Development and validation of novel keloid-derived immortalized fibroblast cell lines.

Keloids are a common connective tissue disorder with an ill-understood etiopathogenesis and no effective treatment. This is exacerbated because of the absence of an animal model. Patient-derived primary keloid cells are insufficient as they age through passaging and have a limited supply. Therefore, there is an unmet need for development of a cellular model that can consistently and faithfully represent keloid's pathognomic features. In view of this, we developed keloid-derived immortalized fibroblast (KDIF) cell lines from primary keloid fibroblasts (PKF) by transfecting the human telomerase reverse transcriptase (hTERT) gene. The TERT gene encodes the catalytic subunit of the telomerase enzyme, which is responsible for maintaining the cellular replicative potential (cellular immortalization). Primary fibroblasts from keloid-specific lesional (peripheral, middle, and top) as well as extralesional sites were isolated and evaluated for cell line development and comparative cellular characteristics by employing qRT-PCR and immunofluorescence staining. Moreover, the immortalized behavior of KDIF cell lines was evaluated by comparing with cutaneous fibrosarcoma and dermatofibrosarcoma protuberans cell lines. Stable KDIF cell lines with elevated expression of hTERT exhibited the cellular characteristics of site-specific keloid fibroblasts. Histochemical staining for ß-galactosidase revealed a significantly lower number of ß-gal-positive cells in all three KDIF cell lines compared with that in PKFs. The cell growth curve pattern was studied over 10 passages for all three KDIF cell lines and was compared with the control groups. The results showed that all three KDIF cell lines grew significantly faster and obtained a fast growing characteristic as compared to primary keloid and normal fibroblasts. Phenotypic behavior in growth potential is an indication of hTERT-mediated immortalized transformation. Cell migration analysis revealed that the top and middle KDIF cell lines exhibited similar migration trend as site-specific PKFs. Notably, peripheral KDIF cell line showed significantly enhanced cell migration in comparison to the primary peripheral fibroblasts. All KDIF cell lines expressed Collagen I protein as a keloid-associated fibrotic marker. Functional testing with triamcinolone inhibited cell migration in KDIF. ATCC short tandem repeat profiling validated the KDIF as keloid representative cell line. In summary, we provide the first novel KDIF cell lines. These cell lines overcome the limitations related to primary cell passaging and tissue supply due to immortalized features and present an accessible and consistent experimental model for keloid research.

Androgenetic alopecia: An update.

Androgenetic alopecia (AGA) is the most common nonscarring alopecia and is characterised by distinct gradual patterned hair loss. AGA is mediated by genetic predisposition and excessive follicular sensitivity to androgens, mainly in males, leading to the progressive conversion of scalp terminal hair into vellus hair. Although highly prevalent, it is not fatal but may have a severe psychosocial impact, especially on females and younger males. Significant advances have been made in understanding AGA's epidemiology and pathophysiology, but only 2 drugs remain approved by the FDA - finasteride and minoxidil. Prolonged use of these drugs, is a prerequisite for enhanced treatment response. However, this leads to poor medication adherence and adverse effects from extended use eg, the "postfinasteride syndrome" which persists beyond stopping the drug. Hence, there is a need for research on more effective alternative treatments for AGA, with fewer side effects. This paper reviewed recent advances in AGA pathophysiology and its treatment options. The recently characterized structure of type 2, 5-alpha reductase holds significance in comprehending present and prospective treatments of AGA.

FAM111B dysregulation promotes malignancy in fibrosarcoma and POIKTMP and a low-cost method for its mutation screening.

