Epithelial cells have been to participate actively in host defense by producing small cationic peptides called defensins. To investigate the biological activity of epithelial defensins in more detail, we expressed two defensins, hBD-1 and HD-5, in eukaryotic cell lines. Defensins were localized in the cytoplasm and in cell culture medium and exhibited strong microbicidal activity toward Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Moreover, our data indicate that the presence of defensins protected the cells from adenoviral infection. The presence of HD-5 or hBD-1 reduced the infectivity of Av1CF2 three- to fivefold. These results imply that defensins must be considered a serious obstacle whenever adenovirus is used to deliver genes to epithelial cells.

Background To date, responsiveness to tumor necrosis factor alpha inhibitors in ulcerative colitis (UC) patients is not predictable. This is partially due to a lack of understanding of the underlying inflammatory processes. The aim of this study was to identify immunological subgroups of patients with UC and to test responsiveness to adalimumab in these subgroups in the mouse model of ulcerative colitis (UC), which is based on NOD/scid IL-2Rγ null (NSG) mice reconstituted with peripheral blood mononuclear cells (PBMCs; NSG-UC). Methods The immunological profiles of 40 UC patients and 16 non-UC donors were determined by flow cytometric analysis of PBMCs in a snapshot and longitudinal study and analyzed by principal component, orthogonal partial least square discrimination (oPLS-DA), and hierarchical clustering analysis. NSG mice were reconstituted 5 times at consecutive time points with PBMCs from a single donor and were analyzed for frequencies of human leukocytes and histological phenotype. The response to adalimumab of 2 identified subgroups was tested in the NSG-UC model. We used the clinical, colon, and histological score, serum levels of glutamic and aspartic acid, and IL-6 and IL-1ß. Response was analyzed by oPLS-DA. Results Analysis revealed a distinction between UC and non-UC donors. Hierarchical clustering identified 2 major subgroups in UC patients. Group I was characterized by TH17 and M1 monocytes, group II by TH2/TH1, and switched B cells. These subgroups reflect the dynamics of inflammation as patients. NSG-UC mice achieved an immunological phenotype reflecting the patient’s immunological phenotype. oPLS-DA revealed that NSG-UC mice reconstituted with PBMCs from group II responded better to adalimumab. Conclusions The combination of profiling and testing of therapeutics in the NSG-UC model may lead to individualized and phase-dependent therapies.

The sedimentation behavior of the halobacterial 7S RNA and bacterioopsin mRNA was ass after application of total cell lysates to sucrose gradients. These two RNAs cosedimented predominantly with membrane-bound polysomes, and the quantity of 7S RNA bound to the ribosomes was directly correlated with the expression of bacterioopsin. Puromycin treatment released the 7S RNA from the polysomes, indicating that it is transiently associated with protein translation. We suggest that halobacteria contain a signalrecognition-like particle involved in translation of membraneassociated proteins.In mammalian cells the 7S RNA is part of the signal recognition particle (SRP), which mediates cotranslational processing mechanisms for membrane and secretory proteins (1,2). SRP is a ribonucleoparticle that recognizes the leader sequence ofthe nascent polypeptide chain as it emerges from the ribosome. The interaction causes a translational arrest that is only released if the complex is targeted to a receptor in the membrane (3). These mechanisms prevent synthesis of hydrophobic proteins in a hydrophilic environment and direct membrane and secretory proteins to their final destinations in the cell. Small ribonucleoproteins resembling SRP have also been found in yeast (Schizosaccharomyces pombe and Yarrowia lipolytica) and in Escherichia coli (4-6), but in contrast to mammalian cells they translocate many proteins posttranslationally; genetic studies do not as yet corroborate a SRP-coupled translocation mechanism (7-9). Genetic analysis in E. coli has shown that the 4.5S RNA, which has been identified as a component of the ribonucleoparticle, is acting on translating ribosomes and perhaps in concert with elongation factor G GTP (10). However, it remains unclear whether it is generally obligatory for translation or only participates in translation of a subset of proteins.Like other archaebacteria, Halobacterium halobium possesses a 7S RNA with a possible secondary structure almost identical to that of the mammalian SRP RNA (11-13). Its function is unknown, but as archaebacterial 7S RNA genes can replace the 4.5S RNA gene in E. coli (14), it is likely that a signal recognition-like particle exists in halobacteria. In this report, we demonstrate that the halobacterial 7S RNA is associated with ribosomes during translation, and our data suggest that it is specifically involved in translation of membrane proteins. (18,19). The precursor is processed in a two-step mechanism (20, 21), but processing is not necessary for correct folding of the protein. Both forms integrate into the-purple membrane with no conformational differences from the mature protein (22). The presequence is unusual and lacks similarities to other prokaryotic and eukaryotic signal sequences (23). This observation raises questions about its functional significance. In this report we show that the N-terminal sequences of six halobacterial integral membrane proteins share a common motif that may be of functional importance. MATERIALS AND METHODSRNAzol was purchase...

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Epithelial cells have been shown to express the antibiotic peptides human beta-defensins-1 and 2. While beta-defensin-2 is known to be up-regulated by bacterial factors and proinflammatory mediators, the expression of beta-defensin-1 does not appear to be affected by these mediators. To determine the regulation and function of beta-defensin-1 we analyzed its expression upon stimulation of inflammatory mediators in vitro and ex vivo. In immortalized human cell lines (HaCaT) and nasal polyps beta-defensin-1 was not induced upon incubation with bacteria or proinflammatory mediators, suggesting that the inertness of beta-defensin-1 expression levels is not the result of the shortcoming of HaCaT cells. As proliferation and regeneration play an important role at sites of inflammation, we examined the expression level of beta-defensin-1 in relation to the differentiation and proliferation of HaCaT cells. beta-defensin-1 mRNA levels remained low during proliferation but were highly induced upon differentiation. In contrast, beta-defensin-2 expression was unaffected under these conditions. To examine the function of beta-defensin-1 in cellular proliferation and differentiation processes beta-defensin-1 was overexpressed in keratinocytes. Protein expression analysis of the differentiation marker keratin 10 revealed that its expression is highly induced in the presence of increased concentrations of beta-defensin-1. Hence our data indicate that high expression of beta-defensin-1 promotes cell differentiation processes of keratinocytes.