Fibroblasts In Tissue Culture Research Articles

Exosomes Released from Senescent Cells and Circulatory Exosomes Isolated from Human Plasma Reveal Aging-associated Proteomic and Lipid Signatures.

Senescence emerged as a significant mechanism of aging and age-related diseases, offering an attractive target for clinical interventions. Senescent cells release a senescence-associated secretory phenotype (SASP), including exosomes that may act as signal transducers between distal tissues, propagating secondary or bystander senescence and signaling throughout the body. However, the composition of exosome SASP remains underexplored, presenting an opportunity for novel unbiased discovery. Here, we present a detailed proteomic and lipidomic analysis of exosome SASP using mass spectrometry from human plasma from young and older individuals and from tissue culture of senescent primary human lung fibroblasts. We identified ~1,300 exosome proteins released by senescent fibroblasts induced by three different senescence inducers causing most exosome proteins to be differentially regulated with senescence. In parallel, a human plasma cohort from young and old individuals revealed over 1,350 exosome proteins and 171 plasma exosome proteins were regulated when comparing old vs young individuals. Of the age-regulated plasma exosome proteins, we observed 52 exosome SASP factors that were also regulated in exosomes from the senescent fibroblasts, including serine protease inhibitors (SERPINs), Prothrombin, Coagulation factor V, Plasminogen, and Reelin. In addition, 247 lipids were identified with high confidence in all exosome samples. Following the senescence inducers, a majority of the identified phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin species increased significantly indicating cellular membrane changes. The most notable categories of significantly changed proteins were related to extracellular matrix remodeling and inflammation, both potentially detrimental pathways that can damage surrounding tissues and even induce secondary or bystander senescence. Our findings reveal mechanistic insights and potential senescence biomarkers, enabling a better approach to surveilling the senescence burden in the aging population and offering promising therapeutic targets for interventions.

A Toxoplasma gondii lipoxygenase-like enzyme is necessary for virulence and changes localization associated with the host immune response.

Lipoxygenases (LOXs) are enzymes that catalyze the deoxygenation of polyunsaturated fatty acids such as linoleic and arachidonic acid. These modifications create signaling molecules that are best characterized for modulating the immune response. Deletion of the first lipoxygenase-like enzyme characterized for Toxoplasma gondii (TgLOXL1) generated a less virulent strain, and infected mice showed a decreased immune response. This virulence defect was dependent on the mouse cytokine interferon gamma IFNγ. TgLOXL1 changes location from inside the parasite in tissue culture conditions to vesicular structures within the host immune cells during mouse infection. These results suggest that TgLOXL1 plays a role in the modification of the host immune response in mice.

Primary Cardiac Fibroblast Cell Culture: Methodological Considerations for Physiologically Relevant Conditions.

Primary cardiac fibroblast (CF) tissue culture is a necessary tool for interrogating specific signaling mechanisms that dictate the phenotypic heterogeneity observed in vivo in different disease states. Traditional approaches that utilize tissue culture plastic and nutrient rich medium have been shown to induce CF activation and therefore alter CF subpopulation composition. This shift away from in vivo phenotypes complicate interpretation of results through the lens of the animal model. As the field works to identify CF diversity, these methodological flaws have begun to be addressed and more studies are focused on the dynamic interaction of CFs with their environment. This review focuses on the aspects of tissue culture that impact CF activation and therefore require consideration when designing in vitro experiments. The complexity of CF biology overlaid onto diverse model systems highlight the need for study-specific optimization and validation.

Simultaneous Effects of a Selective EP2 Agonist, Omidenepag, and a Rho-Associated Coiled-Coil Containing Protein Kinase Inhibitor, Ripasudil, on Human Orbital Fibroblasts.

