Collagen in the scarless fetal skin wound: Detection with Picrosirius-polarization

Our group has developed an ovine model of deep dermal, partial-thickness burn where the fetus heals scarlessly and the lamb heals with scar. The comparison of collagen structure between these two different mechanisms of healing may elucidate the process of scarless wound healing. Picrosirius staining followed by polarized light microscopy was used to visualize collagen fibers, with digital capture and analysis. Collagen deposition increased with fetal age and the fibers became thicker, changing from green (type III collagen) to yellow/red (type I collagen). The ratio of type III collagen to type I was high in the fetus (166), whereas the lamb had a much lower ratio (0.2). After burn, the ratios of type III to type I collagen did not differ from those in control skin for either fetus or lamb. The fetal tissue maintained normal tissue architecture after burn while the lamb tissue showed irregular collagen organization. In conclusion, the type or amount of collagen does not alter significantly after injury. Tissue architecture differed between fetal and lamb tissue, suggesting that scar development is related to collagen cross-linking or arrangement. This study indicates that healing in the scarless fetal wound is representative of the normal fetal growth pattern, rather than a response to burn injury.

An innovative method of increasing early detection for skin cancer in Australia

Objective: To evaluate a family practice intervention to encourage patients to request a skin examination during their consultation. Methods: Family physicians in Queensland, Australia, were randomized to intervention or control groups. In the intervention group, materials were provided by the office receptionist and supported by the family physician. Results: The rate of full-body skin examination was 99.3/ 1000 consultations in intervention-group practices compared to 22.4/ 1000 in control-group practices (p

Simultaneous detection of multiple antigens with triple immunolabeling

Immunolabeling is commonly used to localize antigens within frozen or paraffin tissue sections. We modified existing immunolabeling techniques to allow the detection of three antigens simultaneously within the one tissue section. The approach relies on the use of three monoclonal antibodies in sequential immunoperoxidase staining steps, each with colored substrates, resulting in the deposition of black, brown, and rose stains. The method is rapid and does not require novel techniques or materials. In this report, we demonstrate the colocalization of mast cell tryptase, neurofilament protein, and CD31 (platelet-endothelial cell adhesion molecule) or laminin in normal human skin and normal buccal mucosa, as an illustration of the power and simplicity of the multiple antigen localization technique.

Improved detection of lacZ reporter gene expression in transgenic epithelia by immunofluorescence microscopy

The bacterial lacZ gene is commonly used as a reporter for the in vivo analysis of gene regulation in transgenic mice. However, several laboratories have reported poor detection of beta-galactosidase (the lacZ gene product) using histochemical techniques, particularly in skin. Here we report the difficulties we encountered in assessing lacZ expression in transgenic keratinocytes using classic X-gal histochemical protocols in tissues shown to express the transgene by mRNA in situ hybridization. We found that lacZ reporter gene expression could be reliably detected in frozen tissue sections by immunofluorescence analysis using a beta-galactosidase-specific antibody. Moreover, we were able to localize both transgene and endogenous gene products simultaneously using double-label immunofluorescence. Our results suggest that antibody detection of beta-galactosidase should be used to verify other assays of lacZ expression, particularly where low expression levels are suspected or patchy expression is observed.

Detection of differentially expressed genes in synovial fibroblasts by restriction fragment differential display

Objective. To identify differentially expressed genes in synovial fibroblasts and examine the effect on gene expression of exposure to TNF-alpha and IL-1beta. Methods. Restriction fragment differential display was used to isolate genes using degenerate primers complementary to the lysophosphatidic acid acyl transferase gene family. Differential gene expression was confirmed by reverse transcription-polymerase chain reaction and immunohistochemistry using a variety of synovial fibroblasts, including cells from patients with osteoarthritis and self-limiting parvovirus arthritis. Results. Irrespective of disease process, synovial fibroblasts constitutively produced higher levels of IL-6 and monocyte chemoattractant protein 1 (MCP-1) (CCL2) than skin fibroblasts. Seven genes were differentially expressed in synovial fibroblasts compared with skin fibroblasts. Of these genes, four [tissue factor pathway inhibitor 2 (TFPI2), growth regulatory oncogene beta (GRObeta), manganese superoxide dismutase (MnSOD) and granulocyte chemotactic protein 2 (GCP-2)] were all found to be constitutively overexpressed in synoviocytes derived from patients with osteoarthritis. These four genes were only weakly expressed in other synovial fibroblasts (rheumatoid and self-limiting parvovirus infection). However, expression in all types of fibroblasts was increased after stimulation with TNF-alpha and IL-1beta. Three other genes (aggrecan, biglycan and caldesmon) were expressed at higher levels in all types of synovial fibroblasts compared with skin fibroblasts even after stimulation with TNF-alpha and IL-1. Conclusions. Seven genes have been identified with differential expression patterns in terms of disease process (osteoarthritis vs rheumatoid arthritis), state of activation (resting vs cytokine activation) and anatomical location (synovium vs skin). Four of these genes, TFPI2, GRObeta (CXCL2), MnSOD and GCP-2 (CXCL6), were selectively overexpressed in osteoarthritis fibroblasts rather than rheumatoid fibroblasts. While these differences may represent differential behaviour of synovial fibroblasts in in vitro culture, these observations suggest that TFPI2, GRObeta (CXCL2), MnSOD and GCP-2 (CXCL6) may represent new targets for treatments specifically tailored to osteoarthritis.

