Wall teichoic acid substitution with glucose governs phage susceptibility of Staphylococcus epidermidis.

The species- and clone-specific susceptibility of Staphylococcus cells for bacteriophages is governed by the structures and glycosylation patterns of wall teichoic acid (WTA) glycopolymers. The glycosylation-dependent phage-WTA interactions in the opportunistic pathogen Staphylococcus epidermidis and in other coagulase-negative staphylococci (CoNS) have remained unknown. We report a new S. epidermidis WTA glycosyltransferase TagE whose deletion confers resistance to siphoviruses such as ΦE72 but enables binding of otherwise unbound podoviruses. S. epidermidis glycerolphosphate WTA was found to be modified with glucose in a tagE-dependent manner. TagE is encoded together with the enzymes PgcA and GtaB providing uridine diphosphate-activated glucose. ΦE72 transduced several other CoNS species encoding TagE homologs, suggesting that WTA glycosylation via TagE is a frequent trait among CoNS that permits interspecies horizontal gene transfer. Our study unravels a crucial mechanism of phage-Staphylococcus interaction and horizontal gene transfer, and it will help in the design of anti-staphylococcal phage therapies.IMPORTANCEPhages are highly specific for certain bacterial hosts, and some can transduce DNA even across species boundaries. How phages recognize cognate host cells remains incompletely understood. Phages infecting members of the genus Staphylococcus bind to wall teichoic acid (WTA) glycopolymers with highly variable structures and glycosylation patterns. How WTA is glycosylated in the opportunistic pathogen Staphylococcus epidermidis and in other coagulase-negative staphylococci (CoNS) species has remained unknown. We describe that S. epidermidis glycosylates its WTA backbone with glucose, and we identify a cluster of three genes responsible for glucose activation and transfer to WTA. Their inactivation strongly alters phage susceptibility patterns, yielding resistance to siphoviruses but susceptibility to podoviruses. Many different CoNS species with related glycosylation genes can exchange DNA via siphovirus ΦE72, suggesting that glucose-modified WTA is crucial for interspecies horizontal gene transfer. Our finding will help to develop antibacterial phage therapies and unravel routes of genetic exchange.

Activity of amorolfine or ciclopirox in combination with terbinafine against pathogenic fungi in onychomycosis-Results of an in vitro investigation.

BACKGROUND: Onychomycoses are difficult-to-treat fungal infections with high relapse rates. Combining oral and topical antifungal drugs is associated with higher success rates. Additive or synergistic modes of action are expected to enhance treatment success rates. OBJECTIVES: Investigation of the combined effects of antifungal drugs in vitro with different modes of action and application on clinical isolates from mycotic nails. METHODS: Isolates of Trichophyton rubrum, Trichophyton interdigitale and Scopulariopsis brevicaulis were collected from infected toenail specimens of patients with onychomycosis. Susceptibility testing was performed in 96-well polystyrene plates using a standard stepwise microdilution protocol. Additive or synergistic activity at varying concentrations was investigated by the checkerboard method. RESULTS: Combining terbinafine with amorolfine tended to be more effective than terbinafine in conjunction with ciclopirox. In most combinations, additive effects were observed. Synergy was detected in combinations with involving amorolfine in S. brevicaulis. These additive and synergistic interactions indicate that combined therapy with topical amorolfine and oral terbinafine is justified. Sublimation of amorolfine (and terbinafine) may enhance the penetration in and through the nail plate, and support treatment efficacy. CONCLUSIONS: These in vitro results support the notion that combining oral terbinafine and topical amorolfine is beneficial to patients with onychomycosis, particularly if the pathogen is a non-dermatophyte fungus such as S. brevicaulis.

[18F]pFBC, a Covalent CLIP-Tag Radiotracer for Detection of Viral Reporter Gene Transfer in the Murine Brain.

