Female ageing leads to reduced hair density and thinner fibres. The impact of the ageing dermal environment on the hair follicles (HFs) remains unclear. This study documents in situ changes in human female scalp skin of women (19-81 years (yrs)), and corresponding primary cultures of dermal fibroblast (DF) and dermal sheath (DS) cells. In situ, the papillary/reticular boundary was indistinguishable in young scalp, but delineated over 40yrs, with reduced rete ridges, changes in collagen organisation, reduced podoplanin (PDPN) and increased versican (VCAN) expression. Hyaluronic acid synthase 2 (HAS2) was highly expressed throughout the scalp. Matrix Metallopeptidase 1 (MMP1) and Metallopeptidase inhibitor 1 (TIMP1), cyclin-dependent kinase inhibitor 2A (p16INK4A), 11β-Hydroxysteroid dehydrogenase type 1 and 2 (11β-HSD1/HSD11B1 and 11β-HSD2/HSD11B2) mRNA expression increased in aged DFs. In DS cells, HAS2, Vimentin (VIM) ,PDPN, Lysosomal-associated membrane protein 1 (LAMP1), Sequestosome- 1 (P62) and Protease nexin-1 (SERPINE2) increased, while α-smooth muscle actin (aSMA) decreased. Both cell types exhibited elevated cartilage oligomeric protein (COMP) mRNA expression. Proteomics revealed elevated COMP expression in the DF secretome with age, suggesting a more fibrotic phenotype or DS migration into the dermis. DF and DS lysate protein expression suggests altered extracellular matrix (ECM) remodelling due to increased levels of MMP-2 and Protease inhibitor Plasminogen activator inhibitor-1 (Serpin E1/PAI-1). Cathepsin C/DPPI protein lysate expression decreased in DFs but increased in DS. In summary, ageing female scalp shows striking structural and biological changes in the HF environment that may impact hair growth, due to alterations in ECM, senescence and autophagy associated biomarkers.

Being the longest-lived rodent, naked mole-rats (NMR; Heterocephalus glaber) are an exceptional model for biogerontological research. However, unlike other rodents, not much is known about their wound healing process. To investigate that, full-thickness wounds were created in the back skin of naked mole rats. Our initial data confirmed that wound closure in NMR skin was achieved primarily by reepithelialization and granulation tissue formation, with only ~26% wound contraction, making them an excellent model to study human cutaneous wound healing. Similar to mice and human skin, changes in wound epithelial tongue included progressive enlargement of wound epithelium, increased proliferation and changes in the expression pattern of epidermal markers including K14, K17, integrin α6 and E-cadherin. Further analysis revealed characteristics of reduced scarring in NMR wounds including low collagen I to III ratio, increased HA expression (HMW) and increased fibronectin expression. Transcriptional profiling of TGFβ isoforms and different pro/anti-inflammatory cytokines revealed a balance in the expression and repression of different cytokines, potentially contributing into reduced scarring. Comparison of RNA-seq data from NMR and human fullthickness wounds revealed a delay in the activation of important biological processes and pathways in NMR skin in response to injury. Further analysis based on cultured human and NMR cells revealed differential regulation of TGFβ signalling pathway between both species. 3-D collagen gel contraction assay revealed that NMR fibroblast showed noticeable contraction but independently of TGFβ treatment, while human fibroblast showed marked increased in gel contraction in the presence of TGFβ. In conclusion, NMR can serve as a very useful model to study human cutaneous wound healing. The reduced scarring in NMR could be a result of multiple factors including HMW-HA, balanced cytokine expression and differential regulation of different TGFβ cytokines as observed in the in vitro studies.

Diseases of the aorta rank as the 20th leading cause of mortality in the US, contributing to 10,000 deaths annually. Thoracic aortic aneurysms are typically asymptomatic, often undetected until life-threatening aortic dissection or rupture occurs. Familial cases constitute one in five instances of thoracic aortic aneurysm and dissection (TAAD), with genetic causes being heterogeneous and known risk genes explaining only a small fraction of cases. We hypothesized that additional TAAD risk genes remain undiscovered. This thesis aims to investigate the genetic etiology of TAAD using genetic and genomic approaches. Our methodological approach included: 1) exome sequencing of DNA from TAAD patients with subsequent genomic analysis, integrating clinical data, and 2) single-cell RNA sequencing (scRNA-seq) of the developing (embryonic) mouse aorta. We sequenced 1650 DNA samples from 1429 TAAD patients and, after quality control, analyzed genomic data from 1278 unrelated TAAD patients of European ancestry. For controls, we used 145,103 unrelated individuals of European ancestry from the UK BioBank. We conducted a per-gene and per-domain burden analysis using a binomial test. To improve the power of detection of novel risk genes, we integrated case-control association of rare damaging variants with cell-type specific gene expression data from scRNA-seq of the ascending and descending aorta of 17 mouse embryos (harvested at the E15 stage) with the hypothesis that true risk genes are highly expressed early in development. Our analysis of known TAAD risk genes identified 52 pathogenic or likely pathogenic variants, explaining 4.1% of TAAD cases, and 75 variants of uncertain significance (5.9%). Next, two potential novel candidate genes emerged from the unbiased case-control analysis, which utilized AlphaFold domain-based annotation of protein structure: β-propeller domain of VPS8 (p = 8.8 × 10-9) and UTP11 (p = 3.9 × 10-8). scRNA-seq of the developing mouse aorta revealed significant cell-type-specific expression differences between the ascending and descending aorta, identifying five subtypes of vascular smooth muscle cells in the ascending aorta and four in the descending aorta. Differentially expressed genes between major aortic cell types were also identified. Both, VPS8 and UTP11 were found to expressed in all three major aortic cell types – vascular smooth muscle cells, fibroblasts, and endothelial cells. In conclusion, our case-control association analysis identified two promising candidate risk genes for TAAD (VPS8 and UTP11), warranting further investigation and confirmation in additional cohorts of patients with aortopathy.

