Genetic and Functional Analyses of Cutibacterium Acnes Isolates Reveal the Association of a Linear Plasmid with Skin Inflammation.

Cutibacterium acnes is a commensal bacterium on the skin that is generally well-tolerated, but different strain types have been hypothesized to contribute to the disease acne vulgaris. To understand how some strain types might contribute to skin inflammation, we generated a repository of C. acnes isolates from skin swabs of healthy subjects and subjects with acne and assessed their strain-level identity and capacity to stimulate cytokine release. Phylotype II K-type strains were more frequent on healthy and nonlesional skin of subjects with acne than those isolated from lesions. Phylotype IA-1 C-type strains were increased on lesional skin compared with those on healthy skin. The capacity to induce cytokines from cultured monocyte-derived dendritic cells was opposite to this action on sebocytes and keratinocytes and did not correlate with the strain types associated with the disease. Whole-genome sequencing revealed a linear plasmid in high-inflammatory isolates within similar strain types that had different proinflammatory responses. Single-cell RNA sequencing of mouse skin after intradermal injection showed that strains containing this plasmid induced a higher inflammatory response in dermal fibroblasts. These findings revealed that C. acnes strain type is insufficient to predict inflammation and that carriage of a plasmid could contribute to disease.

Antimicrobial production by perifollicular dermal preadipocytes is essential to the pathophysiology of acne.

Innate immune defense against deep tissue infection by Staphylococcus aureus is orchestrated by fibroblasts that become antimicrobial when triggered to differentiate into adipocytes. However, the role of this process in noninfectious human diseases is unknown. To investigate the potential role of adipogenesis by dermal fibroblasts in acne, a disorder triggered by Cutibacterium acnes, single-cell RNA sequencing was performed on human acne lesions and mouse skin challenged by C. acnes. A transcriptome consistent with adipogenesis was observed within specific fibroblast subsets from human acne and mouse skin lesions infected with C. acnes. Perifollicular dermal preadipocytes in human acne and mouse skin lesions showed colocalization of PREF1, an early marker of adipogenesis, and cathelicidin (Camp), an antimicrobial peptide. This capacity of C. acnes to specifically trigger production of cathelicidin in preadipocytes was dependent on TLR2. Treatment of wild-type mice with retinoic acid (RA) suppressed the capacity of C. acnes to form acne-like lesions, inhibited adipogenesis, and enhanced cathelicidin expression in preadipocytes, but lesions were unresponsive in Camp-/- mice, despite the anti-adipogenic action of RA. Analysis of inflamed skin of acne patients after retinoid treatment also showed enhanced induction of cathelicidin, a previously unknown beneficial effect of retinoids in difficult-to-treat acne. Overall, these data provide evidence that adipogenic fibroblasts are a critical component of the pathogenesis of acne and represent a potential target for therapy.

Antimicrobials from a feline commensal bacterium inhibit skin infection by drug-resistant S. pseudintermedius.

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is an important emerging zoonotic pathogen that causes severe skin infections. To combat infections from drug-resistant bacteria, the transplantation of commensal antimicrobial bacteria as a therapeutic has shown clinical promise. We screened a collection of diverse staphylococcus species from domestic dogs and cats for antimicrobial activity against MRSP. A unique strain (S. felis C4) was isolated from feline skin that inhibited MRSP and multiple gram-positive pathogens. Whole genome sequencing and mass spectrometry revealed several secreted antimicrobials including a thiopeptide bacteriocin micrococcin P1 and phenol-soluble modulin beta (PSMß) peptides that exhibited antimicrobial and anti-inflammatory activity. Fluorescence and electron microscopy revealed that S. felis antimicrobials inhibited translation and disrupted bacterial but not eukaryotic cell membranes. Competition experiments in mice showed that S. felis significantly reduced MRSP skin colonization and an antimicrobial extract from S. felis significantly reduced necrotic skin injury from MRSP infection. These findings indicate a feline commensal bacterium that could be utilized in bacteriotherapy against difficult-to-treat animal and human skin infections.

Mechanisms for control of skin immune function by the microbiome.

