A matter of origin - identification of SEMA3A, BGLAP, SPP1 and PHEX as distinctive molecular features between bone site-specific human osteoblasts on transcription level.

In oral and maxillofacial bone reconstruction, autografts from the iliac crest represent the gold standard due to their superior clinical performance, compared to autografts derived from other extraoral regions. Thus, the aim of our study was to identify putative differences between osteoblasts derived from alveolar (hOB-A) and iliac crest (hOB-IC) bone of the same donor (nine donors) by means of their molecular properties in 2D and 3D culture. We thereby focused on the gene expression of biomarkers involved in osteogenic differentiation, matrix formation and osteoclast modulation. Furthermore, we examined the transcriptional response to Vit.D3 in hOB-A and hOB-IC. Our results revealed different modulation modes of the biomarker expression in osteoblasts, namely cell origin/bone entity-dependent, and culture configuration- and/or time-dependent modulations. SEMA3A, SPP1, BGLAP and PHEX demonstrated the strongest dependence on cell origin. With respect to Vit.D3-effects, BGLAP, SPP1 and ALPL displayed the highest Vit.D3-responsiveness. In this context we demonstrated that the transcriptional Vit.D3-response concerning SPP1 and ALPL in human osteoblasts depended on the cell origin. The results indicate a higher bone remodeling activity of iliac crest than alveolar osteoblasts and support the growing evidence that a high osteoclast activity at the host-/donor bone interface may support graft integration.

Antimicrobial Behavior and Cytotoxicity of Indocyanine Green in Combination with Visible Light and Water-Filtered Infrared A Radiation against Periodontal Bacteria and Subgingival Biofilm.

The widespread increase of antibiotic resistance highlights the need for alternative treatments such as antimicrobial photodynamic therapy (aPDT). This study aimed to evaluate the antimicrobial behavior and cytotoxicity of aPDT with indocyanine green (ICG) in combination with visible light (Vis) and water-filtered infrared A (wIRA). Representative periodontal bacteria (Parvimonas micra, Atopobium riame, Slackia exigua, Actinomyces naeslundii, Porphyromonas gingivalis, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans, and Prevotella nigrescens) and subgingival in situ biofilms from periodontal patients were treated with aPDT for 5 min. ICG was used at different concentrations (50-500 µg/mL) and the number of viable cells was determined in colony forming units (CFU). Untreated negative controls and 0.2% chlorhexidine as a positive control were also prepared. The cytotoxicity test on human keratinocytes in vitro was analyzed with the AlamarBlue assay after 5, 10, and 20 min, with four ICG concentrations, and at two temperatures (room temperature and 37 °C). The tested periodontal pathogens treated with aPDT were eliminated in a range between 1.2 and 6.7 log10 CFU, except for A. naeslundii, which was killed at a lower range. The subgingival biofilm treated with aPDT expressed significant differences to the untreated controls except for at 300 µg/mL ICG concentration. The cytotoxicity was directly related to the concentration of ICG and irradiation time. These observations raise questions concerning the use of this specific aPDT as an adjuvant to periodontal treatments due to its possible toxicity towards human gingival cells.

How Do Polymer Coatings Affect the Growth and Bacterial Population of a Biofilm Formed by Total Human Salivary Bacteria?-A Study by 16S-RNA Sequencing.

Antimicrobial surface modifications are required to prevent biomaterial-associated biofilm infections, which are also a major concern for oral implants. The aim of this study was to evaluate the influence of three different coatings on the biofilm formed by human saliva. Biofilms grown from human saliva on three different bioactive poly(oxanorbornene)-based polymer coatings (the protein-repellent PSB: poly(oxanorbornene)-based poly(sulfobetaine), the protein-repellent and antimicrobial PZI: poly(carboxyzwitterion), and the mildly antimicrobial and protein-adhesive SMAMP: synthetic mimics of antimicrobial peptides) were analyzed and compared with the microbial composition of saliva, biofilms grown on uncoated substrates, and biofilms grown in the presence of chlorhexidine digluconate. It was found that the polymer coatings significantly reduced the amount of adherent bacteria and strongly altered the microbial composition, as analyzed by 16S RNA sequencing. This may hold relevance for maintaining oral health and the outcome of oral implants due to the existing synergism between the host and the oral microbiome. Especially the reduction of some bacterial species that are associated with poor oral health such as Tannerella forsythia and Fusobacterium nucleatum (observed for PSB and SMAMP), and Prevotella denticola (observed for all coatings) may positively modulate the oral biofilm, including in situ.

