Antibiotic peptides are important effector molecules in host-parasite interactions throughout the living world. In vertebrates, they function in first-line host defense by antagonizing a wide range of microbes including bacteria, fungi, and enveloped viruses. The antibiotic activity is thought to be based on their cationic, amphipathic nature, which enables the peptides to impair vital membrane functions. Molecular details for such activities have been elaborated with model membranes; however, there is increasing evidence that these models may not reflect the complex processes involved in the killing of microbes. For example, the overall killing activity of the bacterial peptide antibiotic nisin is composed of independent activities such as the formation of target-mediated pores, inhibition of cell-wall biosynthesis, formation of nontargeted pores, and induction of autolysis. We studied the molecular modes of action of human defense peptides and tried to determine whether they impair membrane functions primarily and whether additional antibiotic activities may be found. We compared killing kinetics, solute efflux kinetics, membrane-depolarization assays, and macromolecular biosynthesis assays and used several strains of Gram-positive cocci as test strains. We found that membrane depolarization contributes to rapid killing of a significant fraction of target cells within a bacterial culture. However, substantial subpopulations appear to survive the primary effects on the membrane. Depending on individual strains and species and peptide concentrations, such subpopulations may resume growth or be killed through additional activities of the peptides. Such activities can include the activation of cell-wall lytic enzymes, which appears of particular importance for killing of staphylococcal strains.

Background: The opportunistic food-borne gram-positive pathogen Listeria monocytogenes can exist as a free-living microorganism in the environment and grow in the cytoplasm of vertebrate and invertebrate cells following infection. The general stress response, controlled by the alternative sigma factor, B , has an important role for bacterial survival both in the environment and during infection. We used quantitative real-time PCR analysis and immuno-blot analysis to examine B expression during growth of L. monocytogenes EGD-e. Whole genome-based transcriptional profiling was used to identify B -dependent genes at different growth phases.

Organotypic culture of human primary bronchial epithelial cells is a useful in vitro system to study normal biological processes and lung disease mechanisms, to develop new therapies, and to assess the biological perturbations induced by environmental pollutants. Herein, we investigate whether the perturbations induced by cigarette smoke (CS) and observed in the epithelium of smokers' airways are reproducible in this in vitro system (AIR-100 tissue), which has been shown to recapitulate most of the characteristics of the human bronchial epithelium. Human AIR-100 tissues were exposed to mainstream CS for 7, 14, 21, or 28 min at the air-liquid interface, and we investigated various biological endpoints [e.g., gene expression and microRNA profiles, matrix metalloproteinase 1 (MMP-1) release] at multiple postexposure time points (0.5, 2, 4, 24, 48 h). By performing a Gene Set Enrichment Analysis, we observed a significant enrichment of human smokers' bronchial epithelium gene signatures derived from different public transcriptomics datasets in CS-exposed AIR-100 tissue. Comparison of in vitro microRNA profiles with microRNA data from healthy smokers highlighted various highly translatable microRNAs associated with inflammation or with cell cycle processes that are known to be perturbed by CS in lung tissue. We also found a dose-dependent increase of MMP-1 release by AIR-100 tissue 48 h after CS exposure in agreement with the known effect of CS on this collagenase expression in smokers' tissues. In conclusion, a similar biological perturbation than the one observed in vivo in smokers' airway epithelium could be induced after a single CS exposure of a human organotypic bronchial epithelium-like tissue culture.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) plays a crucial role in the pathogenesis of hepatic fibrosis and thus may represent an important therapeutic target in the design of anti-fibrotic strategies for chronic liver disease. We present an innovative therapy based on the assignment of inactivated enzymes acting as scavengers for TIMP-1. Hepatic fibrosis was induced in BALB/c mice by repetitive intraperitoneal CCl4 injection. The animals were treated with proteolytic inactive matrix metalloproteinase-9 mutants (E402Q, H401A, E402H/H411E) using adenovirus-mediated gene transfer. Application of these MMP-9 mutants inhibited fibrogenesis, which was indicated by decreasing portal and periportal accumulation of collagen. Total hydroxyproline of liver tissue, the morphometric stage of fibrosis as well as mRNA expression of marker proteins for hepatic fibrosis in livers of E402Q- and H401A-treated mice were significantly reduced. MMP-9 mutants suppressed transdifferentiation of hepatic stellate cells to the myofibroblast like phenotype in vitro and in vivo. Moreover, adenoviral application of the mutants MMP-9-H401A and -E402Q led to increased apoptosis of activated hepatic stellate cells, thought to be the main promoters of hepatic fibrosis. Application of MMP-9 mutants as TIMP-1 scavengers may provide a new therapeutic strategy for hepatic fibrosis.