The skin represents the largest area for direct contact between microbes and host immunocytes and is a site for constant communication between the host and this diverse and essential microbial community. Coagulase-negative staphylococci are an abundant bacterial genus on the human skin and are regulated through various mechanisms that include the epidermal barrier environment and innate and adaptive immune systems within the epidermis and dermis. In turn, some species and strains of these bacteria produce beneficial products that augment host immunity by exerting specifically targeted antimicrobial, anti-inflammatory, or anti-neoplastic activity while also promoting broad innate and adaptive immune responses. The use of selected skin commensals as a therapeutic has shown promise in recent human clinical trials. This emerging concept of bacteriotherapy is defining mechanisms of action and validating the dependence on the microbiome for maintenance of immune homeostasis.

Skin Cancer Incidence in Organ Transplant Recipients Referred for Benign Skin Conditions.

BACKGROUND: Solid organ transplant recipients (SOTRs) are at ∼100-fold increased risk for developing skin cancers compared with the general population, with increased morbidity and mortality. These patients are closely followed by dermatology; however, it is unclear how referral reasons from nondermatologic providers affect care in these patients. OBJECTIVE: This study examines the reason SOTRs are referred to dermatologic care by nondermatologic providers as a potential predictor of nonmelanoma skin cancer (NMSC) outcomes. MATERIALS AND METHODS: A retrospective case-control study was conducted with the records of 353 adult SOTRs referred to a specialized transplant dermatology clinic within an academic tertiary care center between 2007 and mid-2012. RESULTS: Eighty-one patients were diagnosed with 491 total premalignant and malignant skin lesions. A considerable proportion of patients diagnosed with NMSC were referred for benign skin conditions such as rash or acne. CONCLUSION: These results indicate that some SOTRs referred to dermatology for benign skin disorders are incidentally diagnosed with cutaneous malignancies; this is concerning given that referrals for benign skin conditions may delay appropriate care for cutaneous malignancies and preventative care. Better risk stratification, improved interdisciplinary collaboration, and prompt referrals for dermatologic care are needed in the care of SOTRs.

Use of Autologous Bacteriotherapy to Treat Staphylococcus aureus in Patients With Atopic Dermatitis: A Randomized Double-blind Clinical Trial.

IMPORTANCE: Atopic dermatitis (AD) can be negatively affected by Staphylococcus aureus. The skin microbiome of AD is deficient in coagulase-negative Staphylococcus (CoNS) that can kill S aureus. OBJECTIVE: To evaluate if the antimicrobial-producing CoNS (CoNS-AM+) of a patient with AD can be autologously reintroduced to the same patient to inhibit survival of S aureus and improve clinical outcomes. DESIGN, SETTING, AND PARTICIPANTS: This double-blind, vehicle-controlled, single-center randomized clinical trial of 11 adult patients with moderate to severe AD who were randomized to receive either an autologous CoNS-AM+ (n = 5) or the vehicle (n = 6) was conducted between April 2016 and May 2018. The data were analyzed from May 2018 to July 2019. INTERVENTIONS: Autologous CoNS-AM+ was isolated from swabs that were obtained from the nonlesional skin of each patient with AD, expanded by culture, and then reapplied topically to the forearms at a concentration of 107 colony-forming units/g. MAIN OUTCOMES AND MEASURES: The primary end point of this study was to assess S aureus abundance after 1 week of application of autologous CoNS-AM+ on patients with AD by culture-based and DNA-based methods. The secondary end points were to assess the safety and clinical outcomes. RESULTS: Eleven patients (4 men [36.4%] and 7 women [63/6%]) were recruited based on the inclusion criteria. There were no serious adverse events in groups treated with autologous CoNS-AM+ or the vehicle. Staphylococcus aureus colonization on lesional skin at the end of treatment on patients who were treated with autologous CoNS-AM+ (mean of log10 ratio to baseline, -1.702; 95% CI, -2.882 to -0.523) was reduced by 99.2% compared with vehicle treatment (mean of log10 ratio to baseline, 0.671; 95% CI, -0.289 to 1.613; P = .01) and persisted for 4 days after treatment (CoNS-AM+: mean of log10 ratio to baseline, -1.752; 95% CI, -3.051 to -0.453; vehicle: mean of log10 ratio to baseline, -0.003; 95% CI, -1.083 to 1.076; P = .03). Importantly, local Eczema Area And Severity Index scores that were assessed at day 11 on patients who received CoNS-AM+ (mean of percentage change, -48.45; 95% CI, -84.34 to -12.55) were significantly improved compared with vehicle treatment (mean of percentage change, -4.52; 95% CI, -36.25 to 27.22; P = .04). CONCLUSIONS AND RELEVANCE: The data from this randomized clinical trial suggest that bacteriotherapy with an autologous strain of skin commensal bacteria can safely decrease S aureus colonization and improve disease severity. Although larger studies will be needed, this personalized approach for S aureus reduction may provide an alternative treatment for patients with AD beyond antibiotics, immunosuppression, and immunomodulation. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03158012.

