Understanding and treating diabetic foot ulcers: Insights into the role of cutaneous microbiota and innovative therapies.

Background: Notoriously known as the silent pandemic, chronic, non-healing diabetic foot ulcers (DFUs), pose a significant rate of incidence for amputation and are a major cause of morbidity. Alarmingly, the treatment and management strategies of chronic wounds represent a significant economic and health burden as well as a momentous drain on resources with billions per annum being spent in the US and UK alone. Defective wound healing is a major pathophysiological condition which propagates an acute wound to a chronic wound, further propelled by underlying conditions such as diabetes and vascular complications which are more prevalent amongst the elderly. Chronic wounds are prone to infection, which can exacerbate the condition, occasionally resulting in amputation for the patient, despite the intervention of modern therapies. However, amputation can only yield a 5-year survival rate for 50% of patients, highlighting the need for new treatments for chronic wounds. Findings: The dynamic cutaneous microbiota is comprised of diverse microorganisms that often aid wound healing. Conversely, the chronic wound microbiome consists of a combination of common skin commensals such as Staphylococcus aureus and Staphylococcus epidermidis, as well as the opportunistic pathogen Pseudomonas aeruginosa. These bacteria have been identified as the most prevalent bacterial pathogens isolated from chronic wounds and contribute to prolific biofilm formation decreasing the efficiency of antimicrobials and further perpetuating a hyper-inflammatory state. Discussion and Conclusion: Here, we review recent advances and provide a new perspective on alternative treatments including phage and microbiome transplant therapies and how the definitive role of the cutaneous microbiota impacts the aetiology of DFUs.

Nutraceuticals known to promote hair growth do not interfere with the inhibitory action of tamoxifen in MCF7, T47D and BT483 breast cancer cell lines.

BACKGROUND: Hair loss/thinning is a common side effect of tamoxifen in estrogen receptor (ER) positive breast cancer therapy. Some nutraceuticals known to promote hair growth are avoided during breast cancer therapy for fear of phytoestrogenic activity. However, not all botanical ingredients have similarities to estrogens, and in fact, no information exists as to the true interaction of these ingredients with tamoxifen. Therefore, this study sought to ascertain the effect of nutraceuticals (+/- estrogen/tamoxifen), on proliferation of breast cancer cells and the relative expression of ERα/ß. METHODS: Kelp, Astaxanthin, Saw Palmetto, Tocotrienols, Maca, Horsetail, Resveratrol, Curcumin and Ashwagandha were assessed on proliferation of MCF7, T47D and BT483 breast cancer cell lines +/- 17ß-estradiol and tamoxifen. Each extract was analysed by high performance liquid chromatography (HPLC) prior to use. Cellular ERα and ERß expression was assessed by qRT-PCR and western blot. Changes in the cellular localisation of ERα:ERß and their ratio following incubation with the nutraceuticals was confirmed by immunocytochemistry. RESULTS: Estradiol stimulated DNA synthesis in three different breast cancer cell lines: MCF7, T47D and BT483, which was inhibited by tamoxifen; this was mirrored by a specific ERa agonist in T47D and BT483 cells. Overall, nutraceuticals did not interfere with tamoxifen inhibition of estrogen; some even induced further inhibition when combined with tamoxifen. The ERα:ERß ratio was higher at mRNA and protein level in all cell lines. However, incubation with nutraceuticals induced a shift to higher ERß expression and a localization of ERs around the nuclear periphery. CONCLUSIONS: As ERα is the key driver of estrogen-dependent breast cancer, if nutraceuticals have a higher affinity for ERß they may offer a protective effect, particularly if they synergize and augment the actions of tamoxifen. Since ERß is the predominant ER in the hair follicle, further studies confirming whether nutraceuticals can shift the ratio towards ERß in hair follicle cells would support a role for them in hair growth. Although more research is needed to assess safety and efficacy, this promising data suggests the potential of nutraceuticals as adjuvant therapy for hair loss in breast cancer patients receiving endocrine therapy.

