Genetically Modified Dermal Keratinocytes Express High Levels of Transforming Growth Factor-β1

In an attempt to genetically modify cultured keratinocytes with transforming growth factor-β1 (TGF-β1), which has been proven to be one of the most important cytokines involved in wound healing, two constructs were made. One, designated pG3Z:K14-TGF-β1, is a plasmid in which the expression of TGF-β1 is driven by the keratin 14 promoter. The other, designated pLin-TGF-β1, is a retroviral vector in which the retroviral 5' long-terminal repeat promoter drives expression. In both constructs, the deletion of a small fragment of the noncoding region of the TGF-β1 gene was made to differentiate the transcript from that for endogenously expressed TGF-β1. Different types of cells were transfected with the pG3Z:K14-TGF-β1 construct using the calcium phosphate method. The pLin-TGF-β1 construct was propagated in a retroviral packaging cell line and conditioned medium that contained high titers of the virus was used to transduce keratinocytes or other types of cells grown in standard culture. Northern analysis, used to evaluate the expression of TGF-β1 mRNA in the pG3Z:K14-TGF-β1 transfected keratinocyte Cl-177 cell line, showed a smaller TGF-β1 transcript compared with that endogenously expressed by dermal fibroblasts. The level of TGF-β1 protein evaluated by enzyme-linked immunosorbent assay was significantly higher in medium conditioned by either the K14-TGF-β1 transfected or the pLin-TGF-β1 transduced keratinocytes, compared with that obtained from control cells; however, the level of TGF-β1 protein was unchanged in cultures of pG3Z:K14-TGF-β1 transfected nonkeratinocyte cells such as fetal and adult fibroblasts. Using the mink lung epithelial cell growth inhibition assay, we found an increase in TGF-β1 activity in conditioned medium from the pG3Z:K14-TGF-β1 transfected cells. To evaluate possible paracrine effects of the keratinocyte derived TGF-β1, a coculture system was established with pLin-TGF-β1 transduced keratinocytes grown in the upper chamber and dermal fibroblasts in the lower chamber. The results showed that TGF-β1 released from keratinocytes diffused to the lower chamber where it stimulated collagen production by dermal fibroblasts. In summary, we demonstrate here that primary cultured keratinocytes can be genetically modified to express high levels of TGF-β1 and suggest that this offers a potential approach for the therapy of dermal lesions such as nonhealing wounds

Paracrine Factors of Mesenchymal Stem Cells Recruit Macrophages and Endothelial Lineage Cells and Enhance Wound Healing

Bone marrow derived mesenchymal stem cells (BM-MSCs) have been shown to enhance wound healing; however, the mechanisms involved are barely understood. In this study, we examined paracrine factors released by BM-MSCs and their effects on the cells participating in wound healing compared to those released by dermal fibroblasts. Analyses of BM-MSCs with Real-Time PCR and of BM-MSC-conditioned medium by antibody-based protein array and ELISA indicated that BM-MSCs secreted distinctively different cytokines and chemokines, such as greater amounts of VEGF-α, IGF-1, EGF, keratinocyte growth factor, angiopoietin-1, stromal derived factor-1, macrophage inflammatory protein-1alpha and beta and erythropoietin, compared to dermal fibroblasts. These molecules are known to be important in normal wound healing. BM-MSC-conditioned medium significantly enhanced migration of macrophages, keratinocytes and endothelial cells and proliferation of keratinocytes and endothelial cells compared to fibroblast-conditioned medium. Moreover, in a mouse model of excisional wound healing, where concentrated BM-MSC-conditioned medium was applied, accelerated wound healing occurred compared to administration of pre-conditioned or fibroblast-conditioned medium. Analysis of cell suspensions derived from the wound by FACS showed that wounds treated with BM-MSC-conditioned medium had increased proportions of CD4/80-postive macrophages and Flk-1-, CD34- or c-kit-positive endothelial (progenitor) cells compared to wounds treated with pre-conditioned medium or fibroblast-conditioned medium. Consistent with the above findings, immunohistochemical analysis of wound sections showed that wounds treated with BM-MSC-conditioned medium had increased abundance of macrophages. Our results suggest that factors released by BM-MSCs recruit macrophages and endothelial lineage cells into the wound thus enhancing wound healing