INTRODUCTION: Mutations in the uncharacterised human FAM111B gene are associated with POIKTMP, a rare multi-organ fibrosing disease. Recent studies also reported the overexpression of FAM111B in specific cancers. Moreover, FAM111B mutation screening may prove expensive in under-resourced facilities. Therefore, this study investigated its cellular function and dysfunction and described an inexpensive mutation screening method. MATERIALS AND METHODS: FAM111B expression was assessed in silico and validated in vitro in cell lines and primary skin fibroblasts from a South African POIKTMP-patient with the heterozygous FAM111B gene mutation: NM_198947.4: c.1861T>G (p. Tyr621Asp or Y621D) by qPCR and western blot. The cellular function of FAM111B was studied in HT1080 using various cell-based functional assays, and the Y621D mutation was genotyped by PCR-RFLP. RESULTS: Expression studies showed upregulated FAM111B mRNA and protein in the cancer cells. High FAM111B expression with robust nuclear localization occurred in HT1080. Additionally, expression data and cell-based assays indicated that FAM111B led to the upregulation of cell migration, decreased cell apoptosis, and modulatory effects on cell proliferation. Y621D mutation showed similar effects on cell migration but minimal impact on cell apoptosis. FAM111B mRNA and protein expression were markedly downregulated (p ≤ 0.05) in the POIKTMP-patient's fibroblasts. The PCR-RFLP method successfully genotyped Y621D gene mutation. DISCUSSION: FAM111B is a cancer-associated nuclear protein: Its modulation by mutations or overexpression may contribute to the malignancy of cancers and POIKTMP/fibrosis and poor clinical outcomes and represents a viable prognostic marker or therapeutic target. Furthermore, the PCR-RFLP method could prove a valuable tool for FAM111B mutation validation or screening in resource-constrained laboratories.

Advances in surface modifications of the silicone breast implant and impact on its biocompatibility and biointegration.

Silicone breast implants are commonly used for cosmetic and oncologic surgical indications owing to their inertness and being nontoxic. However, complications including capsular contracture and anaplastic large cell lymphoma have been associated with certain breast implant surfaces over time. Novel implant surfaces and modifications of existing ones can directly impact cell-surface interactions and enhance biocompatibility and integration. The extent of foreign body response induced by breast implants influence implant success and integration into the body. This review highlights recent advances in breast implant surface technologies including modifications of implant surface topography and chemistry and effects on protein adsorption, and cell adhesion. A comprehensive online literature search was performed for relevant articles using the following keywords silicone breast implants, foreign body response, cell adhesion, protein adsorption, and cell-surface interaction. Properties of silicone breast implants impacting cell-material interactions including surface roughness, wettability, and stiffness, are discussed. Recent studies highlighting both silicone implant surface activation strategies and modifications to enhance biocompatibility in order to prevent capsular contracture formation and development of anaplastic large cell lymphoma are presented. Overall, breast implant surface modifications are being extensively investigated in order to improve implant biocompatibility to cater for increased demand for both cosmetic and oncologic surgeries.

National suicide management guidelines recommending family-based prevention, intervention and postvention and their association with suicide mortality rates: systematic review.

BACKGROUND: Suicidal behaviour remains a major public health concern and countries have responded by authoring guidelines to help mitigate death by suicide. Guidelines can include family-based recommendations, but evidence for the level and category of family-based involvement that is needed to effectively prevent suicide is unclear. AIMS: To explore the association between family-based recommendations in guidelines and countries' crude suicide rates. PROSPERO registration: CRD42019130195. METHOD: MEDLINE, Embase, PsycInfo, Web of Science and WHO MiNDbank databases and grey literature were searched within the past 20 years (1 January 2000 to 22 June 2020) for national guidelines giving family-based recommendations in any of three categories (prevention, intervention and postvention). RESULTS: We included 63 guidelines from 46 countries. All identified guidelines included at least one family-based recommendation. There were no statistically significant differences seen between mean World Health Organization crude suicide rates for countries that included only one, two or all three categories of family-based recommendations. However, a lower spread of crude suicide rates was seen when guideline recommendations included all three categories (mean crude suicide rates for one category: 11.09 (s.d. = 5.71); for two categories: 13.42 (s.d. = 7.76); for three categories: 10.68 (s.d. = 5.20); P = 0.478). CONCLUSIONS: Countries should work towards a comprehensive national suicide guideline that includes all categories of family-based recommendations. Countries with previously established guidelines should work towards the inclusion of evidence-based recommendations that have clear implementation plans to potentially help lower suicide rates.

