding of the endothelial glycocalyx is prevented by hydrocortisone and antithrombin.

BACKGROUND: Healthy vascular endothelium is clothed by the endothelial glycocalyx. This structure plays a key role in the regulation of inflammation and vascular permeability and is known to be degraded by ischemic and inflammatory stress. Our aim was to show whether hydrocortisone and antithrombin stabilize the glycocalyx and, therefore, the vascular barrier, against damage induced by the inflammatory stimulus TNF-alpha, thus improving the cardio-vascular situation. METHODS: Isolated guinea pig hearts were perfused with Krebs-Henseleit buffer for 20 min at constant flow (baseline perfusion pressure 70 cmH(2)O). Hydrocortisone in a stress dose (10 microg/ml) or antithrombin in a physiological dose (1 U/ml) were then applied for 15 min before infusion of TNF-alpha (4 ng/ml, 10 min). Coronary net fluid filtration was assessed directly by measuring transudate formation on the epicardial surface. Hearts were perfusion-fixed to visualize the glycocalyx. RESULTS: TNF-alpha induced severe degradation of the glycocalyx, increased coronary resistance, heightened vascular leak and permeability to hydroxyethyl starch and caused mast-cell degranulation. Hydrocortisone and antithrombin both reduced all of these effects. Electron microscopy revealed a mostly intact glycocalyx after treatment with either drug. CONCLUSIONS: Both hydrocortisone and antithrombin clearly preserve the endothelial glycocalyx in the face of inflammatory degradation initiated by TNF-alpha, however, with different mechanisms. This is an important new facet in the pathophysiology and therapy of sepsis, since preservation of the glycocalyx should help prevent vasoconstriction, tissue edema as well as leukocyte and platelet adhesion, thus mitigating inflammation and tissue hypoxia.

Exogenous nitric oxide requires an endothelial glycocalyx to prevent postischemic coronary vascular leak in guinea pig hearts.

INTRODUCTION: Postischemic injury to the coronary vascular endothelium, in particular to the endothelial glycocalyx, may provoke fluid extravasation. Shedding of the glycocalyx is triggered by redox stress encountered during reperfusion and should be alleviated by the radical scavenger nitric oxide (NO). The objective of this study was to investigate the effect of exogenous administration of NO during reperfusion on both coronary endothelial glycocalyx and vascular integrity. METHODS: Isolated guinea pig hearts were subjected to 15 minutes of warm global ischemia followed by 20 minutes of reperfusion in the absence (Control group) and presence (NO group) of 4 microM NO. In further experiments, the endothelial glycocalyx was enzymatically degraded by means of heparinase followed by reperfusion without (HEP group) and with NO (HEP+NO group). RESULTS: Ischemia and reperfusion severely damaged the endothelial glycocalyx. Shedding of heparan sulfate and damage assessed by electron microscopy were less in the presence of NO. Compared with baseline, coronary fluid extravasation increased after ischemia in the Control, HEP, and HEP+NO groups but remained almost unchanged in the NO group. Tissue edema was significantly attenuated in this group. Coronary vascular resistance rose by 25% to 30% during reperfusion, but not when NO was applied, irrespective of the state of the glycocalyx. Acute postischemic myocardial release of lactate was comparable in the four groups, whereas release of adenine nucleotide catabolites was reduced 42% by NO. The coronary venous level of uric acid, a potent antioxidant and scavenger of peroxynitrite, paradoxically decreased during postischemic infusion of NO. CONCLUSION: The cardioprotective effect of NO in postischemic reperfusion includes prevention of coronary vascular leak and interstitial edema and a tendency to forestall both no-reflow and degradation of the endothelial glycocalyx.

Differential profile of the OPG/RANKL/RANK-system in degenerative aortic native and bioprosthetic valves.

