Long-Term Antibody Response to SARS-CoV-2 in Children.

Almost 2 years into the pandemic and with vaccination of children significantly lagging behind adults, long-term pediatric humoral immune responses to SARS-CoV-2 are understudied. The C19.CHILD Hamburg (COVID-19 Child Health Investigation of Latent Disease) Study is a prospective cohort study designed to identify and follow up children and their household contacts infected in the early 2020 first wave of SARS-CoV-2. We screened 6113 children < 18 years by nasopharyngeal swab-PCR in a low-incidence setting after general lockdown, from May 11 to June 30, 2020. A total of 4657 participants underwent antibody testing. Positive tests were followed up by repeated PCR and serological testing of all household contacts over 6 months. In total, the study identified 67 seropositive children (1.44%); the median time after infection at first presentation was 83 days post-symptom onset (PSO). Follow-up of household contacts showed less than 100% seroprevalence in most families, with higher seroprevalence in families with adult index cases compared to pediatric index cases (OR 1.79, P = 0.047). Most importantly, children showed sustained seroconversion up to 9 months PSO, and serum antibody concentrations persistently surpassed adult levels (ratio serum IgG spike children vs. adults 90 days PSO 1.75, P < 0.001; 180 days 1.38, P = 0.01; 270 days 1.54, P = 0.001). In a low-incidence setting, SARS-CoV-2 infection and humoral immune response present distinct patterns in children including higher antibody levels, and lower seroprevalence in families with pediatric index cases. Children show long-term SARS-CoV-2 antibody responses. These findings are relevant to novel variants with increased disease burden in children, as well as for the planning of age-appropriate vaccination strategies.

Human epithelial stem cell survival within their niche requires "tonic" cannabinoid receptor 1-signalling-Lessons from the hair follicle.

The endocannabinoid system (ECS) regulates multiple aspects of human epithelial physiology, including inhibition/stimulation of keratinocyte proliferation/apoptosis, respectively. Yet, how the ECS impacts on human adult epithelial stem cell (eSC) functions remains unknown. Scalp hair follicles (HFs) offer a clinically relevant, prototypic model system for studying this directly within the native human stem cell niche. Here, we show in organ-cultured human HFs that, unexpectedly, selective activation of cannabinoid receptor-1 (CB1)-mediated signalling via the MAPK (MEK/Erk 1/2) and Akt pathways significantly increases the number and proliferation of cytokeratin CK15+ or CK19+ human HF bulge eSCs in situ, and enhances CK15 promoter activity in situ. In striking contrast, CB1-stimulation promotes apoptosis in the differentiated progeny of these eSCs (CK6+ HF keratinocytes). Instead, intrafollicular CB1 gene knockdown or CB1 antagonist treatment significantly reduces human HF eSCs numbers and stimulates their apoptosis, while CB1 knockout mice exhibit a reduced bulge eSCs pool in vivo. This identifies "tonic" CB1 signalling as a required survival stimulus for adult human HF eSCs within their niche. This novel concept must be taken into account whenever the human ECS is targeted therapeutically.

Lysosomal proteome analysis reveals that CLN3-defective cells have multiple enzyme deficiencies associated with changes in intracellular trafficking.

Numerous lysosomal enzymes and membrane proteins are essential for the degradation of proteins, lipids, oligosaccharides, and nucleic acids. The CLN3 gene encodes a lysosomal membrane protein of unknown function, and CLN3 mutations cause the fatal neurodegenerative lysosomal storage disorder CLN3 (Batten disease) by mechanisms that are poorly understood. To define components critical for lysosomal homeostasis that are affected by this disease, here we quantified the lysosomal proteome in cerebellar cell lines derived from a CLN3 knock-in mouse model of human Batten disease and control cells. We purified lysosomes from SILAC-labeled, and magnetite-loaded cerebellar cells by magnetic separation and analyzed them by MS. This analysis identified 70 proteins assigned to the lysosomal compartment and 3 lysosomal cargo receptors, of which most exhibited a significant differential abundance between control and CLN3-defective cells. Among these, 28 soluble lysosomal proteins catalyzing the degradation of various macromolecules had reduced levels in CLN3-defective cells. We confirmed these results by immunoblotting and selected protease and glycosidase activities. The reduction of 11 lipid-degrading lysosomal enzymes correlated with reduced capacity for lipid droplet degradation and several alterations in the distribution and composition of membrane lipids. In particular, levels of lactosylceramides and glycosphingolipids were decreased in CLN3-defective cells, which were also impaired in the recycling pathway of the exocytic transferrin receptor. Our findings suggest that CLN3 has a crucial role in regulating lysosome composition and their function, particularly in degrading of sphingolipids, and, as a consequence, in membrane transport along the recycling endosome pathway.