Genetics and Epigenetics of Keloids.

Significance: Keloid scarring is cosmetically disfiguring, psychosocially distressing, and can be physically disabling. The pathophysiology of keloid formation is poorly understood and subsequently, treatment options are ill defined, limited, and largely unsatisfactory. Therefore, in view of its unsatisfactory and recalcitrant management, keloid therapy is often seen as a financial burden affecting both patients and the health care systems. Recent Advances: Increased research on the genetic and epigenetic mechanisms in keloids has broadened our understanding of keloid pathobiology. Epigenetic mechanisms, mainly DNA methylation, histone modification, and noncoding RNAs, are currently being widely investigated. Advances in genetic sequencing technology and reduced cost have aided this endeavor. Studies on blood and patient-derived keloid tissue are being done with therapeutic agents targeting epigenetic and genetic markers with the shared goal of identifying the pathways underlying the initiation and maintenance of keloids. These advances have informed us of multiple complex molecular pathways implicated in keloids, which are yet to be fully elucidated. Critical Issues: Improved understanding of the genetic and epigenetic causes implicated in keloids will enhance our knowledge of this enigmatic disorder and likely lead to the development of therapeutic targets based on the available clinical and experimental studies. Due to the incomplete knowledge of molecular targets involved in keloid scarring pathways, therapeutics is still lagging for this clinically and scientifically important condition. Future Directions: Focused research on the identification of molecular targets and mechanistic pathways implicated in keloids is required to generate novel antifibrotic therapeutic options to decrease or eradicate recurrence of the disease as well as associated morbidity and improve the quality of life of those affected with keloids.

The evolving mechanical response of curly hair fibres subject to fatigue testing.

Cyclic testing of human hair reveals important details about the behaviour of fibres over many cycles of loading. Phenomena which are observed under static tensile tests give important clues about the form and behaviour of hair fibres, but these do not necessarily remain constant on the inevitable march to failure. In previous work, we demonstrated that curly fibres exhibited a toe-region during tensile tests. The form of curly fibres could be altered by mechanical manipulation but the curl could be recovered upon immersion in water. In this study, where straight and curly fibres are subject to cyclic loading, this characteristic toe-region was shown to be present in the first cycle of loading (for curly fibres). As the number of cycles increased (and the curly fibres progressively became straighter), the stress-strain response of curly fibres started to resemble that of straight fibres. This observation supports our previous hypothesis, which states that the toe-region can be attributed to the presence of a hydrogen bonding mechanism, which is present in curly fibres only, and can be altered by mechanical force. Interestingly, the alteration in load-bearing pattern in curly fibres did not necessarily translate to increased endurance, demonstrating that the relationship between fatigue and strength is a complex one in hair fibres.

Antibody-Based Targeted Interventions for the Diagnosis and Treatment of Skin Cancers.