Stromal Vascular Fraction Cells from Individuals Who Have Previously Undergone Radiotherapy Retain Their Pro-Wound Healing Properties.

Beneficial effects have been observed following the transplant of lipoaspirates containing adipose-derived stem cells into chronic wounds caused by oncologic radiotherapy. It is not yet certain whether adipose-derived stem cells are resistant to radiation exposure. Therefore, the aims of this study were to isolate stromal vascular fraction from human breast tissue exposed to radiotherapy and determine the presence of adipose-derived stem cells. Stromal vascular fraction from irradiated donor tissue was compared to commercially sourced pre-adipocytes. Immunocytochemistry was used to determine the presence of adipose-derived stem cell markers. Conditioned media from stromal vascular fraction isolated from irradiated donors was used as a treatment in a scratch wound assay of dermal fibroblasts also isolated from irradiated donors and compared to pre-adipocyte conditioned media and serum free control. This is the first report of human stromal vascular fraction being cultured from previously irradiated breast tissue. Stromal vascular fraction conditioned media from irradiated donors had a similar effect in increasing the migration of dermal fibroblasts from irradiated skin to pre-adipocyte conditioned media from healthy donors. Therefore, the ability of adipose-derived stem cells in the stromal vascular fraction to stimulate dermal fibroblasts in wound healing appears to be preserved following radiotherapy. This study demonstrates that stromal vascular fraction from irradiated patients is viable, functional and may have potential for regenerative medicine techniques following radiotherapy.

Draft Genome Sequence of a Multiple Antibiotic Resistant Staphylococcus aureus NCTC 6571-UB Laboratory Strain.

We report the draft genome sequence of the laboratory strain Staphylococcus aureus NCTC 6571-UB, a strain that was derived from S. aureus NCTC 6571. This strain was selected for sequencing in order to provide information on the genome dynamics and the acquired resistance genes for penicillin G, trimethoprim, and sulfamethoxazole resistance.

Draft Genome Sequence of the Multiple Antibiotic Resistant Pseudomonas aeruginosa PAO1-UB Subline.

We report the draft genome sequence and antibiotic susceptibility of Pseudomonas aeruginosa strain PAO1-UB, a subline of the common reference strain PAO1. This strain was sequenced in order to provide information on the genome dynamics of PAO1 sublines and their genes conferring resistance to multiple antibiotics.

Laser Capture Microdissection on Surgical Tissues to Identify Aberrant Gene Expression in Impaired Wound Healing in Type 2 Diabetes.

The global prevalence Type 2 diabetes mellitus (T2DM) is escalating at a rapid rate. Patients with T2DM suffer from a multitude of complications and one of these is impaired wound healing. This can lead to the development of non-healing sores or foot ulcers and ultimately to amputation. In healthy individuals, wound healing follows a controlled and overlapping sequence of events encompassing inflammation, proliferation, and remodelling. In T2DM, one or more of these steps becomes dysfunctional. Current models to study impaired wound healing in T2DM include in vitro scratch wound assays, skin equivalents, or animal models to examine molecular mechanisms underpinning wound healing and/or potential therapeutic options. However, these do not fully recapitulate the complex wound healing process in T2DM patients, and ex vivo human skin tests are problematic due to the ethics of taking punch biopsies from patients where it is known they will heal poorly. Here, a technique is described whereby expression profiles of the specific cells involved in the (dys)functional wound healing response in T2DM patients can be examined using surplus tissue discarded following amputation or elective cosmetic surgery. In this protocol samples of donated skin are collected, wounded, cultured ex vivo in the air liquid interface, fixed at different time points and sectioned. Specific cell types involved in wound healing (e.g., epidermal keratinocytes, dermal fibroblasts (papillary and reticular), the vasculature) are isolated using laser capture microdissection and differences in gene expression analyzed by sequencing or microarray, with genes of interest further validated by qPCR. This protocol can be used to identify inherent differences in gene expression between both poorly healing and intact skin, in patients with or without diabetes, using tissue ordinarily discarded following surgery. It will yield greater understanding of the molecular mechanisms contributing to T2DM chronic wounds and lower limb loss.

