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Lamellar ichthyosis (LI) is a chronic disease, mostly caused by mutations in the TGM1 gene, marked by impaired skin barrier formation. No definitive therapies are available, and current treatments aim at symptomatic relief. LI mouse models often fail to faithfully replicate the clinical and histopathological features of human skin conditions. To develop advanced therapeutic approaches, such as combined ex vivo cell and gene therapy, we established a human cellular model of LI by efficient CRISPR-Cas9-mediated gene ablation of the TGM1 gene in human primary clonogenic keratinocytes. Gene-edited cells showed complete absence of transglutaminase 1 (TG1) expression and recapitulated a hyperkeratotic phenotype with most of the molecular hallmarks of LI in vitro. Using a self-inactivating γ-retroviral (SINγ-RV) vector expressing transgenic TGM1 under the control of its own promoter, we tested an ex vivo gene therapy approach and validate the model of LI as a platform for pre-clinical evaluation studies. Gene-corrected TGM1-null keratinocytes displayed proper TG1 expression, enzymatic activity, and cornified envelope formation and, hence, restored proper epidermal architecture. Single-cell multiomics analysis demonstrated proviral integrations in holoclone-forming epidermal stem cells, which are crucial for epidermal regeneration. This study serves as a proof of concept for assessing the potential of this therapeutic approach in treating TGM1-dependent LI.

Mycoplasma hominis can be isolated from the human urogenital tract. However, its interaction with the host remains poorly understood. In this study, we aimed to assess the effects of M. hominis infection on primary human keratinocytes (PHKs). Cells were quantified at different phases of the cell cycle. Proteins involved in cell cycle regulation and apoptosis progression were evaluated. The expression of genes encoding proteins that are associated with the DNA damage response and Toll-like receptor pathways was evaluated, and the cytokines involved in inflammatory responses were quantified. A greater number of keratinocytes were observed in the Sub-G0/G1 phase after infection with M. hominis. In the viable keratinocytes, infection resulted in G2/M-phase arrest; GADD45A expression was increased, as was the expression of proteins such as p53, p27, and p21 and others involved in apoptosis regulation and oxidative stress. In infected PHKs, the expression of genes associated with the Toll-like receptor pathways showed a change, and the production of IFN-γ, interleukin (IL) 1β, IL-18, IL-6, and tumour necrosis factor alpha increased. The infection of PHKs by M. hominis causes cellular damage that can affect the cell cycle by activating the response pathways to cellular damage, oxidative stress, and Toll-like receptors. Overall, this response culminated in the reduction of cell proliferation/viability in vitro. [ABSTRACT FROM AUTHOR]

Lamellar ichthyosis (LI) is a chronic disease, mostly caused by mutations in the TGM1 gene, marked by impaired skin barrier formation. No definitive therapies are available, and current treatments aim at symptomatic relief. LI mouse models often fail to faithfully replicate the clinical and histopathological features of human skin conditions. To develop advanced therapeutic approaches, such as combined ex vivo cell and gene therapy, we established a human cellular model of LI by efficient CRISPR-Cas9-mediated gene ablation of the TGM1 gene in human primary clonogenic keratinocytes. Gene-edited cells showed complete absence of transglutaminase 1 (TG1) expression and recapitulated a hyperkeratotic phenotype with most of the molecular hallmarks of LI in vitro . Using a self-inactivating γ-retroviral (SINγ-RV) vector expressing transgenic TGM1 under the control of its own promoter, we tested an ex vivo gene therapy approach and validate the model of LI as a platform for pre-clinical evaluation studies. Gene-corrected TGM1 -null keratinocytes displayed proper TG1 expression, enzymatic activity, and cornified envelope formation and, hence, restored proper epidermal architecture. Single-cell multiomics analysis demonstrated proviral integrations in holoclone-forming epidermal stem cells, which are crucial for epidermal regeneration. This study serves as a proof of concept for assessing the potential of this therapeutic approach in treating TGM1 -dependent LI., Competing Interests: M.D.L. is a consultant for J-TEC-Japan Tissue Engineering, Ltd., (© 2024 The Author(s).)