BACKGROUND: Cutaneous malignancies most commonly arise from skin epidermal cells. These cancers may rapidly progress from benign to a metastatic phase. Surgical resection represents the gold standard therapeutic treatment of non-metastatic skin cancer while chemo- and/or radiotherapy are often used against metastatic tumors. However, these therapeutic treatments are limited by the development of resistance and toxic side effects, resulting from the passive accumulation of cytotoxic drugs within healthy cells. OBJECTIVE: This review aims to elucidate how the use of monoclonal Antibodies (mAbs) targeting specific Tumor Associated Antigens (TAAs) is paving the way to improved treatment. These mAbs are used as therapeutic or diagnostic carriers that can specifically deliver cytotoxic molecules, fluorophores or radiolabels to cancer cells that overexpress specific target antigens. RESULTS: mAbs raised against TAAs are widely in use for e.g. differential diagnosis, prognosis and therapy of skin cancers. Antibody-Drug Conjugates (ADCs) particularly show remarkable potential. The safest ADCs reported to date use non-toxic photo-activatable Photosensitizers (PSs), allowing targeted Photodynamic Therapy (PDT) resulting in targeted delivery of PS into cancer cells and selective killing after light activation without harming the normal cell population. The use of near-infrared-emitting PSs enables both diagnostic and therapeutic applications upon light activation at the specific wavelengths. CONCLUSION: Antibody-based approaches are presenting an array of opportunities to complement and improve current methods employed for skin cancer diagnosis and treatment.

Destruction of the stem cell Niche, Pathogenesis and Promising Treatment Targets for Primary Scarring Alopecias.

The Primary Scarring Alopecias are characterised by the irreversible destruction and fibrosis of hair follicles, leading to permanent and often disfiguring loss of hair. The pathophysiology of these diseases is not well understood. However, follicular-fibrosis and loss of the stem-cell niche appears to be a common theme. This review explores the pathogenesis of primary scarring alopecias, asking what happens to the stem cells of the hair follicle and how they may contribute to the progression of these diseases. Bulge-resident cells are lost (leading to loss of capacity for hair growth) from the follicle either by inflammatory-mediate apoptosis or through epigenetic reprogramming to assume a mesenchymal-like identity. What proportion of bulge cells is lost to which process is unknown and probably differs depending on the individual PCA and its specific inflammatory cell infiltrate. The formation of fibroblast-like cells from follicular stem cells may also mean that the cells of the bulge have a direct role in the pathogenesis. The identification of specific cells involved in the pathogenesis of these diseases could provide unique diagnostic and therapeutic opportunities to prevent disease progression by preventing EMT and specific pro-fibrotic signals.

No difference in the proteome of racially and geometrically classified scalp hair sample from a South African cohort: Preliminary findings.

Differences in the physiological proteome of men of different racial origin is poorly researched, albeit hair is mostly composed of keratins and keratin-associated proteins. Hence, we have carried out label-free, shotgun proteomics analysis on hair samples collected from black African, Caucasian, Asian, and Mixed-Ancestry donors within a heterogeneous population of the Western Cape of South Africa. Further, the same hair was also classified using geometrical measurements. Using both qualitative and quantitative proteomics bioinformatics pipelines, we identified over 450 protein groups (FDR = 0.01). Identified protein classes included keratins, keratin-associated proteins, histone proteins and desmosomes, inter alia. No protein by quantitative proteomic analyses significantly differentiated racial or geometric groups in our cohort. Functional pathway analysis of top-ranking proteins showed enrichment for skin, epidermal and tissue development, as well as intermediate-filament organization. Racial classification is a social construct, and attributing differences in a biologic endpoint to it is both imprecise and valueless in the era of precision medicine. Nonetheless, clarity on the physiological hair proteome could serve as a foundation for using hair proteomics for disease biomarker and targeted therapy identification for precision medicine. For the first time, we established the physiological hair proteome of individuals in a culturally diverse cohort from Africa. SIGNIFICANCE: For the first time we have been able to characterize the physiological human hair proteome in a culturally diverse South African cohort. We have also identified that proteomics differences were not observed in individual hair samples using our quantitative proteomics bioinformatics pipeline. This outcome supports a widely known notion that DNA sequence comparison often shows that people on each continent are not more genetically similar to one another than to people who come from other continents and that there is more genetic variation in Africa. Hence, adaptive traits such as hair and skin phenotype are not scientifically valid distinctions. Racial classification is believed to be a social construct, and attributing differences in a biologic endpoint to it is both imprecise and valueless in the era of precision medicine. Our preliminary finding would serve as a much-needed foundation for establishing a well-annotated, customized hair proteomics repository for Africans.