Purpose: To assess the combined effects of omidenepag (OMD), a selective EP2 agonist, and ripasudil (Rip), an inhibitor of rho-associated coiled-coil containing protein kinases, on the human orbital adipose tissue, two-dimensional (2D) or three-dimensional (3D) cultures of human orbital fibroblasts (HOFs) were employed. Methods: Cellular metabolic functions (2D), physical (3D), lipid staining (3D), and quantitative polymerase chain reaction for adipogenesis-related genes, PPARγ and AP2, and extracellular matrix (ECM) molecules, including collagen (COL)1, 4, and 6, and fibronectin (FN) (3D) were evaluated in the presence of OMD (100 nM) and/or Rip (10 μM). Results: Real-time metabolic analyses revealed that the adipogenic differentiation (DIF+) with OMD significantly shifted an energetic state toward energetic, whereas DIF+ with Rip significantly shifted that toward quiescent. In the case of both drugs upon DIF+, the metabolic effect of OMD was predominant. DIF+ induced enlargement and stiffed 3D spheroid with increased lipid staining and mRNA expression of adipogenesis-related genes, COL4 and COL6, and decreased the expression of COL1. In the presence of OMD and/or Rip to DIF+, (1) the sizes were further increased by Rip and the stiffness was significantly decreased by OMD or Rip and (2) COL4 or AP2 expression was substantially increased by OMD or Rip, respectively. Conclusion: The results presented herein indicate that the metabolic effects of OMD and Rip exerted opposing effects and the effects of OMD toward Ap2 and ECM expressions were distinct from those of Rip, but the effects of OMD toward the physical aspects and adipogenesis of the 3D cultured HOFs were similar to the effects of Rip.

Influence of the prolonged form of lysocyme in the composition of polyurethane implant on cells and tissues in vitro and in vivo.

Background. The development of biocompatible polymeric implant materials and providing them with biological activity is an urgent task that can significantly increase the effectiveness of such materials when used in medical practice. Objective. Investigation of the interaction of connective tissue culture matrix cells with a prolonged form of the proteolytic enzyme lysozyme as part of a polyurethane implant in vitro and study of the effect of this form on cells and tissues during implantation in experimental animals in vivo. Methods. In order to study the possible cytotoxicity of the components of polymer composite materials and the effect of lysozyme on the growth and development of the culture of fibroblastic elements, studies were performed by tissue culture in vitro. Polymeric composite materials based on polyurethaneurea without and with lysozyme were implanted into the body of white laboratory rats of the Wistar line. Cellular responses of the body after implantation were studied by light microscopy by analysis of histological micropreparations. Results. Biological studies have assessed the effect of prolonged lysozyme on cells and tissues in vitro and in vivo. Conclusion. The fibroblast tissue culture method showed the effectiveness of biologically active polyurethaneureas with prolonged release of lysozyme, which was expressed in the prolongation of the dynamics of growth and development of cellular elements in vitro. It has been shown that lysozyme in the composition of polymer composite materials had a biological effect and helped to reduce the cellular response to the implantation of polymer samples, accelerating the formation of connective tissue capsules around the implants.

Extracellular Superoxide Dismutase (EC-SOD) Regulates Gene Methylation and Cardiac Fibrosis During Chronic Hypoxic Stress.

