Comprehensive Analysis of Zinc Derivatives Pro-proliferative, Anti-Apoptotic and Antimicrobial Effect on Human Fibroblasts and Keratinocytes in a Simulated, Nutrient-deficient Environment In Vitro.

Zinc as therapeutic agent in skin and wound care has been known for centuries, but its role is controversial and comprehensive investigations in nutrient-deficient environments are lacking. We aimed to provide a broad analysis of different zinc derivatives on proliferation, apoptosis and antimicrobial properties in a simulated nutrient-deficient environment in vitro. Human fibroblasts (CRL2522) and keratinocytes (HaCaT) were treated with a broad concentration range (10 - 0.0001 µg/mL) of zinc-sulfate (ZnSO4), -gluconate (ZnGluc) and -histidine (ZnHis) for 1-6 days under nutrient-deficient media conditions. Cell proliferation was investigated by XTT assay. Targeted analyzes in proliferation (E2F1, PCNA) and apoptosis (TP53) associated genes were performed via qRT-PCR and apoptosis was determined via FACS (annexin V/7-AAD staining). Antimicrobial efficacy was investigated using a quantitative suspension method against S. aureus, P. aeruginosa, E. coli, and C. albicans. The results indicated that 0.1 to 0.001 µg/mL Zn increased cell proliferation in both cell lines. Fibroblasts were more susceptible with significant proliferation peaks on days 2 & 6, and days 1 & 4 for keratinocytes. No relevant changes in gene expression were detected for E2F1 and PCNA nor for TP53. Annexin-V/7-AAD-staining of fibroblasts revealed a small, yet insignificant reduction of apoptosis induction for ZnGluc and ZnSO4. ZnGluc and ZnSO4 (0.1%) achieved high microbial reductions (4-5 log10 reductions) against tested pathogens. ZnGluc and ZnSO4 showed relevant pro-proliferative and antimicrobial, as well as tendential anti-apoptotic features in a simulated nutrient-deficient microenvironment in vitro. This further validates a potential benefit of local zinc treatment in deficient wound microenvironments.

Effects of Vitamin B Complex and Vitamin C on Human Skin Cells: Is the Perceived Effect Measurable?

OBJECTIVE: Vitamins are essential for human health. In terms of local application for wound healing, vitamins' positive effect remains unclear. However, because of the regular appearance of nutritional deficiency in chronic wound patients, a favorable impact of locally applied vitamins can be hypothesized. METHODS: Vitamins B3, B5, B6, B7, B9, B10, B12, and C individually as well as different combinations of B vitamins were investigated regarding their ability to promote proliferation and migration of human skin fibroblasts and keratinocytes. Proliferation assays with and without bacterial challenge, immunocytochemical staining, and scratch assay were used to determine the most effective combination(s). MAIN RESULTS: Some vitamin combinations showed a positive impact on proliferation, especially for keratinocytes after 72 hours. In terms of wound closure, the combinations B9 and B12; B3, B5, B6, and B10; and B3, B5, and B7 improved closure rates by 25% to 30%. The improved closure rates are also reflected by immunocytochemically detected upregulation of the migration marker CXCR4 for several combinations. CONCLUSIONS: Certain combinations of B vitamins demonstrate a positive influence on human keratinocytes and fibroblasts. Vitamins especially promoted fibroblast migration, and a statistically significant induction of keratinocyte proliferation was observed. Therefore, local vitamin application could benefit the physiologic wound healing process.

Influence of human acute wound fluid on the antibacterial efficacy of different antiseptic polyurethane foam dressings: An in vitro analysis.

