An efficient method for eccrine gland isolation from human scalp.

We describe a simple and efficient method to isolate eccrine sweat glands from the human scalp. This method is inspired by the hair graft harvesting method used in hair transplantation. Based on the recently described anatomical relationship between the scalp hair follicle and the eccrine gland, we have found that scalp follicular unit grafts are an excellent eccrine gland isolation source, especially for the coiled component. In order to make the gland visible for stereoscopic microdissection, the follicular units need to be previously stained with a vital dye like methylene blue or neutral red. The simplicity and efficiency of this isolation method should encourage further research into human eccrine sweat gland function which has always been hindered by the difficulty of gland isolation.

Is the eccrine gland an integral, functionally important component of the human scalp pilosebaceous unit?

The pilosebaceous unit (PSU) and the eccrine sweat gland (ESG) are classically described as completely independent skin appendages. However, careful inspection of scalp follicular units reveals that the secretory segment of the ESG spatially approximates the hair follicle in a position below the sebaceous gland and the insertion of the arrector pili muscle. Therefore, we propose here that, contrary to conventional wisdom, the PSU and the ESG should not be viewed in isolation, and may form instead, along with the arrector pili muscle and the apocrine gland (where present),one functional unit. For this, we suggest the more inclusive term of 'Hair Cluster' (HC). If confirmed, e.g. by 3D imaging techniques, the novel concept of a functional HC, whose individual components may communicate via secreted molecules and may share selected progenitor cell populations for HC repair/regeneration, has major physiological and pathological implications, which are briefly discussed.

Hair follicle-containing punch grafts accelerate chronic ulcer healing: A randomized controlled trial.

BACKGROUND: A prominent role of hair follicle-derived cells in epidermal wound closure is now well established but clinical translation of basic research findings is scarce. Although skin punch grafts have been used as a therapeutic intervention to improve healing of chronic leg ulcers, they are normally harvested from nonhairy areas, thus not taking advantage of the reported role of the hair follicle as a wound-healing promoter. OBJECTIVE: We sought to substantiate the role of hair follicles in venous leg ulcer healing by transplanting hair follicle-containing versus nonhairy punch grafts. METHODS: This was a randomized controlled trial with intraindividual comparison of hair follicle scalp grafts and nonhairy skin grafts transplanted in parallel into 2 halves of the same ulcer. RESULTS: Ulcer healing measured as the average percentage reduction 18 weeks postintervention was significantly increased (P = .002) in the hair follicle group with a 75.15% (SD 23.03) ulcer area reduction compared with 33.07% (SD 46.17) in the control group (nonhairy grafts). LIMITATIONS: Sample size was small (n = 12). CONCLUSION: Autologous transplantation of terminal hair follicles by scalp punch grafts induces better healing than punch grafts harvested from nonhairy areas. Hair punch grafting is a minimally invasive surgical procedure that appears to be effective as a therapeutic tool for chronic venous leg ulcers.

Human epithelial hair follicle stem cells and their progeny: current state of knowledge, the widening gap in translational research and future challenges.

Epithelial hair follicle stem cells (eHFSCs) are required to generate, maintain and renew the continuously cycling hair follicle (HF), supply cells that produce the keratinized hair shaft and aid in the reepithelialization of injured skin. Therefore, their study is biologically and clinically important, from alopecia to carcinogenesis and regenerative medicine. However, human eHFSCs remain ill defined compared to their murine counterparts, and it is unclear which murine eHFSC markers really apply to the human HF. We address this by reviewing current concepts on human eHFSC biology, their immediate progeny and their molecular markers, focusing on Keratin 15 and 19, CD200, CD34, PHLDA1, and EpCAM/Ber-EP4. After delineating how human eHFSCs may be selectively targeted experimentally, we close by defining as yet unmet key challenges in human eHFSC research. The ultimate goal is to transfer emerging concepts from murine epithelial stem cell biology to human HF physiology and pathology.

Reflections on how wound healing-promoting effects of the hair follicle can be translated into clinical practice.