Chronic hypoxic stress induces epigenetic modifications mainly DNA methylation in cardiac fibroblasts, inactivating tumor suppressor genes (RASSF1A) and activating kinases (ERK1/2) leading to fibroblast proliferation and cardiac fibrosis. The Ras/ERK signaling pathway is an intracellular signal transduction critically involved in fibroblast proliferation. RASSF1A functions through its effect on downstream ERK1/2. The antioxidant enzyme, extracellular superoxide dismutase (EC-SOD), decreases oxidative stress from chronic hypoxia, but its effects on these epigenetic changes have not been fully explored. To test our hypothesis, we used an in-vitro model: wild-type C57B6 male mice (WT) and transgenic males with an extra copy of human hEC-SOD (TG). The studied animals were housed in hypoxia (10% O2) for 21 days. The right ventricular tissue was studied for cardiac fibrosis markers using RT-PCR and Western blot analyses. Primary C57BL6 mouse cardiac fibroblast tissue culture was used to study the in-vitro model, the downstream effects of RASSF-1 expression and methylation, and its relation to ERK1/2. Our findings showed a significant increase in cardiac fibrosis markers: Collagen 1, alpha smooth muscle actin (ASMA), and SNAIL, in the WT hypoxic animals as compared to the TG hypoxic group (p < 0.05). The expression of DNA methylation enzymes (DNMT 1&3b) was significantly increased in the WT hypoxic mice as compared to the hypoxic TG mice (p < 0.001). RASSF1A expression was significantly lower and ERK1/2 was significantly higher in hypoxia WT compared to the hypoxic TG group (p < 0.05). Use of SiRNA to block RASSF1A gene expression in murine cardiac fibroblast tissue culture led to increased fibroblast proliferation (p < 0.05). Methylation of the RASSF1A promoter region was significantly reduced in the TG hypoxic group compared to the WT hypoxic group (0.59 vs. 0.75, respectively). Based on our findings, we can speculate that EC-SOD significantly attenuates RASSF1A gene methylation and can alleviate cardiac fibrosis induced by hypoxia.

MigrationStimulating Factor (MSF): Its Role in the Tumour Microenvironment.

Migration Stimulating Factor(MSF) is a 70kDa truncated isoform of fibronectin (FN); its mRNA is generated from the FN gene by an unusual two-stage processing. Unlike full-length FN, MSF is not a matrix molecule but a soluble protein which displays cytokine-like activities not displayed by any other FN isoform due to steric hindrance. There are two isoforms of MSF; these are referred to as MSF+aa and MSF-aa, while the term MSF is used to include both.MSF was first identified as a motogen secreted by foetal and cancer-associated fibroblasts in tissue culture. It is also produced by sprouting (angiogenic) endothelial cells, tumour cells and activated macrophages. Keratinocytes and resting endothelial cells secrete inhibitors of MSF that have been identified as NGAL and IGFBP-7, respectively. MSF+aa and MSF-aa show distinct functionality in that only MSF+aa is inhibited by NGAL.MSF is present in 70-80% of all tumours examined, expressed by the tumour cells as well as by fibroblasts, endothelial cells and macrophages in the tumour microenvironment (TME). High MSF expression is associated with tumour progression and poor prognosis in all tumours examined, including breast carcinomas, non-small cell lung cancer (NSCLC), salivary gland tumours (SGT) and oral squamous cell carcinomas (OSCC). Epithelial and stromal MSF carry independent prognostic value. MSF is also expressed systemically in cancer patients, being detected in serum and produced by fibroblast from distal uninvolved skin. MSF-aa is the main isoform associated with cancer, whereas MSF+aa may be expressed by both normal and malignant tissues.The expression of MSF is not invariant; it may be switched on and off in a reversible manner, which requires precise interactions between soluble factors present in the TME and the extracellular matrix in contact with the cells. MSF expression in fibroblasts may be switched on by a transient exposure to several molecules, including TGFβ1 and MSF itself, indicating an auto-inductive capacity.Acting by both paracrine and autocrine mechanisms, MSF stimulates cell migration/invasion, induces angiogenesis and cell differentiation and alters the matrix and cellular composition of the TME. MSF is also a survival factor for sprouting endothelial cells. IGD tri- and tetra-peptides mimic the motogenic and angiogenic activities of MSF, with both molecules inhibiting AKT activity and requiring αvβ3 functionality. MSF is active at unprecedently low concentrations in a manner which is target cell specific. Thus, different bioactive motifs and extracellular matrix requirements apply to fibroblasts, endothelial cells and tumour cells. Unlike other motogenic and angiogenic factors, MSF does not affect cell proliferation but it stimulates tumour growth through its angiogenic effect and downstream mechanisms.The epithelial-stromal pattern of expression and range of bioactivities displayed puts MSF in the unique position of potentially promoting tumour progression from both the "seed" and the "soil" perspectives.

An In Vitro evaluation of biodegradability of stainless steel crowns at various salivary pH.