Mycoplasma hominis can be isolated from the human urogenital tract. However, its interaction with the host remains poorly understood. In this study, we aimed to assess the effects of M. hominis infection on primary human keratinocytes (PHKs). Cells were quantified at different phases of the cell cycle. Proteins involved in cell cycle regulation and apoptosis progression were evaluated. The expression of genes encoding proteins that are associated with the DNA damage response and Toll-like receptor pathways was evaluated, and the cytokines involved in inflammatory responses were quantified. A greater number of keratinocytes were observed in the Sub-G0/G1 phase after infection with M. hominis. In the viable keratinocytes, infection resulted in G2/M-phase arrest; GADD45A expression was increased, as was the expression of proteins such as p53, p27, and p21 and others involved in apoptosis regulation and oxidative stress. In infected PHKs, the expression of genes associated with the Toll-like receptor pathways showed a change, and the production of IFN-γ, interleukin (IL) 1β, IL-18, IL-6, and tumour necrosis factor alpha increased. The infection of PHKs by M. hominis causes cellular damage that can affect the cell cycle by activating the response pathways to cellular damage, oxidative stress, and Toll-like receptors. Overall, this response culminated in the reduction of cell proliferation/viability in vitro.

[Display omitted] Phospholipids are versatile formulation compounds with high biocompatibility. However, no data on their effect on skin in combination with UVA radiation exist. Thus, it was the aim of this work to (i) develop o/w nanoemulsions (NEs) differing in surfactant type and to investigate their physicochemical stability at different storage temperatures, (ii) establish a standardized protocol for in vitro phototoxicity testing using primary human skin cells and (iii) investigate the phototoxicity of amphoteric phospholipids (S45, S75, E80, S100, LPC80), sodium lauryl ether sulfate (SLES) and polysorbate 80 (PS80). Satisfying systems were developed with all surfactants except S100 due to low zeta potential (-21.4 mV ± 4.69). SLES and PS80-type NEs showed the highest stability after eight weeks; temperature-dependent variations in storage stability were most noticeable for phospholipid surfactants. For phospholipid-based NEs, higher phosphatidylcholine content led to unstable formulations. Phototoxicity assays with primary skin fibroblasts confirmed the lack of UVA-related phototoxicity but revealed cytotoxic effects of LPC80 and SLES, resulting in cell viability as low as 2.7 % ± 0.78 and 1.9 % ± 1.57 compared to the control. Our findings suggest that surfactants S45, S75 and PS80 are the most promising candidates for skin-friendly emulsifiers in sensitive applications involving exposure to UV light. [ABSTRACT FROM AUTHOR]

Herpes simplex virus 1 (HSV-1) must overcome epidermal barriers to reach its receptors on keratinocytes and initiate infection in human skin. The cell-adhesion molecule nectin-1, which is expressed in human epidermis, acts as an efficient receptor for HSV-1 but is not within reach of the virus upon exposure of human skin under nonpathological conditions. Atopic dermatitis skin, however, can provide an entry portal for HSV-1 emphasizing the role of impaired barrier functions. Here, we explored how epidermal barriers impact HSV-1 invasion in human epidermis and influence the accessibility of nectin-1 for the virus. Using human epidermal equivalents, we observed a correlation of the number of infected cells with tight-junction formation, suggesting that mature tight junctions prior to formation of the stratum corneum prevent viral access to nectin-1. Consequently, impaired epidermal barriers driven by Th2-inflammatory cytokines interleukin 4 (IL-4) and IL-13 as well as the genetic predisposition of nonlesional atopic dermatitis keratinocytes correlated with enhanced infection supporting the impact of functional tight junctions for preventing infection in human epidermis. Comparable to E-cadherin, nectin-1 was distributed throughout the epidermal layers and localized just underneath the tight-junctions. While nectin-1 was evenly distributed on primary human keratinocytes in culture, the receptor was enriched at lateral surfaces of basal and suprabasal cells during differentiation. Nectin-1 showed no major redistribution in the thickened atopic dermatitis and IL-4/IL-13-treated human epidermis in which HSV-1 can invade. However, nectin-1 localization toward tight junction components changed, suggesting that defective tight-junction barriers make nectin-1 accessible for HSV-1 which enables facilitated viral penetration. IMPORTANCE Herpes simplex virus 1 (HSV-1) is a widely distributed human pathogen which productively infects epithelia. The open question is which barriers of the highly protected epithelia must the virus overcome to reach its receptor nectin-1. Here, we used human epidermal equivalents to understand how physical barrier formation and nectin-1 distribution contribute to successful viral invasion. Inflammation-induced barrier defects led to facilitated viral penetration strengthening the role of functional tight-junctions in hindering viral access to nectin-1 that is localized just underneath tight junctions and distributed throughout all layers. We also found nectin-1 ubiquitously localized in the epidermis of atopic dermatitis and IL-4/IL-13-treated human skin implying that impaired tight-junctions in combination with a defective cornified layer allow the accessibility of nectin-1 to HSV-1. Our results support that successful invasion of HSV-1 in human skin relies on defective epidermal barriers, which not only include a dysfunctional cornified layer but also depend on impaired tight junctions. [ABSTRACT FROM AUTHOR]