Clinicians have long reported that hair-bearing areas tend to heal more rapidly than those lacking hair follicles. In the past decade, numerous scientific studies have corroborated clinical evidence, showing a direct nexus between the human hair follicle and the wound healing process. The migration of epithelial follicular stem cells to the skin surface to help in the wound re-epithelialization and the effect of the hair cycle on the wound healing rate underline the influence of the hair follicle in the healing process. In clinical practice, non-healing wounds are pathologies of high prevalence with significant associated burden costs for the healthcare system. As the population ages, the prevalence of this pathology is expected to increase in future years. The recent advances in understanding the biology of hair follicle stem cells have created the challenges of using this newly acquired knowledge in practical therapeutic applications. Chronic leg ulcers are an example of the targeted pathologies that urgently need better therapies. In this essay, our aim is to raise interest in this question, reviewing what is known in relation to the connections between hair follicles and wound healing, and elaborating on future directions that the field might take, including implications for clinical practice.

Lichen planopilaris is characterized by immune privilege collapse of the hair follicle's epithelial stem cell niche.

Lichen planopilaris (LPP) is a chronic inflammatory disease of unknown pathogenesis that leads to permanent hair loss. Whilst destruction of epithelial hair follicle stem cells (eHFSCs) that reside in an immunologically protected niche of the HF epithelium, the bulge, is a likely key event in LPP pathogenesis, this remains to be demonstrated. We have tested the hypotheses that bulge immune privilege (IP) collapse and inflammation-induced eHFSC death are key components in the pathogenesis of LPP. Biopsies of lesional and non-lesional scalp skin from adult LPP patients (n = 42) were analysed by quantitative (immuno)histomorphometry, real-time quantitative polymerase chain reaction (qRT-PCR), laser capture microdissection and microarray analysis, or skin organ culture. At both the protein and transcriptional level, lesional LPP HFs showed evidence for bulge IP collapse (ie increased expression of MHC class I and II, ß2microglobulin; reduced TGFß2 and CD200 expression). This was accompanied by a Th1-biased cytotoxic T cell response (ie increased CD8(+) GranzymeB(+) T cells and CD123(+) plasmacytoid dendritic cells, with increased CXCR3 expression) and increased expression of interferon-inducible chemokines (CXCL9/10/11). Interestingly, lesional LPP eHFSCs showed both increased proliferation and apoptosis in situ. Microarray analysis revealed a loss of eHFSC signatures and increased expression of T cell activation/binding markers in active LPP, while bulge PPARγ transcription was unaltered compared to non-lesional LPP HFs. In organ culture of non-lesional LPP skin, interferon-γ (IFNγ) induced bulge IP collapse. LPP is an excellent model disease for studying and preventing immune destruction of human epithelial stem cells in situ. These novel findings raise the possibility that LPP represents an autoimmune disease in whose pathogenesis IFNγ-induced bulge IP collapse plays an important role. Therapeutically, bulge IP protection/restoration may help to better manage this highly treatment-resistant cicatricial alopecia.

Merkel cell carcinoma with divergent differentiation: histopathological and immunohistochemical study of 15 cases with PCR analysis for Merkel cell polyomavirus.

AIMS: To report on 15 cases of Merkel cell carcinoma (MCC) with divergent differentiation, to characterize its clinicopathological spectrum and its relationship with Merkel cell polyomavirus (MCV). METHODS AND RESULTS: Fifteen patients with a mean age of 81 years were included. Follow-up was available for 13 cases (range 12 days to 6 years; median 6 months). Recurrence, metastasis and mortality rates were 15.4%, 53.8% and 61.5%, respectively. All tumours showed the typical histological and immunohistochemical features of MCC, with at least one additional divergent component. Eight cases had a single aberrant component (squamous in six cases, follicular in one case, and porocarcinoma in one case), six cases had two aberrant components (squamous and sarcomatous in three cases, glandular and squamous in two cases, and sarcomatous and neuroblastic in one case), and one case had three aberrant components (glandular, squamous, and sarcomatous). All cases had dysplastic changes in the overlying epithelium, and four of 15 showed epidermotropism. PCR analysis for Merkel cell polyomavirus (MCV) gave negative results in all 12 cases tested. CONCLUSIONS: Merkel cell carcinoma with divergent differentiation is a highly aggressive tumour that might be difficult to recognize, owing to its wide histological variability. Negativity for MCV suggests that the virus is not implicated in the development of this subtype of MCC.

