Stage-specific differences in secretory profile of mesenchymal stromal cells (MSCs) subjected to early- vs late-stage OA synovial fluid.

OBJECTIVE: Although, mesenchymal stromal cells (MSCs) are being clinically investigated for their use in osteoarthritis (OA), it is unclear whether their postulated therapeutic properties are equally effective in the early- and late-stages of OA. In this study we investigated MSC cytokine secretion post-exposure to synovial fluid (SF), obtained from early- vs late-stage knee OA patients to justify a potential patient stratification strategy to maximize MSC-mediated treatment effects. METHOD: Subjects were recruited and categorized into early- [Kellgren-Lawrence (KL) grade I/II, n = 12] and late-stage (KL-III/IV, n = 12) knee OA groups. SF samples were obtained, and their proteome was tested using multiplex assays, after 3-days culture, with and without MSCs. SFs cultured without MSCs were used as a baseline to identify MSC-secreted factors into SFs cultured with MSCs. Linear mixed-effect models and non-parametric tests were used to identify alterations in the MSC secretome during exposure to OA SF (3-days). MSCs cultured for 3-days in 0.5% fetal bovine serum (FBS)-supplemented medium were used to compare SF results with culture medium. RESULTS: Following exposure to OA SF, the MSC secretome contained proteins that are involved in tissue repair, angiogenesis, chemotaxis, matrix remodeling and the clotting process. However, chemokine (C-X-C motif) ligand-8 (CXCL8; chemoattractant), interleukin-6 (IL6) and chemokine (C-C motif) ligand 2 (CCL2) were elevated in the MSC-secretome in response to early- vs late-stage OA SF. CONCLUSION: Early- vs late-stage OA SF samples elicit a differential MSC secretome response, arguing for stratification of OA patients to maximize MSC-mediated therapeutic effects.

Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture.

Stem cells that naturally reside in adult tissues, such as muscle stem cells (MuSCs), exhibit robust regenerative capacity in vivo that is rapidly lost in culture. Using a bioengineered substrate to recapitulate key biophysical and biochemical niche features in conjunction with a highly automated single-cell tracking algorithm, we show that substrate elasticity is a potent regulator of MuSC fate in culture. Unlike MuSCs on rigid plastic dishes (approximately 10(6) kilopascals), MuSCs cultured on soft hydrogel substrates that mimic the elasticity of muscle (12 kilopascals) self-renew in vitro and contribute extensively to muscle regeneration when subsequently transplanted into mice and assayed histologically and quantitatively by noninvasive bioluminescence imaging. Our studies provide novel evidence that by recapitulating physiological tissue rigidity, propagation of adult muscle stem cells is possible, enabling future cell-based therapies for muscle-wasting diseases.

Resuscitation tables: a useful tool in calculating pre-burns unit fluid requirements.

BACKGROUND: There is considerable variation in the standard of initial burn management, particularly burn surface area assessment and application of resuscitation formulae. Early aggressive management of major burns improves survival. Internationally, the Parkland formula employing lactated Ringer's solution is used for fluid resuscitation. This study aimed to assess whether Parkland fluid resuscitation tables could improve the accuracy of initial fluid requirement calculations. METHODS: The burn size had first to be determined for an adult and a child using a preshaded Lund and Browder chart. Fluid requirements then had to be calculated using the conventional Parkland formula. The burn size had to be similarly calculated for two further cases and fluid requirements calculated using resuscitation tables. The study had a sample size of 50, consisting of plastic surgery trainees, anaesthetists and burn nurse specialists. RESULTS: All the participants found the resuscitation tables to be quicker and easier to use. The burn size was correctly calculated in 72% of cases. Fluid resuscitation requirements were correct in only 55% when using the Parkland formula. The use of resuscitation tables improved the accuracy in calculating fluid requirements to 75%. CONCLUSIONS: The use of Parkland fluid resuscitation tables can improve accuracy and ease of calculation of fluid resuscitation requirements.

Return to work after burns: a qualitative research study.

As yet no qualitative research studies looking at return to work following burns have been published. The aim of this study was to investigate the "hows" and "whys" of return to work, by purposively selecting a cross-section of burns patients who returned to the same/similar job, those who returned to work but either on a part-time basis or in a different role/job and those who became or remained unemployed, and using semi-structured interviews to explore their experiences. Using matrix analysis methodology, and with the general themes that emerged from these transcripts, it was possible to place patients into 5 broad groups, the "defeated", the "burdened", the "affected", the "unchanged" and the "stronger". We anticipate that use of these general groups will be useful in targeting multi-disciplinary return to work strategies, and discuss how this qualitative research has changed practice at the Queen Victoria Hospital Burns Centre.

