Radiotherapy for oral cancer decreases the cutaneous expression of host defence peptides.

INTRODUCTION: Bacterial resistance against antibiotics has become an increasing challenge in the treatment of cutaneous infections. Consequences can be severe, especially in infected wounds following previous local radiotherapy. Certain endogenous peptide antibiotics, the host defence peptides (HDPs), exhibit broad-spectrum antimicrobial activity and promote wound healing. Their use as supplements to conventional antibiotics is a current topic of discussion; however, knowledge of their quantities in healthy and compromised tissue is a prerequisite for such discussion. To date, no data concerning HDP quantities in irradiated skin are available. METHODS: Expression profiles of the genes encoding HDPs, namely human beta-defensin-1 (DEFB1, hBD-1), beta-defensin-2 (DEFB4A, hBD-2), beta-defensin-3 (DEFB103, hBD-3) and S100A7, were assessed in samples of non-irradiated and irradiated neck. RESULTS: A reduction in the expression of all of the examined genes was observed in irradiated skin when compared with non-irradiated skin (statistically significant in the case of S100A7, P = 0.013). Immunohistochemistry revealed differences in HDP distribution with respect to the epithelial layers. CONCLUSION: The study demonstrates a significant reduction in HDP gene expression in neck skin as a result of radiotherapy. These findings might represent a starting point for novel treatments of cutaneous infections in irradiated patients, such as topical supplementation of synthetic HDP.

Expression of antimicrobial peptides in cutaneous infections after skin surgery.

BACKGROUND: Increasing numbers of antibiotics have lost efficiency because of bacterial resistance. The consequences can be severe when surgical wounds become infected during postoperative care. Natural peptide antibiotics, the so-called host defence peptides (HDPs), have been investigated since the 1990s in a search for alternative treatment strategies. HDPs build up a protection shield against pathological microorganisms, especially in human epithelium. The use of HDPs is currently being discussed as a new antimicrobial therapeutic strategy. Accordingly, a profound knowledge of the quantitative relationships of the effectors is essential. OBJECTIVES: To evaluate differences in HDP expression between postoperatively inflamed and healthy epithelium. METHODS: Expression profiles of the genes encoding HDP human beta-defensin (hBD)-1 (DEFB1, previously known as HBD-1), hBD-2 (DEFB4A, previously known as HBD-2), hBD-3 (DEFB103A, previously known as HBD-3) and psoriasin (S100A7) were assessed in samples of surgical wound healing disorders (n = 27) and healthy epithelium (n = 16) by using real-time polymerase chain reaction. Immunohistochemical staining was performed in the same samples. RESULTS: A significant overexpression of DEFB4A (P < 0.001), DEFB103A (P = 0.001) and S100A7 (P < 0.001) was found in cutaneous surgical site infections. Immunohistochemistry revealed intensely elevated protein levels of psoriasin in infected wounds, and differences in distribution with respect to the epithelial layers. CONCLUSIONS: The study demonstrates upregulated mRNA expression and protein levels of HDPs in postoperatively inflamed epithelium. The results may be a starting point for novel pharmacological treatments.

[Sonography of enlarged lymph nodes: Pathogenetic categorization using contrast enhanced power Doppler sonography]. / Sonographische Diagnostik vergrösserter Lymphknoten: Pathogenetische Kategorisierung mit der kontrastverstärkten Powerdoppler-Sonographie.

BACKGROUND: The purpose of this study was to categorize enlarged superficial lymph nodes as benign or malignant using sonomorphologic features and vascularization pattern. PATIENTS AND METHODS: Enlarged superficial lymph nodes in 57 patients were assessed with B-mode and contrast-enhanced power Doppler sonography. Morphology and vascularization were evaluated. The lymph nodes were categorized as benign or malignant. Correlation was made with histology and follow-up results. RESULTS: In 55 patients, 40 lymph nodes were correctly categorized as benign and 15 lymph nodes correctly as malignant. The most reliable criteria were shape and vascularization pattern. Intact hilar vessels and branching indicated benign enlargement, destruction of the hilum with vessels running peripherally along the capsule indicated metastatic destruction. Two benign lymph nodes were considered malignant (false positive). CONCLUSION: B-mode ultrasound along with contrast-enhanced power Doppler ultrasound is an easy, cost-effective, and reliable tool for differentiation and categorization of enlarged superficial lymph nodes.

Stimulation of cellular sphingomyelin import by the chemokine connective tissue-activating peptide III.

The selective import of phospholipids into cells could be mediated by proteins secreted from the cells into the extracellular compartment. We observed that the supernatants obtained from suspensions of thrombin-activated platelets stimulated the exchange of pyrene (py)-labeled sphingomyelin between lipid vesicles in vitro. The proteins with sphingomyelin transfer activity were purified and identified as the chemokine connective tissue-activating peptide III (CTAP-III) and platelet basic protein. Isolated CTAP-III stimulated the exchange of py-sphingomyelin between lipid vesicles but did not affect the translocations of py-labeled phosphatidylcholine and phosphatidylethanolamine. CTAP-III rapidly increased the transfer of py-sphingomyelin from low density lipoproteins into peripheral blood lymphocytes, other immune cells, and fibroblasts. In the presence of heparin, CTAP-III was unable to insert sphingomyelin into the peripheral blood lymphocytes. The activation energy of the py-sphingomyelin transfer suggested that the translocation proceeded entirely in a hydrophobic environment. [(3)H]Sphingomyelin transferred to the cells by CTAP-III was hydrolyzed to [(3)H]ceramide and [(3)H]sphingosine after activation with tumor necrosis factor alpha. The generation of the [(3)H]sphingolipid messengers was catalyzed by acid sphingomyelinase. Our results identify CTAP-III as the first mediator of the selective (endocytosis-independent) cellular import of sphingomyelin allowing the paracrine modulation of the sphingolipid signaling.

