Regulation of MAPK activation, AP-1 transcription factor expression and keratinocyte differentiation in wounded fetal skin

Fetal epithelium retains the ability to re-epithelialize a wound in organotypic culture in a manner not dependent on the presence of underlying dermal substrata. This capacity is lost late in the third trimester of gestation or after embryonic day 17 (E-17) in the rat such that embryonic day 19 (E-19) wounds do not re-epithelialize. Moreover, wounds created in E-17 fetuses in utero heal in a regenerative, scar-free fashion. To investigate the molecular events regulating re-epithelialization in fetal skin, the wound-induced expression profile and tissue localization of activator protein 1 (AP-1) transcription factors c-Fos and c-Jun was characterised in E-17 and E-19 skin using organotypic fetal cultures. The involvement of mitogen-activated protein kinase (MAPK) signaling in mediating wound-induced transcription factor expression and wound re-epithelialization was assessed, with the effect of wounding on the expression of keratinocyte differentiation markers determined. Our results show that expression of AP-1 transcription factors was induced immediately by wounding and localized predominantly to the epidermis in E-17 and E-19 skin. c-fos and c-jun induction was transient in E-17 skin with MAPK-dependent c-fos expression necessary for the re-epithelialization of an excisional wound in organotypic culture. In E-19 skin, AP-11 expression persisted beyond 12 h post-wounding, and marked upregulation of the keratinocyte differentiation markers keratin 10 and loricrin was observed. No such changes in the expression of keratin 10 or loricrin occurred in E-17 skin. These findings indicate that re-epithelialization in fetal skin is regulated by wound-induced AP-1 transcription factor expression via MAPK and the differentiation status of keratinocytes.

The tyrosine phosphatase DEP-1 induces cytoskeletal rearrangements, aberrant cell-substratum interactions and a reduction in cell proliferation

The receptor protein tyrosine phosphatase density-enhanced phosphatase-1 (DEP-1) has been implicated in aberrant cancer cell growth and immune cell function, however, its function within cells has yet to be properly elucidated. To investigate the cellular function of DEP-1, stable cell lines inducibly expressing DEP-1 were generated. Induction of DEP-1 expression was found to decrease PDGF-stimulated tyrosine phosphorylation of a number of cellular proteins including the PDGF receptor, and to inhibit growth factor-stimulated phosphorylation of components of the MAPK pathway, indicating that DEP-1 antagonised PDGF receptor signalling. This was supported by data showing that DEP-1 expression resulted in a reduction in cell proliferation. DEP-1-expressing cells had fewer actin-containing microfilament bundles, reduced vinculin and paxillin-containing adhesion plaques, and were defective in interactions with fibronectin. Defective cell-substratum adhesion correlated with lack of activation of FAK in DEP-1-expressing cells. Time-lapse interference reflection microscopy of live cells revealed that although small focal contacts at the leading edge were generated in DEP-1-expressing cells, they failed to mature into stable focal adhesions, as found in control cells. Further motility analysis revealed that DEP-1-expressing cells retained limited random motility, but showed no chemotaxis towards a gradient of PDGF. In addition, cell-cell contacts were disrupted, with a change in the localisation of cadherin from discrete areas of cell-cell contact to large areas of membrane interaction, and there was a parallel redistribution of beta-catenin. These results demonstrate that DEP-1 is a negative regulator of cell proliferation, cell-substratum contacts, motility and chemotaxis in fibroblasts.