The efficacy of a polyhydrated ionogen impregnated dressing in the treatment of recalcitrant diabetic foot ulcers: a multi-centre pilot study.

OBJECTIVE: Assessing the efficacy of a polyhydrated ionogen impregnated dressing in the treatment of recalcitrant diabetic foot ulcers. SUMMARY BACKGROUND DATA: Diabetic Foot Ulcers (DFU) continue to present a formidable challenge in terms of morbidity and health care costs. Increasing evidence ascertains the important role of Matrix MetalloProteinases (MMPs) and their tissue inhibitors, TIMPs, in wound healing. Imbalance of MMPs in the DFU microenvironment has been associated with poor wound healing. Current research is directed towards therapeutic agents that could redress the imbalance of MMPs/TIMPs. Poly Hydrated Ionogen (PHI) formulation is based on metallic ions and citric acid. PHI application aims to positively restore MMP ratios within chronic wounds. This initial multi-centre pilot study aimed to investigate the efficacy of the PHI formulation in achieving stable wound closure in recalcitrant DFUs. MATERIAL AND METHODS: Twenty patients with therapy resistant DFUs of at least 1 cm2 and 3 months duration were treated with PHI formulation in an acetate carrier dressing. Wound debridement, digital imaging and wound perimeter tracing was performed weekly. Off-loading was performed by the use of appropriate shoe-wear (cut-out sandals) and crutches. Patient satisfaction was assessed with a questionnaire. A detailed evaluation sheet was kept for every patient and updated at each visit. RESULTS: Stable wound closure with high patient satisfaction was achieved in 16 (80%) DFUs. The mean time to full closure was 18 weeks. A stable wound epithelization was seen in all full closure patients up to latest follow-up of one year. CONCLUSIONS: Encouraging results of this pilot study prompt us to further investigate the PHI efficacy in DFU treatment in a multi-centre, randomized controlled trial.

The E-Z(easy) clamp--a new instrument to facilitate medial ectropion repair.

The medial spindle/retropunctal diamond procedure is a useful technique to correct medial ectropion. Unfortunately, the procedure is difficult, due to the limited size of the surgical field, the bleeding that is seen when the marginal artery is encountered and because of the ever present risk of damaging the canaliculus. We have developed a clamp that overcomes the anatomical opposition outlined above and at the same time improves access to the surgical field.

Synchronous reconstruction of the exenterated orbit with a pericranial flap, skin graft and osseointegrated implants.

Reconstruction of the exenterated orbit remains a surgical challenge. Here, the authors present a case in which orbital exenteration was performed for an extensive, infiltrating medial canthal basal cell carcinoma; the resulting defect was reconstructed with osseointegrated implants and a pericranial flap onto which a split thickness skin graft was placed. The second stage of the aesthetic rehabilitation of this patient (placement of the transcutaneous abutments) was completed under local anaesthetic 16 weeks later. Subsequently, the patient was fitted with an oculoplastic prosthesis four weeks later. The above technique accelerates the prosthetic rehabilitation of the patient by performing the primary reconstructive procedures simultaneously with the exenteration and by removing the need for secondary surgical procedures under general anaesthetics. The whole process from orbital exenteration to the fitting of an ocular prosthesis was completed in just five months.

So much for percentage, but what about the weight?

The use of resuscitation formulae for burns is advocated for A&E departments. Much care is taken to calculate the percentage of the burn, but this is then multiplied by an approximate weight. How accurate is this figure and should it be more carefully measured? Forty two sets of case notes of patients with resuscitation sized burns were reviewed. In 32, a weight was documented in the A&E notes. This was compared with the measured weight on arrival at the burn centre. In half the cases there was a greater than 5 kg difference and in nine patients over 10 kg of difference. The methods of patient weight assessment in 16 A&E departments were reviewed. The majority have only stand on scales. Three departments have sit on scales; however, if they were assessing the weight of a patient who is unable to sit they would need to ask the patient, relatives, or simply guess. Investment in weighing equipment should be encouraged if resuscitation formulae are to have any place in the A&E management of burn patients.