Prevalence of whole-body skin self-examination in a population at high risk for skin cancer (Australia)

Objective: Whole-body skin self-examination (SSE) with presentation of suspicious lesions to a physician may improve early detection of melanoma. The aim of this study was to establish the prevalence and determinants of SSE in a high-risk population in preparation for a community-based randomised controlled trial of screening for melanoma. Methods: A telephone survey reached 3110 residents older than 30 years (overall response rate of 66.9%) randomly selected from 18 regional communities in Queensland, Australia. Results: Overall, 804 (25.9%) participants reported whole-body SSE within the past 12 months and 1055 (33.9%) within the past three years. Whole-body SSE was associated in multivariate logistic regression analysis with younger age (< 50 years); higher education; having received either a whole-body skin examination, recommendation or instruction on SSE by a primary care physician; giving skin checks a high priority; concern about skin cancer and a personal history of skin cancer. Conclusion: Overall, the prevalence of SSE in the present study is among the highest yet observed in Australia, with about one-third of the adult population reporting whole-body SSE in the past three years. People over 50 years, who are at relatively higher risk for skin cancer, currently perform SSE less frequently than younger people.

Community perceptions of suspicious pigmented skin lesions: are they accurate when compared to general practitioners?

Community responses (n = 925, response rate = 71%) of a series of eight photographs of pigmented skin lesions were compared against those of general practitioners (n = 114, response rate = 77%), considered to be the most relevant gold standard. The eight photographs included three melanomas, two potentially malignant lesions and three benign pigmented lesions. Over the pool of lesions examined, the average probability that community members thought a lesion was likely to be skin cancer (0.68 [99% CI = 0.66-0.69]) was higher (p < 0.0001) than that of the comparison general practitioners 0.58 [99% CI = 0.55-0.62]. This reflects a general (but not consistent) inflated propensity to over-diagnose among community members. The average probability that respondents indicated they would seek medical advice for a lesion was 0.71 [99% CI = 0.70-0.73]. As expected, this was strongly associated with their perceptions of the skin lesion. These results suggest that the community can play a valuable role in assessing the need for medical evaluation of pigmented skin lesions. (c) 2004 International Society for Preventive Oncology. Published by Elsevier Ltd. All rights reserved.

Clinical outcomes from skin screening clinics within a community-based melanoma screening program

Background : Within a randomized trial of population screening for melanoma, primary care physicians conducted whole-body skin examinations and referred all patients with suspect lesions to their own doctor for further treatment. Objective: Our aim was to describe characteristics of skin screening participants, clinical screening diagnoses, management following referral, and specificity and yield of screening examinations. Methods: Information collected from consent forms, referral forms, and histopathological reports of lesions that had been excised or undergone biopsy was analyzed by means of descriptive statistics. Results: A total of 16,383 whole-body skin examinations resulted in 2302 referrals (14.1% overall; 15.5% men, 18.2% >= 50 years of age) for 4129 suspect lesions (including 222 suspected melanoma, 1101 suspected basal cell carcinomas [BCCs], 265 suspected squamous cell carcinomas [SCCs]). Histopathologic results were available for 94.8% of 1417 lesions excised and confirmed 33 melanomas (23 in men; 24 in participants ? 50 years of age), 259 BCCs, and 97 SCCs. The probability of detecting skin cancer of any type within the program was 2.4%. The estimated specificity of whole-body skin examinations for melanoma was 86.1% (95% confidence interval = 85.6-86.6). The positive predictive value (number of confirmed/number of lesions excised or biopsied x 100) for melanoma was 2.5%, 19.3% for BCC, and 7.2% for SCC (overall positive predictive value for skin cancer, 28.9%). Limitations: Follow-up of participants with a negative screening examination has not been conducted for the present investigation. Conclusions: The rate of skin cancer detected per 100 patients screened was higher than previously reported and men and attendees older than 50 years more frequently received a referral and diagnosis of melanoma. The specificity for detection of melanoma through whole-body skin examination by a primary care physician was comparable to that of other screening tests, including mammography.