Dermal fibroblasts cultured from donors with type 2 diabetes mellitus retain an epigenetic memory associated with poor wound healing responses.

The prevalence of Type 2 diabetes mellitus (T2DM) is escalating globally. Patients suffer from multiple complications including the development of chronic wounds that can lead to amputation. These wounds are characterised by an inflammatory environment including elevated tumour necrosis factor alpha (TNF-α). Dermal fibroblasts (DF) are critical for effective wound healing, so we sought to establish whether there were any differences in DF cultured from T2DM donors or those without diabetes (ND-DF). ND- and T2DM-DF when cultured similarly in vitro secreted comparable concentrations of TNF-α. Functionally, pre-treatment with TNF-α reduced the proliferation of ND-DF and transiently altered ND-DF morphology; however, T2DM-DF were resistant to these TNF-α induced changes. In contrast, TNF-α inhibited ND- and T2DM-DF migration and matrix metalloprotease expression to the same degree, although T2DM-DF expressed significantly higher levels of tissue inhibitor of metalloproteases (TIMP)-2. Finally, TNF-α significantly increased the secretion of pro-inflammatory cytokines (including CCL2, CXCL1 and SERPINE1) in ND-DF, whilst this effect in T2DM-DF was blunted, presumably due to the tendency to higher baseline pro-inflammatory cytokine expression observed in this cell type. Collectively, these data demonstrate that T2DM-DF exhibit a selective loss of responsiveness to TNF-α, particularly regarding proliferative and secretory functions. This highlights important phenotypic changes in T2DM-DF that may explain the susceptibility to chronic wounds in these patients.

Age-Related Changes in Female Scalp Dermal Sheath and Dermal Fibroblasts: How the Hair Follicle Environment Impacts Hair Aging.

In women, aging leads to reduced hair density and thinner fibers and can result in female-pattern hair loss. However, the impact of the aging dermal environment on female scalp hair follicles remains unclear. In this study, we document in situ changes in 22 women (aged 19-81 years) and primary cultures of dermal fibroblast and dermal sheath cells. In situ, the papillary reticular boundary was indistinguishable in the young scalp but prominent in the scalp of those aged >40 years, accompanied by reduced podoplanin (PDPN) expression, increased versican expression, and changes in collagen organization. Hair follicles were shorter, not reaching the adipose layer. Hyaluronic acid synthase 2 was highly expressed, whereas matrix metalloproteinase 1 was elevated in the dermal papilla and dermal sheath in situ. Primary dermal fibroblast cultures confirmed that matrix metalloproteinase 1 mRNA, MMP1, increased with aging, whereas in dermal sheath cells, hyaluronic acid synthase 2, HAS2, and PDPN increased and α-smooth muscle actin αSMA mRNA decreased. Both exhibited increased cartilage oligomeric protein, COMP mRNA expression. Proteomics revealed an increase in dermal sheath proteins in the dermal fibroblast secretome with aging. In summary, aging female scalp shows striking structural and biological changes in the hair follicle environment that may impact hair growth.

The Potential Role of Nutraceuticals as an Adjuvant in Breast Cancer Patients to Prevent Hair Loss Induced by Endocrine Therapy.

Nutraceuticals, natural dietary and botanical supplements offering health benefits, provide a basis for complementary and alternative medicine (CAM). Use of CAM by healthy individuals and patients with medical conditions is rapidly increasing. For the majority of breast cancer patients, treatment plans involve 5-10 yrs of endocrine therapy, but hair loss/thinning is a common side effect. Many women consider this significant, severely impacting on quality of life, even leading to non-compliance of therapy. Therefore, nutraceuticals that stimulate/maintain hair growth can be proposed. Although nutraceuticals are often available without prescription and taken at the discretion of patients, physicians can be reluctant to recommend them, even as adjuvants, since potential interactions with endocrine therapy have not been fully elucidated. It is, therefore, important to understand the modus operandi of ingredients to be confident that their use will not interfere/interact with therapy. The aim is to improve clinical/healthcare outcomes by combining specific nutraceuticals with conventional care whilst avoiding detrimental interactions. This review presents the current understanding of nutraceuticals beneficial to hair wellness and outcomes concerning efficacy/safety in breast cancer patients. We will focus on describing endocrine therapy and the role of estrogens in cancer and hair growth before evaluating the effects of natural ingredients on breast cancer and hair growth.

