A randomised controlled trial of amniotic membrane in the treatment of a standardised burn injury in the merino lamb

Burn injury is associated with disabling scar formation which impacts on many aspects of the patient's life. Previously we have shown that the fetus heals a deep dermal burn in a scarless fashion. Amniotic membrane (AM) is the outermost fetal tisue and has beeen used as a dressing in thermal injuries, though there is little data to support this use. To assess the efficacy of AM in scar minimisation after deep dermal burn wound, we conducted a randomised controlled study in the 1-month lamb. Lambs were delivered by caesarian section and the amniotic membranes stored after which lambs were returned to their mothers post-operatively. At 1 month, a standardised deep dermal burn was created under general anaesthesia on both flanks of the lamb. One flank was covered with unmatched AM, the other with paraffin gauze. Animals were sequentially euthanased from Day 3-60 after injury and tissue analysed for histopathology and immunohistochemically for alpha-smooth muscle actin (alphaSMA) content. AM resulted in reduced scar tissue as assessed histopathologically and reduced alphaSMA content. This study provides the first laboratory evidence that AM may reduce scar formation after burn injury.

In vivo and in vitro models demonstrate a role for caveolin-1 in the pathogenesis of ischaemic acute renal failure

Caveolae and their proteins, the caveolins, transport macromolecules; compartmentalize signalling molecules; and are involved in various repair processes. There is little information regarding their role in the pathogenesis of significant renal syndromes such as acute renal failure (ARF). In this study, an in vivo rat model of 30 min bilateral renal ischaemia followed by reperfusion times from 4 h to 1 week was used to map the temporal and spatial association between caveolin-1 and tubular epithelial damage (desquamation, apoptosis, necrosis). An in vitro model of ischaemic ARF was also studied, where cultured renal tubular epithelial cells or arterial endothelial cells were subjected to injury initiators modelled on ischaemia-reperfusion (hypoxia, serum deprivation, free radical damage or hypoxia-hyperoxia). Expression of caveolin proteins was investigated using immunohistochemistry, immunoelectron microscopy, and immunoblots of whole cell, membrane or cytosol protein extracts. In vivo, healthy kidney had abundant caveolin-1 in vascular endothelial cells and also some expression in membrane surfaces of distal tubular epithelium. In the kidneys of ARF animals, punctate cytoplasmic localization of caveolin-1 was identified, with high intensity expression in injured proximal tubules that were losing basement membrane adhesion or were apoptotic, 24 h to 4 days after ischaemia-reperfusion. Western immunoblots indicated a marked increase in caveolin-1 expression in the cortex where some proximal tubular injury was located. In vitro, the main treatment-induced change in both cell types was translocation of caveolin-1 from the original plasma membrane site into membrane-associated sites in the cytoplasm. Overall, expression levels did not alter for whole cell extracts and the protein remained membrane-bound, as indicated by cell fractionation analyses. Caveolin-1 was also found to localize intensely within apoptotic cells. The results are indicative of a role for caveolin-1 in ARF-induced renal injury. Whether it functions for cell repair or death remains to be elucidated.

Phytophotodermatitis : case reports of children presenting with blistering after preparing lime juice

Letter to the Editor We read with interest the case report entitled ‘‘Contact with fig tree sap: An unusual cause of burn injury’’ by Mandalia et al. [1] and would like to report our similar experience with phytophotodermatitis caused by lime juice. Phototoxic dermatitis is understandably easily confused with a burn, particularly when a patient presents with large blisters of unknown mechanism. At the Royal Children’s Hospital Burns Centre, this injury was treated in the same manner as a burn and is described here...

Vascular remodeling in the internal mammary artery graft and association with in situ endothelin-1 and receptor expression