Treating infected acute and/or chronic wounds still represents a major challenge in medical care. Various interactions of antiseptic dressings with wound environments regarding antimicrobial efficacy remain unclear. Therefore, this work aimed to investigate the influence of human acute wound fluid (AWF) on the antimicrobial performance of different antiseptic foam dressings in vitro against typical bacterial wound pathogens. Eight antiseptic polyurethane foam dressings containing either a silver formulation or a polyhexamethylene-biguanide (PHMB) were assessed regarding their antimicrobial potency against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa using a modified time-kill assay based on ISO EN 20743. The antiseptic efficacy was evaluated standardly as well as under the influence of human AWF after 2, 4, 6, and 24 hours. The specific chemical formulation and concentration of the antiseptic substance (ionic or nanocrystalline silver, silver sulfadiazine, PHMB 0.1%/0.5%) embedded within the dressings seemed to play a key role. For certain dressings (two nanocrystalline and one ionic silver dressing), the antimicrobial efficacy was significantly reduced under the influence of AWF compared to unchallenged test series. Unchallenged the efficacy of PHMB was comparable to silver against P. aeruginosa and even significantly superior against S. aureus and E. coli. Challenged with AWF the reduction rates for silver adjusted or even exceeded (P. aeruginosa) those of PHMB. Within a challenging wound environment, especially some silver formulations demonstrated a reduced bacterial reduction. Regarding the presented in vitro results, the biomolecular interactions of antiseptic wound dressings with wound fluid should be part of more extensive investigations, considering varying factors such as bacterial species and wound (micro)environment to develop targeted therapeutic regimes for the individual.

Comparing two polymeric biguanides: chemical distinction, antiseptic efficacy and cytotoxicity of polyaminopropyl biguanide and polyhexamethylene biguanide.

In this study, polyaminopropyl biguanide (PAPB) was compared to the molecularly closely related polyhexamethylene biguanide (PHMB) with respect to chemical relationship, antiseptic efficacy and cytotoxicity in vitro. Cytotoxicity for human keratinocytes (HaCaTs) and murine fibroblasts (L929) was determined according to ISO EN 10993-5 for both substances. Antimicrobial efficacy tests were performed via determination of the MBC, quantitative suspension method for substances and investigation of two PAPB- or PHMB-containing dressings against Staphyloccoccus aureus, Escherichia coli and Pseudomonas aeruginosa, according to international standards. Prior mass spectrometry was performed for chemical differentiation of the investigated substances. PHMB showed high toxicity even in low concentrations for both tested cell lines and a high antimicrobial efficacy against S. aureus and E. coli. In the case of PAPB, no or only low cytotoxicity was detected after 72 h, whilst comparable antibacterial features are lacking, as PAPB showed no relevant antimicrobial effects. Even though chemically closely related, PAPB proved to be ineffective in bacterial eradication, whilst PHMB showed a high efficacy. The discovery and establishment of safe and effective alternative antiseptics are important issues for the treatment of infected wounds. In particular, rising bacterial resistances to established agents, as well as ongoing discussions of potential toxic or carcinogenic effects emphasize this necessity. Nevertheless, the presented results highlight that even small changes in the chemical structure of related agents such as PHMB and PAPB can dramatically affect their efficacy and, therefore, need to be carefully distinguished and assessed side by side.

Comparison of hemostatic dressings for superficial wounds using a new spectrophotometric coagulation assay.

BACKGROUND: Due to demographical changes the number of elderly patients depending on oral anticoagulation is expected to rise. Prolonged bleeding times in case of traumatic injuries represent the drawback of these medications, not only in major trauma, but also in superficial wounds. Therefore, dressings capable of accelerating coagulation onset and shortening bleeding times are desirable for these patients. METHODS: The hemostatic potential and physical properties of different types of superficial wound dressings (standard wound pad, two alginates, chitosan, collagen (Lyostypt(®)), oxidized cellulose, and QuikClot(®)) were assessed in vitro. For this purpose the clotting times of blood under the influence of the named hemostatics from healthy volunteers were compared with Marcumar(®) or ASS(®) treated patients. For that, a newly developed coagulation assay based on spectrophotometric extinction measurements of thrombin activity was used. RESULTS: The fastest coagulation onset was observed for oxidized cellulose (Ø 2.47 min), Lantor alginate-L (Ø 2.50 min) and QuikClot(®) (Ø 3.01 min). Chitosan (Ø 5.32 min) and the collagen Lyostypt(®) (Ø 7.59 min) induced clotting comparatively late. Regarding physical parameters, QuikClot(®) showed the lowest absorption capacity and speed while chitosan and both alginates achieved the highest. While oxidized cellulose displayed the best clotting times, unfortunately it also revealed low absorption capacity. CONCLUSIONS: All tested specimens seem to induce clotting independently from the administered type of oral anticoagulant, providing the possibility to neglect the disadvantage in clotting times arising from anticoagulation on a local basis. QuikClot(®), oxidized cellulose and unexpectedly alginate-L were superior to chitosan and Lyostypt(®). Due to its additional well-known positive effect on wound healing alginate-L should be considered for further investigations.