Anomalous anterior midline cervical tube.

Anterior midline cervical anomalies, excepting thyroglossal tract abnormalities, are rare. Two unusual and almost identical cases of an anomalous congenital tube connecting the chin to the upper sternum are described; only one previous similar report exists in the literature. In all three cases, the tubes occurred as isolated anomalies in otherwise healthy children.

Treatment before transfer: the patient with burns.

OBJECTIVES: To review pre-burns centre management, including assessment, resuscitation, and transfer. METHODS: A retrospective analysis of the notes of all the UK patients admitted to the Burns Centre in 1998, who had a body surface area burn of over 15% in adults (10% in children). RESULTS: There were 31 patients, 21 adults and 10 children, and the average burn size was 32% (12-96%). Fourteen were overestimated (average of 9%) and 13 underestimated by 7.5%. Twenty nine received intravenous fluids, 18 specified a formula, but it was only applied correctly in 10. The average time to the Burns Centre from the burn was 10 hours, and the time for resuscitation and transfer, eight hours. Documentation was generally poor. CONCLUSION: There has previously been considerable variation in the standard of initial burn management and there have been problems with burn percentage assessment and resuscitation formula application. A new proforma has been introduced to tackle these issues.

FYVE domain targets Pib1p ubiquitin ligase to endosome and vacuolar membranes.

Signaling by phosphatidylinositol 3-kinases (PI3Ks) is often mediated by proteins which bind PI3K products directly and are localized to intracellular membranes rich in PI3K products. The FYVE finger domain binds with high specificity to PtdIns3P and proteins containing this domain have been shown to be important components of diverse PI3K signaling pathways. The genome of the yeast Saccharomyces cerevisiae encodes five proteins containing FYVE domains, including Pib1p, whose function is unknown. In addition to a FYVE finger motif, the primary structure of Pib1p contains a region rich in cysteine and histidine residues that we demonstrate binds 2 mol eq of zinc, consistent with this region containing a RING structural domain. The Pib1p RING domain exhibited E2-dependent ubiquitin ligase activity in vitro, indicating that Pib1p is an E3 RING-type ubiquitin ligase. Fluorescence microscopy was used to demonstrate that a GFP-Pib1p fusion protein localized to endosomal and vacuolar membranes and deletional analysis of Pib1p domains indicated that localization of GFP-Pib1p is mediated solely by the FYVE domain. These results suggest that Pib1p mediates ubiquitination of a subset of cellular proteins localized to endosome and vacuolar membranes, and they expand the repertoire of PI3K-regulated pathways identified in eukaryotic cells.

GDP dissociation inhibitor domain II required for Rab GTPase recycling.

Rab GTPases are localized to distinct subsets of organelles within the cell, where they regulate SNARE-mediated membrane trafficking between organelles. One factor required for Rab localization and function is Rab GDP dissociation inhibitor (GDI), which is proposed to recycle Rab after vesicle fusion by extracting Rab from the membrane and loading Rab onto newly formed transport intermediates. GDI is composed of two domains; Rab binding is mediated by Domain I, and the function of Domain II is not known. In this study, Domain II of yeast GDI, encoded by the essential GDI1/SEC19 gene, was targeted in a genetic screen to obtain mutants that might lend insight into the function of this domain. In one gdi1 mutant, the cytosolic pools of all Rabs tested were depleted, and Rab accumulated on membranes, suggesting that this mutant Gdi1 protein has a general defect in extraction of Rab from membranes. In a second gdi1 mutant, the endosomal/vacuolar Rabs Vps21/Ypt51p and Ypt7p accumulated in the cytosol bound to Gdi1p, but localization of Ypt1p and Sec4p were not significantly affected. Using an in vitro assay which reconstitutes Gdi1p-mediated membrane loading of Rab, this mutant Gdi1p was found to be defective in loading of Vps21p but not Ypt1p. Loading of Vps21p by loading-defective Gdi1p was restored when acceptor membranes prepared from a deletion strain lacking Vps21p were used. These results suggest that membrane-associated Rab may regulate recruitment of GDI-Rab from the cytosol, possibly by regulating a GDI-Rab receptor. We conclude that Domain II of Gdi1p is essential for Rab loading and Rab extraction, and confirm that each of these activities is required for Gdi1p function in vivo.