BACKGROUND: The vasoconstricting peptide endothelin-1 (ET-1) has been associated with atherosclerotic cardiovascular disease, vascular smooth muscle cell (VSMC) growth stimulation, and intimal thickening. ET-1 binds 2 receptor subtypes, endothelin A and B, and the ETA receptor mediates vasoconstriction and VSMC growth. This study aims to quantitatively assess arterial remodeling variables and compare them with changes in ET-1, ETA, and ETB expression in the internal mammary artery (IMA). METHODS AND RESULTS: Specimens from 55 coronary artery disease (CAD) patients (45 men, 10 women; mean age 65 years) and 14 control IMA specimens (from 7 men and 7 women; mean age 45 years) were collected. IMA cross sections were assessed by histochemical and immunohistochemical staining methods to quantify the levels of medionecrosis, fibrosis, VSMC growth, ET-1, ETA, ETB, and macrophage infiltration. The percentage area of medionecrosis in the patients was almost double that in the controls (31.85+/-14.52% versus 17.10+/-9.96%, P=0.0006). Total and type 1 collagen was significantly increased compared with controls (65.8+/-18.3% versus 33.7+/-13.7%, P=0.07, and 14.2+/-10.0% versus 4.8+/-2.8%, P=0.01, respectively). Despite ACE and/or statin therapy, ET-1 expression and cell cycling were significantly elevated in the patient IMAs relative to the controls (46.27+/-18.46 versus 8.56+/-8.42, P=0.0001, and 37.29+/-12.88 versus 11.06+/-8.18, P=0.0001, respectively). ETA and ETB staining was elevated in the patient vessels (46.88+/-11.52% versus 18.58+/-7.65%, P=0.0001, and 42.98+/-7.08% versus 34.73+/-5.20%, P=0.0067, respectively). A mild presence of macrophages was noted in all sections. CONCLUSIONS: Elevated distribution of collagen indicative of fibrosis coupled with increased cell cycling and high levels of ET-1 and ETA expression in the absence of chronic inflammation suggests altered IMA VSMC regulation is fundamental to the remodeling process.

Interleukin-1beta induces human proximal tubule cell injury, alpha-smooth muscle actin expression and fibronectin production

BACKGROUND Tubulointerstitial lesions, characterized by tubular injury, interstitial fibrosis and the appearance of myofibroblasts, are the strongest predictors of the degree and progression of chronic renal failure. These lesions are typically preceded by macrophage infiltration of the tubulointerstitium, raising the possibility that these inflammatory cells promote progressive renal disease through fibrogenic actions on resident tubulointerstitial cells. The aim of the present study, therefore, was to investigate the potentially fibrogenic mechanisms of interleukin-1beta (IL-1beta), a macrophage-derived pro-inflammatory cytokine, on human proximal tubule cells (PTC). METHODS Confluent, quiescent, passage 2 PTC were established in primary culture from histologically normal segments of human renal cortex (N = 11) and then incubated in serum- and hormone-free media supplemented with either IL-1beta (0 to 4 ng/mL) or vehicle (control). RESULTS IL-1beta significantly enhanced fibronectin secretion by up to fourfold in a time- and concentration-dependent fashion. This was accompanied by significant (2.5- to 6-fold) increases in alpha-smooth muscle actin (alpha-SMA) expression, transforming growth factor beta (TGF-beta1) secretion, nitric oxide (NO) production, NO synthase 2 (NOS2) mRNA and lactate dehydrogenase (LDH) release. Cell proliferation was dose-dependently suppressed by IL-1beta. NG-methyl-l-arginine (L-NMMA; 1 mmol/L), a specific inhibitor of NOS, blocked NO production but did not alter basal or IL-1beta-stimulated fibronectin secretion. In contrast, a pan-specific TGF-beta neutralizing antibody significantly blocked the effects of IL-1beta on PTC fibronectin secretion (IL-1beta, 268.1 +/- 30.6 vs. IL-1beta+alphaTGF-beta 157.9 +/- 14.4%, of control values, P < 0.001) and DNA synthesis (IL-1beta 81.0 +/- 6.7% vs. IL-1beta+alphaTGF-beta 93.4 +/- 2.1%, of control values, P < 0.01). CONCLUSION IL-1beta acts on human PTC to suppress cell proliferation, enhance fibronectin production and promote alpha-smooth muscle actin expression. These actions appear to be mediated by a TGF-beta1 dependent mechanism and are independent of nitric oxide release.