BACKGROUND AND AIM OF THE STUDY: Although degenerative calcific aortic valve stenosis is the most common valvular disease among the elderly, neither the etiology underlying the condition nor degeneration of the bioprostheses is yet fully understood. The study aim was to assess the expression profile of those OPG/RANKL/RANK-system determinants known to act as key regulators of bone metabolism and the immune system in calcific aortic valve stenosis and porcine aortic bioprostheses. METHODS: Valve probes from a total of 69 patients (41 with end-stage aortic stenosis, 11 with mild-to-moderate aortic sclerosis, 17 with degenerative porcine aortic bioprostheses) were explanted either during surgery or at autopsy. The presence and localization of OPG, RANKL, RANK and NF-kappaB were analyzed by immunostaining and morphometry. RESULTS: The majority of stenotic and sclerotic valves exhibited cell-bound signals of OPG, RANKL, RANK and NF-kappaB, while bioprostheses showed only sparse signaling. As key findings, the percentage of cells labeled by OPG, RANK and NF-kappaB was increased in sclerotic valves compared with stenotic valves (each p < 0.001), whereas the frequency of RANKL was higher in stenotic compared to sclerotic valves (p < 0.001). As a consequence, the OPG/RANKL ratio was decreased in stenotic (0.83) compared to sclerotic valves (20.2). CONCLUSION: The differential expression profile of specific members of the OPG/RANKL/RANK axis suggests an involvement of their determinants in native valve calcification, but not in the degeneration of porcine bioprostheses. Thus, these mediators of bone homeostasis may represent new targets for a more specified prevention and/or therapy of native aortic stenosis.

Shedding of the endothelial glycocalyx in patients undergoing major vascular surgery with global and regional ischemia.

BACKGROUND: The astonishing thickness of the endothelial glycocalyx, which rivals that of endothelial cells in the microvasculature, was disclosed in the last 15 years. As already demonstrated, this structure plays a key role in the regulation of inflammation and vascular permeability. METHODS AND RESULTS: Two components of the glycocalyx, syndecan-1 and heparan sulfate, were measured in arterial blood of 18 patients undergoing surgery of the ascending aorta with cardiopulmonary bypass (n=12 with and n=6 without deep hypothermic circulatory arrest) and of 14 patients undergoing surgery for infrarenal aortic aneurysm. Basal values of syndecan-1 (1.2 microg/dL) and heparan sulfate (590 microg/dL) of patients were similar to those of control subjects. Anesthesia and initiation of surgery caused no changes. Global ischemia with circulatory arrest (n=12) was followed by transient 42- and 10-fold increases in syndecan-1 and heparan sulfate, respectively, during early reperfusion (0 to 15 minutes). After regional ischemia of heart and lungs (cardiopulmonary bypass; n=6), syndecan-1 increased 65-fold, and heparan sulfate increased 19-fold. Infrarenal ischemia was followed by 15- and 3-fold increases, respectively (n=14). The early postischemic rises were positively correlated (r=0.76, P<0.001). Plasma concentrations of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 did not change. Circulating polymorphonuclear granulocytes and the level of postischemic heparan sulfate corresponded negatively. Immunohistochemical imaging and immunoassay of isolated hearts (guinea pig) substantiated syndecan-1 and heparan sulfate as components of the endothelial glycocalyx released into the coronary venous effluent. Electron microscopy revealed shedding of the glycocalyx after ischemia/reperfusion. CONCLUSIONS: This study provides the first evidence in humans for shedding of the endothelial glycocalyx during ischemia/reperfusion procedures.

Secretory phenomena in the non-lactating human mammary gland.

The normal non-lactating premenopausal human mammary gland has been shown by immunohistochemistry and transmission electron microscopy to secrete a number of antimicrobial peptides such as beta-defensins, the cathelicidin LL37, lactoferrin and adrenomedullin. In addition, the non-lactating gland elaborates a prominent glycocalyx at the apical membrane of the glandular epithelial cells, parts of which are shed into the lumen of endpieces and ducts. This glycocalyx includes the mucins MUC 1 and MUC 4, a strongly Alcian Blue positive palyanionic component and sulfated material stained with Aldehyde Fuchsin. MUC 1 and the Alcian Blue positive material are considered to play an antimicrobial role, too. Lactalbumin and lipid droplets also occur in the non-lactating gland. At the EM-level secretory phenomena operating by exocytosis and by means of the apocrine mechanism have been observed. Cytoskeletal components presumably play a role in apocrine secretion. Apart from secretion at the cellular apex, secretion at the cellular basis also occurs regularly, which may represent the production of para- or endocrine factors.

Human ceruminous gland: ultrastructure and histochemical analysis of antimicrobial and cytoskeletal components.