Development of a human skin commensal microbe for bacteriotherapy of atopic dermatitis and use in a phase 1 randomized clinical trial.

Staphylococcus aureus colonizes patients with atopic dermatitis (AD) and exacerbates disease by promoting inflammation. The present study investigated the safety and mechanisms of action of Staphylococcus hominis A9 (ShA9), a bacterium isolated from healthy human skin, as a topical therapy for AD. ShA9 killed S. aureus on the skin of mice and inhibited expression of a toxin from S. aureus (psmα) that promotes inflammation. A first-in-human, phase 1, double-blinded, randomized 1-week trial of topical ShA9 or vehicle on the forearm skin of 54 adults with S. aureus-positive AD (NCT03151148) met its primary endpoint of safety, and participants receiving ShA9 had fewer adverse events associated with AD. Eczema severity was not significantly different when evaluated in all participants treated with ShA9 but a significant decrease in S. aureus and increased ShA9 DNA were seen and met secondary endpoints. Some S. aureus strains on participants were not directly killed by ShA9, but expression of mRNA for psmα was inhibited in all strains. Improvement in local eczema severity was suggested by post-hoc analysis of participants with S. aureus directly killed by ShA9. These observations demonstrate the safety and potential benefits of bacteriotherapy for AD.

Staphylococcus epidermidis protease EcpA can be a deleterious component of the skin microbiome in atopic dermatitis.

BACKGROUND: Staphylococcus aureus and Staphylococcus epidermidis are the most abundant bacteria found on the skin of patients with atopic dermatitis (AD). S aureus is known to exacerbate AD, whereas S epidermidis has been considered a beneficial commensal organism. OBJECTIVE: In this study, we hypothesized that S epidermidis could promote skin damage in AD by the production of a protease that damages the epidermal barrier. METHODS: The protease activity of S epidermidis isolates was compared with that of other staphylococcal species. The capacity of S epidermidis to degrade the barrier and induce inflammation was examined by using human keratinocyte tissue culture and mouse models. Skin swabs from atopic and healthy adult subjects were analyzed for the presence of S epidermidis genomic DNA and mRNA. RESULTS: S epidermidis strains were observed to produce strong cysteine protease activity when grown at high density. The enzyme responsible for this activity was identified as EcpA, a cysteine protease under quorum sensing control. EcpA was shown to degrade desmoglein-1 and LL-37 in vitro, disrupt the physical barrier, and induce skin inflammation in mice. The abundance of S epidermidis and expression of ecpA mRNA were increased on the skin of some patients with AD, and this correlated with disease severity. Another commensal skin bacterial species, Staphylococcus hominis, can inhibit EcpA production by S epidermidis. CONCLUSION: S epidermidis has commonly been regarded as a beneficial skin microbe, whereas S aureus has been considered deleterious. This study suggests that the overabundance of S epidermidis found on some atopic patients can act similarly to S aureus and damage the skin by expression of a cysteine protease.

Innate Immune Dysfunction in Rosacea Promotes Photosensitivity and Vascular Adhesion Molecule Expression.

