148 research outputs found
The Impact of Blue Light Irradiation on Keratinocytes in Vitro
ABSTRACT
Background: This study examined the effects of irradiation with blue
light on HaCaT keratinocytes. As irradiation with blue light is known to be
antimicrobial, it offers a promising alternative therapy for contaminated
wounds. There is evidence that red light promotes wound healing, but
the potential benefits of irradiation with blue light have not yet been ad-
equately investigated.
Methods: The rate of wound closure in sterile and contaminated cells
was measured using an in vitro scratch assay wound-healing model. Ad-
ditionally, cell viability after treatment was determined using a Sulforho-
damine B (SRB) assay.
Results: In both the sterile and contaminated groups, treated cells
showed delayed wound closure when compared with cells not irradiated
with blue light. Additionally, treatment with blue light resulted in poorer
viability in the treatment groups.
Conclusion: Although irradiation with blue light may offer a promising
alternative therapy for reducing bacterial colonization, our data indicate
that re-epithelization may be negatively influenced by blue light. Further
research is needed to clarify possible wound healing applications
Human Sterol Regulatory Element-Binding Protein 1a Contributes Significantly to Hepatic Lipogenic Gene Expression
Background/Aims:Sterol regulatory element-binding protein (SREBP) 1, the master regulator of lipogenesis, was shown to be associated with non-alcoholic fatty liver disease, which is attributed to its major isoform SREBP1c. Based on studies in mice, the minor isoform SREBP1a is regarded as negligible for hepatic lipogenesis. This study aims to elucidate the expression and functional role of SREBP1a in human liver. Methods: mRNA expression of both isoforms was quantified in cohorts of human livers and primary human hepatocytes. Hepatocytes were treated with PF-429242 to inhibit the proteolytic activation of SREBP precursor protein. SREBP1a-specific and pan-SREBP1 knock-down were performed by transfection of respective siRNAs. Lipogenic SREBP-target gene expression was analyzed by real-time RT-PCR. Results: In human liver, SREBP1a accounts for up to half of the total SREBP1 pool. Treatment with PF-429242 indicated SREBP-dependent auto-regulation of SREBP1a, which however was much weaker than of SREBP1c. SREBP1a-specific knock-down also reduced significantly the expression of SREBP1c and of SREBP-target genes. Regarding most SREBP-target genes, simultaneous knock-down of both isoforms resulted in effects of only similar extent as SREBP1a-specific knock-down. Conclusion: We here showed that SREBP1a is significantly contributing to the human hepatic SREBP1 pool and has a share in human hepatic lipogenic gene expression. Copyright (C) 2015 S. Karger AG, Base
Reduced NK-Cell Activity in Patients with Metastatic Colon Cancer
Natural killer cells (NK-cells) are believed to play an essential role in the immune surveillance against tumors and infectious diseases. The role of NK-cells in colon cancer remains obscure, since increased as well as decreased percentages and/or activity of NK-cells in comparison to control patients have been reported. Percentage and cytolytic activity of NKcells in the peripheral blood were analyzed in 42 patients with colon cancer before surgery and one year thereafter in comparison to control patients with non-malignant diseases. Patients without distant metastasis at the time of diagnosis displayed a significantly increased percentage of NK-cells as well as sustained NK-cell activity in the peripheral blood prior to surgery when compared to control patients. In contrast, patients with metastatic disease at the time of diagnosis displayed significantly decreased NK-cell activity in the peripheral blood when compared to control patients. One year after surgery, patients who remained free of metastasis still displayed sustained NK-cell activity, whereas patients who developed metastasis presented with profoundly decreased levels of NK-cell activity. Further analysis of these patients revealed that patients who developed metastasis within the first year after surgery already displayed reduced NK-cell activity prior to curative colorectal surgery. These observations indicate that metastatic spread of colorectal cancer is associated with decreased NK-cell activity. It remains speculative whether decreased NK-cell activity precedes the development of metastasis and thus may help to identify patients with a high risk of rapid tumor progression following curative colorectal surgery
Bone Healing Gone Wrong : Pathological Fracture Healing and Non-Unions—Overview of Basic and Clinical Aspects and Systematic Review of Risk Factors