To evaluate the biodegradability of preformed stainless steel crowns at varying salivary pH and the cytotoxic effect of leached out elements on fibroblasts. A total of 243 stainless steel crowns were selected and were divided into 3 groups (I, II, III) based on Ph of immersion media. The pH of samples in group I, II, III were 4.3, 5.5 and 6.3 with 81 crowns in each group. Each group has 9 samples with 8 crowns in each sample. All samples were immersed in polyethylene bottles containing 10ml of artificial saliva and incubated at 37°C for 4 weeks. All the samples were analyzed on 1,7,14 and 21 days by atomic absorption spectrophotometer for the quantitative assement of Ni, Cr and Fe. Fibroblast tissue culture was used to assess the cytotoxicity of the samples. Analysis of variance. Maximum release of Ni, Cr, Fe ions were observed at pH 4.3 followed by pH 5.5 and least release of ions were observed at pH 6.3 from SS crowns. The cytotoxic results showed that the least cell viability of cells was seen at pH 4.3. With decrease in pH, there is an increase in ion release from stainless steel crowns and the mean release of nickel, chromium and iron were very much below the average dietary intake. But the allergic manifestations of ions like nickel can't be ruled out.

Indigenous irrigants as potent antimicrobials in endodontic treatment: An in vitro study.

The success of an endodontic treatment depends on effective disinfection and complete sealing of root canal. The irrigants that are currently used in the field of endodontics have certain limitations, so the quest for an ideal root canal irrigant continues. Nowadays, the use of herbal extracts such as Triphala and liquorice are used for their potent antimicrobial activity and less side effects. This study aims (1) to evaluate the antimicrobial efficacy of Triphala and liquorice against Enterococcus faecalis.(2) To determine any cytotoxic effect on isolated human periodontal ligament (PDL) fibroblasts. The antimicrobial efficacy of Triphala and liquorice extracts was analyzed at different concentrations (12.5, 25, 50, 75, and 100 mg/ml) using the well-diffusion method. Three percent sodium hypochlorite (NaOCl) and distilled water were taken as positive and negative controls. Minimum inhibitory concentration of the active extract was determined by the broth dilution assay. Human PDL fibroblast tissue culture was used to assess the cytotoxicity of the preparations. The data thus obtained were subjected to statistical analysis. The results showed that the mean antimicrobial efficacy of Triphala and liquorice at 50 mg/ml is 20.33 and 9.33, respectively, which are statistically significant (P < 0.0001) as compared with a concentration 12.5 and 25 mg/ml. 50 mg/ml showed significant results (P < 0.001) on comparing with hypochlorite. Triphala and liquorice showed no cytotoxic effect as compared to NaOCl on human PDL fibroblasts. Among the three tested materials Triphala showed the highest antimicrobial efficacy followed by NaOCl and liquorice.

Prolonged skin grafts survival time by IFN-γ in allogeneic skin transplantation model during acute rejection through IFN-γ/STAT3/IDO pathway in epidermal layer

Allogeneic skin transplantation is the life-saving therapy for multiple diseases, including extensive burn, large-scale trauma and certain post-surgical complications. However, acute rejection impedes clinical application of allogeneic skin transplantation. Although a lot of novel immunosuppressant drugs have been developed, there is still great need for ideal therapy with less complication and more therapeutic effects. Here, we found interferon gamma (IFN-γ) as an immunomodulatory cytokine prolonged the survival time of allografts from (8.50 ± 1.517) days to (14.83 ± 2.714) days at best. Indoleamine-2, 3-dioxygenase (IDO) has been proposed to play key roles in induction of immune tolerance. Using in vitro tissue culture and primary keratinocytes and fibroblasts, we investigated the regulatory effects of IFN-γ on the IDO expression. IFN-γ upregulated IDO expression through STAT3 phosphorylation and this upregulation was reduced by abolition of STAT3 phosphorylation through a STAT3 phosphorylation inhibitor. Interestingly, IFN-γ induced IDO expression predominately in epidermis rather than dermis. In consistent with these results, IFN-γ significantly triggered IDO expression in keratinocytes but not fibroblasts. Taken together, this suggests that IFN-γ might be a potential immunomodulatory drug in acute rejection and keratinocytes in epidermis may play a main role in immune tolerance after allogeneic skin transplantation.

