Assessing the effects of bladder decellularization protocols on extracellular matrix (ECM) structure, mechanics, and biology.

INTRODUCTION: Acellular matrices have historically been applied as biologic scaffolds in surgery, wound care, and tissue engineering, albeit with inconsistent outcomes. One aspect that varies widely between products is the selection of decellularization protocol, yet few studies assess comparative effectiveness of these protocols in preserving mechanics, and protein content. This study characterizes bladder acellular matrix (BAM) using two different detergent and enzymatic protocols, evaluating effects on nuclei and DNA removal (≥90%), structure, tensile properties, and maintenance of extracellular matrix proteins. METHODS: Porcine bladders were decellularized with 0.5% Sodium Dodecyl Sulfate (SDS) or 0.25% Trypsin-hypotonic-Triton X-100 hypertonic (TT)-based agitation protocols, followed by DNase/RNase agents. Characterization of BAM included decellularization efficacy (DAPI, DNA quantification), structure (histology and scanning electron microscopy), tensile testing (Instron 345C-1 mechanical tester), and protein presence and diversity (mass spectrometry). SDS and TT data was directly compared to the same native bladder using two-tailed paired t-tests. Native, TT, and SDS cohorts for tensile testing were compared using one-way ANOVA; Tukey's post-hoc tests for among group differences. RESULTS: Effective nuclei removal was achieved by SDS- and TT-based protocols. However, target DNA removal was achieved with SDS but not TT. SDS more effectively maintained qualitative tissue architecture compared to TT. The tensile modulus of the TT cohort increased, and stretchability decreased after decellularization in both SDS and TT. UTS was unaffected by either protocol. Higher overall diversity and quantity of core matrisome and matrisome-associated proteins was maintained in the SDS vs TT cohort post-decellularization. CONCLUSION: The results indicated that detergent selection affects multiple aspects of the resultant BAM biologic product. In the selected protocols, SDS was superior to TT efficacy, and maintenance of gross tissue architecture as well as maintenance of ECM proteins. Decellularization increased scaffold resistance to deformation in both cohorts. Future studies applying biologic scaffolds must consider the processing method and agents used to ensure that materials selected are optimized for characteristics that will facilitate effective translational use.

The interplay of sun damage and genetic risk in Australian multiple and single primary melanoma cases and controls.

BACKGROUND: Skin phenotype, host genotype and ultraviolet (UV) damage play a role in the development of melanoma. OBJECTIVES: To ascertain whether the level of UV damage at the site of melanomas was associated with genetic polymorphisms. METHODS: Deep phenotyping was performed on 1244 individuals; 281 with multiple primary melanomas (MPMs), 304 with single primary melanoma (SPM) and 659 convenience controls. Genotype data was generated using the Illumina CoreExome microarray platform, assaying over 500 000 single-nucleotide polymorphisms. A subset of variants were combined to assess a polygenic risk score (PRS) for melanoma. RESULTS: Most MPM cases were diagnosed in patients aged > 40 years, in sites with visible chronic UV damage. Women and those diagnosed at age ≤ 40 years were less likely to have perilesional UV damage. Patients with MPM had higher frequencies of MITF E318K, MC1R R-alleles and the ASIP risk haplotype. Individuals who had melanoma in a visibly UV-damaged site were more likely to carry MC1R rs75570604 [odds ratio (OR) 2·5], 9q31.2 rs10816595 (OR 1·4) and MTAP rs869329 (OR 1·4). These same alleles were more common in patients with MPM who were diagnosed at age ≤ 40 years. The mean PRS was significantly higher in MPM than in SPM and controls. Naevus count was comparable in early-onset MPM cases and those diagnosed at age > 40 years. CONCLUSIONS: Our cohort demonstrated higher frequencies of previously reported alleles associated with melanoma. MPM melanomas more commonly occur in UV-damaged areas, and these individuals are more likely to carry MC1R red hair colour alleles. Awareness of the interplay of genetic vulnerability with UV damage can stratify risk and guide recommendations for melanoma screening. What's already known about this topic? Skin phenotype, host genotype and ultraviolet (UV) damage all play a role in melanoma development. One of the main risk factors is a personal history of melanoma; second and subsequent primary melanomas account for over 20% of all melanomas registered in Queensland. Multiple loci are associated with melanoma risk, including many low-penetrance loci, which may have a cumulatively significant risk. Population-wide screening programmes for melanoma are not yet economically viable. What does this study add? Patients diagnosed with melanoma at age ≤ 40 years were more likely than older patients to have melanomas in non-UV-damaged sites. Patients with multiple melanomas had higher frequencies of MITF E318K, MC1R R-alleles, and the ASIP extended risk haplotype than patients with single melanoma. CDKN2A, MC1R and MTAP variants were more frequent in patients who developed melanomas at a younger age, but also in those whose melanomas were all on visibly UV-damaged sites. What is the translational message? Incorporating these genetic findings into the known risk factors of skin phenotype and visible UV damage may allow for a more customized and economically feasible approach to early detection of melanoma, particularly in younger patients. Plain language summary available online.