Preclinical models of neurological diseases and gene therapy are essential for neurobiological research. However, the evaluation of such models lacks reliable reporter systems for use with noninvasive imaging methods. Here, we report the development of a reporter system based on the CLIP-tag enzyme and [18F]pFBC, an 18F-labeled covalent CLIP-tag-ligand synthesized via a DoE-optimized and fully automated process. We demonstrated its specificity using a subcutaneous xenograft model and a model of viral vector-mediated brain gene transfer by engineering HEK293 cells and striatal neurons to express membrane-tethered CLIP-tag protein. After in vitro characterization of the reporter, mice carrying either CLIP-tag expressing or control subcutaneous xenografts underwent dynamic [18F]pFBC PET imaging. The CLIP-tag expressing xenografts showed a significantly higher uptake than control xenografts (tumor-to-muscle ratio 5.0 vs 1.7, p = 0.0379). In vivo, metabolite analysis by radio-HPLC from plasma and brain homogenates showed only one radio-metabolite in plasma and none in the brain. In addition, [18F]pFBC showed fast uptake and rapid clearance from the brain in animals injected with adeno-associated virus (AAV)-CLIP in the right striatum but no right-to-left (R-L) uptake difference in the striata in the acquired PET data. In contrast, autoradiography showed a clear accumulation of radioactivity in the AAV-CLIP-injected right striatum compared to the sham-injected left striatum control. CLIP-tag expression and brain integrity were verified by immunofluorescence and light sheet microscopy. In conclusion, we established a novel reporter gene system for PET imaging of gene expression in the brain and periphery and demonstrated its potential for a wide range of applications, particularly for neurobiological research and gene therapy with viral vectors.

Augmenting MEK inhibitor efficacy in BRAF wild-type melanoma: synergistic effects of disulfiram combination therapy.

BACKGROUND: MEK inhibitors (MEKi) were shown to be clinically insufficiently effective in patients suffering from BRAF wild-type (BRAF WT) melanoma, even if the MAPK pathway was constitutively activated due to mutations in NRAS or NF-1. Thus, novel combinations are needed to increase the efficacy and duration of response to MEKi in BRAF WT melanoma. Disulfiram and its metabolite diethyldithiocarbamate are known to have antitumor effects related to cellular stress, and induction of endoplasmic reticulum (ER) stress was found to synergize with MEK inhibitors in NRAS-mutated melanoma cells. Therefore, we investigated the combination of both therapeutics to test their effects on BRAF-WT melanoma cells and compared them with monotherapy using the MEKi trametinib. METHODS: The effects of combined therapy with disulfiram or its metabolite diethyldithiocarbamate and the MEKi trametinib were evaluated in a series of BRAF-WT melanoma cell lines by measuring cell viability and apoptosis induction. Cytotoxicity was additionally assessed in 3D spheroids, ex vivo melanoma slice cultures, and in vivo xenograft mouse models. The response of melanoma cells to treatment was studied at the RNA and protein levels to decipher the mode of action. Intracellular and intratumoral copper measurements were performed to investigate the role of copper ions in the antitumor cytotoxicity of disulfiram and its combination with the MEKi. RESULTS: Diethyldithiocarbamate enhanced trametinib-induced cytotoxicity and apoptosis induction in 2D and 3D melanoma culture models. Mechanistically, copper-dependent induction of oxidative stress and ER stress led to Janus kinase (JNK)-mediated apoptosis in melanoma cells. This mechanism was also detectable in patient-derived xenograft melanoma models and resulted in a significantly improved therapeutic effect compared to monotherapy with the MEKi trametinib. CONCLUSIONS: Disulfiram and its metabolite represent an attractive pharmaceutical approach to induce ER stress in melanoma cells that potentiates the antitumor effect of MEK inhibition and may be an interesting candidate for combination therapy of BRAF WT melanoma.

SARS-CoV-2-Induced Vasculitic Skin Lesions Are Associated with Massive Spike Protein Depositions in Autophagosomes.