Differential marker expression by cultures rich in mesenchymal stem cells

BackgroundMesenchymal stem cells have properties that make them amenable to therapeutic use. However, the acceptance of mesenchymal stem cells in clinical practice requires standardized techniques for their specific isolation. To date, there are no conclusive marker (s) for the exclusive isolation of mesenchymal stem cells. Our aim was to identify markers differentially expressed between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. We compared and contrasted the phenotype of tissue cultures in which mesenchymal stem cells are rich and rare. By initially assessing mesenchymal stem cell differentiation, we established that bone marrow and breast adipose cultures are rich in mesenchymal stem cells while, in our hands, foreskin fibroblast and olfactory tissue cultures contain rare mesenchymal stem cells. In particular, olfactory tissue cells represent non-stem cell mesenchymal cells. Subsequently, the phenotype of the tissue cultures were thoroughly assessed using immuno-fluorescence, flow-cytometry, proteomics, antibody arrays and qPCR.ResultsOur analysis revealed that all tissue cultures, regardless of differentiation potential, demonstrated remarkably similar phenotypes. Importantly, it was also observed that common mesenchymal stem cell markers, and fibroblast-associated markers, do not discriminate between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. Examination and comparison of the phenotypes of mesenchymal stem cell and non-stem cell mesenchymal cell cultures revealed three differentially expressed markers – CD24, CD108 and CD40.ConclusionWe indicate the importance of establishing differential marker expression between mesenchymal stem cells and non-stem cell mesenchymal cells in order to determine stem cell specific markers.

Quantitative Model of Cell Cycle Arrest and Cellular Senescence in Primary Human Fibroblasts

Primary human fibroblasts in tissue culture undergo a limited number of cell divisions before entering a non-replicative “senescent” state. At early population doublings (PD), fibroblasts are proliferation-competent displaying exponential growth. During further cell passaging, an increasing number of cells become cell cycle arrested and finally senescent. This transition from proliferating to senescent cells is driven by a number of endogenous and exogenous stress factors. Here, we have developed a new quantitative model for the stepwise transition from proliferating human fibroblasts (P) via reversibly cell cycle arrested (C) to irreversibly arrested senescent cells (S). In this model, the transition from P to C and to S is driven by a stress function γ and a cellular stress response function F which describes the time-delayed cellular response to experimentally induced irradiation stress. The application of this model based on senescence marker quantification at the single-cell level allowed to discriminate between the cellular states P, C, and S and delivers the transition rates between the P, C and S states for different human fibroblast cell types. Model-derived quantification unexpectedly revealed significant differences in the stress response of different fibroblast cell lines. Evaluating marker specificity, we found that SA-β-Gal is a good quantitative marker for cellular senescence in WI-38 and BJ cells, however much less so in MRC-5 cells. Furthermore we found that WI-38 cells are more sensitive to stress than BJ and MRC-5 cells. Thus, the explicit separation of stress induction from the cellular stress response, and the differentiation between three cellular states P, C and S allows for the first time to quantitatively assess the response of primary human fibroblasts towards endogenous and exogenous stress during cellular ageing.