Silver deposits in cutaneous burn scar tissue is a common phenomenon following application of a silver dressing

BACKGROUND Silver dressings have been widely and successfully used to prevent cutaneous wounds, including burns, chronic ulcers, dermatitis and other cutaneous conditions, from infection. However, in a few cases, skin discolouration or argyria-like appearances have been reported. This study investigated the level of silver in scar tissue post-burn injury following application of Acticoat, a silver dressing. METHODS A porcine deep dermal partial thickness burn model was used. Burn wounds were treated with this silver dressing until completion of re-epithelialization, and silver levels were measured in a total of 160 scars and normal tissues. RESULTS The mean level of silver in scar tissue covered with silver dressings was 136 microg/g, while the silver level in normal skin was less than 0.747 microg/g. A number of wounds had a slate-grey appearance, and dissection of the scars revealed brown-black pigment mostly in the middle and deep dermis within the scar. The level of silver and the severity of the slate-grey discolouration were correlated with the length of time of the silver dressing application. CONCLUSIONS These results show that silver deposition in cutaneous scar tissue is a common phenomenon, and higher levels of silver deposits and severe skin discolouration are correlated with an increase in the duration of this silver dressing application.

Fetuin-A : a major fetal serum protein that promotes "wound closure" and scarless healing [Letter to the Editor]

Burn-wound healing is a dynamic, interactive process involving a number of cellular and molecular events and is characterized by inflammation, granulation tissue formation, re-epithelialization, and tissue remodeling (Greenhalgh, 2002; Linares, 2002). Unlike incisional-wound healing, it also requires extensive re-epithelialization due to a predominant horizontal loss of tissue and often heals with abnormal scarring when burns involve deep dermis. The early mammalian fetus has the remarkable ability to regenerate normal epidermis and dermis and to heal dermal incisional wounds with no signs of scarring. Extensive research has indicated that scarless healing appears to be intrinsic to fetal skin (McCallion and Ferguson, 1996; Ferguson and O’Kane, 2004). Previously, we reported a fetal burn model, in which 80-day-old ovine fetuses (gestation¼ 145–153 days) healed deep dermal partial thickness burns without scars, whereas postnatal lambs healed equal depth burns with significant scarring (Cuttle et al., 2005; Fraser et al., 2005). This burn model provided early evidence that fetal skin has the capacity to repair and restore dermal horizontal loss, not just vertical injuries.

The evaluation of a clinical scar scale for porcine burn scars

This study describes the evaluation of a clinical scar scale for our porcine burn scars, which includes scar cosmetic outcome, colour, height and hair, supplemented with reference porcine scar photographs representing each scar outcome and scar colour scores. A total of 72 porcine burn scars at week 6 after burn were rated in vivo and/or on photographs. Good agreements were achieved for both intra-rater reliability (correlation is 0.86-0.98) and inter-rater reliability (ICC=80-85%). The results showed statistically significant correlations for each pair in this clinical scar scale (p<0.01), with the best correlation found between scar cosmetic outcome and scar colour. A multivariate principle components analysis revealed that this clinical scar assessment was highly correlated with scar histology, wound size, and re-epithelialisation data (p<0.001). More severe scars are clinically characterised by darker purple colouration, more elevation, no presence of hair, histologically by thicker scar tissue, thinner remaining normal dermis, are more likely to have worse contraction, and slower re-epithelialisation. This study demonstrates that our clinical scar scale is a reliable, independent and valuable tool for assessing porcine burn outcome and truthfully reflects scar appearance and function. To our knowledge, this is the first study demonstrating a high correlation between clinical scar assessment and scar histology, wound contraction and re-epithelialisation data on porcine burn scars. We believe that the successful use of porcine scar scales is invaluable for assessing potential human burn treatments.

Typing early Australian healthcare-associated MRSA : confirmation of major clones and emergence of ST1-MRSA-IV and novel ST2249-MRSA-III