RFXB and its splice variant RFXBSV mediate the antagonism between IFNγ and TGFβ on COL1A2 transcription in vascular smooth muscle cells

Cytokines secreted by infiltrating immune cells during atherogenesis modulate vascular remodeling. One exemplary event is the antagonism between transformed growth factor (TGF-β) and interferon gamma (IFN-γ) on the transcriptional control of type I collagen gene (COL1A2). Previously we have reported that IFN-γ up-regulates regulatory factor for X-box B (RFXB) to repress collagen transcription while down-regulates the expression of RFXBSV, a splice variant of RFXB that blocks collagen repression in fibroblasts. Here we demonstrate that TGF-β abrogated COL1A2 repression by IFN-γ through altering the relative expression of RFXB and RFXBSV. Unlike RFXB, RFXBSV did not bind to the collagen promoter and competed with RFXB for the co-repressor histone deacetylase 2 (HDAC2), limiting HDAC2 recruitment to the collagen transcription start site as evidenced by chromatin immunoprecipitation assays. Over-expression of RFXB by lentiviral infection in HASMCs enhanced HDAC2 enlistment, promoted histone deacetylation surrounding the collagen site by IFN-γ, and blocked the TGF-β antagonism, a pattern reversed by RFXBSV infection. On the contrary, silencing of RFXB, but not both RFXB and RFXBSV, expression promoted the TGF-β antagonism. Thus, we have identified a novel mechanism whereby TGF-β antagonizes the IFN-γ repression of collagen transcription in HASMCs and as such provided new insights into antiatherogenic strategies

Add-on topiramate reduces weight in overweight patients with affective disorders: a clinical case series

BACKGROUND: The weight-gain caused by many psychotropic drugs is a major cause for poor compliance with such medications and could also increase cardio-vascular morbidity among psychiatric patients. Recent reports have shown that the anticonvulsant topiramate causes weight loss in various patient groups. The drug has also shown effectiveness in open trials as a mood stabilizer in patients with affective disorders, but not in controlled trials in the acute treatment of mania. We used topiramate to treat 12 patients with affective disorders who had a body-mass index >30 kg/m(2). METHODS: Topiramate was prescribed as part of our routine clinical practice, as an add-on medication, or as a replacement of a mood stabilizer. Patients' weight was recorded in 1 to 2 monthly intervals. Patients were followed up for between 6 and 12 months. The final dose of topiramate varied from 200 to 600 mg/day. RESULTS: Topiramate was effective in reducing the weight in 10 out of the 12 patients. At six months the 12 patients had lost a mean of 7.75 kg (SD = 6.9 kg, p < 0.001) and at 12 months 9 patients had lost a mean of 9.61 kg (SD = 6.7 kg, p = 0.003). Three patients stopped the treatment: one due to side effects, one due to possible side effects, and one suffered a manic relapse and showed no sustained weight loss. There were no other clear changes in the course of illness of the patients. CONCLUSION: The evidence of a strong weight-reducing potential of topiramate is indisputable and clinically significant. Topiramate could be considered in the treatment of bipolar patients who are overweight, or whose concerns about weight gain compromise their compliance with long-term prophylactic medication. So far there is no evidence that topiramate has anti-manic effect and it should not be used as monotherapy

Gram Negative Wound Infection in Hospitalised Adult Burn Patients-Systematic Review and Metanalysis-