Adipose Tissue: A Source of Stem Cells with Potential for Regenerative Therapies for Wound Healing.

Interest in adipose tissue is fast becoming a focus of research after many years of being considered as a simple connective tissue. It is becoming increasingly apparent that adipose tissue contains a number of diverse cell types, including adipose-derived stem cells (ASCs) with the potential to differentiate into a number of cell lineages, and thus has significant potential for developing therapies for regenerative medicine. Currently, there is no gold standard treatment for scars and impaired wound healing continues to be a challenge faced by clinicians worldwide. This review describes the current understanding of the origin, different types, anatomical location, and genetics of adipose tissue before discussing the properties of ASCs and their promising applications for tissue engineering, scarring, and wound healing.

Getting under the skin of hair aging: the impact of the hair follicle environment.

Like the skin, our hair shows striking changes with age, producing hairs with altered diameter, lustre and texture. The biology of hair aging has focused predominately on various aspects of the hair cycle, follicle size and the fibre produced, but surprisingly the impact of the aging scalp dermal environment on the hair follicle and fibre has been generally overlooked. Hair loss affects both sexes with incidence increasing with age. In men, male pattern-balding (androgenetic alopecia) is driven by androgens and follows a specific pattern of frontotemporal and vertex regression. Women also experience female pattern hair loss (FPHL), presenting as more general, diffuse hair thinning. Hair thinning in women is commonly associated with the menopause, corresponding with other age-related changes in skin. The rapidly growing hair follicle undergoes continued renewal throughout the life span of an individual, where it is exposed to a substantial number of extrinsic and intrinsic stressors. As the hair follicle sits deep within the dermis with its bulb residing in the hypodermis, detrimental age-related changes in the surrounding scalp skin may likely disrupt the hair follicle machinery. The impacts of these changes are unknown, but evidence suggests that scalp skin aging and hair follicle aging go hand-in-hand. Herein, we summarize the evidence that the age-related changes observed in sun-exposed human skin also occur in scalp skin and that these changes are likely to play a contributing role in the aging hair phenotype.

Isolation of Epidermal Keratinocytes from Human Skin: The Scratch-Wound Assay for Assessment of Epidermal Keratinocyte Migration.

The migration of epidermal keratinocytes is the basis for skin reepithelialization during wound healing. The in vitro scratch-wound assay using monolayers of primary human epidermal keratinocytes is a straightforward and effective method to assess their migratory capacity. The mechanical scratch of a confluent monolayer directly disrupts the adhesion of the keratinocytes to one another and to the underlying matrix, resembling the physical trauma of a wound in an in vitro assay. The keratinocytes will undergo an epithelial-to-mesenchymal transition, which will confer an ability to migrate toward each other to cover the gap by restructuring cell-cell and cell-extracellular matrix connections. However, a good scratch-wound method and protocol to ensure scratch reproducibility is essential, particularly when using primary cell cultures where donor variability may also impact on results.

Isolation of Different Dermal Fibroblast Populations from the Skin and the Hair Follicle.

The establishment of primary cells from fresh tissue is a widely used method for investigating human tissue in vitro. The skin harbors different cell populations in the dermis and the hair follicle, which can be isolated for downstream analysis. Here we describe the isolation of four dermal fibroblast populations from human haired skin and their maintenance in culture. The four cell populations for which isolation is described are papillary dermal fibroblast cells, reticular dermal fibroblast cells, hair follicle dermal sheath cells, and hair follicle dermal papilla cells.

