Jane Sterling

Efficient generation of neural precursors from adult human skin: astrocytes promote neurogenesis from skin-derived stem cells

BACKGROUND Neural stem cells are a potential source of cells for drug screening or cell-based treatments for neurodegenerative diseases. However, ethical and practical considerations limit the availability of neural stem cells derived from human embryonic tissue. An alternative source of human neural stem cells is needed; a source that is readily accessible, easily expanded, and reliably induced to a neural fate. METHODS Dermis isolated from biopsy samples of adult human skin was cultured and expanded in the presence of the mitogens epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF 2), and then by serum. We used immunocytochemical techniques, clonal analysis, and physiological characterisation to assess neural differentiation after the treatment of expanded cells with novel induction media. FINDINGS Initial characterisation of skin samples confirmed the absence of nestin, a neural precursor marker. Sequential culture in EGF and FGF 2 followed by adherent expansion in serum, and re-exposure to mitogens in substrate-free conditions resulted in large numbers of nestin-positive/musashi-positive neural precursors. Subsequent exposure of these precursors to hippocampal-astrocyte-derived signals resulted in cells of neuronal morphology that had stable expression of markers of neuronal differentiation (neurofilament, beta tubulin). We also show the presence of voltage-dependent calcium transients, and demonstrate monoclonal neural potential. INTERPRETATION We describe the isolation and characterisation of cells derived from adult human dermis that can be expanded for extended periods of time in vitro, while retaining inducible neural potential. The generation of almost limitless numbers of neural precursors from a readily accessible autologous adult human source provides a platform for further experimental studies and has potential therapeutic implications.

A Highly Enriched Niche of Precursor Cells with Neuronal and Glial Potential Within the Hair Follicle Dermal Papilla of Adult Skin

Skin‐derived precursor cells (SKPs) are multipotent neural crest‐related stem cells that grow as self‐renewing spheres and are capable of generating neurons and myelinating glial cells. SKPs are of clinical interest because they are accessible and potentially autologous. However, although spheres can be readily isolated from embryonic and neonatal skin, SKP frequency falls away sharply in adulthood, and primary sphere generation from adult human skin is more problematic. In addition, the culture‐initiating cell population is undefined and heterogeneous, limiting experimental studies addressing important aspects of these cells such as the behavior of endogenous precursors in vivo and the molecular mechanisms of neural generation. Using a combined fate‐mapping and microdissection approach, we identified and characterized a highly enriched niche of neural crest‐derived sphere‐forming cells within the dermal papilla of the hair follicle of adult skin. We demonstrated that the dermal papilla of the rodent vibrissal follicle is 1,000‐fold enriched for sphere‐forming neural crest‐derived cells compared with whole facial skin. These “papillaspheres” share a phenotypic and developmental profile similar to that of SKPs, can be readily expanded in vitro, and are able to generate both neuronal and glial cells in response to appropriate cues. We demonstrate that papillaspheres can be efficiently generated and expanded from adult human facial skin by microdissection of a single hair follicle. This strategy of targeting a highly enriched niche of sphere‐forming cells provides a novel and efficient method for generating neuronal and glial cells from an accessible adult somatic source that is both defined and minimally invasive.

A prospective study on the natural course of low-grade squamous intraepithelial lesions and the presence of HPV16 E2-, E6- and E7-specific T-cell responses.

This study investigates the clinical course of low grade squamous intraepithelial lesions (LSIL), HPV status and HPV16‐specific immune response in a large prospective study of 125 women with LSIL followed cytologically, virologically and histologically. Women with low‐grade abnormal smears were recruited and followed‐up for one year. Colposcopy, cervical biopsy for histology and brushings for HPV typing was performed at recruitment, 6 months (no biopsy) and upon completion of the study at one year. HPV16‐specific T‐cell responses were analysed by interferon‐γ ELISPOT at entry, 6 and 12 months. Infection with multiple HPV types was detected in 70% of all patients, HPV16 was found in 42% of the patients. LSIL lesions progressed to HSIL in 24%, persisted in 60% and regressed to normal in 16% of the patients. No difference was observed in the clearance rate of infections with single or multiple HPV types among the groups with a different histological outcome. HPV16‐specific type 1 T‐cell responses were detected in only half of the patients with an HPV16+ LSIL, and predominantly reactive to HPV16 E2 and E6. Interestingly, the presence of HPV16 E2‐specific T‐cell responses correlated with absence of progression of HPV16+ lesions (p = 0.005) while the detection of HPV16 E6 specific reactivity was associated with persistence (p = 0.05). This large prospective study showed that the majority of LSIL persisted or progressed within the first year.This was paralleled by immune failure as most of the patients with an HPV16+ LSIL failed to react to peptides of HPV16 E2, E6 or E7.