Aortic Adventitial Fibroblasts Participate in Angiotensin-Induced Vascular Wall Inflammation and Remodeling

Background/Aims: The role of adventitial fibroblasts in the vascular inflammation observed in the adventitia of large vessels in numerous cardiovascular diseases remains unclear. Our objective was to explore the contribution of these cells to angiotensin II (Ang II)-induced aortic inflammation and adventitial expansion. Methods: Cytokine production by primary human aortic adventitial fibroblasts (AoAF) in tissue culture was detected using multiplex ELISA, and increases in cytokine mRNA following Ang II stimulation were quantitated by real-time PCR. The ability of AoAF-derived MCP-1 to attract monocytes was studied in vitro using Boyden assays, and the resulting effect of the monocyte-AoAF interaction on fibroblast proliferation was measured in vitro using proliferation and ³H-thymidine incorporation assays. Ang II-induced fibroblast proliferation was measured in vivo using aortic digestion of single cells followed by flow cytometric quantification of fibroblast numbers as well as fibroblast and PCNA immunostaining. The ability of monocytes to induce AoAF proliferation was demonstrated in vivo using CCR2^+/+ wild-type monocyte adoptive transfer into Ang II-stimulated CCR2-null mice which can produce MCP-1 but have cells lacking the MCP-1 receptor – CCR2. Results: AoAF constitutively secreted numerous proinflammatory cytokines, particularly IL-6 and MCP-1, whose gene expressions were further upregulated in response to Ang II stimulation. AoAF-derived MCP-1 was potent in recruiting THP-1 monocytes in vitro, and these monocytes stimulated AoAF proliferation based on a flow cytometric assessment of cell number and ³H-thymidine incorporation in tissue culture. In vivo, Ang II induced fibroblast proliferation, increased fibroblast and PCNA adventitial staining, and blunted inflammatory responses in the CCR2^–/– background. Injection of CCR2^+/+ monocytes into Ang II-treated CCR2^–/– mice restored adventitial thickening which resulted in increased fibrosis secondary to adventitial fibroblast proliferation. Conclusions: Our results suggest that Ang II-stimulates AoAF to recruit monocytes via fibroblast-derived MCP-1, and the recruited monocytes further activate fibroblast proliferation, adventitial thickening, and additional cytokine production. This fibroblast-monocyte amplification loop may critically mediate hallmarks of adventitial inflammation common to many cardiovascular diseases.

A laboratory evaluation of the antibacterial and cytotoxic effect of Liquorice when used as root canal medicament

To evaluate the antibacterial and cytotoxic effects of Liquorice as a root canal medicament and to compare its action to the commonly used root canal medicament calcium hydroxide Ca(OH)(2). The antibacterial effect of Liquorice and Ca(OH)(2) either separately or in combination was investigated against Enterococcus faecalis. Agar-well diffusion methods, broth microdilution tests and biofilm susceptibility assays were used to determine the antibacterial activity. Human periodontal ligament fibroblast tissue culture was used to assess the cytotoxicity of the preparations under investigation. Liquorice extract either by itself or in combination with Ca(OH)(2) had a significant inhibitory effect against Enterococcus faecalis compared with that of Ca(OH)(2) alone. The use of Liquorice extract followed by Liquorice/Ca(OH)(2) mixture retained significantly more viable periodontal ligament cells than Ca(OH)(2) , which had a strong lethal effect on the cells. Liquorice extract either separately or as Liquorice/Ca(OH)(2) mixture had a potent bactericidal effect against Enterococcus faecalis and retained compatibility with fibroblasts in tissue culture compared to the commonly used root canal medicament Ca(OH)(2).

Marginal Adaptation and Cytotoxicity of Bone Cement Compared with Amalgam and Mineral Trioxide Aggregate as Root-end Filling Materials

Introduction Bone cements have a well-established place in the practice of orthopedic surgery. It has many characteristics that make it well-suited as a root-end filling material. The purpose of this study was to evaluate the marginal adaptation and cytotoxic effect of polymethylmethacrylate (PMMA) bone cement, mineral trioxide aggregate (MTA), and amalgam as root-end filling materials. Methods Thirty extracted human single-rooted teeth were cleaned, shaped, and obturated with gutta-percha and AH 26 sealer. The roots tips were removed; root-end cavities were prepared and filled with the 3 tested materials (bone cement, MTA, and amalgam). Impressions of retrofilled root ends were taken by polysiloxane impression material, and positive replicas were fabricated by using epoxy resin. The original roots were longitudinally sectioned into 2 halves; both the replicas and longitudinal sections were prepared for scanning electron microscope to measure the gaps at the material/dentin interface. Human periodontal ligament fibroblast tissue culture was used to assess the cytotoxicity of the 3 tested materials. Results The obtained data revealed that both bone cement and MTA exhibited a better adaptation to the dentinal walls than that of amalgam. Also, the cytotoxicity testing showed that bone cement had a comparable cytotoxic effect on fibroblast cells with MTA; both root-end filling materials showed less cytotoxicity than that of amalgam. Conclusions PMMA bone cement could be considered as a promising root-end filling material.