Advances in TERS (tip-enhanced Raman scattering) for biochemical applications.

TERS (tip-enhanced Raman scattering) provides exceptional spatial resolution without any need for labelling and has become a versatile tool for biochemical analysis. Two examples will be highlighted here. On the one hand, TERS measurements on a single mitochondrion are discussed, monitoring the oxidation state of the central iron ion of cytochrome c, leading towards a single protein characterization scheme in a natural environment. On the other hand, a novel approach of single molecule analysis is discussed, again based on TERS experiments on DNA and RNA, further highlighting the resolution capabilities of this method.

Mucolipidosis II is caused by mutations in GNPTA encoding the alpha/beta GlcNAc-1-phosphotransferase.

Mucolipidosis II (ML II) is a fatal lysosomal storage disorder resulting from defects in the multimeric GlcNAc-1-phosphotransferase responsible for the initial step in the generation of the mannose 6-phosphate (M6P) recognition marker. M6P residues on oligosaccharides of newly synthesized lysosomal enzymes are essential for efficient receptor-mediated transport to lysosomes. We used the recombinant GlcNAc-1-phosphotransferase gamma subunit as an affinity matrix to purify an unknown protein identified as the product of GNPTA (encoding GNPTA, previously known as MGC4170). The cDNA encodes a protein of 1,256 amino acids with two putative transmembrane domains and a complex preserved modular structure comprising at least six domains. The N-terminal domain of GNPTA, interrupted by a long insertion, shows similarities to bacterial capsule biosynthesis proteins. We identified seven mutations in GNPTA that lead to premature translational termination in six individuals with ML II. Retroviral transduction of fibroblasts from an individual with ML II resulted in the expression and localization of GNPTA in the Golgi apparatus, accompanied by the correction of hypersecretion of lysosomal enzymes. Our results provide evidence that GNPTA encodes a subunit of GlcNAc-1-phosphotransferase defective in individuals with ML II.

Promising Effect of High Dose Ambroxol Treatment on Neurocognition and Motor Development in a Patient With Neuropathic Gaucher Disease 2.

Gaucher Disease (GD) 2 is a rare inherited lysosomal disorder. Early-onset and rapid progression of neurovisceral symptoms lead to fatal outcome in early childhood. Treatment is symptomatic, a curative therapy is currently not available. This prospective study describes the clinical and biochemical outcome of a GD 2 patient treated with high dose ambroxol from the age of 4 months. Due to progressive hepatosplenomegaly additional enzyme replacement therapy was required 1 year after ambroxol monotherapy was initiated. Detailed clinical follow-up data demonstrated an age-appropriate neurocognitive and motor development but no clear benefit on peripheral organs. Glucosylsphingosine (Lyso-GL1) in cerebrospinal fluid decreased remarkably compared to pre-treatment, whereas Lyso-GL1 and chitotriosidase in blood increased. Ambroxol treatment of patient fibroblasts revealed a significant increase in ß-glucocerebrosidase activity in vitro. To our knowledge, this is the first report of a GD 2 patient with age-appropriate cognitive and motor development at 3 years of age. Combination of high dose ambroxol with ERT proved to be a successful approach to manage both visceral and neurological manifestations.

Proteolytic processing of the gamma-subunit is associated with the failure to form GlcNAc-1-phosphotransferase complexes and mannose 6-phosphate residues on lysosomal enzymes in human macrophages.