Rosacea is a chronic skin disease characterized by photosensitivity, abnormal dermal vascular behavior, inflammation, and enhanced expression of the antimicrobial peptide LL-37. We observed that dermal endothelial cells in rosacea had an increased expression of VCAM1 and hypothesized that LL-37 could be responsible for this response. The digestion of double-stranded RNA from keratinocytes exposed to UVB blocked the capacity of these cells to induce adhesion molecules on dermal microvascular endothelial cells. However, a synthetic noncoding snoU1RNA was only capable of increasing adhesion molecules on endothelial cells in the presence of LL-37, suggesting that the capacity of UVB exposure to promote both double-stranded RNA and LL-37 was responsible for the endothelial response to keratinocytes. Sequencing of RNA from the endothelial cells uncovered the activation of Gene Ontology (GO) pathways relevant to the human disease, such as type I and II interferon signaling, cell-cell adhesion, leukocyte chemotaxis, and angiogenesis. Functional relevance was demonstrated as double-stranded RNA and LL-37 promoted adhesion and transmigration of monocytes across the endothelial cell monolayers. Gene knockdown of TLR3, RIGI, or IRF1 decreased monocyte adhesion in endothelial cells, confirming the role of the double-stranded RNA recognition pathways. These observations show how the expression of LL-37 can lead to enhanced sensitivity to UVB radiation in rosacea.

Lymphomatoid papulosis in patients with chronic lymphocytic leukemia/small lymphocytic lymphoma: case report and literature review.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is a B cell lymphoproliferative disorder that characteristically presents in older individuals. Small lymphocytic lymphoma (SLL) occurs when CLL cells infiltrate lymph nodes and other tissues but spare peripheral blood and bone marrow. Lymphomatoid papulosis (LyP) is an indolent cutaneous CD30+ lymphoproliferative disorder characterized by papules and nodules that develop and spontaneously regress over weeks to months. METHODS: An 84-year-old man with CLL who developed LyP is described. The features of other patients who concurrently had both of these conditions are reviewed. RESULTS: A man was diagnosed with CLL at age 50 years. At 84 years of age, he presented with red papules on his buttocks, which demonstrated a CD30+ lymphoproliferative disorder on biopsy. Correlation of the lesion history, morphology, and histopathology established the diagnosis of LyP. LyP and CLL/SLL, including in this patient, has only been reported in 11 individuals, to our knowledge. CONCLUSION: The concurrent expression of LyP and CLL/SLL is rare. Since the conditions derive from different lymphocyte subsets, the concurrent expression may be merely coincidental. However, the development of both conditions in the same individual may provide additional insight into the pathogenesis of these disorders.

Cutaneous Squamous Cell Carcinomas in Solid Organ Transplant Recipients Compared With Immunocompetent Patients.

Importance: Solid organ transplant recipients (SOTRs) have a 100-fold increased risk of squamous cell carcinoma (SCC), and they may develop more aggressive SCCs compared with immunocompetent individuals. Objective: To compare outcomes associated with aggressive behavior of SCC in SOTRs and high-risk immunocompetent patients. Design, Setting, and Participants: A retrospective cohort study of 58 SOTRs and 40 immunocompetent patients evaluated at the Yale Transplant Dermatology Clinic in New Haven, Connecticut, who had at least 1 SCC confirmed histopathologically between January 1, 2008, and December 31, 2015. Cumulative follow-up time for this study was 369 patient-years. Exposure: Immunosuppressive medication regimen for SOTRs. Main Outcomes and Measures: The primary outcome measure was tumor depth of SCC. Secondary outcome measures that reflected tumor aggressiveness included perineural invasion, regional metastases, nodal metastases, disease-specific death, and overall death. Results: Of the 58 SOTR study participants, 14 were women and 44 were men; the mean (SD) age was 61.3 (8.4) years. Of the 40 immunocompetent study participants, 16 were women and 24 were men; the mean (SD) age was 69.8 (10.9) years, resulting in a statistically significant difference from the SOTR group. The mean (SD) number of years that SOTRs were immunosuppressed was 14.6 (9.2) years (range, 2-37 years). The SOTR and immunocompetent groups were statistically comparable regarding race and sex, patient care, follow-up time, numbers of skin lesions, and field cancerization and chemopreventive therapies. The SOTR group had a significantly higher annual frequency of visits (mean [SD], 4 [2] vs 3 [2] office visits per patient per year, P = .02) and annual biopsy rates (mean [SD], 6 [4] vs 5 [3] biopsies per patient per year, P = .04). The SOTRs developed SCCs that did not appear to be significantly more aggressive than those found in the immunocompetent control group. These SOTRs also did not develop significantly thicker tumors than the immunocompetent control group (median [IQR] tumor depth, 1.30 [0.90-1.60] mm in 35 SOTRs vs 1.22 [1.10-1.60] mm in 20 immunocompetent patients). Conclusions and Relevance: The increased risk and the potential for aggressive behavior of SCCs in SOTRs may be successfully managed at a level comparable to that in high-risk immunocompetent individuals through close adherence to current dermatologic surveillance recommendations and a marginally lower threshold for biopsy of suspicious lesions for SOTRs.

