Primary cultured fibroblasts derived from patients with chronic wounds: a methodology to produce human cell lines and test putative growth factor therapy such as GMCSF

<p>Abstract</p> <p>Background</p> <p>Multiple physiologic impairments are responsible for chronic wounds. A cell line grown which retains its phenotype from patient wounds would provide means of testing new therapies. Clinical information on patients from whom cells were grown can provide insights into mechanisms of specific disease such as diabetes or biological processes such as aging.</p> <p>The objective of this study was 1) To culture human cells derived from patients with chronic wounds and to test the effects of putative therapies, Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) on these cells. 2) To describe a methodology to create fibroblast cell lines from patients with chronic wounds.</p> <p>Methods</p> <p>Patient biopsies were obtained from 3 distinct locations on venous ulcers. Fibroblasts derived from different wound locations were tested for their migration capacities without stimulators and in response to GM-CSF. Another portion of the patient biopsy was used to develop primary fibroblast cultures after rigorous passage and antimicrobial testing.</p> <p>Results</p> <p>Fibroblasts from the non-healing edge had almost no migration capacity, wound base fibroblasts were intermediate, and fibroblasts derived from the healing edge had a capacity to migrate similar to healthy, normal, primary dermal fibroblasts. Non-healing edge fibroblasts did not respond to GM-CSF. Six fibroblast cell lines are currently available at the National Institute on Aging (NIA) Cell Repository.</p> <p>Conclusion</p> <p>We conclude that primary cells from chronic ulcers can be established in culture and that they maintain their <it>in vivo </it>phenotype. These cells can be utilized for evaluating the effects of wound healing stimulators <it>in vitro</it>.</p

Treatment of streptococcal pharyngitis with once-daily amoxicillin versus intramuscular benzathine penicillin g in low-resource settings : A randomized controlled trial

Background: Primary prevention of acute rheumatic fever is achieved by proper antibiotic treatment of group A β -hemolytic streptococcal (GAS) pharyngitis. Methods: To assess noninferiority of oral amoxicillin to intramuscular benzathine penicillin G (IM BPG). Children (2 to 12 years) meeting enrollment criteria were randomized 1:1 to receive antibiotic treatment in 2 urban outpatient clinics in Egypt and Croatia. Results: A total of 558 children (Croatia = 166, Egypt = 392) were randomized, with 368 evaluable in an intention-to-treat (ITT) analysis, and 272 evaluable in the per protocol (PP) analysis. In Croatia, ITT and PP treatment success rates were comparable for IM BPG and amoxicillin (2.5% difference vs 1.1% difference, respectively). In Egypt, amoxicillin was not comparable with IM BPG in ITT analysis (15.1% difference), but was comparable in PP analysis (-9.3% difference). Conclusion: If compliance is a major issue, a single dose of IM BPG may be preferable for treatment of GAS pharyngitis.publishersversionPeer reviewe

Novel workpiece clamping method for increased machining performance

Naprave kod kojih se sile i momenti nastali u procesu rezanja uravnotežuju silama trenja, nastalim na kontaktima elemenata za baziranje i stezanje s izratkom, su vrlo zastupljene u industriji. Spomenute kontakte, pored ostalog, karakterizira određena popustljivost veza koja je složena funkcija makro i mikrogeometrije kontaktnih parova u sustavu izradak-naprava, sila stezanja i sila rezanja. Pogreške obrade uvelike su posljedica upravo popustljivosti spomenutih veza. U radu se razmatra popustljivost veza između elemenata za stezanje i izratka u slučajevima stezanja uređajem za stezanje s ravnim čelom i specijalno dizajniranog elementa za stezanje. Nakon teorijskih razmatranja izloženi su rezultati eksperimentalnih istraživanja. Rezultati ukazuju na značajne prednosti specijalno dizajniranog elementa za stezanje u odnosu na standardni oblik elementa za stezanje s ravnim čelom. Dobiveni rezultati otvaraju prostor za istraživanja u smislu optimizacije elemenata naprava za stezanje i mogućnosti njihove industrijske primjene.Fixtures which balance cutting forces and torques by the friction forces generated on contact surfaces of locating, clamping elements and workpiece surfaces are widespread in industrial practice. Among other things, these friction contacts are characterized by a certain amount of interface compliance which is a complex function of macro- and microgeometry of contact pairs in the workpiece-fixture system, as well as the clamping and cutting forces. Workpiece machining errors are mostly the consequence of that interface compliance. This paper investigates workpiece-fixture interface compliance in cases where clamping is performed using a standard, and specially designed clamping element. Theoretical considerations are presented, followed by results of experimental investigation. Considerable advantages of the specially designed clamping element compared to its standard counterpart are demonstrated by experiments. The results are a good starting point for a research into optimization of special fixture clamping elements and their wider industrial application

Stress Signals, Mediated by Membranous Glucocorticoid Receptor, Activate PLC/PKC/GSK-3β/β-catenin Pathway to Inhibit Wound Closure