Isolation of Epidermal and Hair Follicle Melanocytes.

Here we describe the isolation of epidermal melanocytes and hair follicle melanocytes from human skin tissue. Epidermal and hair follicle melanocytes are two distinct populations of melanocytes which are contained within the skin and the hair follicle, with differing yet overlapping roles. Epidermal melanocytes are normally isolated from the epidermis of vellus-haired skin tissue (e.g., face, breast, abdomen), while hair follicle melanocytes are derived from the outer root sheath (ORS) of the middle/lower terminal anagen hair follicles of the scalp. These methods utilize ethically sourced human skin tissue obtained from donors undergoing plastic surgery.

Does blue light restore human epidermal barrier function via activation of Opsin during cutaneous wound healing?

BACKGROUND AND OBJECTIVE: Visible light has beneficial effects on cutaneous wound healing, but the role of potential photoreceptors in human skin is unknown. In addition, inconsistency in the parameters of blue and red light-based therapies for skin conditions makes interpretation difficult. Red light can activate cytochrome c oxidase and has been proposed as a wound healing therapy. UV-blue light can activate Opsin 1-SW, Opsin 2, Opsin 3, Opsin 4, and Opsin 5 receptors, triggering biological responses, but their role in human skin physiology is unclear. MATERIALS AND METHODS: Localization of Opsins was analyzed in situ in human skin derived from face and abdomen by immunohistochemistry. An ex vivo human skin wound healing model was established and expression of Opsins confirmed by immunohistochemistry. The rate of wound closure was quantitated after irradiation with blue and red light and mRNA was extracted from the regenerating epithelial tongue by laser micro-dissection to detect changes in Opsin 3 (OPN3) expression. Retention of the expression of Opsins in primary cultures of human epidermal keratinocytes and dermal fibroblasts was confirmed by qRT-PCR and immunocytochemistry. Modulation of metabolic activity by visible light was studied. Furthermore, migration in a scratch-wound assay, DNA synthesis and differentiation of epidermal keratinocytes was established following irradiation with blue light. A role for OPN3 in keratinocytes was investigated by gene silencing. RESULTS: Opsin receptors (OPN1-SW, 3 and 5) were similarly localized in the epidermis of human facial and abdominal skin in situ. Corresponding expression was confirmed in the regenerating epithelial tongue of ex vivo wounds after 2 days in culture, and irradiation with blue light stimulated wound closure, with a corresponding increase in OPN3 expression. Expression of Opsins was retained in primary cultures of epidermal keratinocytes and dermal fibroblasts. Both blue and red light stimulated the metabolic activity of cultured keratinocytes. Low levels of blue light reduced DNA synthesis and stimulated differentiation of keratinocytes. While low levels of blue light did not alter keratinocyte migration in a scratch wound assay, higher levels inhibited migration. Gene silencing of OPN3 in keratinocytes was effective (87% reduction). The rate of DNA synthesis in OPN3 knockdown keratinocytes did not change following irradiation with blue light, however, the level of differentiation was decreased. CONCLUSIONS: Opsins are expressed in the epidermis and dermis of human skin and in the newly regenerating epidermis following wounding. An increase in OPN3 expression in the epithelial tongue may be a potential mechanism for the stimulation of wound closure by blue light. Since keratinocytes and fibroblasts retain their expression of Opsins in culture, they provide a good model to investigate the mechanism of blue light in wound healing responses. Knockdown of OPN3 led to a reduction in early differentiation of keratinocytes following irradiation with blue light, suggesting OPN3 is required for restoration of the barrier function. Understanding the function and relationship of different photoreceptors and their response to specific light parameters will lead to the development of reliable light-based therapies for cutaneous wound healing. Lasers Surg. Med. © 2018 Wiley Periodicals, Inc.

Intracrine sex steroid synthesis and signaling in human epidermal keratinocytes and dermal fibroblasts.