The ceruminous glands in the skin of the human external auditory canal are modified apocrine glands, which, together with sebaceous glands, produce the cerumen, the ear wax. Cerumen plays an important role in the protection of the ear canal against physical damage and microbial invasion. We studied the morphology of the glandular cells by light and electronmicroscopy. Antimicrobial and cytoskeletal components of the ceruminous glands were investigated by immunohistochemical methods. Numerous antimicrobial proteins and peptides are present in the ceruminous glandular cells: beta-defensin-1, beta-defensin-2, cathelicidin, lysozyme, lactoferrin, MUC1, secretory component of IgA. These data indicate a crucial role in the innate host defense against diverse pathogens. The apocrine secretion mechanism is a special mode of secretion by which the apical part of the cell cytoplasm surrounded by a membrane is pinched off. We could show that the presence of actin filaments, CK 19 and CK 7, seems to play a role in the pinching-off mechanism. Finally, we showed the secretion of lipid vesicles from the ceruminous gland. We could extend the number of detected antimicrobial peptides and proteins in human ceruminous glandular cells that protect the surface of the external auditory meatus. In addition, we detected proteins involved in the apocrine secretion mode of the ceruminous gland.

Antimicrobial peptides are present in immune and host defense cells of the human respiratory and gastrointestinal tracts.

Previous studies have implicated antimicrobial peptides in the host defense of the mammalian intestinal and respiratory tract. The aim of the present study has been to characterize further the expression of these molecules in non-epithelial cells of the human pulmonary and digestive systems by detailed immunohistochemical analysis of the small and large bowel and of the large airways and lung parenchyma. Additionally, cells obtained from bronchoalveolar lavage were analyzed by fluorescent activated cell sorting and immunostaining of cytospin preparations. hBD-1, hBD-2, and LL-37 were detected in lymphocytes and macrophages in the large airways, lung parenchyma, duodenum, and colon. Lymphocytes positive for the peptides revealed a staining pattern and distribution that largely matched that of CD3-positive and CD8-positive T-cells. Macrophages with positive staining for the antimicrobial peptides also stained positively for CD68 and CD74. In view of the morphology of the LL-37-positive and hBD-2-positive mucosal lymphocytes, they are probably also B-cells. Thus, antimicrobial peptides of the defensin and cathelicidin families are present in a variety of non-epithelial cells of mucosal organs. These findings confirm that antimicrobial peptides have multiple functions in the biology of the mucosa of these organs.

Cells of primarily extra-valvular origin in degenerative aortic valves and bioprostheses.

AIMS: We assessed aortic valves from patients with non-rheumatic aortic valve stenosis (AS) and with degenerative aortic valve bioprostheses (BP) for the presence of progenitor cell and leukocyte subtype-specific markers. METHODS AND RESULTS: Diseased valve probes from a total of 87 patients (60 AS and 27 BP) were studied. We assessed presence and localization of endothelial progenitor cells (EPCs: CD34, CD133), dendritic cells (DCs: S100), T-lymphocytes (CD3), and macrophages (CD68) by immunohistochemical and morphometric analyses. In the majority of valves, we detected cell-bound signals of CD34 (48% of AS, 74% of BP, respectively), CD133 (58%/81%), S100 (58%/93%), CD3 (62%/81%), and CD68 (78%/93%). Labelled cells were predominantly localized within the valvular fibrosa. As key results, frequency of EPCs, DCs, macrophages, and lymphocytes was found significantly higher in BP when compared with AS (CD34: 19.2+/-23.2 vs. 5.7+/-13.0%; CD133: 13.7+/-12.4 vs. 5.5+/-8.3%; S100: 15.2+/-12.2 vs. 5.7+/-8.9%; CD3: 3.3+/-2.7 vs. 1.1+/-1.4%; CD68: 35.3+/-26.6 vs. 3.4+/-4.1%; each P

Insights into the mechanism of magnetofection using PEI-based magnetofectins for gene transfer.