Investigation of in vitro transdermal absorption of fentanyl from patches placed on skin samples obtained from various anatomic regions of dogs

Objective-To investigate in vitro transdermal absorption of fentanyl from patches through skin samples obtained from various anatomic regions of dogs. Sample Population-Skin samples from 5 Greyhounds. Procedure-Skin samples from the dogs' thoracic, neck, and groin regions were collected postmortem and frozen. After samples were thawed, circular sections were cut and placed in Franz-type diffusion cells in a water bath (32degreesC). A commercial fentanyl patch, attached to an acetate strip with a circular hole, was applied to each skin sample. Cellulose strips were used as control membranes. Samples of receptor fluid in the diffusion cells were collected at intervals for 48 hours, and fentanyl concentrations were analyzed by use of high-performance liquid chromatography. Results-Mean +/- SD release rate of fentanyl from the patch, defined by its absorption rate through the non-rate-limiting cellulose membrane, was linear during the first 8 hours (2.01 +/- 0.05 pg/cm(2) of cellulose membrane/h) and then decreased. Fentanyl passed through skin from the groin region at a faster rate and with a significantly shorter lag time, compared with findings in neck or thoracic skin samples. Conclusions and Clinical Relevance-In vitro, fentanyl from a patch was absorbed more quickly and to a greater extent through skin collected from the groin region of dogs, compared with skin samples from the thoracic and neck regions. Placement of fentanyl patches in the groin region of dogs may decrease the lag time to achieve analgesia perioperatively; however, in vivo studies are necessary to confirm these findings.

The effect of region of application on absorption of ethanol and hexanol through canine skin

The effect of region of application on the percutaneous penetration of solutes with differing lipophilicity was investigated in canine skin. Skin from the thorax, neck, back, groin, and axilla regions was harvested from Greyhound dogs and placed in Franz-type diffusion cells. Radiolabelled (C-14) ethanol (Log P 0.19) or hexanol (Log P 1.94) was applied to each skin section for a total of 5 h. The permeability coefficient (k(P), cm h(-1)) and residue of alcohol remaining in the skin were significantly (P = 0.001) higher for hexanol compared to ethanol. In contrast, ethanol had a far greater maximum flux (J(max), mol (cm(2))(-1) h(-1)) than hexanol (P = 0.001). A comparison of regional differences shows the k(P) and Jmax for ethanol in the groin was significantly lower (P = 0.035) than the back. The k(P) and Jmax for hexanol were significantly higher (P = 0.001) in the axilla than the other four skin sites. An understanding of factors influencing percutaneous drug movement is important when formulating topical preparations for the dog. (C) 2003 Elsevier Ltd. All rights reserved.

Treatment of partial-thickness burns: A prospective, randomized trial using Transcyte (TM)

Background: The purpose of the present study was to compare the effectiveness of three burns dressings (TransCyte, a bio-engineered skin substitute; Biobrane; and Silvazine cream (silver sulphadiazine and 0.2% chlorhexidine)), in treating children with partial-thickness burns. The primary objective was to determine the days until greater than or equal to90% re-epithelialization. The secondary objectives were to evaluate the number of wounds requiring autografting and the number of dressing changes/local wound care required. Methods: Study wounds were identified on each patient and the patients were randomized to receive TransCyte or Biobrane or Silvazine. Assessment of study wound closure began at 2 days after treatment and continued at least every other day thereafter until the wounds re-epithelialized or were autografted. A laser Doppler imaging system was used as an adjunct to assessing the depth of the burn. Results: Thirty-three patients with 58 wound sites enrolled in the study (TransCyte, n = 20, Biobrane, n = 17; Silvazine, n = 21). Mean time to re-epithelialization was 7.5 days for TransCyte, 9.5 days for Biobrane, and 11.2 days for Silvazine. The number of wounds requiring autografting were 5/21 (24%) for Silvazine, 3/17 (17%) for Biobrane, and 1/20 (5%) for TransCyte. Conclusions: When used in partial-thickness burns in children, TransCyte promotes fastest re-epithelialization and required less overall dressings then Biobrane or Silvazine. Patients who received Silvazine or Biobrane require more autografting than those treated with TransCyte.