Background: Chemotherapeutic approaches to the control of HPV infection suffer from a lack of specificity. For most existing HPV inhibitors, the weak antiviral effects observed in cellular assays suggest that further improvements in selecting targets, in drug potency, and in bioavailability and cell uptake are required. Objective: To synthesize novel 1,3-oxazole derivatives and define their antiviral activities against the human papillomavirus (HPV) in vitro. Methods: Determination of transient replication of an HPV-11 in transfected HEK293 cells, and HPV-18 DNA amplification in an organotypic squamous epithelial raft culture of primary human keratinocytes (PHKs), and cytotoxicity assays were used. Results: Bioassays showed that the synthesized compounds 2, 4, 5, and 9 exhibited potent antiviral activity against low-risk HPV-11 (IC50 = 1.7-9.6 µM) in a transient DNA replication assay and exhibited low cytotoxicity in HEK293 cells compared to cidofovir (CDV), antiviral agent in clinical use. Selectivity indices of compounds 4 and 5 were 20-40 times greater than that of CDV. However, compounds 4 and 9 did not exhibit a significant antiviral effect against high-risk HPV-18 infections in organotypic epithelial raft cultures. Although prophylactic HPV vaccines are now available to protect against primary infections by the seven genotypes most commonly found in cervical, penile, anal and oro-pharyngeal cancers (HPV16, 18, 31, 33, 45, 52, and 58) and two genotypes (HPV6 and 11) that cause benign anogenital warts and laryngeal papillomas, they do not protect against infections by other HPV types. Moreover, individuals already infected with HPV will not benefit from the vaccines. Thus, the need for antiviral agents to treat HPV-associated diseases remains great, but few options currently exist. Conclusions: We show that substituted 1,3-oxazole derivatives are a promising structure class of chemical compounds for the development of antiviral drugs against HPV lesions. [ABSTRACT FROM AUTHOR]

The prevalent human papillomaviruses (HPVs) infect human epithelial tissues. Infections by the mucosotropic HPV genotypes cause hyperproliferative ano-genital lesions. Persistent infections by high-risk (HR) HPVs such as HPV-16, HPV-18 and related types can progress to high grade intraepithelial neoplasias and cancers. Prophylactic HPV vaccines are based on DNA-free virus-like particles (VLPs) composed of the major capsid protein L1 of HPV-16, -18, -6 and -11 (Gardasil) or HPV-16 and -18 (Cervarix). Sera from vaccinated animals effectively prevent HPV pseudovirions to infect cell lines and mouse cervical epithelia. Both vaccines have proven to be highly protective in people. HPV pseudovirions are assembled in HEK293TT cells from matched L1 and L2 capsid proteins to encapsidate a reporter gene. Pseudovirions and genuine virions have structural differences and they infect cell lines or primary human keratinocytes (PHKs) with different efficiencies. In this study, we show that sera and isolated IgG from women immunized with Gardasil prevent authentic HPV-18 virions from infecting PHKs, whereas non-immune sera and purified IgG thereof are uniformly ineffective. Using early passage PHKs, neutralization is achieved only if immune sera are added within 2–4 h of infection. We attribute the timing effect to a conformational change in HPV virions, thought to occur upon initial binding to heparan sulfate proteoglycans (HSPG) on the cell surface. This interpretation is consistent with the inability of immune IgG bound to or taken up by PHKs to neutralize the virus. Interestingly, the window of neutralization increases to 12–16 h in slow growing, late passage PHKs, suggestive of altered cell surface molecules. In vivo , this window might be further lengthened by the time required to activate the normally quiescent basal cells to become susceptible to infection. Our observations help explain the high efficacy of HPV vaccines. [ABSTRACT FROM AUTHOR]