Yes / Interferon lambda (IFNλ) is important for epidermal defence against viruses. It is produced by, and acts on, keratinocytes, whereas fibroblasts were previously considered to be unresponsive to this type III IFN. Herein we report findings revealing cell type-specific differences in IFNλ signalling and function in skin resident cells. In dermal fibroblasts, IFNλ induced the expression of MxA, a potent antiviral factor, but not other IFN signature genes as it does in primary keratinocytes. In contrast to its effect on keratinocytes, IFNλ did not phosphorylate STAT1 in fibroblasts, but instead activated MAPKs. Accordingly, inhibition of MAPK activation (p38 and p42/44) blocked the expression of MxA protein in fibroblasts but not in keratinocytes. Functionally, IFNλ inhibited proliferation in keratinocytes but not in fibroblasts. Moreover, IFNλ upregulated the expression of TGFβ1-induced collagens in fibroblasts. Taken together, our findings identify primary human dermal fibroblasts as responder cells to IFNλ. Our study shows cutaneous cell type-specific IFN signalling and suggests that IFNλ, whilst important for epidermal anti-viral competence, may also have a regulatory role in the dermal compartment balancing type I IFN-induced inhibition of tissue repair processes.

No / The development of hair follicle organ culture techniques is a significant milestone in cutaneous biology research. The hair follicle, or more accurately the "pilo-sebaceous unit", encapsulates all the important physiologic processes found in the human body; controlled cell growth/death, interactions between cells of different histologic type, cell differentiation and migration, and hormone responsitivity to name a few. Thus, the value of the hair follicle as a model for biological scientific research goes way beyond its scope for cutaneous biology or dermatology alone. Indeed, the recent and dramatic upturn in interest in hair follicle biology has focused principally on the pursuit of two of biology's holy grails; post-embryonic morphogenesis and control of cyclical tissue activity. The hair follicle organ culture model, pioneered by Philpott and colleagues, ushered in an exceptionally accessible way to assess how cells of epithelial (e.g., keratinocytes), mesenchymal (e.g., fibroblasts), and neuroectodermal (e.g., melanocytes) origin interact in a three-dimensional manner. Moreover, this assay system allows us to assess how various natural and pharmacologic agents affect complex tissues for growth modulation. In this article, I focus on the culture of the human hair follicle mini-organ, discussing both the practical issues involved and some possible research applications of this assay.

Yes / Alopecia areata (AA) is an autoimmune hair loss disease with infiltration of proinflammatory cells into hair follicles. Current therapeutic regimens are unsatisfactory mainly because of the potential for side effects and/or limited efficacy. Here we report that cultured, transduced fibroblasts, which express the immunomodulatory molecule indoleamine 2,3-dioxygenase (IDO), can be applied to prevent hair loss in an experimental AA model. A single intraperitoneal (IP) injection of IDO-expressing primary dermal fibroblasts was given to C3H/HeJ mice at the time of AA induction. While 60–70% of mice that received either control fibroblasts or vehicle injections developed extensive AA, none of the IDO-expressing fibroblast-treated mice showed new hair loss up to 20 weeks post injection. IDO cell therapy significantly reduced infiltration of CD4+ and CD8+ T cells into hair follicles and resulted in decreased expression of TNF-α, IFN-γ and IL-17 in the skin. Skin draining lymph nodes of IDO fibroblast-treated mice were significantly smaller, with more CD4+ CD25+ FoxP3+ regulatory T cells and fewer Th17 cells than those of control fibroblast and vehicle-injected mice. These findings indicate that IP injected IDO-expressing dermal fibroblasts can control inflammation and thereby prevent AA hair loss. / Canadian Institutes of Health Researches (Funding Reference Number: 134214 and 136945).