High naevus count and MC1R red hair alleles contribute synergistically to increased melanoma risk.

BACKGROUND: A high total body naevus count is the highest risk factor for melanoma; the phenotype of red hair colour, freckling and pale skin that burns easily, produced by MC1R R alleles, also predisposes to melanoma. OBJECTIVES: To determine whether the known melanoma risk factors of high naevus count and red hair or MC1R R alleles act synergistically to increase melanoma risk. METHODS: The Brisbane Naevus Morphology Study involved 1267 participants from volunteers presenting at a melanoma unit, dermatology outpatient clinic, private dermatology clinics, the Brisbane Longitudinal Twin Study and the QSkin Study. We examined pigmentation characteristics, total body naevus ≥ 5 mm count, and MC1R, ASIP and CDKN2A genotype in participants with and without a personal history of melanoma, living in Queensland, Australia, which is an area of high ultraviolet radiation. RESULTS: Cases were older than controls (median 57 vs. 33 years). Compared with individuals with dark brown hair and zero to four naevi, individuals with red hair and ≥ 20 naevi had a melanoma odds ratio of 10·0 (95% confidence interval 4·2-24·3). Individuals with MC1R R/R genotype and ≥ 20 naevi (≥ 5 mm diameter) had a melanoma odds ratio of 25·1 (95% confidence interval 8·4-82·7) compared with wild-type (WT)/WT individuals with zero to four naevi. The highest risk group is Australian men with the MC1R R/R genotype and ≥ 20 moles, who have an absolute risk of melanoma to age 75 years of 23·3%, compared with 0·8% for men with the WT/WT genotype and zero to four naevi. CONCLUSIONS: Patients who live in areas of high ultraviolet radiation, and have many large naevi and the red hair colour phenotype, particularly those with the MC1R R/R genotype, have a high risk of melanoma above the threshold recommended for screening in other cancers. Therefore, they should undergo intensive physician-led surveillance. What's already known about this topic? A high number of acquired melanocytic naevi, the red hair phenotype and MC1R R alleles all independently increase melanoma risk. Women with atypical naevi have an increasing melanoma risk gradient from darker hair to lighter hair. Women with many naevi have an increasing melanoma risk gradient from those with no elements of the red hair phenotype, to those with freckles but not red hair, to those with red hair. What does this study add? In Queensland, Australia, people with ≥ 20 naevi (≥ 5 mm diameter) and MC1R R/R genotype have a 25-fold increased melanoma risk, relative to people with zero to four naevi and the MC1R WT/WT genotype. In Queensland, individuals with ≥ 20 naevi and the MC1R R/R genotype have an absolute melanoma risk to age 75 years of 23·3% for men and 19·3% for women. This effect is independent of CDKN2A genotype. Further research is required to determine the effect of areas of lower ultraviolet radiation, as this study took place in the Queensland, Australia, which is an area of high ultraviolet radiation. MC1R R/r genotype is associated with increased total body naevus count but this is not the case for R/R. What is the translational message? Patients with many large naevi and the red hair colour phenotype, particularly those with an MC1R R/R genotype, have an unusually high risk of melanoma. In a high ultraviolet environment, this risk exceeds the threshold recommended for screening in other cancers, and such individuals should undergo intensive, regular, physician-led surveillance. Patients with many large naevi but with non-red colour hair may benefit further from clinical MC1R genotyping.