HPV infection, anal intra-epithelial neoplasia (AIN) and anal cancer: current issues

BackgroundHuman papillomavirus (HPV) is well known as the major etiological agent for ano-genital cancer. In contrast to cervical cancer, anal cancer is uncommon, but is increasing steadily in the community over the last few decades. However, it has undergone an exponential rise in the men who have sex with men (MSM) and HIV + groups. HIV + MSM in particular, have anal cancer incidences about three times that of the highest worldwide reported cervical cancer incidences.DiscussionThere has therefore traditionally been a lack of data from studies focused on heterosexual men and non-HIV + women. There is also less evidence reporting on the putative precursor lesion to anal cancer (AIN – anal intraepithelial neoplasia), when compared to cervical cancer and CIN (cervical intraepithelial neoplasia). This review summarises the available biological and epidemiological evidence for HPV in the anal site and the pathogenesis of AIN and anal cancer amongst traditionally non-high risk groups.SummaryThere is strong evidence to conclude that high-grade AIN is a precursor to anal cancer, and some data on the progression of AIN to invasive cancer.

Doxycycline versus prednisolone as an initial treatment strategy for bullous pemphigoid: a pragmatic, non-inferiority, randomised controlled trial

Summary Background Bullous pemphigoid is a blistering skin disorder with increased mortality. We tested whether a strategy of starting treatment with doxycycline gives acceptable short-term blister control while conferring long-term safety advantages over starting treatment with oral corticosteroids. Methods We did a pragmatic, multicentre, parallel-group randomised controlled trial of adults with bullous pemphigoid (three or more blisters at two or more sites and linear basement membrane IgG or C3). Participants were randomly assigned to doxycycline (200 mg per day) or prednisolone (0·5 mg/kg per day) using random permuted blocks of randomly varying size, and stratified by baseline severity (3–9, 10–30, and >30 blisters for mild, moderate, and severe disease, respectively). Localised adjuvant potent topical corticosteroids (<30 g per week) were permitted during weeks 1–3. The non-inferiority primary effectiveness outcome was the proportion of participants with three or fewer blisters at 6 weeks. We assumed that doxycycline would be 25% less effective than corticosteroids with a 37% acceptable margin of non-inferiority. The primary safety outcome was the proportion with severe, life-threatening, or fatal (grade 3–5) treatment-related adverse events by 52 weeks. Analysis (modified intention to treat [mITT] for the superiority safety analysis and mITT and per protocol for non-inferiority effectiveness analysis) used a regression model adjusting for baseline disease severity, age, and Karnofsky score, with missing data imputed. The trial is registered at ISRCTN, number ISRCTN13704604. Findings Between March 1, 2009, and Oct 31, 2013, 132 patients were randomly assigned to doxycycline and 121 to prednisolone from 54 UK and seven German dermatology centres. Mean age was 77·7 years (SD 9·7) and 173 (68%) of 253 patients had moderate-to-severe baseline disease. For those starting doxycycline, 83 (74%) of 112 patients had three or fewer blisters at 6 weeks compared with 92 (91%) of 101 patients on prednisolone, an adjusted difference of 18·6% (90% CI 11·1–26·1) favouring prednisolone (upper limit of 90% CI, 26·1%, within the predefined 37% margin). Related severe, life-threatening, and fatal events at 52 weeks were 18% (22 of 121) for those starting doxycycline and 36% (41 of 113) for prednisolone (mITT), an adjusted difference of 19·0% (95% CI 7·9–30·1), p=0·001. Interpretation Starting patients on doxycycline is non-inferior to standard treatment with oral prednisolone for short-term blister control in bullous pemphigoid and significantly safer in the long-term. Funding NIHR Health Technology Assessment Programme.

Analyses of human papillomavirus genotypes and viral loads in anogenital warts.

Condylomata acuminata (genital warts) are the most common sexually transmitted viral diseases. These lesions are caused by infection with mucosal human papillomaviruses (HPVs). However, there is limited information on HPV strain distribution involved in the molecular pathogenesis of these lesions. To address this, the strain prevalence and the frequency of multiple HPV infections were determined in wart tissue obtained from 31 patients attending a wart clinic. These lesions were bisected and subjected to parallel DNA and mRNA extractions. HPV‐type prevalence and incidence of multiple infections were determined by the Roche Linear Array assay. qPCR compared HPV 6, 11, 16, and 18 viral loads and RT‐qPCR measured HPV 6 and 11 E6 genomic expression levels. Seventy‐one percent of these samples were infected with multiple HPVs. Only one sample was negative for HPV 6 or 11 DNA. Forty‐eight percent of samples were positive for a high risk (oncogenic) HPV. The results show that multiple infections in tissue are frequent and the subsequent analysis of HPV 6 and 11 E6 DNA viral loads suggested that other HPVs could be causing lesions. Further analysis of HPV 6/11 E6 mRNA levels showed that there was no discernable relationship between HPV 6 E6 DNA viral load and relative HPV 6 or 11 E6 mRNA levels thereby questioning the relevance of viral load to lesion causality. J. Med. Virol. 83:1345–1350, 2011.