Understanding Curly Hair Mechanics: Fiber Strength.

The relationship between the geometric and mechanical profiles of hair fibers has been studied, with special focus on curly samples. Incidental observations pointed to a significantly different viscoelastic character with varying curliness. Further investigations confirmed initial observations, showing an initial distinct toe region behavior for curly fibers on the stress-strain plot, which is absent for straight fibers. This behavior suggested a difference in the viscoelastic nature of the curly fiber that is linked to mechanical energy stored in the fiber. Results also suggest that the strength of hair depends on two main components, and further pointed out that de facto methods of tensile testing may erode curly fiber strength during preparation. The main outcome of this study is that the tensile strength (σT) of hair fibers is composed of two (rather than one main) components, namely the toe region (σt) and the elastic region (σε), so that: σT=σt+σε. For noncurly fibers, the greatest part of fiber strength is derived from σε, while σt ≈ 0. For curly fibers, σt (i.e., springiness) adds significantly to the overall strength, even though σε remains the major contributor. Although these results require validation in larger studies, they are significant in the current understanding of curly hair. Also, they may represent a fundamental shift from the current understanding of tensile testing of human hair in general.

The what, why and how of curly hair: a review.

An attempt to understand and explain a peculiarity that was observed for curly fibres during experimentation revealed disparate literature reporting on several key issues. The phenotypical nature of curly fibres is only accurately understood within the larger scope of hair fibres, which are highly complex biological structures. A brief literature search produced thousands of research items. Besides the large amount of information on the topic, there was also great variability in research focus. From our review, it appeared that the complexity of hair biology, combined with the variety of research subtopics, often results in uncertainty when relating different aspects of investigation. During the literature investigation, we systematically categorized elements of curly hair research into three basic topics: essentially asking why fibres curl, what the curly fibre looks like and how the curly fibre behaves. These categories were subsequently formalized into a curvature fibre model that is composed of successive but distinctive tiers comprising the elements in curly hair research. The purpose of this paper is twofold: namely to present (i) a literature review that explores the different aspects of curly human scalp hair and (ii) the curvature fibre model as a systemized approach to investigating curly hair.

In Vitro and Ex Vivo Models for Functional Testing of Therapeutic Anti-scarring Drug Targets in Keloids.

Significance: Keloids are benign fibro-proliferative raised dermal lesions that spread beyond the original borders of the wound, continue to grow, rarely regress, and are the most common in pigmented individuals after an abnormal wound healing response. The current treatment failure and respective challenges involved highlighting the underlying issue that the etiopathogenesis of keloids is still not well understood. Disease models are required to better understand the disease pathogenesis. It is not possible to establish keloids in animals because of the uniqueness of this disease to human skin. To address this challenge, along these lines, non-animal reproducible models are vital in investigating molecular mechanisms of keloid pathogenesis and therapeutics development. Recent Advances: Various non-animal models have been developed to better understand the molecular mechanisms involved in keloid scarring and aid in identifying and evaluating the therapeutic potential of novel drug candidates. In this scenario, the current review aims at describing in vitro monocultures, co-cultures, organotypic cultures, and ex vivo whole skin keloid tissue organ culture models. Critical Issues and Future Directions: Current treatment options for keloids are far from securing a cure or preventing disease recurrence. Identifying universally accepted effective therapy for keloids has been hampered by the absence of appropriate disease model systems. Animal models do not accurately mimic the disease, thus non-animal model systems are pivotal in keloid research. The use of these models is essential not only for a better understanding of disease biology but also for identifying and evaluating novel drug targets.

Reliability of Histopathology for the Early Recognition of Fibrosis in Traction Alopecia: Correlation with Clinical Severity.