[Monkeypox with eccrine duct involvement]. / Viruela del mono con afectación de conductos ecrinos.

Monkeypox was historically considered a zoonotic disease restricted to areas with an animal reservoir and with limited possibilities of human transmission. However, the recent increase in incidence in non-endemic areas, together with the demonstration of human transmission, has led to more attention being paid to this disease. We present the case of a 27-year-old man with cutaneous lesions and perianal ulcers, clinically suggestive of a viral disease. Monkeypox was demonstrated with PCR analysis. The histological features and differential diagnoses of monkeypox are discussed and the characteristic histopathological pattern of eccrine gland epithelium is described which, if found in an ulcerated lesion, should raise suspicion of monkeypox.

A pilot clinical study of hair grafting in chronic leg ulcers.

Epidermal sheets spread centrifugally postinjury from the hair follicle infundibulum to reepithelialize the wound bed. Healing progresses faster in skin areas rich in terminal hair follicles. These observations are consistent with the role of the hair follicle as a major reservoir for progenitor cells. To evaluate the feasibility and potential healing capacity of autologous scalp follicular grafts transplanted into the wound bed of chronic leg ulcers, 10 patients with ulcers of an average 36.8 cm(2) size and a 10.5-year duration were included in this pilot study. Within each ulcer we randomly assigned a 2 × 2 cm "experimental" square to receive 20 hair grafts and a nongrafted "control" square of equal size. The procedure seemed to be safe, although major unrelated complications occurred in two patients. At the 18-week end point, we observed a 27.1% ulcer area reduction in the experimental square as compared with 6.5% in the control square (p = 0.046) with a maximum 33.5% vs. 9.7% reduction at week 4 (p = 0.007). Histological analyses showed enhanced epithelialization, neovascularization, and dermal reorganization. We conclude that terminal hair follicle grafting into wound beds is feasible in an outpatient setting and represents a promising therapeutic alternative for nonhealing chronic leg ulcers.

The utility of a gross dissection anatomical model for simulation-based learning in pathology.

INTRODUCTION: The examination of morphological alterations in tissues is fundamental in Pathology. Traditional training in gross dissection has several limitations, including the risk of transmissible diseases, formaldehyde exposure and limited specimen availability. We describe a teaching method using anatomical simulators. METHODS: Liquid silicone-based artisan neoplastic anatomical models were used in conjunction with clinical scenarios. Eighty-five medical students participated in a gross dissection experience and were asked to complete a feedback questionnaire. Additionally, a workshop was organized for students to compare three different teaching methods. The first one used still images (Group1-G1), the second a video explanation (Group2-G2), and the third directly observed a pathologist while grossing (Group3-G3). RESULTS: The knowledge acquisition questionnaire showed an average value of 4.4 out of 5 (1-5) (range 3.4-4.7, σ0.89). The categories 'knowledge of resection margins' and 'macroscopic diagnosis' received the highest values (4.8, σ0.11 and 4.7, σ0.32, respectively), followed by 'understanding of handling and gross examination of the surgical specimen' (4.5, σ0.49), 'prognosis' (4.3, σ0.67) and 'understanding of a tumor resection' (3.9, σ0.96) (p<0.05). Regarding teaching methods, G3 spent less time than G2 and G1 with mean times of 15'39″ (σ2'12″), 16'50″ (σ3'45″), and 17'52″ (σ2'12″), respectively (p<0.05). Gross dissection marks (0-5) showed statistically significant differences (p<0.05). G2 obtained better results (3.7;σ0.54) than G3 (3.4;σ0.94) or G1 (3.1;σ0.8). CONCLUSIONS: This preliminary study demonstrates that it is possible to implement a gross dissection simulation module at medical school and thus enable the acquisition of skills in a secure environment.

Comparison of muscle activity while using different input devices in digital pathology.