The APOBEC3 family of antiviral DNA cytosine deaminases is implicated as the second largest source of mutation in cancer. This mutational process may be a causal driver or inconsequential passenger to the overall tumor phenotype. We show that human APOBEC3A expression in murine colon and liver tissues increases tumorigenesis. All other APOBEC3 family members, including APOBEC3B, fail to promote liver tumor formation. Tumor DNA sequences from APOBEC3A-expressing animals display hallmark APOBEC signature mutations in TCA/T motifs. Bioinformatic comparisons of the observed APOBEC3A mutation signature in murine tumors, previously reported APOBEC3A and APOBEC3B mutation signatures in yeast, and reanalyzed APOBEC mutation signatures in human tumor datasets support cause-and-effect relationships for APOBEC3A-catalyzed deamination and mutagenesis in driving multiple human cancers.

Matrix metalloproteinases (MMPs) are responsible for the turnover and degradation of extracellular matrix. They play a crucial role in the growth and migration of colorectal carcinoma cells. Colorectal carcinomas are characterized by enhanced expression of MMP-2, MMP-9, MMP-7, and MMP-13. The aim of this study was to determine the expression levels of MMP-2, MMP-9, MMP-7, MMP-13, and MMP-14 and their specific inhibitor TIMP-1 in inflammatory bowel diseases and precancerous lesions of the colon, i.e., Crohn's disease and ulcerative colitis, and in adenomatous polyps (APs) for comparison. Biopsy samples of pathological and healthy tissue were obtained from 40 patients with inflammatory bowel disease (ulcerative colitis, n = 17; Crohn's disease, n = 23) and from 19 patients with APs. mRNA was measured by quantitative real-time polymerase chain reaction to study MMP and TIMP-1 gene expression in both pathological and normal mucosal specimens. For MMP-2, MMP-9, and TIMP-1, protein expression also was quantified with sandwich enzyme-linked immunosorbent assay. In biopsy specimens of Crohn's disease and ulcerative colitis, significantly increased levels of MMP-2, MMP-7, and MMP-13 mRNA were found. MMP-2 and MMP-9 showed enhanced secretion on the protein level. AP revealed an increased transcription of MMP-7 and MMP-13 genes. MMP-14 mRNA was decreased in APs. MMPs, especially MMP-7 and MMP-13, which are expressed primarily on the tumor cell surface, are elevated in inflammatory bowel disease, which may have more chance to evolve into malignancy than normal tissue. In APs, increased expression of MMP-7 and MMP-13 may serve as an early indicator for colorectal carcinogenesis.

BackgroundOnly a few exposure systems are presently available that enable cigarette smoke exposure of living cells at the air–liquid interface, of which one of the most versatile is the Vitrocell® system (Vitrocell® Systems GmbH). To assess its performance and optimize the exposure conditions, we characterized a Vitrocell® 24/48 system connected to a 30-port carousel smoking machine. The Vitrocell® 24/48 system allows for simultaneous exposure of 48 cell culture inserts using dilution airflow rates of 0–3.0 L/min and exposes six inserts per dilution. These flow rates represent cigarette smoke concentrations of 7–100%.ResultsBy characterizing the exposure inside the Vitrocell® 24/48, we verified that (I) the cigarette smoke aerosol distribution is uniform across all inserts, (II) the utility of Vitrocell® crystal quartz microbalances for determining the online deposition of particle mass on the inserts, and (III) the amount of particles deposited per surface area and the amounts of trapped carbonyls and nicotine were concentration dependent. At a fixed dilution airflow of 0.5 L/min, the results showed a coefficient of variation of 12.2% between inserts of the Vitrocell® 24/48 module, excluding variations caused by different runs. Although nicotine and carbonyl concentrations were linear over the tested dilution range, particle mass deposition increased nonlinearly. The observed effect on cell viability was well-correlated with increasing concentration of cigarette smoke.ConclusionsOverall, the obtained results highlight the suitability of the Vitrocell® 24/48 system to assess the effect of cigarette smoke on cells under air–liquid interface exposure conditions, which is closely related to the conditions occurring in human airways.

Therapeutic options for the treatment of colorectal cancer (CRC) are diverse but still not always satisfying. Recent success of immune checkpoint inhibition treatment for the subgroup of CRC patients suffering from hyper-mutated tumors suggests a permanent role of immune therapy in the clinical management of CRC. Substantial improvement in treatment outcome could be achieved by development of efficient patient-individual CRC vaccination strategies. This mini-review summarizes the current knowledge on the two general classes of targets: tumor-associated antigens (TAAs) and tumor-specific antigens. TAAs like carcinoembryonic antigen and melanoma associated antigen are present in and shared by a subgroup of patients and a variety of clinical studies examined the efficacy of different TAA-derived peptide vaccines. Combinations of several TAAs as the next step and the development of personalized TAA-based peptide vaccines are discussed. Improvements of peptide-based vaccines achievable by adjuvants and immune-stimulatory chemotherapeutics are highlighted. Finally, we sum up clinical studies using tumor-specific antigens - in CRC almost exclusively neoantigens - which revealed promising results; particularly no severe adverse events were reported so far. Critical progress for clinical outcomes can be expected by individualizing neoantigen-based peptide vaccines and combining them with immune-stimulatory chemotherapeutics and immune checkpoint inhibitors. In light of these data and latest developments, truly personalized neoantigen-based peptide vaccines can be expected to fulfill modern precision medicine’s requirements and will manifest as treatment pillar for routine clinical management of CRC.