Traction alopecia (TA) is hair loss caused by prolonged pulling or repetitive tension on scalp hair; it belongs to the biphasic group of primary alopecia. It is non-scarring, typically with preservation of follicular stem cells and the potential for regrowth of early lesions especially if traction hairstyles are stopped. However, the alopecia may become permanent (scarring) and fail to respond to treatment if the traction is excessive and prolonged. Hence, the ability to detect fibrosis early in these lesions could predict patients who respond to treatment. Histopathological diagnosis based on scalp biopsies has been used as a gold standard to delineate various forms of non-scarring alopecia and to differentiate them from scarring ones. However, due to potential discrepant reporting as a result of the type of biopsy, method of sectioning, and site of biopsy, histopathology often tends to be unreliable for the early recognition of fibrosis in TA. In this study, 45 patients were assessed using the marginal TA severity scoring system, and their biopsies (both longitudinal and transverse sections) were systematically assessed by three dermatopathologists, the aim being to correlate histopathological findings with clinical staging. Intraclass correlation coefficients were used to determine the level of agreement between the assessors. We found poor agreement of the identification and grading of perifollicular and interfollicular fibrosis (0.55 [0.23-0.75] and 0.01 [2.20-0.41], respectively), and no correlation could be drawn with the clinical severity score. Better methods of diagnosis are needed for grading and for recognition of early fibrosis in TA.

Systems Approach to Human Hair Fibers: Interdependence Between Physical, Mechanical, Biochemical and Geometric Properties of Natural Healthy Hair.

Contextual interpretation of hair fiber data is often blind to the effects of the dynamic complexity between different fiber properties. This intrinsic complexity requires systems thinking to decipher hair fiber accurately. Hair research, studied by various disciplines, follows a reductionist research approach, where elements of interest are studied from a local context with a certain amount of detachment from other elements or contexts. Following a systems approach, the authors are currently developing a cross-disciplinary taxonomy to provide a holistic view of fiber constituents and their interactions within large-scale dynamics. Based on the development process, this paper presents a review that explores the associated features, interrelationships and interactive complexities between physical, mechanical, biochemical and geometric features of natural, healthy hair fibers. Through the review, the importance of an appropriate taxonomy for interpreting hair fiber data across different disciplines is revealed. The review also demonstrates how seemingly unrelated fiber constituents are indeed interdependent and that these interdependencies may affect the behavior of the fiber. Finally, the review highlights how a non-integrative approach may have a negative impact on the reliability of hair data interpretation.

Multi-dimensional models for functional testing of keloid scars: In silico, in vitro, organoid, organotypic, ex vivo organ culture, and in vivo models.

Keloid scars are described as benign fibro-proliferative dermal outgrowths that commonly occur in pigmented skin post cutaneous injury, and continue to grow beyond the boundary of the original wound margin. There is a lack of thorough understanding of keloid pathogenesis and thus keloid therapeutic options remain ill-defined. In view of the poor response to current therapy and high recurrence rates, there is an unmet need in improving our knowledge and therefore in identifying targeted and effective treatment strategies in management of keloids. Keloid research however, is hampered by a lack of relevant animal models as keloids do not spontaneously occur in animals and are unique to human skin. Therefore, developing novel animal models and nonanimal models for functional evaluation of keloid cells and tissue for better understanding their pathobiology and response to putative candidate therapies are essential. Here, we present the key concepts and relevant emerging research on two-dimensional and three-dimensional cell and tissue models for functional testing of keloid scars. We will describe in detail current models including in vitro mono- and co-cultures, multi-cellular spheroids (organoids) and organotyopic cultures, ex vivo whole skin keloid tissue organ culture models as well as in vivo human patient models. Finally, we discuss the role played by time as the fourth dimension in a novel model that involves sequential temporal biopsies of human patients with keloids (a so called 4D in vivo human model). The use of these unique models will no doubt prove pivotal in identification of new drug targets as well as biomarkers, in functional testing of emerging novel therapeutics, and in enhancing our understanding of keloid disease biology.