GlcNAc-1-phosphotransferase is a Golgi-resident 540-kDa complex of three subunits, alpha(2)beta(2)gamma(2), that catalyze the first step in the formation of the mannose 6-phosphate (M6P) recognition marker on lysosomal enzymes. Anti-M6P antibody analysis shows that human primary macrophages fail to generate M6P residues. Here we have explored the sorting and intracellular targeting of cathepsin D as a model, and the expression of the GlcNAc-1-phosphotransferase complex in macrophages. Newly synthesized cathepsin D is transported to lysosomes in an M6P-independent manner in association with membranes whereas the majority is secreted. Realtime PCR analysis revealed a 3-10-fold higher GlcNAc-1-phosphotransferase subunit mRNA levels in macrophages than in fibroblasts or HeLa cells. At the protein level, the gamma-subunit but not the beta-subunit was found to be proteolytically cleaved into three fragments which form irregular 97-kDa disulfide-linked oligomers in macrophages. Size exclusion chromatography showed that the gamma-subunit fragments lost the capability to assemble with other GlcNAc-1-phosphotransferase subunits to higher molecular complexes. These findings demonstrate that proteolytic processing of the gamma-subunit represents a novel mechanism to regulate GlcNAc-1-phosphotransferase activity and the subsequent sorting of lysosomal enzymes.

Prolactin--a novel neuroendocrine regulator of human keratin expression in situ.

The controls of human keratin expression in situ remain to be fully elucidated. Here, we have investigated the effects of the neurohormone prolactin (PRL) on keratin expression in a physiologically and clinically relevant test system: organ-cultured normal human hair follicles (HFs). Not only do HFs express a wide range of keratins, but they are also a source and target of PRL. Microarray analysis revealed that PRL differentially regulated a defined subset of keratins and keratin-associated proteins. Quantitative immunohistomorphometry and quantitative PCR confirmed that PRL up-regulated expression of keratins K5 and K14 and the epithelial stem cell-associated keratins K15 and K19 in organ-cultured HFs and/or isolated HF keratinocytes. PRL also up-regulated K15 promoter activity and K15 protein expression in situ, whereas it inhibited K6 and K31 expression. These regulatory effects were reversed by a pure competitive PRL receptor antagonist. Antagonist alone also modulated keratin expression, suggesting that "tonic stimulation" by endogenous PRL is required for normal expression levels of selected keratins. Therefore, our study identifies PRL as a major, clinically relevant, novel neuroendocrine regulator of both human keratin expression and human epithelial stem cell biology in situ.

Endocrine controls of keratin expression.

Keratins are a family of intermediate filaments that serve various crucial roles in skin physiology. For mammalian skin to function properly, and to produce epidermal and hair keratins that are optimally adapted for their environment, it is critical that keratin gene and protein expression are stringently controlled. Given that the skin is not only targeted by multiple hormones, but also constitutes a veritable peripheral endocrine organ, it is not surprizing that intracutaneous keratin expression is underlined by tight endocrine controls. These controls encompass thyroid hormones, steroid hormones such as glucocorticoids (GCs), retinoic acid (RA) and vitamin D, and several neuroendocrine mediators. Here, we review why a better understanding of the endocrine controls of keratin expression is not only required for an improved insight into normal human skin and hair function, but may also open new therapeutic avenues in a wide range of skin and hair diseases.

UDP-GlcNAc-1-Phosphotransferase Is a Clinically Important Regulator of Human and Mouse Hair Pigmentation.

UDP-GlcNAc-1-phosphotransferase, a product of two separate genes (GNPTAB, GNPTG), is essential for the sorting and transportation of lysosomal enzymes to lysosomes. GNPTAB gene defects cause extracellular missorting of lysosomal enzymes resulting in lysosomal storage diseases, namely mucolipidosis type II and mucolipidosis type III alpha/beta, which is associated with hair discoloration. Yet, the physiological functions of GNPTAB in the control of hair follicle (HF) pigmentation remain unknown. To elucidate these, we have silenced GNPTAB in organ-cultured human HFs as a human ex vivo model for mucolipidosis type II. GNPTAB silencing profoundly inhibited intrafollicular melanin production, the correct sorting of melanosomes, tyrosinase activity, and HMB45 expression in the HF pigmentary unit and altered HF melanocyte morphology in situ. In isolated primary human HF melanocytes, GNPTAB knockdown significantly reduced melanogenesis, tyrosinase activity, and correct tyrosinase protein sorting as well as POMC expression and caused the expected lysosomal enzyme missorting in vitro. Moreover, transgenic mice overexpressing an inserted missense mutation corresponding to that seen in human mucolipidosis type II and mucolipidosis type III alpha/beta showed significantly reduced HF pigmentation, thus corroborating the in vivo relevance of our ex vivo and in vitro findings in the human system. This identifies GNPTAB as a clinically important enzymatic control of human HF pigmentation, likely by directly controlling tyrosinase sorting and POMC transcription in HF melanocytes.