In patients infected with severe acute respiratory syndrome coronavirus 2, vasculopathic changes of the skin are associated with a severe prognosis. However, the pathogenesis of this vasculopathy is not conclusively clarified. In this study, 25 prospectively collected skin samples from patients with COVID-19-related skin lesions were examined for vasculopathic changes and, in case of vasculitis, were further analyzed with electron microscopy and immunohistochemistry. Vasculopathy was observed in 76% of all COVID-19-related inflammatory skin lesions. Visual endothelial changes without manifest leukocytoclastic vasculitis were found in 60% of the COVID-19-related skin lesions, whereas leukocytoclastic vasculitis was diagnosed in 16%. In the cases of vasculitis, there were extensive spike protein depositions in microvascular endothelial cells that colocalized with the autophagosome proteins LC3B and LC3C. The autophagy protein complex LC3-associated endocytosis in microvascular endothelial cells seems to be an important pathogenic factor for severe acute respiratory syndrome coronavirus 2-related vasculitis in the skin. On ultrastructural morphology, the vasculitic process was dominated by intracellular vesicle formation and endothelial cell disruption. Direct presence of severe acute respiratory syndrome coronavirus 2 particles in the skin was not observed. Therefore, our results suggest that instead of direct viral infection, dermal vasculitic lesions in COVID-19 are caused by severe acute respiratory syndrome coronavirus 2 spike protein deposition followed by endothelial damage with activation of autophagy.

Acidosis-mediated increase in IFN-γ-induced PD-L1 expression on cancer cells as an immune escape mechanism in solid tumors.

Immune checkpoint inhibitors have revolutionized cancer therapy, yet the efficacy of these treatments is often limited by the heterogeneous and hypoxic tumor microenvironment (TME) of solid tumors. In the TME, programmed death-ligand 1 (PD-L1) expression on cancer cells is mainly regulated by Interferon-gamma (IFN-γ), which induces T cell exhaustion and enables tumor immune evasion. In this study, we demonstrate that acidosis, a common characteristic of solid tumors, significantly increases IFN-γ-induced PD-L1 expression on aggressive cancer cells, thus promoting immune escape. Using preclinical models, we found that acidosis enhances the genomic expression and phosphorylation of signal transducer and activator of transcription 1 (STAT1), and the translation of STAT1 mRNA by eukaryotic initiation factor 4F (elF4F), resulting in an increased PD-L1 expression. We observed this effect in murine and human anti-PD-L1-responsive tumor cell lines, but not in anti-PD-L1-nonresponsive tumor cell lines. In vivo studies fully validated our in vitro findings and revealed that neutralizing the acidic extracellular tumor pH by sodium bicarbonate treatment suppresses IFN-γ-induced PD-L1 expression and promotes immune cell infiltration in responsive tumors and thus reduces tumor growth. However, this effect was not observed in anti-PD-L1-nonresponsive tumors. In vivo experiments in tumor-bearing IFN-γ-/- mice validated the dependency on immune cell-derived IFN-γ for acidosis-mediated cancer cell PD-L1 induction and tumor immune escape. Thus, acidosis and IFN-γ-induced elevation of PD-L1 expression on cancer cells represent a previously unknown immune escape mechanism that may serve as a novel biomarker for anti-PD-L1/PD-1 treatment response. These findings have important implications for the development of new strategies to enhance the efficacy of immunotherapy in cancer patients.

[Recurrent tinea corporis generalisata due to Terbinafine-resistant Trichophyton rubrum strain : Long-term treatment with super bioavailability itraconazole]. / Rezidivierende Tinea corporis generalisata durch einen Terbinafin-resistenten Trichophyton-rubrum-Stamm : Langzeitbehandlung mit Super-Bioavailability-Itraconazol.