Plectranthus tenuiflorus (Shara) Promotes Wound Healing: In vitro and in vivo Studies

The present study proved that bothPlectranthus tenuiflorus juice and essential oil exerted a healing promoting effect in rat wound model. The effect was shown to be mainly via their ability to stimulate fibroblasts proliferation in addition to an anti-bacterial effect of its thymol content. Leaves of the plant were collected from different regions. The whole leave juice or essential oil were extracted by chemical steam distillation method. Different concentrations were tested for their effects on the proliferation of human foreskin fibroblasts in tissue culture. Its efficiency in enhancing wound healing processes using excision wound model in rat was also designed. The results revealed complete wound healing (100% contraction) at day 14(10% juice), day 17 (80% juice) and day 18(10% essential oil) compared to 22 days. Histological studies showed that at day 14 complete epithelization, well formed small sized scar tissue and reappearance of cutaneous appendages were evident in wounds painted with 10% essential oil, followed by 80% juice. In vitro study proved a stimulatory effect of plant extracts on human fibroblasts which may explain the speeding of healing process. The healing promoting effect or P. tenuiflorus may be attributed to the high content of calcium (903.16333±0.21); zinc (0.37933±0.05). Essential amino acids (Ala, Leu, Glu, Asp, Asn, Phe and His) seemed also to have a role. On the other hand, thymol was known to have an anti-bacterial effect. Thymol found in this study to be the main component (82.16%) of P. tenuiflorus extracts. © 2010 Asian Network for Scientific Information.

Identification of a novel betaherpesvirus in Mus musculus

Rodent betaherpesviruses vary considerably in genomic content, and these variations can result in a distinct pathogenicity. Therefore, the identification of unknown betaherpesviruses in house mice (Mus musculus), the most important rodent host species in basic research, is of importance. During a search for novel herpesviruses in house mice using herpesvirus consensus PCR and attempts to isolate viruses in tissue culture, we identified a previously unknown betaherpesvirus. The primary PCR search in mouse organs revealed the presence of known strains of murine cytomegalovirus (Murid herpesvirus 1) and of Mus musculus rhadinovirus 1 only. However, the novel virus was detected after incubation of organ pieces in fibroblast tissue culture and subsequent PCR analysis of the supernatants. Long-distance PCR amplification including the DNA polymerase and glycoprotein B genes revealed a 3.4 kb sequence that was similar to sequences of rodent cytomegaloviruses. Pairwise sequence comparisons and phylogenetic analyses showed that this newly identified murine virus is most similar to the English isolate of rat cytomegalovirus, thereby raising the possibility that two distinct CMV lineages have evolved in both Mus musculus and Rattus norvegicus.