Abstract: The pathogenicity of Mycobacterium ulcerans (Buruli ulcer) depends on cytotoxic effect of its exotoxin mycolactone. Since epidermis represents a barrier against infectious agents and balanced apoptosis is essential in epidermal homeostasis, we explored if mycolactone A/B induces apoptosis on two human keratinocyte populations, stem cells (KSC) and transit amplifying cells (TAC), and on human keratinocyte line, HaCaT. Treatment of TAC with 1 and 10 ng/ml mycolactone-induced 60 and 90% apoptosis. KSC were more resistant than TAC: 50 and 75% of cells underwent apoptosis after 10 and 100 ng/ml toxin-treatment. Higher doses (1000 ng/ml) induced about 30% apoptosis on HaCaT. In contrast, mycolactone A/B was devoid of toxicity neither on human hepatoma HuH7 nor on human embryonic kidney HEK 293 T cell lines. In conclusion, mycolactone induces apoptosis in human keratinocytes, thus contributing to Buruli ulcer lesions development. [ABSTRACT FROM AUTHOR]

Stem cell-like keratinocytes are responsible for the high regenerative potential of the skin. For clinical applications using keratinocytes in artificial skin constructs, it is suitable to work with serum-free medium under defined conditions. This is also true for the preceding expansion of the stem cell-like keratinocyte population. Therefore, we analyzed the effect of a serum-free medium on the population distribution in comparison to an established serum-containing standard medium for keratinocyte culture. We quantified the freshly isolated as well as cultured primary human keratinocytes by their expression of the β1 integrin (CD29) in combination with the expression of the α6 integrin (CD49f) and the transferrin receptor (CD71) by flow cytometric methods. We were able to show that cultivation with serum-free medium induces a switch of the cell population to higher expression of the β1 integrin. In addition, the proportion of the α6bri/ CD71dim-expressing keratinocyte cell population was enhanced about 35.4 ± 6.56% after cultivation with serum-free medium. Culture in serum-containing medium increased this proportion of the keratinocyte cell population only about 17.3 ± 8.06%, when compared to the α6bri/ CD71dim-expressing keratinocyte cell population measured directly after isolation. Our data show that the applied culture conditions already have an enormous impact on the development of a stem cell-like phenotype of keratinocytes. This work demonstrates that the serum-free medium significantly increases the proportion of β1bri/α6bri/CD71dim-expressing keratinocytes. In conclusion, these findings implicate new applications in keratinocyte stem cell research and regenerative medicine. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

A gene therapy clinical trial for treatment of growth hormone (GH) deficiency has not been reached yet, but several strategies using different gene transfer methodologies and animal models have been developed and showed successful results. We have set up an ex vivo gene therapy protocol using primary human keratinocytes transduced with an efficient retroviral vector (LXSN) encoding the human (hGH) or mouse GH (mGH) genes. These stably modified cells presented high in vitro expression levels of hGH (7 μg/106 cells/d) and mGH (11 μg/106 cells/d) after selection with geneticin. When the hGH-secreting keratinocytes were grafted onto immunodeficient dwarf mice ( lit/scid), hGH levels in the circulation were about 0.2–0.3 ng/mL during a 12-d assay and these animals presented a significant body weight increase ( p<0.01) compared to the control. Substitution of conventional grafting methodologies with organotypic raft cultures revealed a peak value of up to 20 ng mGH/mL in the circulation of grafted lit/scid mice at 1 h postimplantation, followed by a rapid decline to baseline (≈2 ng/mL) within 24 h. One week after grafting, however, the cultured excised implants still presented approx 45% of their original in vitro secretion efficiency. Further studies are being carrier out to identify the main factor(s) that still constitute one of the major impediments to the success of this promising model of cutaneous gene therapy. [ABSTRACT FROM AUTHOR]

• Six daily 1 h topical applications of 2 mg/ml NVN1000, a NO-releasing polymer, abrogated HPV-18 progeny virus production. • NVN1000 inhibits terminal squamous differentiation of both infected and uninfected PHK raft cultures. • NVN1000 reduces HPV-18 E6 and E7 proteins and activities. • NVN1000 impairs S-phase progression and induces DNA damage in infected cultures. • NVN1000 is a promising candidate for sustained inhibition of HPV DNA replication and progeny virus production. [ABSTRACT FROM AUTHOR]