Increase in skin cancer screening during a community-based randomized intervention trial

Survival from cutaneous melanoma is mainly dependent on the thickness of the lesion at diagnosis. Skin screening may increase detection of thin lesions and hence improve survival. Within a community-based randomized controlled trial of a population screening program for melanoma in Queensland, Australia, 9 communities were randomly assigned to the 3-year intervention and 9 communities to the control group. Skin screening prevalence was monitored by cross-sectional surveys at baseline, 1, 2 and 3 years into the intervention and 2 years later. At baseline, prevalence of whole-body clinical skin examination was similar in intervention and control communities. In intervention communities, the prevalence of whole-body skin examinations increased to 29.2%, an absolute difference of 18% from baseline, with a peak of 34.8% 2 years after baseline, and began to decline again at the end of the intervention period. The largest increases were seen in men and women ≥50 years. Uptake of screening did not differ according to melanoma risk factors; however, the decline in screening was less in participants who reported a number of melanoma risk factors. The prevalence of skin self-examination remained stable during the intervention program. No changes were observed in the control communities. These results indicate that the intervention program significantly increased the prevalence of whole-body clinical skin examinations in intervention communities. Once the intervention program ceased, and particularly after skin clinics ceased, levels of skin screening began to decline. The provision of specialized skin screening clinics may be needed to achieve sufficient screening rates should population based screening for skin cancer be considered. © 2005 Wiley-Liss, Inc.

Presentation and detection of invasive melanoma in a high-risk population

Background: Early detection of melanoma has been encouraged in Queensland for many years, yet little is known about the patterns of detection and the way in which they relate to tumor thickness. Objective: Our purpose was to describe current patterns of melanoma detection in Queensland. Methods: This was a population-based study, comprising 3772 Queensland residents diagnosed with a histologically confirmed melanoma between 2000 and 2003. Results: Almost half (44.0%) of the melanomas were detected by the patients themselves, with physicians detecting one fourth (25.3%) and partners one fifth (18.6%). Melanomas detected by doctors were more likely to be thin (\0.75 mm) than those detected by the patient or other layperson. Melanomas detected during a deliberate skin examination were thinner than those detected incidentally. Limitations: Although a participation rate of 78% was achieved, as in any survey, nonresponse bias cannot be completely excluded, and the ability of the results to be generalized to other geographical areas is unknown. Conclusion: There are clear differences in the depth distribution of melanoma in terms of method of detection and who detects the lesions that are consistent with, but do not automatically lead to, the conclusion that promoting active methods of detection may be beneficial. ( J Am Acad Dermatol 2006;54:783-92.)

Quantitative analysis of MC1R gene expression in human skin cell cultures

To address the issue of melanocortin-1 receptor (MC1R) expression in non-melanocytic cells, we have quantitatively evaluated the relative expression levels of both MC1R mRNA and protein in a subset of different cell types. Using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) at high cycle numbers, we detected MC1R mRNA in all cell types examined, including human embryonic kidney-293 (HEK 293) cells, a cell type widely used as a negative control in melanocortin expression studies. Quantitative real-time PCR revealed the highest levels of MC1R transcripts were in melanocytic cells, whereas the keratinocyte and fibroblast cell cultures examined had only a low level of expression, similar to that of HEK 293 cells. Antibody mediated detection of MC1R protein in membrane extracts demonstrated exogenous receptor in MC1R transfected cell lines, as well as endogenous MC1R in melanoma cells. However, radioligand binding procedures were required to detect MC1R protein of normal human melanocytes and no surface expression of MC1R was detected in any of the non-melanocytic cells examined. This was consistent with their low level of mRNA, and suggests that, if present, the levels of surface receptor are significantly lower than that in melanocytes. The capacity of such limited levels of MC1R protein to influence non-melanocytic skin cell biology would likely be severely compromised. Indeed, the MC1R agonist [NIe(4), D-Phe(7)] alpha-melanocyte stimulating hormone (NDP-MSH) was unable to elevate intracellular cyclic adenosine monophosphate (cAMP) levels in the keratinocyte and fibroblast cells examined, whereas a robust increase was elicited in melanocytes. Although there are a variety of cell types with detectable MC1R mRNA, the expression of physiologically significant levels of the receptor may be more restricted than the current literature indicates, and within epidermal tissue may be limited to the melanocyte

A method to lower the detection limit for optical beat signals from a frequency-dithered stabilized laser

A narrow absorption feature in an atomic or molecular gas (such as iodine or methane) is used as the frequency reference in many stabilized lasers. As part of the stabilization scheme an optical frequency dither is applied to the laser. In optical heterodyne experiments, this dither is transferred to the RF beat signal, reducing the spectral power density and hence the signal to noise ratio over that in the absence of dither. We removed the dither by mixing the raw beat signal with a dithered local oscillator signal. When the dither waveform is matched to that of the reference laser the output signal from the mixer is rendered dither free. Application of this method to a Winters iodine-stabilized helium-neon laser reduced the bandwidth of the beat signal from 6 MHz to 390 kHz, thereby lowering the detection threshold from 5 pW of laser power to 3 pW. In addition, a simple signal detection model is developed which predicts similar threshold reductions.