INTRODUCTION: The high prevalence of musculoskeletal disorders in pathologists, together with the current trend towards the digitization of pathology, prompted us to study the different types of input devices employed during the revision of whole slide images, in order to investigate the pattern and extent of muscle activity involved in their use. MATERIAL AND METHODS: A comparative study was made of 10 input devices (conventional and vertical mouse, three trackballs, the Ergopointer™, the Rollermouse™, an optical pen mouse, a touchpad, and the Leap Motion™). Six medical students performed a standardized circuit using a Fitts' Law based tissue array, digitized. The electrical activity of seven upper limb muscles (adductor pollicis, extensor pollicis longus, extensor digitorum, flexor digitorum, middle deltoid, upper trapezius, and middle trapezius) was measured using surface electromyography. RESULTS: Statistically significant differences in the overall electrical activity among the different input devices, both absolute values in mV as well as normalized values to the upper limb at rest, were observed (p<0.001); the Rollermouse™ (0.1027mV; 139%), Logitech M570 trackball (0.1053mV; 145%), Ergopointer™ (0.1151mV; 167%), conventional mouse (0.1251mV; 191%), and vertical mouse (0.1312mV; 205%) required less activity, while the optical pen mouse (0.1717mV; 299%), Leap Motion™ (0.1803mV; 319%), Expert Mouse trackball (0.1845mV; 329%), EIGIIS trackball (0.2442mV; 468%) and the touchpad (0.2560mV; 496%) required greater muscle mobilization. CONCLUSION: We designed a system based on Fitts' Law to compare input devices in digital pathology. Variability between compared devices and muscle activity was found. Long-term use could result in different muscular fatigue patterns. Even though the selection of an input device is a matter of personal preference, its impact on ergonomics should be considered.

Morphometric analysis of the human scalp hair follicle: practical implications for the hair transplant surgeon and hair regeneration studies.

BACKGROUND: The bulge stem cell region is a structure important for the regeneration of the pilosebaceous unit. Measurements of the different compartments of a hair follicle may have implications in hair transplantation and hair regeneration studies. OBJECTIVE: To measure the length of the different portions of the occipital scalp hair and to estimate at what depth they are located. METHODS AND MATERIAL: Hair follicles from the occipital scalp were obtained from 29 individuals. Measurements were performed on digital pictures using a software imaging system. Antibody anticytokeratin (CK), 15 was used as a bulge stem cell marker. RESULTS: The mean length of a scalp hair follicle is 4.16 mm. The infundibulum measures 0.76 mm, the isthmus 0.89 mm, and the inferior portion 2.5 mm. The insertion of the arrector pili muscle is located 1.65 mm deep. CK15 immunoreactivity starts at a depth of 1 mm and extends down to 1.8 mm. CONCLUSION: The ideal depth for the trichophytic procedure is to cut the wound edge at a depth of less than 1 mm to avoid the bulge zone. The data provided can serve as an objective anatomical reference in hair regeneration studies using horizontally transected follicles.

Functional characterization of highly adherent CD34+ keratinocytes isolated from human skin.

Compared to murine models, data on cells responsible for the homeostasis of human epidermis are scarce and often contradictory. Given the conflicting results and the availability of clinical grade protocols to purify CD34 cells from a given tissue, we pursued to phenotypically characterize human epidermal CD34+ population. After magnetic separation of whole skin CD34+ and CD34- cell fractions and selection for cells highly adherent to extracellular matrix, both CD34+/- fractions retained the ability to form a stratified epidermis in organotypic cultures and presented similar in vitro migratory phenotypes. However CD34- cells showed higher clonogenic potential and in vitro proliferative capacity. These results indicated that CD34- cell fraction contains stem/early progenitor cells, while CD34+ cells might be a transit-amplifying precursor for hair follicle (HF) sheath cells. The ability to isolate living cells using differential cell adhesion and surface markers provides an opportunity to study cells from different morphological regions of the HF.

Head-tracking as an interface device for image control in digital pathology: a comparative study.