Peripheral intracrine sex steroid synthesis from adrenal precursors dehydroepiandrosterone (DHEA) and DHEA-sulfate has evolved in humans. We sought to establish if there are differences in intracrine, paracrine, and endocrine regulation of sex steroids by primary cultures of human skin epidermal keratinocytes and dermal fibroblasts. Microarray analysis identified multifunctional genes modulated by steroids, quantitative RT-PCR (qRT-PCR) mRNA expression, enzymatic assay aromatase activity, scratch assay cell migration, immunocytochemistry α-smooth muscle actin (α-SMA), and collagen gel fibroblast contraction. All steroidogenic components were present, although only keratinocytes expressed the organic anion organic anion transporter protein (OATP) 2B1 transporter. Both expressed the G-protein-coupled estrogen receptor (GPER1). Steroids modulated multifunctional genes, up-regulating genes important in repair and aging [angiopoietin-like 4 (ANGPTL4), chemokine (C-X-C motif) ligand 1 (CXCL1), lamin B1 (LMNB1), and thioredoxin interacting protein (TXNIP)]. DHEA-sulfate (DHEA-S), DHEA, and 17ß-estradiol stimulated keratinocyte and fibroblast migration at early (4 h) and late (24-48 h) time points, suggesting involvement of genomic and nongenomic signaling. Migration was blocked by aromatase and steroid sulfatase (STS) inhibitors confirming intracrine synthesis to estrogen. Testosterone had little effect, implying it is not an intermediate. Steroids stimulated fibroblast contraction but not α-SMA expression. Mechanical wounding reduced fibroblast aromatase activity but increased keratinocyte activity, amplifying the bioavailability of intracellular estrogen. Cultured fibroblasts and keratinocytes provide a biologically relevant model system to investigate the complex pathways of sex steroid intracrinology in human skin.

Estrogens and aging skin.

Estrogen deficiency following menopause results in atrophic skin changes and acceleration of skin aging. Estrogens significantly modulate skin physiology, targeting keratinocytes, fibroblasts, melanocytes, hair follicles and sebaceous glands, and improve angiogenesis, wound healing and immune responses. Estrogen insufficiency decreases defense against oxidative stress; skin becomes thinner with less collagen, decreased elasticity, increased wrinkling, increased dryness and reduced vascularity. Its protective function becomes compromised and aging is associated with impaired wound healing, hair loss, pigmentary changes and skin cancer.   Skin aging can be significantly delayed by the administration of estrogen. This paper reviews estrogen effects on human skin and the mechanisms by which estrogens can alleviate the changes due to aging. The relevance of estrogen replacement, selective estrogen receptor modulators (SERMs) and phytoestrogens as therapies for diminishing skin aging is highlighted. Understanding estrogen signaling in skin will provide a basis for interventions in aging pathologies.

Effects of oestrogen agonists on human dermal fibroblasts in an in vitro wounding assay.

Oestrogen and dehydroepiandrosterone (DHEA) improve wound healing, but circulating levels decline significantly with age. Recently, the selective oestrogen receptor modulators (SERMs) tamoxifen and raloxifene have been shown to improve age-associated impaired wound healing. Therefore, we have evaluated the effects of 17beta-oestradiol, ER alpha and ER beta agonists, tamoxifen, raloxifene and DHEA on human dermal fibroblasts using an in vitro wound assay. An ER alpha agonist, 17beta-oestradiol and DHEA all significantly accelerated cell migration; the DHEA effect was blocked with an aromatase inhibitor. Tamoxifen, raloxifene and DHEA all significantly increased DNA synthesis; the DHEA stimulatory effect was reversed by an aromatase inhibitor. This study demonstrates that 17beta-oestradiol, an ER alpha agonist, tamoxifen, raloxifene and DHEA (following conversion to oestrogen) all have significant effects on human fibroblasts, the key mesenchymal cell involved in the wound healing process. Further understanding of the mechanisms involved may have important implications for the management of age-related impaired wound healing.