Cytotoxicity of topical antimicrobial agents used in burn wounds in Australasia

Background: Burn sepsis is a leading cause of mortality and morbidity in patients with major burns. The use of topical antimicrobial agents has helped improve the survival of these patients. Silvazine (Sigma Pharmaceuticals, Melbourne, Australia) (1% silver sulphadiazine and 0.2% chlorhexidine digluconate) is used exclusively in Australasia, and there is no published study on its cytotoxicity. This study compared the relative cytotoxicity of Silvazine with 1% silver sulphadiazine (Flamazine (Smith & Nephew Healthcare. Hull. UK)) and a silver-based dressing (Acticoat (Smith & Nephew Healthcare, Hull, UK)). Methods: Dressings were applied to the centre of culture plates that were then seeded with keratinocytes at an estimated 25% confluence. The plates were incubated for 72 h and culture medium and dressings then removed. Toluidine blue was added to stain the remaining keratinocytes. Following removal of the dye, the plates were photographed under standard conditions and these digital images were analysed using image analysis software. Data was analysed using Student's t-test. Results: In the present study, Silvazine is the most cytotoxic agent. Seventy-two hour exposure to Silvazine in the present study results in almost no keratinocyte survival at all and a highly statistically significant reduction in cell survival relative to control, Acticoat and Flamazine (P

An in vitro study of the anti-microbial efficacy of a 1% silver sulphadiazine and 0.2% chlorhexidine digluconate cream Silvazine (TM), 1% silver sulphadiazine cream Flamazine (TM) and a silver coated dressing Acticoat (TM)

Burn sepsis is a leading cause of mortality and morbidity in patients with major burns. The use of topical anti-microbial agents has helped improve the survival in these patients. There are a number of anti-microbials available, one of which, Silvazine(TM) (1% silver sulphadiazine (SSD) and 0.2% chlorhexidine digluconate), is used only in Australasia. No study, in vitro or clinical, had compared Silvazine(TM) with the new dressing Acticoat(TM). This study compared the anti-microbial activity of Silvazine(TM), Acticoa(TM) and 1% silver sulphadiazine (Flamazine(TM)) against eight common burn wound pathogens. Methods: Each organism was prepared as a suspension. A 10 mul inoculum of the chosen bacterial isolate (representing approximately between 104 and 105 total bacteria) was added to each of four vials, followed by samples of each dressing and a control. The broths were then incubated and 10 mul loops removed at specified intervals and transferred onto Horse Blood Agar. These plates were then incubated for 18 hours and a colony count was performed. Results: The data demonstrates that the combination of 1% SSD and 0.2% chlorhexidine digluconate (Silvazine(TM)) results in the most effective killing of all bacteria. SSD and Acticoat(TM) had similar efficacies against a number of isolates, but Acticoat(TM) seemed only bacteriostatic against E. faecalis and methicillin-resistant Staphylococcus aureus. Viable quantities of Enterobacter cloacae and Proteus mirabilis rei named at 24 h. Conclusion: The combination of 1% SSD and 0.2% chlorhexidine digluconate (Silvazine(TM)) is a more effective anti-microbial against a number of burn wound pathogens in this in vitro study. A clinical study of its in vivo anti-microbial efficacy is required. (C) 2003 Elsevier Ltd and ISBI. All rights reserved.

Symptoms and diagnosis of borderline, early and advanced epithelial ovarian cancer

Objective. To document symptoms associated with borderline, early and advanced ovarian cancer and identify personal characteristics associated with early versus late diagnosis. Methods. Information concerning symptoms and diagnosis history was available from 811 women with ovarian cancer who took part in an Australian case–control study in the early 1990s. Women were classified into three groups for comparison based on their diagnosis: borderline, early (stage I–II) and advanced (stage III–IV) invasive cancer. Results. Sixteen percent of women with borderline tumors, 7% with early cancer and 4% with advanced cancer experienced no symptoms before diagnosis (P < 0.0001). Among women with symptoms, abdominal pain (44%) or swelling (39%) were most frequently reported; an abdominal mass (12%) and gynecological symptoms (12%) were less common. Compared to advanced stage cancer, women with early stage cancer were more likely to report an abdominal mass or urinary symptoms but less likely to report gastrointestinal problems or general malaise. General malaise and ‘other’ symptoms were least common in borderline disease. Older women, and those with higher parity or a family history of breast or ovarian cancer, were more likely to be diagnosed at an advanced stage of disease. Conclusions. Women who experience persistent or recurrent abdominal symptoms, particularly swelling and/or pain should be encouraged to seek medical attention and physicians should be alert to the possibility of ovarian cancer even in the absence of an abdominal mass. Further information about the prevalence of these symptoms in the general population is essential to assist physicians in patient management.