Compensatory expression of human N-acetylglucosaminyl-1-phosphotransferase subunits in mucolipidosis type III gamma.

The N-Acetylglucosaminyl-1-phosphotransferase plays a key role in the generation of mannose 6-phosphate (M6P) recognition markersessential for efficient transport of lysosomal hydrolases to lysosomes. The phosphotransferase is composed of six subunits (alpha2, beta2, gamma2). The alpha- and beta-subunits are catalytically active and encoded by a single gene, GNPTAB, whereas the gamma-subunit encoded by GNPTG is proposed to recognize conformational structures common to lysosomal enzymes. Defects in GNPTG cause mucolipidosis type III gamma, which is characterized by missorting and cellular loss of lysosomal enzymes leading to lysosomal accumulation of storage material. Using plasmon resonance spectrometry, we showed that recombinant gamma-subunit failed to bind the lysosomal enzyme arylsulfatase A. Additionally, the overexpression of the gamma-subunit in COS7 cells did not result in hypersecretion of newly synthesized lysosomal enzymes expected for competition for binding sites of the endogenous phosphotransferase complex. Analysis of fibroblasts exhibiting a novel mutation in GNPTG (c.619insT, p.K207IfsX7) revealed that the expression of GNPTAB was increased whereas in gamma-subunit overexpressing cells the GNPTAB mRNA was reduced. The data suggest that the gamma-subunit is important for the balance of phosphotransferase subunits rather for general binding of lysosomal enzymes.

Nonviral in situ green fluorescent protein labeling and culture of primary, adult human hair follicle epithelial progenitor cells.

In this article we show that cloning of the human K15 promoter before a green fluorescence protein (GFP)/geneticin-resistance cassette and transfection of microdissected, organ-cultured adult human scalp hair follicles generates specific K15 promoter-driven GFP expression in their stem cell-rich bulge region. K15-GFP+ cells can be visualized in situ by GFP fluorescence and 2-photon laser scanning microscopy. Vital K15-GFP+ progenitor cells can then be selected by using the criteria of their green fluorescence, adhesion to collagen type IV and fibronectin, and geneticin resistance. Propagated K15-GFP+ cells express epithelial progenitor markers, show the expected differential gene expression profile of human bulge epithelium, and form holoclones. This application of nonretroviral, K15 promoter-driven, GFP labeling to adult human hair follicles facilitates the characterization and manipulation of human epithelial stem cells, both in situ and in vitro, and should be transferable to other complex human tissues.

An improved, standardised protocol for the isolation, enrichment and targeted neural differentiation of Nestin+ progenitors from adult human dermis.

Human skin-derived Nestin+ cells serve as a convenient source for autologous, adult, pluripotent progenitor cells that offer new therapeutic possibilities in cell-based regenerative medicine. However, the isolation of human Nestin+ cells has tended to be of very low efficiency and to produce highly variable cell yields. Here we report a standardised protocol that facilitates the isolation and enrichment of Nestin+ progenitor cells from enzymatically digested adult human scalp dermis. The use of distinct media like Dulbecco's modified Eagle medium supplemented with foetal bovine serum or, alternatively, serum-free, supplemented neural stem cell medium greatly affected cell morphology, proliferation and differentiation (e.g. towards a neural versus mesenchymal phenotype). Finally, Nestin+ cells were isolated from a heterogeneous dermis-derived progenitor cell population, which proliferates within clones or floating microspheres under defined serum-free culture conditions. Supplementation of the medium with epidermal growth factor and basic fibroblast growth factor as well as coating with fibronectin allowed the highest enrichment level of Nestin+ progenitors and differentiation towards neural fate. These methodological advances should greatly facilitate the isolation, culture and targeted differentiation of primary, adult human scalp skin dermis-derived Nestin+ cells.

Leptin and the skin: a new frontier.