BACKGROUND: Inasmuch as the conventional mouse is not an ideal input device for digital pathology, the aim of this study was to evaluate alternative systems with the goal of identifying a natural user interface (NUI) for controlling whole slide images (WSI). DESIGN: Four pathologists evaluated three webcam-based, head-tracking mouse emulators: Enable Viacam (eViacam, CREA Software), Nouse (JLG Health Solutions Inc), and Camera Mouse (CM Solutions Inc). Twenty WSI dermatopathological cases were randomly selected and examined with Image Viewer (Ventana, AZ, USA). The NASA-TLX was used to rate the perceived workload of using these systems and time was recorded. In addition, a satisfaction survey was used. RESULTS: The mean total time needed for diagnosis with Camera Mouse, eViacam, and Nouse was 18'57", 19'37" and 22'32", respectively (57/59/68seconds per case, respectively). The NASA-TLX workload score, where lower scores are better, was 42.1 for eViacam, 53.3 for Nouse and 60.62 for Camera Mouse. This correlated with the pathologists' degree of satisfaction on a scale of 1-5: 3.4 for eViacam, 3 for Nouse, and 2 for Camera Mouse (p<0.05). CONCLUSIONS: Head-tracking systems enable pathologists to control the computer cursor and virtual slides without their hands using only a webcam as an input device. - Of the three software solutions examined, eViacam seems to be the best of those evaluated in this study, followed by Nouse and, finally, Camera Mouse. - Further studies integrating other systems should be performed in conjunction with software developments to identify the ideal device for digital pathology.

A novel simulator model and standardized assessment tools for fine needle aspiration cytology training.

OBJECTIVES: Fine needle aspiration (FNA) is an invaluable diagnostic procedure for evaluation of lesions; however, acquisition of diagnostic material is dependent on the skill of the practitioner. We report a novel patient simulator for teaching the FNA procedure and structured assessment tools for educators and learners. METHODS: We created a novel simulator model for FNA training, employed a standardized teaching module, and assessed procedure utility in medical students. Groups of students completed training using a commercial version of the model, and underwent structured evaluation using an Objective Structured Assessment of Technical Skills (OSATS) form, and the Debriefing Assessment for Simulation in Healthcare (DASH) tool. RESULTS: In the initial phase, 178 students rated the training workshop between valuable and essential (4.2 on a 5-point Likert scale). In the second phase, for students evaluated with the OSATS form, the mean overall score was 33 out of 50 (range 26-43). The areas of weakness for the participants were: (a) compression after the FNA procedure, (b) completion of the informed consent, and (c) correct explanation of the procedure to the patient. For the group of students that completed the DASH questionnaire, the results were: 6.2 (assessment by students) and 6.7 (assessment by instructor) out of a maximum of 7. CONCLUSION: A realistic simulation model, in combination with a standardized training program with formal assessment methods is a valuable tool to teach FNA. We here describe a process for teaching the FNA procedure to interested educators and learners.

Human papillomavirus-associated penile sarcomatoid carcinoma.

Sarcomatoid carcinomas are rare tumors predominantly composed of spindle cells. This report describes two cases of penile sarcomatoid carcinoma with similar clinicopathological findings. Distinctive features of these tumors were the focal immunostaining that showed the sarcoma-like cells with keratin, smooth muscle actin and p16, and the absence of immunostaining of these cells with p53, S100 protein and desmin. Polymerase chain reaction (PCR) using the GP5+/GP6+ set of primers was positive in both cases. The sequences of the amplified products showed that the implicated genotypes were Human papillomavirus (HPV) 16 and HPV18. To the best of our knowledge, there has been no report in the English literature of HPV-associated penile sarcomatoid carcinoma. These cases might represent an unusual presentation of dedifferentiated carcinoma in which HPV could be shown by a sensitive technique of PCR.

An update on the biology of cancer stem cells in breast cancer.

Breast cancer stem cells are defined as cancer cells with self-renewal capacity. These cells represent a small subpopulation endowed with the ability to form new tumours when injected in nude mice. Markers of differentiation have been used to identify these cancer cells. In the case of breast cancer, CD44+/CD24- select a population with stem cell properties. The fact that these cells have self-renewal ability has suggested that this population could be responsible for new tumour formation and cancer relapse. These cells have been shown to be more resistant to chemotherapy and radiotherapy than normal cancer cells. The identification of the molecular druggable alterations responsible for the initiation and maintenance of cancer stem cells is an important goal. In this article we will review all these points with special emphasis on the possible role of new drugs designed to interact with molecular pathways of cancer stem cells.