Characterising the local immune responses in cervical intraepithelial neoplasia: a cross-sectional and longitudinal analysis

Introduction  Immunological competence influences the progression of cervical intraepithelial neoplasia (CIN) to invasive cancer. Information on the local immunological changes during the natural course of CIN is central for the development of new therapies.

High prevalence of HPV in non-cervical sites of women with abnormal cervical cytology.

BackgroundHuman papillomaviruses (HPV) are causally associated with ano-genital and a subset of head and neck cancers. Rising incidence of HPV+ anal cancers and head and neck cancers have now been demonstrated in the developed world over the last decade. The majority of published data on HPV prevalence at the anal and oro-pharyngeal sites are from studies of higher-risk populations. There is a paucity of data on the prevalence of HPV at non-cervical sites in lower risk, non-HIV+ women and this study was designed to provide initial pilot data on a population of women recalled for colposcopy as part of the UK cervical screening programme.Methods100 non-HIV+ women with abnormal cervical cytology, attending clinic for colposcopic examination were recruited. Swabs from the oro-pharyngeal, anal and cervical sites were taken and DNA extracted. HPV detection and genotyping were performed using a standardised, commercially available PCR-line blot assay, which is used to genotype 37 HPV subtypes known to infect the ano-genital and oro-pharyngeal areas. Strict sampling and laboratory precautions were taken to prevent cross-contamination.ResultsThere was a very high prevalence of HPV infection at all three sites: 96.0%, 91.4% and 92.4% at the cervix, anus and oro-pharynx, respectively. Multiple HPV subtype infections were dominant at all 3 mucosal sites. At least one or more HR genotype was present at both the cervix/anus in 39/52 (75.0%) patients; both the cervix/oro-pharynx in 48/56 (85.7%) patients; and both the anus/oro-pharynx in 39/52 (75.0%) patients. HPV 16 infection was highly dominant across all mucosal sites, with over a 2-fold increase over the next most prevalent subtype (HPV 31).ConclusionsWomen with abnormal smears have widespread infection with high-risk HPV at the cervical, anal and oro-pharyngeal mucosal sites and may represent a higher risk population for HPV disease in the future.

HIF isoforms in the skin differentially regulate systemic arterial pressure

Significance The differential expression of the hypoxia-inducible factor-alpha (HIF-α) isoforms in the skin of mice influences vascular resistance and is correlated with homeostatic regulation of nitric oxide synthesis. A correlation between HIF isoform expression and hypertension was found in skin biopsies from human subjects, and may indicate a mechanism in the etiology of idiopathic hypertension. Vascular flow through tissues is regulated via a number of homeostatic mechanisms. Localized control of tissue blood flow, or autoregulation, is a key factor in regulating tissue perfusion and oxygenation. We show here that the net balance between two hypoxia-inducible factor (HIF) transcription factor isoforms, HIF-1α and HIF-2α, is an essential mechanism regulating both local and systemic blood flow in the skin of mice. We also show that balance of HIF isoforms in keratinocyte-specific mutant mice affects thermal adaptation, exercise capacity, and systemic arterial pressure. The two primary HIF isoforms achieve these effects in opposing ways that are associated with HIF isoform regulation of nitric oxide production. We also show that a correlation exists between altered levels of HIF isoforms in the skin and the degree of idiopathic hypertension in human subjects. Thus, the balance between HIF-1α and HIF-2α expression in keratinocytes is a control element of both tissue perfusion and systemic arterial pressure, with potential implications in human hypertension.

Host Responses to Infection with Human Papillomavirus

Human papillomavirus (HPV) infections of cutaneous and genital mucosae are very common but the majority of individuals clear the infection without overt clinical disease. Those who develop lesions, also in most cases, mount an effective cell-mediated immune response and the lesions regress. The increased prevalence of HPV infections in individuals with a range of immunodeficiencies, including immunosuppression as a consequence of HIV infection, demonstrates the central importance of the CD4 T cell population in the control of established HPV infections. Failure to induce an effective immune response is related to inefficient activation of innate immunity and ineffective priming of the adaptive immune response; this defective immune response facilitates viral persistence, a key feature of high-risk HPV infection. This milieu becomes operationally HPV antigen tolerant, and the hosts defences become irrevocably compromised. HPV antigen-specific effector cells are poorly recruited to the infected focus, and their activity is downregulated; neoplastic HPV-containing cervical keratinocytes expressing high levels of E6 and E7 oncoproteins are not killed in this immunosuppressive, tolerant milieu, and progression to high-grade disease and cancer can result. Highly efficacious prophylactic HPV L1 virus-like particle (VLP) vaccines circumvent viral epithelial evasion strategies since they are delivered by intramuscular injection. The stromal dendritic cells of the muscle that encounter the highly immunogenic repeat structure of the VLP then migrate with their cargo to the lymph node, initiating an immune cascade that results in a robust T cell-dependent B cell response, which generates high levels of L1-specific serum neutralizing antibodies and immune memory.