Sirolimus-Associated Pruritus: Case Report and Review.

Sirolimus is an immunosuppressant drug used to prevent organ rejection in transplant patients. We describe a man with sirolimus-associated pruritus and review the features of this adverse event in other individuals receiving this drug. The patient was a 67-year-old heart transplant recipient receiving sirolimus as part of his immunosuppressive regimen. He developed severe pruritus over the distal extremities, face, and earlobes six months after starting the drug. The symptoms became progressively worse as he continued to receive this medication. Temporary elimination of the drug resulted in cessation of his itching. Subsequently, sirolimus was discontinued and everolimus was started; this provided temporary relief of his pruritus. PubMed was used to review the following terms: "sirolimus", "itch", and "pruritus." Relevant papers and their references were reviewed. We are aware of only one other patient in whom pruritus necessitated cessation of treatment with sirolimus. Systemic pruritus is a rare adverse event associated with sirolimus. It can occur in both heart and liver transplant patients, beginning several months after transplant, and typically persists. Dose reduction may improve symptoms. Discontinuation of the medication or use of alternative immunosuppressants may be necessary for complete symptom relief.

A novel FOXO1-mediated dedifferentiation blocking role for DKK3 in adrenocortical carcinogenesis.

BACKGROUND: Dysregulated WNT signaling dominates adrenocortical malignancies. This study investigates whether silencing of the WNT negative regulator DKK3 (Dickkopf-related protein 3), an implicated adrenocortical differentiation marker and an established tumor suppressor in multiple cancers, allows dedifferentiation of the adrenal cortex. METHODS: We analyzed the expression and regulation of DKK3 in human adrenocortical carcinoma (ACC) by qRT-PCR, immunofluorescence, promoter methylation assay, and copy number analysis. We also conducted functional studies on ACC cell lines, NCI-H295R and SW-13, using siRNAs and enforced DKK3 expression to test DKK3's role in blocking dedifferentiation of adrenal cortex. RESULTS: While robust expression was observed in normal adrenal cortex, DKK3 was down-regulated in the majority (>75%) of adrenocortical carcinomas (ACC) tested. Both genetic (gene copy loss) and epigenetic (promoter methylation) events were found to play significant roles in DKK3 down-regulation in ACCs. While NCI-H295R cells harboring ß-catenin activating mutations failed to respond to DKK3 silencing, SW-13 cells showed increased motility and reduced clonal growth. Conversely, exogenously added DKK3 also increased motility of SW-13 cells without influencing their growth. Enforced over-expression of DKK3 in SW-13 cells resulted in slower cell growth by an extension of G1 phase, promoted survival of microcolonies, and resulted in significant impairment of migratory and invasive behaviors, largely attributable to modified cell adhesions and adhesion kinetics. DKK3-over-expressing cells also showed increased expression of Forkhead Box Protein O1 (FOXO1) transcription factor, RNAi silencing of which partially restored the migratory proficiency of cells without interfering with their viability. CONCLUSIONS: DKK3 suppression observed in ACCs and the effects of manipulation of DKK3 expression in ACC cell lines suggest a FOXO1-mediated differentiation-promoting role for DKK3 in the adrenal cortex, silencing of which may allow adrenocortical dedifferentiation and malignancy.