For more than 30 years, an 82-year-old man has been suffering from tinea corporis generalisata in the sense of Trichophyton rubrum syndrome. The patient received long-term treatment with terbinafine. Fluconazole had no effect. There was an increase in liver enzymes with itraconazole. Super bioavailability (SUBA) itraconazole was initially not tolerated. A therapy attempt with voriconazole was successful, but was stopped due to side effects. The Trichophyton (T.) rubrum strain isolated from skin scales was tested for terbinafine resistance using the breakpoint method and found to be (still) sensitive. Sequencing of the squalene epoxidase (SQLE) gene revealed a previously unknown point mutation of the codon for isoleucine ATC→ACC with amino acid substitution I479T (isoleucine479 threonine). Long-term therapy with terbinafine 250 mg had been given every 3 days since 2018. In addition, bifonazole cream, ciclopirox solution, and occasionally terbinafine cream were used. The skin condition was stable until an exacerbation of the dermatophytosis in 2021. There were erythematosquamous, partly atrophic, centrifugal, scaly, confluent plaques on the integument and the extremities. Fingernails and toenails had white to yellow-brown discoloration, and were hyperkeratotic and totally dystrophic. T. rubrum was cultured from skin scales from the integument, from the feet, from nail shavings from the fingernails and also toenails and detected by PCR. In the breakpoint test, the T. rubrum isolates from tinea corporis and nail samples showed a minimum inhibitory concentration (MIC) of 0.5 µg ml-1 (terbinafine resistance in vitro). Sequencing of the SQLE gene of the T. rubrum isolate revealed evidence of a further point mutation that led to amino acid substitution I479V (isoleucine 479 valine). Long-term therapy was started with SUBA itraconazole: 14 days 2â€¯× 1 capsule daily, then twice weekly administration of 2â€¯× 50 mg. During breaks in therapy, the mycosis regularly flared up again. Finally, 50 mg SUBA itraconazole was given 5 days a week, which completely suppressed the dermatophytosis. Topically, ciclopirox and miconazole cream were used alternately. In conclusion, in the case of recurrent and therapy-refractory dermatophytoses caused by T. rubrum, terbinafine resistance must also be considered in individual cases. An in vitro resistance test and point mutation analysis of the squalene epoxidase gene confirms the diagnosis. Itraconazole, also in the form of SUBA itraconazole, is the drug of choice for the oral antifungal treatment of these patients.

Neutrophil extracellular traps enhance S. aureus skin colonization by oxidative stress induction and downregulation of epidermal barrier genes.

Staphylococcus aureus is the most common cause of bacterial skin infections in humans, including patients with atopic dermatitis (AD). Polymorphonuclear neutrophils (PMNs) are the first cells to infiltrate an infection site, where they usually provide an effective first line of defense, including neutrophil extracellular trap (NET) formation. Here, we show that infiltrating PMNs in inflamed human and mouse skin enhance S. aureus skin colonization and persistence. Mechanistically, we demonstrate that a crosstalk between keratinocytes and PMNs results in enhanced NET formation upon S. aureus infection, which in turn induces oxidative stress and expression of danger-associated molecular patterns such as high-mobility-group-protein B1 (HMGB1) in keratinocytes. In turn, HMGB1 enhances S. aureus skin colonization and persistence by promoting skin barrier dysfunctions by the downregulation of epidermal barrier genes. Using patient material, we show that patients with AD exhibit enhanced presence of PMNs, NETs, and HMGB1 in the skin, demonstrating the clinical relevance of our finding.

Case Report: Paraneoplastic psoriasis in thymic carcinoma.

Thymic carcinomas are exceedingly rare and very aggressive malignancies of the anterior mediastinum. While thymomas exhibit a high association with paraneoplastic syndromes, these phenomena are a rarity in thymic carcinomas. In general, acanthotic syndromes such as acroceratosis neoplastica and acanthosis nigricans maligna are commonly observed as paraneoplastic phenomena in patients with carcinomas. In contrast, psoriasis vulgaris, another acanthotic disease, rarely occurs as a paraneoplasia. We report the case of a 36-year-old patient with progressive thymic carcinoma (undifferentiated carcinoma, T3N2M1a) and paraneoplastic psoriasis occurring ten months before the initial diagnosis of the carcinoma. Over the course of the disease, new psoriatic flares heralded relapse or progression of the carcinoma. To our knowledge, this is the first reported case of paraneoplastic psoriasis in thymic carcinoma.

Treatment of aseptic facial granuloma as a manifestation of pediatric rosacea with oral macrolides.

Aseptic facial granuloma is a rare pediatric disease, presenting with asymptomatic facial nodules on the cheeks or the eyelids and may represent a form of granulomatous rosacea in children. In this retrospective case series, 12 children with aseptic facial granuloma were treated with oral macrolides (erythromycin or roxithromycin) resulting in a healing of the lesions within a mean treatment time of 5.25 months with no recurrences. The treatment was mainly well tolerated. Oral macrolides may be effective in the treatment of patients with aseptic facial granuloma.