Poly(oxanorbornene)-Coated CdTe Quantum Dots as Antibacterial Agents.

In this study, synthetic mimics of antimicrobial peptides based on poly(oxanorbornene) molecules (or PONs) were used to coat CdTe quantum dots (QDs). These PONs-CdTe QDs were investigated for their activity against Escherichia coli, a bacterium with antibiotic resistant strains. At the same time, the antibacterial activity of the PONs-CdTe QDs was compared to the antibacterial activity of free PONs and free CdTe QDs. The observed antibacterial activity of the PONs-CdTe QDs was additive and concentration dependent. The conjugates had a significantly lower minimum inhibitory concentration (MIC) than the free PONs and QDs, particularly for PONs-CdTe QDs which contained PONs of high amine density. The maximum activity of PONs-CdTe QDs was not realized by conjugating PONs with the highest intrinsic antibacterial activity (i.e., the lowest MIC in solution as free PONs), indicating that the mechanism of action for free PONs and PONs-CdTe QDs is different. Equally important, conjugating PONs to CdTe QDs decreased their hemolytic activity against red blood cells compared to free PONs, lending to higher therapeutic indices against E. coli. This could potentially enable the use of higher, and therefore more effective, PONs-QDs concentrations when addressing bacterial contamination, without concerns of adverse impacts on mammalian cells and organisms.

Stimulus-Responsive Polyzwitterionic Surfaces Made from Itaconic Acid: Self-Triggered Antimicrobial Activity, Protein Repellency, and Cell Compatibility.

A functional monomer carrying a carboxylate and a protected primary ammonium group is synthesized from itaconic acid. When copolymerized with dimethyl acrylamide and 4-methacryloyloxybenzophenone, cross-linkable polyzwitterions are obtained. These are converted to surface-attached polyzwitterion networks by simultaneous UV-triggered C,H insertion reactions. The resulting polyzwitterion-coated substrates were studied by surface plasmon resonance spectroscopy measurements, ζ potential and various biological assays. They were (expectedly) protein repellent, yet at the same time (and unexpectedly) cell-adhesive and antimicrobially active. This was attributed to stimulus-responsiveness of the polyzwitterion (confirmed by the ζ potential measurements), which enables charge adjustment at different pH values. When protonated, the polyzwitterions become amphiphilic polycations and, in this state, kill bacteria upon contact like their parent structures (polymer-based synthetic mimics of antimicrobial peptides, SMAMPs).

Surface Structuring Combined with Chemical Surface Functionalization: An Effective Tool to Manipulate Cell Adhesion.

In this study, we investigate how a surface structure underneath a surface-attached polymer coating affects the bioactivity of the resulting material. To that end, structured surfaces were fabricated using colloidal lithography (lateral dimensions: 200 nm to 1 µm, height ~15 to 50 nm). The surface structures were further functionalized either with antimicrobial, cell-adhesive polycations or with protein-repellent polyzwitterions. The materials thus obtained were compared to non-functionalized structured surfaces and unstructured polymer monolayers. Their physical properties were studied by contact-angle measurements and atomic force microscopy (AFM). Protein adhesion was studied by surface plasmon resonance spectroscopy, and the antimicrobial activity against Escherichia coli bacteria was tested. The growth of human mucosal gingiva keratinocytes on the materials was analyzed using the Alamar blue assay, optical microscopy, and live-dead staining. The data shows that the underlying surface structure itself reduced protein adhesion and also bacterial adhesion, as evidenced by increased antimicrobial activity. It also enhanced cell adhesion to the surfaces. Particularly in combination with the adhesive polycations, the surfaces increased the cell growth compared to the unstructured reference materials. Thus, functionalizing structured surfaces with adhesive polymer could be a valuable tool for improved tissue integration.

CD40 inhibits replication of hepatitis C virus in primary human hepatocytes by c-Jun N terminal kinase activation independent from the interferon pathway.

