Mucosal secretions contain a range of defense effector molecules including antimicrobial peptides and proteinase inhibitors. These molecules play a central role in host defense against infection, and in a variety of immune and inflammatory reactions. The aim of this study was to analyze the levels of neutrophil defensins, the cathelicidin hCAP-18/LL-37, and the proteinase inhibitors secretory leukocyte proteinase inhibitor, SKALP/elafin and cystatin M/E in various mucosal secretions and urine. We show here that especially seminal plasma is characterized by high concentrations of hCAP-18/LL-37, SLPI, SKALP/elafin and cystatin M/E. The results of this study demonstrate that each mucosal secretion is characterized by a unique profile of effector molecules, which may supply individual mucosal secretions with specific properties related to the control of local infection and inflammation.

Homozygosity for Cst6 null alleles causes the phenotype of the ichq mouse, which is a model for human harlequin ichthyosis (OMIM 242500), a genetically heterogeneous group of keratinization disorders. Here we report evidence for the mechanism by which deficiency of the cysteine protease inhibitor cystatin M/E (the Cst6 gene product) leads to disturbed cornification, impaired barrier function and dehydration. Absence of cystatin M/E causes unrestricted activity of its target protease legumain in hair follicles and epidermis, which is the exact location where cystatin M/E is normally expressed. Analysis of stratum corneum proteins revealed a strong decrease of soluble loricrin monomers in skin extracts of ichq mice, although normal levels of loricrin were present in the stratum granulosum and stratum corneum of ichq mice, as shown by immunohistochemistry. This suggested a premature or enhanced crosslinking of loricrin monomers in ichq mice by transglutaminase 3 (TGase 3). In these mice, we indeed found strongly increased levels of TGase 3 that was processed into its activated 30 and 47 kDa subunits, compared to wild-type mice. This study shows that cystatin M/E and legumain form a functional dyad in epidermis in vivo. Disturbance of this protease-antiprotease balance causes increased enzyme activity of TGase 3 that could explain the observed abnormal cornification.

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BackgroundEpithelia are barrier-forming tissues that protect the organism against external noxious stimuli. Despite the similarity in function of epithelia, only few common protective mechanisms that are employed by these tissues have been systematically studied. Comparative analysis of genome-wide expression profiles generated by means of Serial Analysis of Gene Expression (SAGE) is a powerful approach to yield further insight into epithelial host defense mechanisms. We performed an extensive comparative analysis of previously published SAGE data sets of two types of epithelial cells, namely bronchial epithelial cells and keratinocytes, in which the response to pro-inflammatory cytokines was assessed. These data sets were used to elucidate a common denominator in epithelial host defense.ResultsBronchial epithelial cells and keratinocytes were found to have a high degree of overlap in gene expression. Using an in silico approach, an epithelial-specific molecular signature of gene expression was identified in bronchial epithelial cells and keratinocytes comprising of family members of keratins, small proline-rich proteins and proteinase inhibitors. Whereas some of the identified genes were known to be involved in inflammation, the majority of the signature represented genes that were previously not associated with host defense. Using polymerase chain reaction, presence of expression of selected tissue-specific genes was validated.ConclusionOur comparative analysis of gene transcription reveals that bronchial epithelial cells and keratinocytes both express a subset of genes that is likely to be essential in epithelial barrier formation in these cell types. The expression of these genes is specific for bronchial epithelial cells and keratinocytes and is not seen in non-epithelial cells. We show that bronchial epithelial cells, similar to keratinocytes, express components that are able to form a cross-linked protein envelope that may contribute to an effective barrier against noxious stimuli and pathogens.

Cutaneous squamous (pre)malignancies can be classified according to the keratinocytic intraepidermal neoplasia (KIN) classification. Aneuploidy can be seen as the result of chromosomal aberrations leading to altered DNA content and has been strongly associated with malignancy. Hyperproliferation is also strongly associated with tumorigenesis. The aim of the study was to analyse the presence and the amount of aneuploidy and proliferation in the progression from intraepithelial neoplasm to microinvasive carcinoma (miSCC). For this purpose, nuclei were isolated from 116 formalin-fixed KIN lesions from 68 patients in which DNA content was measured by flow cytometry. Proliferation was assessed by immunohistochemical staining for Ki67 as well as by flow cytometry. Aneuploidy was increasingly found in higher KIN lesions, but not in normal skin. However, in miSCC aneuploidy was relatively less frequently found. DNA indices (mean +/- SE) of KIN III-lesions (1.57 +/- 0.05) were significantly lower compared with KIN I/II lesions (1.71 +/- 0.05). Ki67 expression was strongly positively correlated with KIN grade, and proved to be a good adjunct in the classification of KINs. Thus, aneuploidy occurred more frequently in higher KIN lesions, indicating cumulative damage during KIN progression. The lower frequency of aneuploidy in miSCC compared with KIN III may point at alternative routes towards invasive carcinoma besides serial progression through all three KIN stages. Ki67 expression appears a valuable marker in the classification of KINs.