BACKGROUND: Gram negative infection is a major determinant of morbidity and survival. Traditional teaching suggests that burn wound infections in different centres are caused by differing sets of causative organisms. This study established whether Gram-negative burn wound isolates associated to clinical wound infection differ between burn centres. METHODS: Studies investigating adult hospitalised patients (2000-2010) were critically appraised and qualified to a levels of evidence hierarchy. The contribution of bacterial pathogen type, and burn centre to the variance in standardised incidence of Gram-negative burn wound infection was analysed using two-way analysis of variance. PRIMARY FINDINGS: Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumanni, Enterobacter spp., Proteus spp. and Escherichia coli emerged as the commonest Gram-negative burn wound pathogens. Individual pathogens' incidence did not differ significantly between burn centres (F (4, 20) = 1.1, p = 0.3797; r2 = 9.84). INTERPRETATION: Gram-negative infections predominate in burn surgery. This study is the first to establish that burn wound infections do not differ significantly between burn centres. It is the first study to report the pathogens responsible for the majority of Gram-negative infections in these patients. Whilst burn wound infection is not exclusive to these bacteria, it is hoped that reporting the presence of this group of common Gram-negative "target organisms" facilitate clinical practice and target research towards a defined clinical demand.peer-reviewe

Phenotypic differences between dermal fibroblasts from different body sites determine their responses to tension and TGFβ1

BACKGROUND: Wounds in the nonglabrous skin of keloid-prone individuals tend to cause large disordered accumulations of collagen which extend beyond the original margins of the wound. In addition to abnormalities in keloid fibroblasts, comparison of dermal fibroblasts derived from nonwounded glabrous or nonglabrous skin revealed differences that may account for the observed location of keloids. METHODS: Fibroblast apoptosis and the cellular content of α-smooth-muscle actin, TGFβ1 receptorII and ED-A fibronectin were estimated by FACS analysis. The effects of TGFβ1 and serum were examined. RESULTS: In monolayer cultures non-glabrous fibroblasts were slower growing, had higher granularity and accumulated more α-smooth-muscle actin than fibroblasts from glabrous tissues. Keloid fibroblasts had the highest level of α-smooth-muscle actin in parallel with their expression level of ED-A fibronectin. TGFβ1 positively regulated α-smooth-muscle actin expression in all fibroblast cultures, although its effects on apoptosis in fibroblasts from glabrous and non-glabrous tissues were found to differ. The presence of collagen I in the ECM resulted in reduction of α-smooth-muscle actin. A considerable percentage of the apoptotic fibroblasts in attached gels were α-smooth-muscle actin positive. The extent of apoptosis correlated positively with increased cell and matrix relaxation. TGFβ1 was unable to overcome this apoptotic effect of matrix relaxation. CONCLUSION: The presence of myofibroblasts and the apoptosis level can be regulated by both TGFβ1 and by the extracellular matrix. However, reduction of tension in the matrix is the critical determinant. This predicts that the tension in the wound bed determines the type of scar at different body sites

Focus on collagen: in vitro systems to study fibrogenesis and antifibrosis _ state of the art

Fibrosis represents a major global disease burden, yet a potent antifibrotic compound is still not in sight. Part of the explanation for this situation is the difficulties that both academic laboratories and research and development departments in the pharmaceutical industry have been facing in re-enacting the fibrotic process in vitro for screening procedures prior to animal testing. Effective in vitro characterization of antifibrotic compounds has been hampered by cell culture settings that are lacking crucial cofactors or are not holistic representations of the biosynthetic and depositional pathway leading to the formation of an insoluble pericellular collagen matrix. In order to appreciate the task which in vitro screening of antifibrotics is up against, we will first review the fibrotic process by categorizing it into events that are upstream of collagen biosynthesis and the actual biosynthetic and depositional cascade of collagen I. We point out oversights such as the omission of vitamin C, a vital cofactor for the production of stable procollagen molecules, as well as the little known in vitro tardy procollagen processing by collagen C-proteinase/BMP-1, another reason for minimal collagen deposition in cell culture. We review current methods of cell culture and collagen quantitation vis-à-vis the high content options and requirements for normalization against cell number for meaningful data retrieval. Only when collagen has formed a fibrillar matrix that becomes cross-linked, invested with ligands, and can be remodelled and resorbed, the complete picture of fibrogenesis can be reflected in vitro. We show here how this can be achieved. A well thought-out in vitro fibrogenesis system represents the missing link between brute force chemical library screens and rational animal experimentation, thus providing both cost-effectiveness and streamlined procedures towards the development of better antifibrotic drugs