AIMS: To investigate the evolutionary origins of Australian healthcare-associated (HCA) methicillin-resistant Staphylococcus aureus (MRSA) strains from a panel of historical isolates typed using current genotyping techniques. METHODS: Nineteen MRSA isolates from 1965 to 1981 were examined and antibiotic susceptibility profiles determined. Genetic characterisation included real-time (RT) polymerase chain reaction (PCR) assays to identify single nucleotide polymorhpism (SNP) clonal complexes (SNP CC) and sequence type (SNP ST), multi locus sequence typing (MLST) and staphylococcal chromosomal cassette mec typing. RESULTS: All SNP CC30 isolates belonged to a novel sequence type, ST2249. All SNP CC239 isolates were confirmed as ST239-MRSA-III, except for a new single locus variant of ST239, ST2275. A further new type, ST2276, was identified. CONCLUSIONS: The earliest MRSA examined from 1965 was confirmed as ST250-MRSA-I, consistent with archaic European types. Identification of ST1-MRSA-IV in 1981 is the earliest appearance of this clinically important lineage which manifested in Australia and the United States in the 1990s. A previously unknown multi-resistant clone, ST2249-MRSA-III, was identified from 1973. Gentamicin resistance first appeared in this novel strain from 1976 and not ST239 as previously suspected. Thus, ST2249 was present in the earliest phase of the HCA MRSA epidemic in eastern Australia and was perhaps related to the emergence of the globally epidemic strain ST239.

Corneal confocal microscopy detects early nerve regeneration in diabetic neuropathy after simultaneous pancreas and kidney transplantation

Diabetic neuropathy is associated with increased morbidity and mortality. To date, limited data in subjects with impaired glucose tolerance and diabetes demonstrate nerve fiber repair after intervention. This may reflect a lack of efficacy of the interventions but may also reflect difficulty of the tests currently deployed to adequately assess nerve fiber repair, particularly in short-term studies. Corneal confocal microscopy (CCM) represents a novel noninvasive means to quantify nerve fiber damage and repair. Fifteen type 1 diabetic patients undergoing simultaneous pancreas-kidney transplantation (SPK) underwent detailed assessment of neurologic deficits, quantitative sensory testing (QST), electrophysiology, skin biopsy, corneal sensitivity, and CCM at baseline and at 6 and 12 months after successful SPK. At baseline, diabetic patients had a significant neuropathy compared with control subjects. After successful SPK there was no significant change in neurologic impairment, neurophysiology, QST, corneal sensitivity, and intraepidermal nerve fiber density (IENFD). However, CCM demonstrated significant improvements in corneal nerve fiber density, branch density, and length at 12 months. Normalization of glycemia after SPK shows no significant improvement in neuropathy assessed by the neurologic deficits, QST, electrophysiology, and IENFD. However, CCM shows a significant improvement in nerve morphology, providing a novel noninvasive means to establish early nerve repair that is missed by currently advocated assessment techniques.

Response to re : putting vital stains in context

A response to "Re: Putting vital stains in context" by Charles W McMonnies, published in the same issue of this journal. "I thank Professor McMonnies for his thoughtful comments,1 which rightly forcemeto more directly address the clinical ramifications of solution-induced corneal staining (SICS). I concur with his observation that determining whether the staining can be attributed to preservative-associated transient hyperfluorescence (PATH) or true pathology can be difficult in a typical clinical situation, perhaps requiring two visits in a single day. There is no easy answer to this dilemma..."

The TFOS International Workshop on Contact Lens Discomfort: report of the contact lens interactions with the ocular surface and adnexa subcommittee

The report of this subcommittee concerns the impact of contact lenses (CLs) on the ocular surface, with a particular emphasis on CL discomfort (CLD). We define the ocular surface, its regional anatomy, and the physiological responses of each region to CL wear.

Fasting increases serum bilirubin levels in clinically normal, healthy males but not females : a retrospective study from phase I clinical trial participants

Aim: To examine if fasting affects serum bilirubin levels in clinical healthy males and females. Methods: We utilised retrospective data from phase 1 clinical trials where blood was collected in either a fed or fasting state at screening and pre-dosing time points and analysed for total bilirubin levels as per standard clinical procedures. Participants were clinically healthy males (n = 105) or females (n = 30) aged 18 to 48 inclusive who participated in a phase 1 clinical trial in 2012 or 2013. Results: We found a statistically significant increase in total serum bilirubin levels in fasting males as compared to non-fasting males. The fasting time correlated positively with increased bilirubin levels. The age of the healthy males did not correlate with their fasting bilirubin level. We found no correlation between fasting and bilirubin levels in clinically normal females. Conclusions: The recruitment and screening of volunteers for a clinical trial is a time-consuming and expensive process. This study clearly demonstrates that testing for serum bilirubin should be conducted on non-fasting male subjects. If fasting is required, then participants should not be excluded from a trial based on an elevated serum bilirubin that is deemed non-clinically significant.