S1 Guideline onychomycosis.

Onychomycosis is a fungal infection of the fingernails and toenails. In Europe, tinea unguium is mainly caused by dermatophytes. The diagnostic workup comprises microscopic examination, culture and/or molecular testing (nail scrapings). Local treatment with antifungal nail polish is recommended for mild or moderate nail infections. In case of moderate to severe onychomycosis, oral treatment is recommended (in the absence of contraindications). Treatment should consist of topical and systemic agents. The aim of this update of the German S1 guideline is to simplify the selection and implementation of appropriate diagnostics and treatment. The guideline was based on current international guidelines and the results of a literature review conducted by the experts of the guideline committee. This multidisciplinary committee consisted of representatives from the German Society of Dermatology (DDG), the German-Speaking Mycological Society (DMykG), the Association of German Dermatologists (BVDD), the German Society for Hygiene and Microbiology (DGHM), the German Society of Pediatric and Adolescent Medicine (DGKJ), the Working Group for Pediatric Dermatology (APD) and the German Society for Pediatric Infectious Diseases (DGPI). The Division of Evidence-based Medicine (dEBM) provided methodological assistance. The guideline was approved by the participating medical societies following a comprehensive internal and external review.

Radiolabeled GPVI-Fc for PET Imaging of Multiple Extracellular Matrix Fibers: A New Look into Pulmonary Fibrosis Progression.

Invariably fatal and with a particularly fast progression, pulmonary fibrosis (PF) is currently devoid of curative treatment options. Routine clinical diagnosis relies on breathing tests and visualizing the changes in lung structure by CT, but anatomic information is often not sufficient to identify early signs of progressive PF. For more efficient diagnosis, additional imaging techniques were investigated in combination with CT, such as 18F-FDG PET, although with limited success because of lack of disease specificity. Therefore, novel molecular targets enabling specific diagnosis are investigated, in particular for molecular imaging techniques. Methods: In this study, we used a 64Cu-radiolabeled platelet glycoprotein VI fusion protein (64Cu-GPVI-Fc) targeting extracellular matrix (ECM) fibers as a PET tracer to observe longitudinal ECM remodeling in a bleomycin-induced PF mouse model. Results: 64Cu-GPVI-Fc showed significant uptake in fibrotic lungs, matching histology results. Contrary to 18F-FDG PET measurements, 64Cu-GPVI-Fc uptake was linked entirely to the fibrotic activity of tissue and not was susceptible to inflammation. Conclusion: Our study highlights 64Cu-GPVI-Fc as a specific tracer for ECM remodeling in PF, with clear therapy-monitoring and clinical translation potential.

Novel GATA1 Variant Causing a Bleeding Phenotype Associated with Combined Platelet α-/δ-Storage Pool Deficiency and Mild Dyserythropoiesis Modified by a SLC4A1 Variant.

Germline defects in the transcription factor GATA1 are known to cause dyserythropoiesis with(out) anemia and variable abnormalities in platelet count and function. However, damaging variants closely located to the C-terminal zinc finger domain of GATA1 are nearly unknown. In this study, a 36-year-old male index patient and his 4-year-old daughter suffered from moderate mucocutaneous bleeding diathesis since birth. Whole exome sequencing detected a novel hemizygous GATA1 missense variant, c.886A>C p.T296P, located between the C-terminal zinc finger and the nuclear localization sequence with non-random X-chromosome inactivation in the heterozygous daughter. Blood smears from both patients demonstrated large platelet fractions and moderate thrombocytopenia in the index. Flow cytometry and electron microscopy analysis supported a combined α-/δ (AN-subtype)-storage pool deficiency as cause for impaired agonist-induced platelet aggregation (light transmission aggregometry) and granule exocytosis (flow cytometry). The absence of BCAM in the index (Lu(a-b-)) and its low expression in the daughter (Lu(a-b+)) confirmed a less obvious effect of defective GATA1 also on erythrocytes. Borderline anemia, elevated HbF levels, and differential transcription of GATA1-regulated genes indicated mild dyserythropoiesis in both patients. Furthermore, a mild SLC4A1 defect associated with a heterozygous SLC4A1 c.2210C>T p.A737V variant maternally transmitted in the daughter may modify the disease to mild spherocytosis and hemolysis.