BACKGROUND: Gene delivery by the use of magnetic forces, so-called magnetofection, has been shown to enhance transfection efficiency of viral and non-viral systems up to several-hundred-fold. For this purpose gene carriers, such as polyethylenimine (PEI), are associated with superparamagnetic nanoparticles and complexed with plasmid DNA. Gene delivery is targeted by the application of a magnetic field. METHODS: To investigate the underlying mechanism, we studied the impact of the applied magnetic field on the transfection process of PEI-coated superparamagnetic iron oxide gene vectors (magnetofectins) using various cell lines. In particular, we addressed the question whether accelerated sedimentation of magnetofectins is the driving force or if the magnetic field itself directly influences the endocytic processing of the magnetofectins. The cellular uptake mechanism of magnetofectins was studied by electron microscopy and transfection experiments in the presence of various inhibitors that operate at different steps of endocytosis. RESULTS: In this study we could show that cellular uptake of magnetofectins proceeds obviously by endocytosis. Cellular uptake of magnetofectins behaves almost analogously as compared with PEI polyplexes. Besides unspecific endocytosis, apparently clathrin-dependent as well as caveolae-mediated endocytic uptake is involved. CONCLUSIONS: The magnetic field itself does not alter the uptake mechanism of magnetofectins. Obviously, the magnetic forces lead to an accelerated sedimentation of magnetofectins on the cell surface and do not directly affect the endocytic uptake mechanism. So further improvement of magnetic field application could lead to efficient targeting of gene expression into the desired organ and tissue in vivo.

Round window application of D-methionine, sodium thiosulfate, brain-derived neurotrophic factor, and fibroblast growth factor-2 in cisplatin-induced ototoxicity.

HYPOTHESIS: In this study we tested the effect of local administration of D-methionine, sodium thiosulfate, brain-derived neurotrophic factor, and fibroblast growth factor-2 on cisplatin ototoxicity in guinea pigs to the round window membrane. BACKGROUND: Cisplatin is an important antineoplastic agent in the therapy of many malignancies. Its clinical utility is limited by severe side effects, including ototoxicity. Recent studies have shown protection against cisplatin ototoxicity in animal experiments by the systemic administration of D-methionine and sodium thiosulfate. Growth factors such as brain-derived neurotrophic factor and fibroblast growth factor-2 also have shown otoprotective effects in in vitro studies. METHODS: Osmotic pumps (Alzet) were implanted unilaterally in 30 guinea pigs. Five groups of six animals received either D-methionine, sodium thiosulfate, fibroblast growth factor-2, brain-derived neurotrophic factor, or saline 0.9%. Cisplatin was administered intraperitoneally for 5 consecutive days. Distortion product otoacoustic emissions were recorded every day. The animals were killed on day 6, and their cochleae were removed and analyzed by transmission electron microscopy. RESULTS: Compared with control animals, guinea pigs treated with D-methionine showed better otoacoustic emissions on days 3 and 4 (Mann-Whitney test, p < 0.05). The differences were not evident on days 5 and 6. Sodium thiosulfate, brain-derived neurotrophic factor, and fibroblast growth factor-2 showed no significant protective effect. CONCLUSION: Local application to the round window membrane can be used as an effective treatment in the prevention of cisplatin toxicity. Local application may avoid systemic side effects and reduce the antineoplastic effects of cisplatin.

Release of TNF-alpha during myocardial reperfusion depends on oxidative stress and is prevented by mast cell stabilizers.

OBJECTIVES: Our study sought to elucidate the role of oxidative stress for shedding of tumor necrosis factor-alpha (TNF-alpha) and for activating TNF-alpha-converting enzyme (TACE). BACKGROUND: TNF-alpha, a central inflammatory cytokine, is discussed as one of the mediators of reperfusion injury. Shedding of membrane-bound pro-TNF-alpha is thought to be largely due to TNF-alpha-converting enzyme (TACE). METHODS: Release of TNF-alpha and TACE dependency were studied in isolated rat hearts and in the human mast cell line HMC-1. RESULTS: In reperfused hearts, interstitial release of TNF-alpha occurred in two phases (2-10 and >45 min). It depended on the presence of oxygen during reperfusion and was attenuated by reduced glutathione. Infusion of the oxidants H(2)O(2) or HOCl elicited release in non-ischemic hearts. TNF-alpha release was inhibited in hearts treated with degranulation inhibitors ketotifen or cromoglycate, suggesting mast cells as major source for myocardial TNF-alpha. This was confirmed by tissue staining. Post-ischemic release of histamine, however, did not parallel that of TNF-alpha. Heart tissue contained mainly mature TACE. HMC-1 expressed abundant pro-TACE and cleaved the pro-TNF-alpha-peptide Ac-SPLAQAVRSSSR-NH(2). However, cleavage was nonspecific and only partly inhibited by TACE inhibitor TAPI-2 (10-100 micromol/l), while it was stimulated by H(2)O(2) and HOCl and fully blocked by the nonspecific metalloprotease inhibitor o-phenanthroline. CONCLUSIONS: The mechanism underlying TNF-alpha release from post-ischemic myocardium is oxidation-dependent but largely independent of activation of TACE. Mast cell stabilizers may be useful in preventing TNF-alpha release during reperfusion.