S'ha avaluat mitjançant estudis in vitro la viabilitat de la teràpia fotodinàmica antifúngica per al tractament clínic d'infeccions causades per les espècies fúngiques patògenes Candida albicans, Candida parapsilosis, Candida krusei i Trichophyton mentagrophytes. L'objectiu és optimitzar aquesta tècnica per proporcionar una alternativa als fàrmacs antifúngics utilitzats en els tractaments clàssics actuals i eradicar així les micosis superficials. S'ha avaluat la capacitat citotòxica de tres fotosensibilitzadors amb propietats fotoquímiques diferents, la hipericina, la 5,10,15,20-tetraquis(N-metil-4-piridil)-21H,23H-porfina i el clorur de 3,7-Bis(etilamino)-2,8-dimetilfenotiacina-5-io, induint tots la inactivació cel.lular sobre les diferents espècies fúngiques. Per avaluar si la la tècnica produeix danys significatius sobre les cèl.lules epidèrmiques humanes s'han realitzat estudis sobre queratinòcits i fibroblasts humans. S'han avaluat els mecanismes d'acció dels tres fotosensibilitzadors mitjançant estudis espectroscòpics i cinètics de formació i desaparició dels seus estats excitats i oxigen singlet. En el cas de l'hipericina també s'han descrit els intermedis que es formen en irradiar, caracteritzant l'accessibilitat dels triplets a l'oxigen. / Se ha evaluado mediante estudios in vitro la viabilidad de la terapia fotodinámica antifúngica para el tratamiento clínico de infecciones causadas por las especies fúngicas patógenas Candida albicans, Candida parapsilosis, Candida krusei y Trichophyton mentagrophytes. El objetivo es optimizar esta técnica para proporcionar una alternativa a los fármacos antifúngicos usados en los tratamientos clásicos actuales y erradicar así las micosis superficiales. Se ha evaluado la capacidad citotóxica de tres fotosensibilizadores con propiedades fotoquímicas distintas, la hipericina, la 5,10,15,20-tetraquis(N-metil-4-piridil)-21H,23H-porfina y el cloruro de 3,7-Bis(etilamino)-2,8-dimetilfenotiacina-5-io, induciendo todos la inactivación celular sobre las distintas especies fúngicas. Para evaluar si la la técnica produce daños significativos sobre las células epidérmicas humanas se han realizado estudios sobre queratinocitos y fibroblastos humanos. Se han evaluado los mecanismos de acción de los tres fotosensibilizadores mediante estudios espectroscópicos y cinéticos de formación y desaparición de sus estados excitados y oxígeno singlete. En el caso de la hipericina también se han descrito los intermedios que se forman al irradiar, caracterizándose la accesibilidad de los tripletes al oxígeno. / Has been evaluated by in vitro viability studies of photodynamic therapy for clinical fungal infections caused by pathogenic fungal species Candida albicans, Candida parapsilosis, Candida krusei and Trichophyton mentagrophytes. The goal is to optimize this technique to provide an alternative to the antifungal drugs used in current classical treatments and thus eliminate superficial mycoses. We assessed the cytotoxic capacity of three photosensitizers with different photochemical properties, the hypericin, 5,10,15,20-tetrakis(N-methyl-4-pyridyl)-21H,23H-porphine and 3,7-Bis(ethylamino)-2,8-dimethylphenothiazin-5-ium chloride, inducing all cell inactivation on different fungal species. To assess if the technique produces significant damage to human epidermal cells, studies with human fibroblasts and keratinocytes were carried. We evaluated the mechanisms of action of the three photosensitisers by spectroscopic and kinetic studies of formation and disappearance of excited states and singlet oxygen. In the case of hypericin have also been described intermediates formed by irradiating, characterized accessibility of triplet oxygen.

Diss. (sammanfattning) Linköping : Univ., 2001. / Härtill 4 uppsatser.

No description available.

Mitochondrial isocitrate dehydrogenase 2 (IDH2) catalyzes reductive carboxylation (RC, reverse Krebs cycle pathway) and 2HG synthesis (2HG) - metabolite of which many scientists are interested. 2HG may be concurrently synthetized in cytosol by IDH1. RC is involved in anabolic reactions necessary for cell proliferation - produces citrate, fatty acid precursor - especially in hypoxia. IDH2 and IDH1 are not the only enzymes that are involved in 2HG synthesis. Recently, several enzymes, which participate in 2HG production, have been discovered. 2HG is useful in cancer diagnostics due to its overproduction by transformed cells. Moreover, 2HG may cause epigenetic changes via inhibition of 2-oxoglutarate dependent dioxygenase. In this work, the importance of RC and 2HG synthesis in cancer and healthy cells was investigated by gas chromatography with mass spectrometry detection as well as IDH2 influence. We found that IDH2 significantly participates in reverse RC and 2HG synthesis in breast cancer cell lines and uses glutaminolysis as a supplementary anaplerotic pathway. RC is increased by hypoxia, inhibition of respiration, and decreased by activation of respiration or hypocapnia. We confirmed 2HG synthesis and RC in healthy cells (fibroblasts, breast epithelial cells etc.) as well as in cancer cells....