Cetylpyridinium chloride and miramistin as antiseptic substances in chronic wound management - prospects and limitations.

The antimicrobial activity of cetylpyridinium chloride (CPC) and miramistin (MST) solutions at different concentrations (5×10(-5) to 0.4%) and a dressing, containing 0.15% CPC, were tested against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli after 30 (solutions) and 60 min (fleece) incubation, respectively. Furthermore, the cytotoxic effects of CPC and MST were examined in human keratinocyte (HaCaT) and murine fibroblast (L929) cell lines. A dose of 3×10(-3)% CPC or MST was sufficient to entirely eradicate S. aureus after 30 min incubation. To achieve the same effect, higher concentrations were required against E. coli (0.025% CPC; 0.0125% MST) and P. aeruginosa (0.5% CPC; 0.05% MST). The CPC-fleece showed a high antiseptic effect against all three bacterial strains, although it did not completely eliminate P. aeruginosa. Both substances showed a high cytotoxic impact at higher tested concentrations (CPC >3×10(-3)%; MST >8×10(-4)%). CPC showed high antimicrobial potency at low concentrations against S. aureus, accompanied by low cytotoxic (side) effects at these concentrations, whilst the required minimal concentration to eradicate E. coli and P. aeruginosa was shown to be cytotoxic for keratinocytes and fibroblasts. The necessary antibacterial amounts of MST were lower, but also cytotoxic in direct contact with typical human wound cells. With regard to demographic changes and increasing bacterial resistance, new effective antiseptics, such as CPC and MST, incorporated in wound dressings without releasing an active substance could help to improve the treatment and healing rates of chronic wounds.

Functional capacity of reconstituted blood in 1:1:1 versus 3:1:1 ratios: a thrombelastometry study.

INTRODUCTION: Different transfusion ratio concepts of packed red blood cells (pRBCs), fresh frozen plasma (FFP) and platelets (PLTs) have been implemented in trauma care, but the optimal ratios are still discussed. In this study the hemostatic potential of two predefined ratios was assessed by using an in vitro thrombelastometric approach. Furthermore, age effects of reconstituted blood were analyzed. METHODS: Whole blood (WB) of voluntary donors was separated into pRBCs, FFP and PLTs and reconstituted into the ratios 1:1:1 and 3:1:1 at day 1, 4, 14, and 24. Standard blood count, electrolytes and coagulation proteins were quantified. The functional coagulation in ratio- and age-specific groups was evaluated using rotational thromboelastometry (ROTEM). RESULTS: Several coagulation factors reduced significantly in the 3:1:1 ratio and were consistent with increased INR, decelerated clot formation times and A10 (amplitude 10 minutes after clotting time (CT)), flattened α-angle during the EXTEM and diminished MCF for distinct time points during the INTEM, FIBTEM and APTEM assays. With rising age of pRBCs the pH, sodium and potassium reached non-physiological levels. CONCLUSION: Under standardized in vitro conditions the higher amount of pRBCs in the 3:1:1 ratio diluted coagulation factors significantly on the expense of its functional coagulation capacity as revealed by ROTEM results. Thus, the coagulation functionality of the 1:1:1 ratio predominated.

Dynamics of mRNA and polysomal abundance in early 3T3-L1 adipogenesis.