Here, we examine the currently available information which supports that the adipokine, leptin, is a major player in the biology and pathology of mammalian skin and its appendages. Specifically, the potent metabolic effects of leptin and its mimetics may be utilized to improve, preserve and restore skin regeneration and hair cycle progression, and may halt or even partially reverse some aspects of skin ageing. Since leptin can enhance mitochondrial activity and biogenesis, this may contribute to the wound healing-promoting and hair growth-modulatory effects of leptin. Leptin dependent intracellular signalling by the Janus kinase 2 dependent signal transducer and activator of transcription 3, adenosine monophosphate kinase, and peroxisome proliferator-activated receptor (PPAR) gamma coactivator/PPAR converges to mediate mitochondrial metabolic activation and enhanced cell proliferation which may orchestrate the potent developmental, trophic and protective effects of leptin. Since leptin and leptin mimetics have already been clinically tested, investigative dermatology is well-advised to place greater emphasis on the systematic exploration of the cutaneous dimensions and dermatological potential of this pleiotropic hormone.

Exploring the role of stem cells in cutaneous wound healing.

The skin offers a perfect model system for studying the wound healing cascade, which involves a finely tuned interplay between several cell types, pathways and processes. The dysregulation of these factors may lead to wound healing disorders resulting in chronic wounds, as well as abnormal scars such as hypertrophic and keloid scars. As the contribution of stem cells towards tissue regeneration and wound healing is increasingly appreciated, a rising number of stem cell therapies for cutaneous wounds are currently under development, encouraged by emerging preliminary findings in both animal models and human studies. However, we still lack an in-depth understanding of the underlying mechanisms through which stem cells contribute to cutaneous wound healing. The aim of this review is, therefore, to present a critical synthesis of our current understanding of the role of stem cells in normal cutaneous wound healing. In addition to summarizing wound healing principles and related key molecular and cellular players, we discuss the potential participation of different cutaneous stem cell populations in wound healing, and list corresponding stem cells markers. In summary, this review delineates current strategies, future applications, and limitations of stem cell-based or stem cell-targeted therapy in the management of acute and chronic skin wounds.

Nestin+ progenitor cells isolated from adult human sweat gland stroma promote reepithelialisation and may stimulate angiogenesis in wounded human skin ex vivo.

The combination of an aging population and an increasing prevalence of diseases associated with impaired-wound healing, including obesity, peripheral vascular disease and diabetes, is likely to result in a dramatic increase in the incidence and prevalence of chronic skin wounds. Indeed, systemic reviews are now not only trying to establish both the prevalence and the often under-estimated socio-economic costs of chronic skin wounds, but most importantly are addressing the impact that chronic wounds have on quality of life. Given the clear need for novel approaches to the management of chronic skin ulceration, ideally developed and tested in the human system in a manner that can be rapidly translated into clinical practice, we examined the effects of multipotent primary human nestin+ progenitor cells on human wound healing in an ex vivo model. Human sweat gland-derived nestin+ cells demonstrated the capacity to significantly promote two key wound healing parameters, i.e., both reepithelialisation and angiogenesis in experimentally wounded, organ-cultured human skin. The current data further support the use of full-thickness human skin wound-healing models ex vivo to pre-clinically test wound healing-promoting candidate agents. Whilst larger studies are required to substantiate a firm "proof-of-concept," our preliminary studies encourage further efforts to systemically determine the potential of cell-based regenerative medicine strategies in general, and the use of skin appendage-associated human nestin+ cells in particular, as novel treatment strategies for chronic skin ulceration.

Skin-derived human adult stem cells surprisingly share many features with human pancreatic stem cells.

Multiple tissue niches in the human body are now recognised to harbour stem cells. Here, we have asked how different adult stem cell populations, isolated from two ontogenetically distinct human organs (skin, pancreas), actually are with respect to a panel of standard markers/characteristics. Here we show that an easily accessible adult human tissue such as skin may serve as a convenient source of adult stem cell-like populations that share markers with stem cells derived from an internal, exocrine organ. Surprisingly, both, human pancreas- and skin-derived stem/progenitor cells demonstrate differentiation patterns across lineage boundaries into cell types of ectoderm (e.g. PGP 9.5+ and GFAP+), mesoderm (e.g. alpha-SMA+) and entoderm (e.g. amylase+ and albumin+). This intriguing differentiation capability warrants systemic follow-up, since it raises the theoretical possibility that an adult human skin-derived progenitor cell population could be envisioned for possible application in cell replacement therapies.

(Neuro-)endocrinology of epithelial hair follicle stem cells.