Histo-physiology of the scent-marking glands of the penile pad, anal pouch, and the forefoot in the aardwolf (Proteles cristatus).

The scentmarking glands of the anal pouch, penile pad, and the forefoot of the aardwolf (Proteles cristatus) were studied by histological, histochemical, immunohistochemical methods, and by electron microscopy. The morphological observations are correlated with eco-ethological aspects of this nocturnal animal. In all studied regions there was a superficial layer of holocrine sebaceous glands and a deeper layer of apocrine scent glands; these two types of glands apparently function in concert. Only in the forefoot were additional tubular glands, resembling eccrine sweat glands found, which may improve the frictional capacities of the paw, while apocrine and holocrine glands serve scent-marking functions of the forefoot. Penile pad and anal pouch are exclusively scent marking organs. The secretion modus of the apocrine glands is both via exocytosis and apocrine mechanism. Homogeneous apical, secretory granules, which contain glycoproteinaceous material, represent evidence for exocytosis. In the anal pouch, additional variably sized granules contain endogenous pigments which are probably responsible for the brownish coloration of the secretory product of the male animals. Variable heights of the glandular cells, frequent apical tall protrusions as well as pinched-off pieces of cytoplasm in the glandular tubules support the concept of an apocrine secretion in the scent glands. The immunohistochemical staining pattern of actin points to the involvement of actin filaments in the pinching-off process of the apical cell protrusion, which does not contain any cell organelles. The variable actin staining patterns suggest a dynamic process during which actin filaments form a ring or sheet at the basis of the pinching-off bleb. Proliferative and apoptotic phenomena show no preference for active and inactive glandular cells suggesting that replacement of cells occurs independently of the functional status of the glands.

Platelet agonists enhance the import of phosphatidylethanolamine into human platelets.

It is unknown whether the endocytosis-independent transfer of phospholipids from lipoproteins to platelets is regulated by platelet agonists such as thrombin. The movements of the choline phospholipids phosphatidylcholine and sphingomyelin (labeled with either 14C or the fluorescent pyrenedecanoic acid) between low density lipoproteins and platelets were unaffected by thrombin (0.5 unit/ml). In contrast, thrombin accelerated the import of diacyl phosphatidylethanolamine (PE) and alkenylacyl phosphatidylethanolamine into platelets by about 4-fold. Similarly, thrombin receptor-activating peptide (15 microM), collagen (10 microgram/ml), and ADP (10 microM) enhanced PE uptake. High density lipoprotein particles and egg phosphatidylcholine vesicles were also donors for stimulation of platelet PE import. Part of the [14C]arachidonic acid-labeled PE transferred from low density lipoprotein to platelets activated by thrombin and collagen was metabolized to 14C-eicosanoids. Inhibitors of protein kinase C partially prevented thrombin-induced [14C]PE uptake, while direct activators of protein kinase C increased incorporation of [14C]PE into platelets. Proteinaceous factor(s) recovered in the extracellular medium from ADP- and thrombin-activated platelet suspensions were found to accelerate the transfer of pyrenedecanoic acid-labeled PE between donor and acceptor lipid vesicles. The stimulation of import of ethanolamine phospholipids led to a 2-fold enhancement of the prothrombinase activity of thrombin-activated platelets. Our study demonstrates that physiological platelet stimuli increase specifically the transfer of ethanolamine phospholipids from lipoproteins to platelets through a secretion-dependent mechanism. This might contribute to the increase of procoagulant activity of stimulated platelets.

Structure/function studies on the pH-dependent actin-binding protein hisactophilin in Dictyostelium mutants.

Our previous studies have shown that the actin-binding protein hisactophilin from Dictyostelium discoideum is a candidate for organizing the actin cytoskeleton at the plasma membrane in a pH-dependent manner. To further characterize this interaction we isolated hisactophilin overexpression (hisII+) and hisactophilin minus (his-) mutants. D. discoideum contains two hisactophilin isoforms; both genes are independently transcribed and carry a short intron at the same position of the coding region. The deduced amino acid sequence of hisactophilin II showed a characteristic high content of 35 histidine residues out of a total 118 amino acids. After transformation of Dictyostelium AX2 wild-type cells with a genomic fragment designed to inactivate the hisactophilin I gene we obtained hisactophilin II overexpressing mutants (hisII+). Multiple integration of the vector led to strong overexpression of hisactophilin II which even outnumbered the actin concentration by a factor of two. Hisactophilin II protein showed the same biochemical properties as hisactophilin I during purification and in its pH-dependent binding to F-actin; as shown by mass spectrometry the hisactophilin II fraction was almost completely myristoylated despite of this high overexpression. The inactivation of both hisactophilin genes was achieved by gene replacement with a vector construct encompassing parts of gene I and gene II connected by a geneticin cassette. The properties of the hisII+ and his- cells with regard to growth in shaking culture and on Klebsiella plates, development, chemotaxis and morphology were not affected under normal conditions. However, the hisII+ transformants revealed a significant difference to wild-type cells and his- cells when the cytoplasmic pH was lowered by diethylstilbestrol (DES), a proton pump inhibitor. HisII+ cells were more resistant to the acidification; in contrast to AX2 wild-type cells and his- cells they did not form plasma membrane protrusions, showed an increase in F-actin content, and contained large clusters of F-actin. Lowering the internal pH caused an accumulation of hisactophilin below the plasma membrane. The fact that cells deficient in hisactophilin again lose resistance to acidification is in good agreement with the hypothesis that hisactophilin functions as a pH sensor at the plasma membrane by reversibly connecting the membrane with the actin cortical network upon local changes of the proton concentration.