Phenotypic and genotypic analysis of amelanotic and hypomelanotic melanoma patients.

BACKGROUND: Amelanotic/hypomelanotic melanoma is associated with poorer outcomes due to a more advanced disease stage at diagnosis. OBJECTIVE: To determine phenotypic risks and genotypic associations with amelanotic/hypomelanotic melanoma to develop a clinical and genetic profile that could assist in identifying high-risk individuals. METHODS: The Brisbane Naevus Morphology Study conducted from 2009 to 2016 has recruited a core of 1254 participants. Participants were drawn from a combination of volunteers from dermatology outpatient clinics, private dermatology clinics, the Brisbane Longitudinal Twin Study and QSkin study. Case participants had a personal history of melanoma and control participants no personal history of melanoma. We specifically examined seven known candidate pigmentation and melanoma genes and pigmentary phenotypic characteristics in participants with amelanotic/hypomelanotic melanoma compared to pigmented melanomas. This assayed single nucleotide polymorphisms in MC1R, TYR, HERC/OCA2, IRF4, MTAP, PLA2G6 and MITF. RESULTS: Forty-seven participants had at least one amelanotic/hypomelanotic melanoma, and 389 had pigmented melanomas, with amelanotic/hypomelanotic melanoma patients significantly older than pigmented melanoma participants (63.3 ± 13.0 vs. 54.6 ± 15.3 years; P < 0.001). Amelanotic/hypomelanotic melanoma patients were more likely than pigmented melanoma patients to have red hair (34% vs. 15%; P = 0.01), severe hand freckling (13% vs. 5%; P = 0.01) and propensity to sunburn (63% vs. 44%; P = 0.01). MC1R R/R genotype was much more frequent in our amelanotic/hypomelanotic melanoma population (31.1% vs. 11%; P < 0.001; OR 26.4 vs. 5.9; control 1.0). Amelanotic/hypomelanotic melanoma was associated with TYR rs1126809*A/A [OR (CI 95%) 2.7 (1.1-6.8) vs. 1.2 (0.8-1.9)] and PLA2G6 rs11570734*A/A [OR (CI 95%) 3.7 (1.0-13.6) vs. 1.3 (0.9-2.0)]. The MTAP melanoma risk SNP genotype, associated with darker pigmentation, (rs4636294*A/A) was less common in amelanotic/hypomelanotic melanoma patients [OR (CI 95%) 0.8 (0.3-2.1) vs. 2.0 (1.3-3.1)]. CONCLUSIONS: Knowledge of phenotypic and genotypic associations of amelanotic/hypomelanotic melanoma can help predict risks and associations of this difficult to diagnose melanoma, which may ultimately assist clinical management and patient skin self-examination.

Iris pigmented lesions as a marker of cutaneous melanoma risk: an Australian case-control study.

BACKGROUND: Iris naevi and iris freckles have a frequency of 4% and 50% in the European population, respectively. They are associated with dysplastic naevi, but few studies have examined their link to cutaneous melanoma. OBJECTIVES: To assess whether iris pigmented lesions are a predictive indicator for cutaneous melanoma. METHODS: This is a melanoma case-control study of 1254 European-background Australians. Sun exposure and melanoma history, a saliva sample for DNA analysis and eye photographs taken with a digital camera were collected from 1117 participants. Iris images were assessed by up to four trained observers for the number of iris pigmented lesions. The data were analysed for correlations between iris pigmented lesions and melanoma history. RESULTS: Case participants over the age of 40 had similar numbers of iris pigmented lesions to age matched controls (mean 5·7 vs. 5·2, P = 0·02), but in younger case and control participants there was a greater difference (mean 3·96 vs. 2·19, P = 0·004). A logistic regression adjusted for age, sex, skin, hair and eye colour, skin freckling and naevus count found that the presence of three or more iris pigmented lesions increases the melanoma risk 1·45-fold [95% confidence interval (CI) 1·07-1·95]. HERC2/OCA2 rs12913832 and IRF4 rs12203592 influenced both eye colour and the number of iris pigmented lesions. On the HERC2/OCA2 A/A and A/G genotype background there was an increasing proportion of blue eye colour when carrying the IRF4 T allele (P = 3 × 10-4 ) and a higher number of iris pigmented lesions with the IRF4 T/T homozygote (P = 3 × 10-9 ). CONCLUSIONS: Iris pigmented lesion count provides additional predictive information for melanoma risk above that from conventional risk factors.