An in vitro evaluation of the cell toxicity of honey and silver dressings

To establish whether honey and silver-impregnated dressings used by wound-healing practitioners are cytotoxic in vitro to human skin keratinocytes and dermal fibroblasts. Human keratinocyte and fibroblast tissue cultures were established in vitro. Untreated cultures served as controls (group I). Small dressing implants of monofloral, medicinal honey (L-Mesitran) (group 2) and nanocrystalline silver (Acticoat) (group 3) were placed in test wells and co-cultured with each of the two cell lines. Morphological changes, including cell toxicity, were assessed using inverted microscopy, trypan blue staining and the Rosdy and Clauss cell toxicity scoring system. Untreated cultures consisting of both keratinocytes and fibroblasts (group 1) were established in 90% of all cases. In group 2, cultures with honey-impregnated implants, cell proliferation remained present at two and four months. Cell viability remained intact and cell toxicity was not evident at four months after continuous tissue culture. In group 3, marked toxicity was observed with high non-viability staining and cell-scoring counts compared with groups 1 and 2 (p<0.05). This demonstrates that the silver interfered with epidermal cell proliferation and migration, implying that it contains cytotoxic material. The honey-based product showed excellent cytocompatibility with tissue cell cultures compared with the silver dressing, which demonstrated consistent culture and cell toxicity. Further studies are needed to assess if these comparative in-vitro findings should influence a clinician's choice of wound dressing.

Biocompatibility of acrylic resin after being soaked in sodium hypochlorite

Background: Acrylic resin as basic material for denture will stay on oral mucosa for a very long time. The polymerization of acrylic resin can be performed by conventional method and microwave, both produce different residual monomer at different toxicity. Acrylic resin can absorb solution, porous and possibly absorb disinfectantt as well, that may have toxic reaction with the tissue. Sodium Hypochlorite as removable denture disinfectant can be expected to be biocompatible to human body. The problem is how biocompatible acrylic resin which has been processed by conventional method and microwave method after being soaked in sodium hypochlorite solution. Purpose: The aim of this study was to understand in vitro biocompatibility of acrylic resin which has polimerated by conventional method and microwave after being soaked in sodium hypochlorite using tissue culture. Methods: Four groups of acrylic resin plate were produced, the first group was acrylic resin plate with microwave polymeration and soaked in sodium hypochlorite, the second group was acrylic resin plate with microwave polymeration but not soaked, the thirdwas one with conventional method and soaked and the last group was one with conventional method but not soaked, and in 1 control group. Each group consists of 7 plates. Biocompatibility test was performed in-vitro on each material using fibroblast tissue culture (BHK-21 cell-line). Result: The percentage between living cells and dead cells from materials which was given acrylic plate was wounted. The data was analyzed statistically with T test. Conclusion: The average value of living cells is higher in acrylic resin poimerization using microwave method compared to conventional method, in both soaked and non soaked (by sodium hypochlorite) group. This means that sodium hypochlorite 0.5% was biocompatible to the mouth mucosa as removable denture disinfectant for 10 minutes soaking and washing afterwards.

Interferon-Induced ISG15 Conjugation Inhibits Influenza A Virus Gene Expression and Replication in Human Cells

The ubiquitin-like ISG15 protein, as well as its conjugating enzymes, is induced by type I interferons (IFNs). Experiments using ISG15 knockout (ISG15(-/-)) mice established that ISG15 and/or its conjugation inhibits the replication of influenza A virus. However, in contrast to the virus inhibition results for mice, the rates of virus replication in ISG15(+/+) and ISG15(-/-) mouse embryo fibroblasts in tissue culture were similar. Here we focus on human tissue culture cells and on the effect of ISG15 and/or its conjugation on influenza A virus gene expression and replication in such cells. We demonstrate that IFN-induced antiviral activity against influenza A virus in human cells is significantly alleviated by inhibiting ISG15 conjugation using small interfering RNAs directed against ISG15-conjugating enzymes. IFN-induced antiviral activity against influenza A virus protein synthesis was reduced 5- to 20-fold by suppressing ISG15 conjugation. The amounts of the viral proteins that were restored by these siRNA treatments were approximately 40 to 50% of the amounts produced in cells that were not pretreated with IFN. Further, we show that ISG15 conjugation inhibits influenza A virus replication 10- to 20-fold at early times after infection in human cells. These results show that ISG15 conjugation plays a substantial role in the antiviral state induced by IFN in human cells. In contrast, we show that in mouse embryo fibroblasts ISG15 conjugation not only does not affect influenza A virus replication but also does not contribute to the IFN-induced antiviral activity against influenza A virus gene expression.