Fourier-domain optical coherence tomography: optimization of signal-to-noise ratio in full space

Optical coherence tomography (OCT) is an emerging coherence-domain technique capable of in vivo imaging of sub-surface structures at millimeter-scale depth. Its steady progress over the last decade has been galvanized by a breakthrough detection concept, termed spectral-domain OCT, which has resulted in a dramatic improvement of the OCT signal-to-noise ratio of 150 times demonstrated for weakly scattering objects at video-frame-rates. As we have realized, however, an important OCT sub-system remains sub-optimal: the sample arm traditionally operates serially, i.e. in flying-spot mode. To realize the full-field image acquisition, a Fourier holography system illuminated with a swept-source is employed instead of a Michelson interferometer commonly used in OCT. The proposed technique, termed Fourier-domain OCT, offers a new leap in signal-to-noise ratio improvement, as compared to flying-spot OCT systems, and represents the main thrust of this paper. Fourier-domain OCT is described, and its basic theoretical aspects, including the reconstruction algorithm, are discussed. (C) 2004 Elsevier B.V. All rights reserved.

Diagnostic accuracy of and patient satisfaction with telemedicine for the follow-up of paediatric burns patients

Videoconferencing has become a routine technique for the post-acute burns care of children in Queensland. We compared the agreement between clinical assessments conducted via videoconference and assessments conducted in the conventional, face-to-face manner (FTF). A total of 35 children with a previous burn injury were studied. Twenty-five children received three consecutive assessments: first FTF by a consultant in the outpatient department, then by a second consultant who reviewed the patient via videoconference, and then by the second consultant in person. The second consultant also reviewed another 10 children twice. At each review, the following variables were measured: scar colour, scar thickening, contractures, range of motion, the patient's level of general activity, any breakdown of the graft site, and adequacy of the consultation. Agreement between the two consultants when seeing patients FTF was moderately high, with an overall concordance of 85%. When videoconferencing was used, the level of agreement was almost the same, at 84%. If one consultant reviewed patients FTF first and then via videoconference, the overall concordance was 98%; if the process was reversed, the overall concordance was 97%. This study confirms that the quality of information collected during a videoconference appointment is comparable to that collected during a traditional, FTF appointment for a follow-up burns consultation.

Paediatric palliative home care with Internet-based video-phones: lessons learnt

We have designed and tested an Internet-based video-phone suitable for use in the homes of families in need of paediatric palliative care services. The equipment uses an ordinary telephone line and includes a PC, Web camera and modem housed in a custom-made box. In initial field testing, six clinical consultations were conducted in a one-month trial of the videophone with a family in receipt of palliative care services who were living in the outer suburbs of Brisbane. Problems with variability in call quality-namely audio and video freezing, and audio break-up-prompted further laboratory testing. We completed a programme of over 250 test calls. Fixing modem connection parameters to use the V.34 modulation protocol at a set bandwidth of 24 kbit/s improved connection stability and the reliability of the video-phone. In subsequent field testing 47 of 50 calls (94%) connected without problems. The freezes that did occur were brief (with greatly reduced packet loss) and had little effect on the ability to communicate, unlike the problems arising in the home testing. The low-bandwidth Internet-based video-phone we have developed provides a feasible means of doing telemedicine in the home.

Analyzing checkpoint controls in human skin

A short-term whole-skin organ culture model has been established to enable the investigation of cell cycle perturbations in epidermal layer cells following exposure to ultraviolet radiation (UVR). This model affords the opportunity to manipulate the growth and nutrient conditions, and to perform detailed biochemical and immunohistochemical analysis of skin cells in their normal epidermal layer microenvironment. The use of this model is described in this chapter.