Dendritic cells in neointima formation after rat carotid balloon injury: coordinated expression withanti-apoptotic Bcl-2 and HSP47 in arterial repair.

OBJECTIVES: We sought to evaluate: 1) the contribution of dendritic cells (DCs); and 2) the impact of B-cell lymphoma 2 protein (Bcl-2), a central anti-apoptotic protooncogene, and of heat shock protein 47 (HSP47), indicating subsequent collagen deposition, in neointima formation after angioplasty. BACKGROUND: The origin of neointimal cells and the factors that promote their accumulation are still unclear. Previous studies reported intimal presence of DCs and suggested cells of primarily extravascular origin to contribute to arterial repair. METHODS: Sprague-Dawley rats underwent carotid balloon angioplasty. At different times after angioplasty, tissue sections were analyzed by immunohistochemistry using OX-62 and S100 as DC markers and antibodies against Bcl-2 and HSP47, supplemented by electron microscopic analysis of cell type and apoptosis. RESULTS: Four days after injury, DCs adhered along the internal elastic lamina and demonstrated intense Bcl-2 and HSP47 expression, consistent with low apoptosis. With ongoing neointima enlargement, luminal DCs remained prevalent and were colocalized with Bcl-2 and HSP47, while signaling decreased to basal regions. Media showed no DCs and only low Bcl-2 and HSP47 immunoreactivity. Adventitia transiently revealed a structural separation between day 4 and 7. Whereas the inner layer demonstrated sparse cellularity, apoptosis and no DC, Bcl-2, and HSP47 labeling, the outer layer was characterized by high myofibroblast density with strong Bcl-2 and HSP47 expression but absence of DCs. CONCLUSIONS: We identify DCs as novel components in early neointima formation, promoted by coordinated anti-apoptotic Bcl-2 and HSP47 expression. Despite intense adventitial remodeling, there is no evidence of adventitial cell transmigration.

Human glands of Moll: histochemical and ultrastructural characterization of the glands of Moll in the human eyelid.

The function of the human gland of Moll of the eyelid is not exactly known. We studied the secretory and cytoskeletal components of these apocrine glands in males and females by immunohistochemical methods, and the ultrastructural organization of the glandular cells with an electron microscope. The glands of Moll are exclusively located at the margin of the eyelids and their ducts empty into the lash follicle. Immunohistochemical staining for actin and cytokeratins CK19 and CK7 points to the involvement of actin in the pinching-off mechanism of the apical cell protrusion during apocrine secretion and to a stabilizing role for the cytokeratins in this apical region of the glandular cells. The presence of the bacteriolytic enzyme lysozyme, the membrane-associated mucin 1, and the immunoglobulin A and its secretory component within the gland suggest a function in local immune defense. The presence of a variety of sugar components in the secretory product was verified by lectin histochemistry and periodic acid Schiff and Alcian blue stain. We suppose that these apocrine glands are active from birth in producing agents against pathogenic microorganisms in the eyelid shaft and on the ocular surface.

An angiogenic role for the human peptide antibiotic LL-37/hCAP-18.

Antimicrobial peptides are effector molecules of the innate immune system and contribute to host defense and regulation of inflammation. The human cathelicidin antimicrobial peptide LL-37/hCAP-18 is expressed in leukocytes and epithelial cells and secreted into wound and airway surface fluid. Here we show that LL-37 induces angiogenesis mediated by formyl peptide receptor-like 1 expressed on endothelial cells. Application of LL-37 resulted in neovascularization in the chorioallantoic membrane assay and in a rabbit model of hind-limb ischemia. The peptide directly activates endothelial cells, resulting in increased proliferation and formation of vessel-like structures in cultivated endothelial cells. Decreased vascularization during wound repair in mice deficient for CRAMP, the murine homologue of LL-37/hCAP-18, shows that cathelicidin-mediated angiogenesis is important for cutaneous wound neovascularization in vivo. Taken together, these findings demonstrate that LL-37/hCAP-18 is a multifunctional antimicrobial peptide with a central role in innate immunity by linking host defense and inflammation with angiogenesis and arteriogenesis.