The hair follicle is a repository of different types of somatic stem cells. However, even though the hair follicle is both a prominent target organ and a potent, non-classical site of production and/or metabolism of numerous polypetide- and steroid hormones, neuropeptides, neurotransmitters and neurotrophins, the (neuro-)endocrine controls of hair follicle epithelial stem cell (HFeSC) biology remain to be systematically explored. Focussing on HFeSCs, we attempt here to offer a "roadmap through terra incognita" by listing key open questions, by exploring endocrinologically relevant HFeSC gene profiling and mouse genomics data, and by sketching several clinically relevant pathways via which systemic and/or locally generated (neuro-)endocrine signals might impact on HFeSC. Exemplarily, we discuss, e.g. the potential roles of glucocorticoid and vitamin D receptors, the hairless gene product, thymic hormones, bone morphogenic proteins (BMPs) and their antagonists, and Skg-3 in HFeSC biology. Furthermore, we elaborate on the potential role of nerve growth factor (NGF) and substance P-dependent neurogenic inflammation in HFeSC damage, and explore how neuroendocrine signals may influence the balance between maintenance and destruction of hair follicle immune privilege, which protects these stem cells and their progeny. These considerations call for a concerted research effort to dissect the (neuro-)endocrinology of HFeSCs much more systematically than before.

Immunophenotyping of the human bulge region: the quest to define useful in situ markers for human epithelial hair follicle stem cells and their niche.

Since the discovery of epithelial hair follicle stem cells (eHFSCs) in the bulge of human hair follicles (HFs) an important quest has started: to define useful markers. In the current study, we contribute to this by critically evaluating corresponding published immunoreactivity (IR) patterns, and by attempting to identify markers for the in situ identification of human eHFSCs and their niche. For this, human scalp skin cryosections of at least five different individuals were examined, employing standard immunohistology as well as increased sensitivity methods. Defined reference areas were compared by quantitative immunohistochemistry for the relative intensity of their specific IR. According to our experience, the most useful positive markers for human bulge cells turned out to be cytokeratin 15, cytokeratin 19 and CD200, but were not exclusive, while beta1 integrin and Lhx2 IR were not upregulated by human bulge keratinocytes. Absent IR for CD34, connexin43 and nestin on human bulge cells may be exploited as negative markers. alpha6 integrin, fibronectin, nidogen, fibrillin-1 and latent transforming growth factor (TGF)-beta-binding protein-1 were expressed throughout the connective tissue sheath of human HFs. On the other hand, tenascin-C was upregulated in the bulge and may thus constitute a component of the bulge stem cell niche of human HFs. These immunophenotyping results shed further light on the in situ expression patterns of claimed follicular 'stem cell markers' and suggest that not a single marker alone but only the use of a limited corresponding panel of positive and negative markers may offer a reasonable and pragmatic compromise for identifying human bulge stem cells in situ.

Exoproteome Analysis of Human Pathogenic Dermatophyte Species and Identification of Immunoreactive Proteins.

PURPOSE: Increasing incidence of onychomycosis and tinea pedis in humans of industrialized countries together with deep tissue infections are a therapeutic challenge in clinical mycology. For a better understanding of the pathology and immunology of infection, the authors analyze the exoproteomes of three reference strains of the most common clinical dermatophyte species (Trichophyton rubrum, Trichophyton interdigitale, Arthroderma benhamiae) and of Trichophyton strains isolated from affected patients. EXPERIMENTAL DESIGN: Extracellular proteins of those in vitro grown strains are separated via 2D High Performance Electrophoresis and identified by mass spectrometry to find proteins with provoked host immune reactivity. RESULTS: More than 80 secreted proteins including virulence factors such as peptidases and other hydrolases are identified. By Western blotting with respective patient sera, up to 31 proteins with significant antigen-antibody reactions are detected in comparison with control sera, for example, peptidases as well as several oxidoreductases. One protein, beta-glucosidase F2SZI9 seems to be a commonly processed antigen in all Trichophyton infections. CONCLUSIONS AND CLINICAL RELEVANCE: These first global exoproteome data of three dermatophyte species can be a stepping stone on the way to further study the molecular mechanisms of Trichophyton pathogenicity-associated traits. Possible candidates for potential new diagnostic methods or vaccination have to be validated in further investigations.