Bone healing is a multifarious process involving mesenchymal stem cells, osteoprogenitor
cells, macrophages, osteoblasts and -clasts, and chondrocytes to restore the osseous tissue. Particularly
in long bones including the tibia, clavicle, humerus and femur, this process fails in 2–10% of all
fractures, with devastating effects for the patient and the healthcare system. Underlying reasons for
this failure are manifold, from lack of biomechanical stability to impaired biological host conditions
and wound-immanent intricacies. In this review, we describe the cellular components involved in
impaired bone healing and how they interfere with the delicately orchestrated processes of bone
repair and formation. We subsequently outline and weigh the risk factors for the development of
non-unions that have been established in the literature. Therapeutic prospects are illustrated and put
into clinical perspective, before the applicability of biomarkers is finally discussed
Subtoxic Concentrations of Hepatotoxic Drugs Lead to Kupffer Cell Activation in a Human In Vitro Liver Model
Drug induced liver injury (DILI) is an idiosyncratic adverse drug reaction
leading to severe liver damage. Kupffer cells (KC) sense hepatic tissue
stress/damage and therefore could be a tool for the estimation of consequent
effects associated with DILI. Aim of the present study was to establish a
human in vitro liver model for the investigation of immune-mediated signaling
in the pathogenesis of DILI. Hepatocytes and KC were isolated from human liver
specimens. The isolated KC yield was cells/g liver tissue with a purity of
>80%. KC activation was investigated by the measurement of reactive oxygen
intermediates (ROI, DCF assay) and cell activity (XTT assay). The initial KC
activation levels showed broad donor variability. Additional activation of KC
using supernatants of hepatocytes treated with hepatotoxic drugs increased KC
activity and led to donor-dependent changes in the formation of ROI compared
to KC incubated with supernatants from untreated hepatocytes. Additionally, a
compound- and donor-dependent increase in proinflammatory cytokines or in
anti-inflammatory cytokines was detected. In conclusion, KC related immune
signaling in hepatotoxicity was successfully determined in a newly established
in vitro liver model. KC were able to detect hepatocyte stress/damage and to
transmit a donor- and compound-dependent immune response via cytokine
production
A systems biology approach to dynamic modeling and inter-subject variability of statin pharmacokinetics in human hepatocytes
A dynamic model for the biotransformation of atorvastatin has been developed using quantitative metabolite measurements in primary human hepatocytes. The model comprises kinetics for transport processes and metabolic enzymes as well as population liver expression data allowing us to assess the impact of inter-individual variability of concentrations of key proteins. Application of computational tools for parameter sensitivity analysis enabled us to considerably improve the validity of the model and to create a consistent framework for precise computer-aided simulations in toxicology
Posttranslational Modification of Vesicular Stomatitis Virus Glycoprotein, but Not JNK Inhibition, Is the Antiviral Mechanism of SP600125
Vesicular stomatitis virus (VSV), a negative-sense single-stranded-RNA rhabdovirus, is an extremely promising oncolytic agent for cancer treatment. Since oncolytic virotherapy is moving closer to clinical application, potentially synergistic combinations of oncolytic viruses and molecularly targeted antitumor agents are becoming a meaningful strategy for cancer treatment. Mitogenactivated protein kinase (MAPK) inhibitors have been shown to impair liver cell proliferation and tumor development, suggesting their potential use as therapeutic agents for hepatocellular carcinoma (HCC). In this work, we show that the impairment of MAPK in vitro did not interfere with the oncolytic properties of VSV in HCC cell lines. Moreover, the administration of MAPK inhibitors did not restore the responsiveness of HCC cells to alpha/beta interferon (IFN-α/β). In contrast to previous reports, we show that JNK inhibition by the inhibitor SP600125 is not responsible for VSV attenuation in HCC cells and that this compound acts by causing a posttranslational modification of the viral glycoprotein
Differentiation of In Vitro–Modified Human Peripheral Blood Monocytes Into Hepatocyte–like and Pancreatic Islet-like Cells
BACKGROUND & AIMS:
Adult stem cells provide a promising alternative for the treatment of diabetes mellitus and end-stage liver diseases. We evaluated the differentiation potential of human peripheral blood monocytes into hepatocyte-like and pancreatic islet-like cells.