Glucocorticoids (GCs), key mediators of stress signals, are also potent wound healing inhibitors. To understand how stress signals inhibit wound healing, we investigated the role of membranous glucocorticoid receptor (mbGR) by using cell-impermeable BSA-conjugated dexamethasone. We found that mbGR inhibits keratinocyte migration and wound closure by activating a Wnt-like phospholipase (PLC)/ protein kinase C (PKC) signaling cascade. Rapid activation of mbGR/PLC/PKC further leads to activation of known biomarkers of nonhealing found in patients, β-catenin and c-myc. Conversely, a selective inhibitor of PKC, calphostin C, blocks mbGR/PKC pathway, and rescues GC-mediated inhibition of keratinocyte migration in vitro and accelerates wound epithelialization of human wounds ex vivo. This novel signaling mechanism may have a major impact on understanding how stress response via GC signaling regulates homeostasis and its role in development and treatments of skin diseases, including wound healing. To test tissue specificity of this nongenomic signaling mechanism, we tested retinal and bronchial human epithelial cells and fibroblasts. We found that mbGR/PLC/PKC signaling cascade exists in all cell types tested, suggesting a more general role. The discovery of this nongenomic signaling pathway, in which glucocorticoids activate Wnt pathway via mbGR, provides new insights into how stress-mediated signals may activate growth signals in various epithelial and mesenchymal tissues

Novel genomic effects of glucocorticoids in epidermal keratinocytes: inhibition of apoptosis, interferon-gamma pathway, and wound healing along with promotion of terminal differentiation

Glucocorticoids (GCs) have a long history of use as therapeutic agents for numerous skin diseases. Surprisingly, their specific molecular effects are largely unknown. To characterize GC action in epidermis, we compared the transcriptional profiles of primary human keratinocytes untreated and treated with dexamethasone (DEX) for 1, 4, 24, 48, and 72 h using large scale microarray analyses. The majority of genes were found to be regulated only after 24 h and remained regulated throughout treatment. In addition to regulation of the expected pro-inflammatory genes, we found that GCs regulate cell fate, tissue remodeling, cell motility, differentiation, and metabolism. GCs suppress the expression of essentially all IFNgamma-regulated genes, including IFNgamma receptor and STAT-1, an effect that was previously unknown. GCs also block STAT-1 activation and nuclear translocation. Unexpectedly, GCs induce the expression of anti-apoptotic genes and repress pro-apoptotic ones, preventing UV-induced keratinocyte apoptosis. Consequently, treatment with GCs blocked UV-induced apoptosis of keratinocytes. GCs have profound effect on wound healing by inhibiting cell motility and the expression of the proangiogenic factor, vascular endothelial growth factor. They play an important role in tissue remodeling and scar formation by suppressing the expression of TGFbeta1 and -2 and MMP1, -2, -9, and -10 and inducing TIMP-2. Finally, GCs promote terminal epidermal differentiation while simultaneously inhibiting early stage differentiation. These results provide new insights into the beneficial and adverse effects of GCs in the epidermis, defining the participating genes and mechanisms that coordinate the cellular responses important for GC-based therapies

Surgical pathology to describe the clinical margin of debridement of chronic wounds using a wound electronic medical record

Chronic wounds, including diabetic foot ulcers (DFU), pressure ulcers (PU), and venous ulcers (VU) result from multiple physiologic impairments. Operative debridement is a mainstay of treatment to remove nonviable tissue and to stimulate wound healing. Unlike tumor resection, however, operative wound specimens are not routinely sent for pathology. The objective of this study was to describe the pathology present in chronic wounds. Pathology reports of the skin edge and wound base from 397 initial debridements in 336 consecutive patients with chronic wounds were retrospectively reviewed. All data were entered and stored in a Wound Electronic Medical Record. Pathology data were extracted from the Wound Electronic Medical Record, coded, and quantified. Up to 15 distinct histopathologic findings across 7 tissue types were observed after review of pathology reports from chronic wounds. Specifically, the pathology of epidermis revealed hyperkeratosis: 66% in DFUs, 31% in PUs, and 29% in VUs. Dermal pathology revealed fibrosis in 49% of DFUs, 30% of PUs, and 15% of VUs. Wound bed pathology revealed necrosis in the subcutaneous tissue in 67% of DFUs, 55% of PUs, and 19% of VUs. Fibrosis was reported in between 19% and 52% of all wound types. Acute osteomyelitis was present in 39% of DFUs, 33% of PUs, and 29% of VUs. This observational study of the histopathology of initial surgical debridement of chronic wounds revealed a wide range of findings across multiple tissue levels. Although certain findings such as osteomyelitis and gangrene have been shown to directly relate to impaired wound healing and amputation, other findings require additional investigation. To rigorously define a margin of debridement, a prospective study relating histopathology and clinical outcomes such as healing rates and amputation is needed