The BRAF and NRAS mutation prevalence in dermoscopic subtypes of acquired naevi reveals constitutive mitogen-activated protein kinase pathway activation.

BACKGROUND: Acquired naevi can have unique dermoscopic patterns that correspond to distinct microanatomical growth patterns. Previous studies on acquired naevi stratified according to dermoscopic pattern focused on the frequency of somatic BRAF mutations, whereas NRAS mutations remained to be elucidated. OBJECTIVES: To investigate the BRAF and NRAS mutation prevalence and activation of the mitogen-activated protein kinase (MAPK) pathway in distinct dermoscopic subtypes of acquired naevi. METHODS: Common mutations present in BRAF and NRAS were assessed in 40 globular, reticular and peripheral rim of globules (PG) subtypes of acquired naevi from 27 participants (19 male, 8 female; mean age 46·7 years) selected from 1261 eligible volunteers. Mutations were determined using the highly sensitive and quantitative QX200 droplet digital™ polymerase chain reaction (ddPCR) system. RESULTS: The BRAF V600E (c.1799T>A or c.1799_1800delTGinsA) and BRAF V600K mutations were detected in 85% (n = 34/40) of naevi. All BRAF wild-type naevi (15%; n = 6/40) harboured an NRAS codon 12/13 or 61 mutation. BRAF mutations were present in 92% (n = 12/13) of globular and 100% (n = 12/12) of PG naevi, whereas reticular naevi were 67% (n = 10/15) BRAF- and 33% (n = 5/15) NRAS-mutant (P = 0·037). CONCLUSIONS: We discovered that 100% of the assessed acquired naevi had either a BRAF or NRAS mutation. Using sensitive techniques capable of single-cell mutation detection, it is likely that all acquired naevi will be mutated for BRAF or NRAS. Because both of these mutations are prevalent in distinct dermoscopic naevus subsets, our study supports the role of the MAPK pathway in the development of benign melanocytic proliferations, indicating that additional genomic events besides somatic mutations in BRAF or NRAS are required for melanoma development.

Heritability of naevus patterns in an adult twin cohort from the Brisbane Twin Registry: a cross-sectional study.

BACKGROUND: Heritability of naevi counts is widely acknowledged as a potential surveillance parameter for prevention purposes. The contribution of heritability to the changes seen in naevus number and morphology over time and their corresponding dermoscopic characteristics is unknown, but is important to understand in order to account for adequate prevention measures. OBJECTIVES: To identify naevus characteristics that are strongly influenced by heritability. METHODS: This cross-sectional study included 220 individuals [76 monozygotic (MZ), 144 dizygotic (DZ)], recruited from the Brisbane Twin Naevus Study. Participants received full body imaging and dermoscopy of naevi ≥ 5 mm in diameter. Dermoscopic type, total naevus count (TNC), change in TNC with age, and naevus distribution, size, colour and profile were compared between MZ and DZ twins. Heritability of these traits was assessed via Falconer's estimate. RESULTS: Significant differences were found in comparing MZ and DZ twins for TNC, numbers of naevi 5·0-7·9 mm in diameter, counts of light-brown naevi, naevi on the back and sun-protected sites, and naevi with the 'nonspecific' dermoscopic pattern. CONCLUSIONS: This study strongly supports a heritable component to TNC, as well as changes in TNC, and the number of medium-sized naevi, light-brown naevi, specific sites and certain dermoscopic features in adults. These characteristics should be taken into account by naevus surveillance programmes and further studied to identify candidate gene associations for clinical and dermoscopic patterns in conjunction with melanoma risk stratification.