BACKGROUND: Adipogenesis is a complex process, in which immature pre-adipocytes change morphology, micro-anatomy and physiology to become mature adipocytes. These store and accumulate fat and release diverse hormones. Massive changes in protein content and protein composition of the transforming cell take place within a short time-frame.In a previous study we analyzed changes in the abundance of free and polysomal, i.e. ribosome bound, RNAs in the first hours of adipogenesis in the murine cell line 3T3-L1. Here we analyze changes of mRNA levels and their potential contribution to the changing protein pool by determination of mRNA levels and ribosome binding to mRNAs in 3T3-L1 cells stimulated for adipogenesis. We grouped mRNA species into categories with respect to up- or down-regulated transcription and translation and analyzed the groups regarding specific functionalities based on Gene Ontology (GO). RESULTS: A shift towards up-regulation of gene expression in early adipogenesis was detected. Genes up-regulated at the transcriptional (TC:up) and translational (TL:up) level (TC:up/TL:up) are very likely involved in control and logistics of translation. Many of them are known to contain a TOP motif. In the TC:up/TL:unchanged group we detected most of the metal binding proteins and metal transporters. In the TC:unchanged/TL:up group several factors of the olfactory receptor family were identified, while in TC:unchanged/TL:down methylation and repair genes are represented. In the TC:down/TL:up group we detected many signaling factors. The TC:down/TL:unchanged group mainly consists of regulatory factors. CONCLUSIONS: Within the first hours of adipogenesis, changes in transcriptional and translational regulation take place. Notably, genes with a specific biological or molecular function tend to cluster in groups according to their transcriptional and translational regulation.

Extrinsic and intrinsic regulation of DOR/TP53INP2 expression in mice: effects of dietary fat content, tissue type and sex in adipose and muscle tissues.

BACKGROUND: DOR/TP53INP2 acts both at the chromosomal level as a nuclear co-factor e.g. for the thyroid hormone receptor and at the extrachromosomal level as an organizing factor of the autophagosome. In a previous study, DOR was shown to be down-regulated in skeletal muscle of obese diabetic Zucker fa/fa rats. METHODS: To identify sites of differential DOR expression in metabolically active tissues, we measured differences in DOR expression in white adipose tissue (WAT), brown adipose tissue (BAT), skeletal muscle (SM) and heart muscle (HM) by qPCR. To assess whether DOR expression is influenced in the short term by nutritional factors, NMRI mice were fed different fat rich diets (fat diet, FD: 18% or high fat diet, HFD: 80% fat) for one week and DOR expression was compared to NMRI mice fed a control diet (normal diet, ND: 3.3% fat). Additionally, DOR expression was measured in young (45 days old) and adult (100 days old) genetically obese (DU6/DU6i) mice and compared to control (DUKs/DUKsi) animals. RESULTS: ANOVA results demonstrate a significant influence of diet, tissue type and sex on DOR expression in adipose and muscle tissues of FD and HFD mice. In SM, DOR expression was higher in HFD than in FD male mice. In WAT, DOR expression was increased compared to BAT in male FD and HFD mice. In contrast, expression levels in female mice were higher in BAT for both dietary conditions.DOR expression levels in all tissues of 100 days old genetically obese animals were mainly influenced by sex. In HM, DOR expression was higher in male than female animals. CONCLUSIONS: DOR expression varies under the influence of dietary fat content, tissue type and sex. We identified target tissues for further studies to analyze the specific function of DOR in obesity. DOR might be part of a defense mechanism against fat storage in high fat diets or obesity.

Phenotype selection reveals coevolution of muscle glycogen and protein and PTEN as a gate keeper for the accretion of muscle mass in adult female mice.