Assessing experimental models in myocardial injury: lack of activation of the proteases TACE and calpain in brief ischaemia and reperfusion.

BACKGROUND: Calpain inhibitors are reportedly cardioprotective. Furthermore, oxidative stress may acutely activate the sheddase tumour necrosis factor (TNF)-alpha-cleaving enzyme (TACE). The aim of this study was to examine whether myocardial reperfusion leads to activation of the proteases mu- and m-calpain, and to evaluate which cardiac cells act as a source of TNF-alpha. METHODS: Isolated hearts (guinea pig) were subjected to global ischaemia (15 min) and reperfused. Calpain activity was determined by zymography. Calpastatin (inhibitor) and troponin I (substrate) were quantified by western blotting. Immunohistology of hearts and a human mast cell line (HMC-1) was used to localise expression of TNF-alpha and TACE. Shedding of TNF-alpha was assessed in Mono Mach, Jurkat-T, HMC-1 and peripheral blood leucocytes with and without oxidative stress. RESULTS: Neither of the ubiquitous calpains (mu- and m-calpain) was significantly activated by brief ischaemia/reperfusion, nor were calpastatin and troponin degraded more than in extracts of control hearts. Cardiac TNF-alpha immunoreactivity was localised to mast cells. None of the tested cell lines shed TNF-alpha in response to non-toxic amounts of oxidants. However, HMC-1 cells showed poor expression of proTNF-alpha, while TACE was abundant. CONCLUSIONS: Although the severity of ischaemia in the current model may have been insufficient, activation of calpain by ischaemia/reperfusion cannot be demonstrated simply in the Langendorff-mode perfused isolated heart. Mast cells are the prime source of myocardial TNF-alpha. A suitable whole-cell model remains to be found to demonstrate acute oxidative activation of TACE.

Evaluation of acute traumatic changes of the coronary artery wall after robotically assisted endoscopic coronary artery bypass grafting.

BACKGROUND: There is concern that the technical limitations of robotic systems used in endoscopic coronary artery bypass grafting (CABG) may lead to increased trauma of the anastomotic site. To examine this issue, we compared the acute traumatic changes of the coronary artery wall caused by conventional manual suturing and robotically assisted suturing for anastomoses using the ZEUS telemanipulator (Computer Motion Inc., Goleta, CA) in a laboratory setting. METHODS: Coronary artery bypass grafting was performed on isolated porcine hearts. Fifteen anastomoses (with harvested porcine right coronary artery (RCA) segments) were carried out using the ZEUS microsurgical telemanipulator (group Z), while 15 further anastomoses were performed with a conventional manual technique (group M) using Gore-Tex CV-8 suture material. Specimens were taken from each anastomotic site and from native parts of the left anterior descending artery (LAD) (control group). Morphological changes of the cellular and fibrous components of the lamina intima and lamina media, and the shape and maximum diameter of the puncture mark, were examined by light microscopy (LM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Vascular endothelial damage and denudation were graded on a score from 1 to 5. RESULTS: In each group, 14 specimens were evaluated. SEM findings showed a significantly higher degree of endothelial denudation in group Z and group M compared to the control group, while group Z was significantly more affected than group M. Likewise, the maximum diameter of the puncture mark was significantly larger in group Z than in group M. TEM and LM studies supported these results. In addition, LM revealed that in five specimens of group Z the shape of the stitch through the artery wall was not cylindrical, as in the other cases, but was asymmetrical and displayed a superficial furrow on the side of the vascular lumen. CONCLUSION: The results indicate that there is an increased incidence of damage to the coronary artery wall caused by the microsurgical telemanipulator. Further studies are necessary to determine whether the differences between conventional and robotic-assisted suturing techniques will have an effect on the long-term outcome of coronary artery bypass grafting.