Comparison of clinical outcomes between "ideal" and "nonideal" transobturator male sling patients for treatment of postprostatectomy incontinence.

OBJECTIVE: To review the clinical outcomes of "ideal" vs "nonideal" postprostatectomy stress urinary incontinence (PPI) patients who underwent male sling placement. METHODS: The medical records of 95 consecutive patients with PPI who underwent male sling insertion (AdVance male sling, American Medical Systems, Minnetonka, MN) were reviewed. Patients were divided into "ideal" vs "nonideal" cohorts. The ideal group consisted of patients with mild to moderate incontinence (<4 pads/day or <300 g daily pad weight), ability to volitionally contract the external urinary sphincter, no history of pelvic radiation or cryotherapy, no history of previous anti-incontinence surgical procedures, the ability to generate a volitional detrusor contraction when voiding, and a postvoid residual urine volume <100 mL. Patients in the nonideal group did not satisfy all these criteria. RESULTS: Significant reductions in daily pad usage and weight were noted in both cohorts. In the ideal patient group, 66 of 72 patients (92%) would undergo the procedure again. Conversely, only 7 of 23 nonideal men (30%) would undergo the procedure again. CONCLUSION: Preoperative patient selection can influence favorable outcomes after the treatment of PPI with AdVance male slings. Attention to ideal vs nonideal patient characteristics should be used when counseling men considering male sling surgery.

The effects of obesity, smoking, and excessive alcohol intake on healthcare expenditure in a comprehensive medical scheme.

BACKGROUND: Health risks such as tobacco use, excessive alcohol consumption and unhealthy body weight contribute to the development of chronic health problems. OBJECTIVE: To estimate the associations of tobacco use, excessive alcohol consumption and obesity with healthcare expenditure and chronic diseases among South Africans on a comprehensive medical scheme. METHODS: We performed a cross-sectional analysis of health survey and medical claims data for 70 000 South Africans during 2010. RESULTS: Moderately obese individuals, with a body mass index (BMI) of 30 - 35 kg/m2 averaged R2 300 (11%) higher annual medical expenditure in the year 2010 than never-smokers with a BMI <30 kg/m2. This increase is comparable with being a current or past smoker (expenditure increase by R2 600; 13%). Severely obese individuals (BMI >35 kg/m2), however, had increased healthcare costs of R4 400 (23% increase). This exceeds the difference between a 40- and a 50-year-old (increase of R3 200). Being overweight or excessive consumption of alcohol was not significantly associated with medical expenditures. Absolute and relative excess expenditures associated with these health risks are higher among older individuals. In the 54 - 69-year age group, estimated additional expenses were R6 200 for smoking (20% increase over never-smokers with BMI <30 kg/m2), R6 600 for moderate obesity (21%) and R15 800 for severe obesity (51% increase). Overweight or heavy drinking was not statistically significantly associated with healthcare expenditure. CONCLUSION: Obesity and tobacco use are associated with significantly increased healthcare expenditure. Severe obesity doubles these excess costs.

Provincial screening rates for chronic diseases of lifestyle, cancers and HIV in a health-insured population.

BACKGROUND: Screening for asymptomatic diseases can reduce the burden of morbidity and mortality in all population groups. There is widespread geographical variation in the quality of care. Few data are available on national screening rates in South Africa and how these vary across the provinces. OBJECTIVE: To examine screening rates for chronic diseases of lifestyle (CDL), HIV and cancer in a privately insured population for a single insurer across all nine provinces in South Africa, and to determine whether or not there are any differences between the provinces. METHOD: Screening rates were calculated as the proportion of eligible members who had received screening tests during 2011 in each province. Mean screening rates were compared between Gauteng and the other eight provinces. RESULTS: Nationwide screening rates were 20.5% for CDL, 8.2% for HIV and 31.9% for cancer. Despite similar insurance coverage, screening rates ranged from 0.3% to 0.95% lower in other provinces compared with Gauteng. Of all the provinces, Gauteng had the highestannual screening rates for CDL, breast cancer, prostate cancer and HIV (p < 0.001), while the Western Cape had the highest rate for cervical cancer (p < 0.001). CONCLUSION: There is much variation in preventive care utilisation across the provinces within this health-insured population. Provinces with more abundant healthcare resources have higher screening rates. Further research is required to understand the reasons for the variation, given equal payment access.