METHODS:
Monocytes were treated with macrophage colony-stimulating factor and interleukin 3 for 6 days, followed by incubation with hepatocyte and pancreatic islet-specific differentiation media. Cells were characterized by flow cytometry, gene-expression analysis, metabolic assays, and transplantation for their state of differentiation and tissue-specific functions.
RESULTS:
In response to macrophage colony-stimulating factor and interleukin 3, monocytes resumed cell division in a CD115-dependent fashion, which was associated with a down-regulation of the PRDM1 and ICSBP genes. These programmable cells of monocytic origin were capable of differentiating into neohepatocytes, which closely resemble primary human hepatocytes with respect to morphology, expression of hepatocyte markers, and specific metabolic functions. After transplantation into the liver of severe combined immunodeficiency disease/nonobese diabetic mice, neohepatocytes integrated well into the liver tissue and showed a morphology and albumin expression similar to that of primary human hepatocytes transplanted under identical conditions. Programmable cells of monocytic origin-derived pancreatic neoislets expressed beta cell-specific transcription factors, secreted insulin and C peptide in a glucose-dependent manner, and normalized blood glucose levels when xenotransplanted into immunocompetent, streptozotocin-treated diabetic mice. Programmable cells of monocytic origin retained monocytic characteristics, notably CD14 expression, a monocyte-specific methylation pattern of the CD115 gene, and expression of the transcription factor PU.1.
CONCLUSIONS:
The ability to reprogram, expand, and differentiate peripheral blood monocytes in large quantities opens the real possibility of the clinical application of programmable cells of monocytic origin in tissue repair and organ regeneration
Gene networks and transcription factor motifs defining the differentiation of stem cells into hepatocyte-like cells
Background & AimsThe differentiation of stem cells to hepatocyte-like cells (HLC) offers the perspective of unlimited supply of human hepatocytes. However, the degree of differentiation of HLC remains controversial. To obtain an unbiased characterization, we performed a transcriptomic study with HLC derived from human embryonic and induced stem cells (ESC, hiPSC) from three different laboratories.MethodsGenome-wide gene expression profiles of ESC and HLC were compared to freshly isolated and up to 14days cultivated primary human hepatocytes. Gene networks representing successful and failed hepatocyte differentiation, and the transcription factors involved in their regulation were identified.ResultsGene regulatory network analysis demonstrated that HLC represent a mixed cell type with features of liver, intestine, fibroblast and stem cells. The “unwanted” intestinal features were associated with KLF5 and CDX2 transcriptional networks. Cluster analysis identified highly correlated groups of genes associated with mature liver functions (n=1057) and downregulated proliferation associated genes (n=1562) that approach levels of primary hepatocytes. However, three further clusters containing 447, 101, and 505 genes failed to reach levels of hepatocytes. Key TF of two of these clusters include SOX11, FOXQ1, and YBX3. The third unsuccessful cluster, controlled by HNF1, CAR, FXR, and PXR, strongly overlaps with genes repressed in cultivated hepatocytes compared to freshly isolated hepatocytes, suggesting that current in vitro conditions lack stimuli required to maintain gene expression in hepatocytes, which consequently also explains a corresponding deficiency of HLC.ConclusionsThe present gene regulatory network approach identifies key transcription factors which require modulation to improve HLC differentiation
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