We have investigated molecular mechanisms for muscle mass accretion in a non-inbred mouse model (DU6P mice) characterized by extreme muscle mass. This extreme muscle mass was developed during 138 generations of phenotype selection for high protein content. Due to the repeated trait selection a complex setting of different mechanisms was expected to be enriched during the selection experiment. In muscle from 29-week female DU6P mice we have identified robust increases of protein kinase B activation (AKT, Ser-473, up to 2-fold) if compared to 11- and 54-week DU6P mice or controls. While a number of accepted effectors of AKT activation, including IGF-I, IGF-II, insulin/IGF-receptor, myostatin or integrin-linked kinase (ILK), were not correlated with this increase, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was down-regulated in 29-week female DU6P mice. In addition, higher levels of PTEN phosphorylation were found identifying a second mechanism of PTEN inhibition. Inhibition of PTEN and activation of AKT correlated with specific activation of p70S6 kinase and ribosomal protein S6, reduced phosphorylation of eukaryotic initiation factor 2α (eIF2α) and higher rates of protein synthesis in 29-week female DU6P mice. On the other hand, AKT activation also translated into specific inactivation of glycogen synthase kinase 3ß (GSK3ß) and an increase of muscular glycogen. In muscles from 29-week female DU6P mice a significant increase of protein/DNA was identified, which was not due to a reduction of protein breakdown or to specific increases of translation initiation. Instead our data support the conclusion that a higher rate of protein translation is contributing to the higher muscle mass in mid-aged female DU6P mice. Our results further reveal coevolution of high protein and high glycogen content during the selection experiment and identify PTEN as gate keeper for muscle mass in mid-aged female DU6P mice.

Novel polysome messages and changes in translational activity appear after induction of adipogenesis in 3T3-L1 cells.

BACKGROUND: Control of translation allows for rapid adaptation of the cell to stimuli, rather than the slower transcriptional control. We presume that translational control is an essential process in the control of adipogenesis, especially in the first hours after hormonal stimulation. 3T3-L1 preadipocytes were cultured to confluency and adipogenesis was induced by standard protocols using a hormonal cocktail. Cells were harvested before and 6 hours after hormonal induction. mRNAs attached to ribosomes (polysomal mRNAs) were separated from unbound mRNAs by velocity sedimentation. Pools of polysomal and unbound mRNA fractions were analyzed by microarray analysis. Changes in relative abundance in unbound and polysomal mRNA pools were calculated to detect putative changes in translational activity. Changes of expression levels of selected genes were verified by qPCR and Western blotting. RESULTS: We identified 43 genes that shifted towards the polysomal fraction (up-regulated) and 2 genes that shifted towards free mRNA fraction (down-regulated). Interestingly, we found Ghrelin to be down-regulated. Up-regulated genes comprise factors that are nucleic acid binding (eIF4B, HSF1, IRF6, MYC, POLR2a, RPL18, RPL27a, RPL6, RPL7a, RPS18, RPSa, TSC22d3), form part of ribosomes (RPL18, RPL27a, RPL6, RPL7a, RPS18, RPSa), act on the regulation of translation (eIF4B) or transcription (HSF1, IRF6, MYC, TSC22d3). Others act as chaperones (BAG3, HSPA8, HSP90ab1) or in other metabolic or signals transducing processes. CONCLUSIONS: We conclude that a moderate reorganisation of the functionality of the ribosomal machinery and translational activity are very important steps for growth and gene expression control in the initial phase of adipogenesis.

Multiplex analysis of genetic markers related to body mass index (BMI) and bone mineral density (BMD).

The multiplex analysis system described here allows simultaneous typing of one short tandem repeat (STR) and three single nucleotide polymorphisms (SNPs) that are associated with obesity and/or osteoporosis. Genes that are related to a high body mass index (BMI) and/or a high bone mineral density (BMD) are presumed to give an advantage in surviving famines. This analysis system makes it possible to genotype the (TTTA)n polymorphism of CYP19 and three SNPs, namely the rs1800795 polymorphism of IL6, the rs373 6228 polymorphism of LRP5 and the rs993 9609 polymorphism of FTO, in a single PCR amplification in recent and ancient DNA samples. Furthermore, it allows a synchronous authentication of the results with the (TATC)n polymorphism of D13S317, the (TCTA)n polymorphism of D21S11 and the (TTTC)n polymorphism of FGA in a partial genetic fingerprinting. For this purpose, PCR products for fragment-length analysis, as well as those for sequence analysis, were amplified together. After amplification, the PCR product was split into two aliquots. The first aliquot was used for fragment-length analysis and the second one for sequence analysis. The analysis system described here has been optimized for analysing ancient samples, since only minimal amounts of material are available.