Morbid obesity rates continue to rise rapidly in the United States.

Clinically severe or morbid obesity (body mass index (BMI) >40 or 50 kg m(-2)) entails far more serious health consequences than moderate obesity for patients, and creates additional challenges for providers. The paper provides time trends for extreme weight categories (BMI >40 and >50 kg m(-2)) until 2010, using data from the Behavioral Risk Factor Surveillance System. Between 2000 and 2010, the prevalence of a BMI >40 kg m(-2) (type III obesity), calculated from self-reported height and weight, increased by 70%, whereas the prevalence of BMI >50 kg m(-2) increased even faster. Although the BMI rates at every point in time are higher among Hispanics and Blacks, there were no significant differences in trends between them and non-Hispanic Whites. The growth rate appears to have slowed down since 2005. Adjusting for self-report biases, we estimate that in 2010 15.5 million adult Americans or 6.6% of the population had an actual BMI >40 kg m(-2). The prevalence of clinically severe obesity continues to be increasing, although less rapidly in more recent years than prior to 2005.

Is there a robust relationship between neighbourhood food environment and childhood obesity in the USA?

OBJECTIVES: To examine the robustness of the relationship between neighbourhood food environment and youth body mass index (BMI) percentile using alternative measures of food environment and model specifications. STUDY DESIGN: Observational study using individual-level longitudinal survey data of children in fifth and eighth grades merged with food outlet data based on student residential census tracts. METHODS: The relationship between food environment and BMI was examined with two individual outcomes (BMI percentile in eighth grade and change in BMI percentile from fifth to eighth grade) and three alternative measures of food environment (per-capita counts of a particular outlet type, food environment indices, and indicators for specific combinations of outlet types). RESULTS: No consistent evidence was found across measures (counts of a particular type of food outlet per population, food environment indices, and indicators for the presence of specific combinations of types of food stores) and outcomes to support the hypothesis that improved access to large supermarkets results in lower youth BMI; or that greater exposure to fast food restaurants, convenience stores and small food stores increases BMI. CONCLUSIONS: To the extent that there is an association between food environment and youth BMI, the existence of more types of food outlets in an area, including supermarkets, is associated with higher BMI.

Osteopontin expression in plasma of melanoma patients and in melanocytic tumours.

BACKGROUND: While the serological tumour marker S100 is well established for the detection of metastatic melanoma, the extracellular matrix protein osteopontin (OPN) seems to be a promising novel marker for invasive melanoma. OBJECTIVES: We analysed the potential of OPN as a serological tumour marker for metastatic melanoma and evaluated its combination with S100 and lactate dehydrogenase (LDH) levels to increase the reliability of these biomarkers for the detection of metastatic disease. METHODS: We examined OPN in the peripheral blood of 110 melanoma patients using enzyme-linked immunosorbent assay and combined it with S100 and LDH levels. In addition, the protein expression of OPN was analysed in tissue sections of melanocytic nevi and melanomas of different progression stages by immunohistochemistry. RESULTS: The independent comparison of S100 and OPN levels in metastatic vs. non-metastatic patients revealed a P-value <0.001 respectively. The predictiveness of OPN, S100 and LDH was 0.85, 0.89 and 0.69 as measured by the area under the receiver operating curve (AUC) respectively, while the combination of the two biomarkers OPN and S100 showed an AUC of 0.97. The optimal cut-off of the combination of OPN and S100 yielded a specificity of 85.9% and a sensitivity of 95.5%. By immunohistochemistry, OPN protein expression was detected in 29% (7/24) of melanocytic nevi, 67% (30/45) of primary melanomas and 39% (7/18) of metastatic melanomas. CONCLUSIONS: Together, OPN seems to be a promising novel biomarker for the detection of metastatic disease in melanoma patients, showing elevated plasma levels in metastatic disease and increased protein expression in melanocytic lesions. The combination of OPN with the well-established tumour marker S100 might increase the prediction of metastases.