Ivermectin treatment in rosacea: How novel smartphone technology can support monitoring rosacea-associated signs and symptoms.

Rosacea lessens patients' quality of life not only by visible symptoms like erythema, papules, and pustules but also by invisible symptoms like stinging, burning, and dryness. Ivermectin 1% cream has recently been introduced as an efficient therapy for papules and pustules in rosacea patients. To investigate the potential of ivermectin 1% cream to improve rosacea-associated erythema and invisible symptoms by combining established questionnaires with the novel photography and analysis tool Scarletred®Vision. We performed an open monocentric pilot study including 25 Caucasian patients presenting with moderate to severe rosacea with erythema, less than 10 papules and/or pustules, and ≥ 15 Demodex mites/cm2 . Patients applied 1 g of ivermectin 1% cream (Soolantra®) once a day for ≥16 weeks. Skin symptoms were recorded at baseline, week 8 and ≥ week 16. Grade of erythema was determined by clinician erythema assessment (CEA) and patient self-assessment (PSA). Severity of invisible skin symptoms (stinging and/or burning, dryness, itching) were assessed by questionnaire. Erythema and skin texture were additionally quantified using Scarletred®Vision. Ivermectin 1% cream significantly reduced invisible symptoms of rosacea (stinging and/or burning, dryness: p < 0.0001; itching p < 0.001; at ≥16 weeks). Analysis with Scarletred®Vision confirmed CEA and PSA results for improvement of erythema (p < 0.0001; at ≥16 weeks) and skin roughness (p < 0.001; at ≥16 weeks). Treatment with ivermectin 1% cream is efficient in treating not only rosacea-associated papules and pustules but also erythema and invisible skin symptoms.

S2k guideline: Rosacea.

This updated and upgraded S2k guideline deals with the diagnosis and treatment of rosacea, which is a common, chronic inflammatory skin disease mostly affecting the face. Initially, rosacea is characterized by recurrent erythema, telangiectasia and flushing. Later, the inflammatory component predominates, with persistent erythema with follicular papules, papulopustules and pustules. The development of phyma, which usually occurs on the acral localizations, is the most severe manifestation. For the treatment of rosacea, the interdisciplinary guideline committee, with representatives of the German Dermatological Society (DDG), the Professional Association of German Dermatologists (BVDD), the German Opthalmological Society (DOG), the Society for Dermopharmacy (GD), the Swiss Society for Dermatology and Venereology (SGDV) and the German Rosacea Aid e. V., recommends the avoidance of trigger factors and topical applications of metronidazole, azelaic acid or ivermectin. For symptomatic treatment of persistent centrofacial erythema, the topical vasoconstrictors brimonidine or oxymetazoline can also be used. Systemic therapy is recommended for therapy-resistant and severe forms of rosacea papulopustulosa. The drug of choice is low-dose doxycycline. Alternatively, low-dose isotretinoin can be recommended. Ocular rosacea should be treated with lid margin hygiene. For topical treatment, ciclosporin eye drops, azithromycin, ivermectin or metronidazole are suggested.

How to Optimize Rosacea Treatment for Better Patient Outcomes: An Opinion Piece.

Rosacea is a chronic disease requiring long-term management. However, it is often treated according to package label instructions, which reflect the conditions of a Phase III study rather than a chronic disease. Furthermore, due to a lack of clinical data or guidelines on long-term treatment, many clinicians choose to discontinue treatment once success has been reached, rather than continuing with maintenance therapy. As experienced practicing dermatologists and investigators in the field, in this article we address the current evidence gaps in rosacea management and provide practical advice to clinicians on how optimal outcomes can be achieved and maintained in patients with rosacea in real-world practice, based on our own experience and the available clinical data.