Incidence of and Risk Factors for Skin Cancer in Organ Transplant Recipients in the United States.

Importance: Skin cancer is the most common malignancy occurring after organ transplantation. Although previous research has reported an increased risk of skin cancer in solid organ transplant recipients (OTRs), no study has estimated the posttransplant population-based incidence in the United States. Objective: To determine the incidence and evaluate the risk factors for posttransplant skin cancer, including squamous cell carcinoma (SCC), melanoma (MM), and Merkel cell carcinoma (MCC) in a cohort of US OTRs receiving a primary organ transplant in 2003 or 2008. Design, Setting, and Participants: This multicenter retrospective cohort study examined 10 649 adult recipients of a primary transplant performed at 26 centers across the United States in the Transplant Skin Cancer Network during 1 of 2 calendar years (either 2003 or 2008) identified through the Organ Procurement and Transplantation Network (OPTN) database. Recipients of all organs except intestine were included, and the follow-up periods were 5 and 10 years. Main Outcomes and Measures: Incident skin cancer was determined through detailed medical record review. Data on predictors were obtained from the OPTN database. The incidence rates for posttransplant skin cancer overall and for SCC, MM, and MCC were calculated per 100 000 person-years. Potential risk factors for posttransplant skin cancer were tested using multivariate Cox regression analysis to yield adjusted hazard ratios (HR). Results: Overall, 10 649 organ transplant recipients (mean [SD] age, 51 [12] years; 3873 women [36%] and 6776 men [64%]) contributed 59 923 years of follow-up. The incidence rates for posttransplant skin cancer was 1437 per 100 000 person-years. Specific subtype rates for SCC, MM, and MCC were 812, 75, and 2 per 100 000 person-years, respectively. Statistically significant risk factors for posttransplant skin cancer included pretransplant skin cancer (HR, 4.69; 95% CI, 3.26-6.73), male sex (HR, 1.56; 95% CI, 1.34-1.81), white race (HR, 9.04; 95% CI, 6.20-13.18), age at transplant 50 years or older (HR, 2.77; 95% CI, 2.20-3.48), and being transplanted in 2008 vs 2003 (HR, 1.53; 95% CI, 1.22-1.94). Conclusions and Relevance: Posttransplant skin cancer is common, with elevated risk imparted by increased age, white race, male sex, and thoracic organ transplantation. A temporal cohort effect was present. Understanding the risk factors and trends in posttransplant skin cancer is fundamental to targeted screening and prevention in this population.

Impaired consciousness in epilepsy investigated by a prospective responsiveness in epilepsy scale (RES).

PURPOSE: Impaired consciousness in epileptic seizures has a major negative impact on patient quality of life. Prior work on epileptic unconsciousness has mainly used retrospective and nonstandardized methods. Our goal was to validate and to obtain initial data using a standardized prospective testing battery. METHODS: The responsiveness in epilepsy scale (RES) was used on 52 patients during continuous video-electroencephalography (EEG) monitoring. RES begins with higher-level questions and commands, and switches adaptively to more basic sensorimotor responses depending on patient performance. RES continues after seizures and includes postictal memory testing. Scoring was conducted based on video review. KEY FINDINGS: Testing on standardized seizure simulations yielded good intrarater and interrater reliability. We captured 59 seizures from 18 patients (35% of participants) during 1,420 h of RES monitoring. RES impairment was greatest during and after tonic-clonic seizures, less in partial seizures, and minimal in auras and subclinical seizures. In partial seizures, ictal RES impairment was significantly greater if EEG changes were present. Maximum RES impairment (lowest ictal score) was also significantly correlated with long postictal recovery time, and poor postictal memory. SIGNIFICANCE: We found that prospective testing of responsiveness during seizures is feasible and reliable. RES impairment was related to EEG changes during seizures, as well as to postictal memory deficits and recovery time. With a larger patient sample it is hoped that this approach can identify brain networks underlying specific components of impaired consciousness in seizures. This may allow the development of improved treatments targeted at preventing dysfunction in these networks.