UNLABELLED: CD40, a member of the tumor necrosis factor receptor family, and its ligand, CD40L (CD154), are important regulators of the antiviral immune response. CD40L is up-regulated on lymphocytes and CD40 on hepatocytes during infection with hepatitis C virus (HCV); we investigated the role of CD40 signaling during HCV replication in hepatocytes. Viral replication was studied in primary human hepatocytes (PHH) and Huh7.5 cells using the infectious HCV Japanese fulminate hepatitis 1 isolate (JFH1) culture system, and in coculture with HCV antigen-specific CD8+ T cells. CD40L rapidly and transiently inhibits expression of the HCV nonstructural proteins NS3 and NS5A as well as HCV structural proteins core and E2 in Huh7.5 cells. Similarly, CD40L prevented replication of HCV in PHH, in synergy with interferon (IFN)-alpha. In Huh7.5 cells with replicating HCV, CD40L prevented production of infectious viral particles. When HCV antigen-specific CD8+ T cells were cocultured with HLA-A2-expressing Huh7 cells that had replicating virus, the T cells became activated, up-regulated CD40L, and inhibited HCV replication. Inhibition of CD40L partially prevented the antiviral activity of the CD8+ T cells. The antiviral effect of CD40L required activation of c-Jun N terminal kinases (JNK)1/2, but not induction of apoptosis or the JAK/STAT pathway that is necessary for the antiviral effects of IFNs. CONCLUSION: CD40 inhibits HCV replication by a novel, innate immune mechanism. This pathway might mediate viral clearance, and disruptions might be involved in the pathogenesis of HCV infection.

Low perforin expression of early differentiated HCV-specific CD8+ T cells limits their hepatotoxic potential.

BACKGROUND & AIMS: Perforin plays a central role in the immunopathogenesis of different viral infections. However, its role in hepatitis C virus (HCV) infection has not been fully understood. Here, we analyzed two closely related questions: first, is CD8+ T cell-mediated killing of HCV-replicating human hepatoma cells mediated by perforin? Second, if so, do HCV-specific CD8+ T cells obtained from chronically HCV infected patients express and upregulate perforin? METHODS: Susceptibility of HCV-replicating human hepatoma cells to the cytotoxic pathway was tested in vitro by addition of perforin substitute streptolysin O and granzyme B and by co-culture experiments with a perforin-expressing HCV-specific CD8+ T cell clone in the presence of perforin or caspase inhibitors. HCV-specific CD8+ T cells were obtained and analyzed for perforin expression and differentiation markers ex vivo from 12 chronically infected patients and 12 patients with resolved HCV infection. RESULTS: HCV-replicating human hepatoma cells were susceptible to cytotoxic killing in vitro and a dominant role of perforin in HCV-specific CD8+ T cell-mediated cytolysis was observed. However, HCV-specific CD8+ T cells obtained ex vivo from chronically HCV infected patients expressed only low levels of perforin and showed an impaired ability to upregulate perforin. This was tightly linked to the distinct differentiation stage of HCV-specific CD8+ T cell differentiation ex vivo since early and intermediate differentiated HCV-specific CD8+ T cells only showed weak perforin expression in contrast to late differentiated CD8+ T cells that displayed strong perforin expression. CONCLUSIONS: Our results suggest that perforin plays a dominant role in CD8+ T cell-mediated lysis of HCV-replicating human hepatoma cells but that lysis may be limited in human chronic viral infection by the low perforin expression of early/intermediate differentiated HCV-specific CD8+ T cells.

Hepatitis C virus infection sensitizes human hepatocytes to TRAIL-induced apoptosis in a caspase 9-dependent manner.

Apoptosis of infected cells represents a key host defense mechanism against viral infections. The impact of apoptosis on the elimination of hepatitis C virus (HCV)-infected cells is poorly understood. The TRAIL has been implicated in the death of liver cells in hepatitis-infected but not in normal liver cells. To determine the impact of TRAIL on apoptosis of virus-infected host cells, we studied TRAIL-induced apoptosis in a tissue culture model system for HCV infection. We demonstrated that HCV infection sensitizes primary human hepatocytes and Huh7.5 hepatoma cells to TRAIL induced apoptosis in a dose- and time-dependent manner. Mapping studies identified the HCV nonstructural proteins as key mediators of sensitization to TRAIL. Using a panel of inhibitors targeting different apoptosis pathways, we demonstrate that sensitization to TRAIL is caspase-9 dependent and mediated in part via the mitochondrial pathway. Sensitization of hepatocytes to TRAIL-induced apoptosis by HCV infection represents a novel antiviral host defense mechanism that may have important implications for the pathogenesis of HCV infection and may contribute to the elimination of virus-infected hepatocytes.