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T cells in human skin play an important role in the immune defense against pathogens and tumors. T cells are present already in fetal skin, where little is known about their cellular phenotype and biological function. Using single-cell analyses, we identified a naive T cell population expressing αβ and γδ T cell receptors (TCRs) that was enriched in fetal skin and intestine but not detected in other fetal organs and peripheral blood. TCR sequencing data revealed that double-positive (DP) αβγδ T cells displayed little overlap of CDR3 sequences with single-positive αβ T cells. Gene signatures, cytokine profiles and in silico receptor-ligand interaction studies indicate their contribution to early skin development. DP αβγδ T cells were phosphoantigen responsive, suggesting their participation in the protection of the fetus against pathogens in intrauterine infections. Together, our analyses unveil a unique cutaneous T cell type within the native skin microenvironment and point to fundamental differences in the immune surveillance between fetal and adult human skin., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2021 Reitermaier et al.)

Energy dissipation through the promotion of brown adipose tissue (BAT) or browning of white adipose tissue has recently evolved as novel promising concept in the fight against metabolic disease. New evidence suggests that hormones can contribute to the thermogenic programming of adipocytes through paracrine or endocrine actions. Recent studies in rodents identified parathyroid hormone (PTH) and PTH-related peptide as mediators of energy wasting in cachexia models due to adipocyte browning. However, the effects of PTH on human adipocyte thermogenesis and metabolic activity are unknown. Here we isolated subcutaneous white adipocyte precursor cells (APCs) from human donors followed by stimulation with recombinant PTH. Our data show that acute and chronic PTH administration in primary in vitro differentiated human subcutaneous adipocytes induces a molecular thermogenic program with increased mitochondrial activity and oxidative respiratory capacity. PTH also enhances hormone sensitive lipase activity and lipolysis in human adipocytes which may contribute to the observed thermogenic effects. In summary, we demonstrate here that PTH is a novel mediator of human adipocyte browning, suggesting a hitherto unknown endocrine axis between the parathyroid gland and adipose tissue in humans.

Skin resident T cells provide immediate immunologic responses at their specific location and play a role in the pathogenesis of skin diseases such as psoriasis. Recently, IL-9-producing T cells were described as a major T-cell subtype present in the skin, but knowledge on the biology and in situ regulation of this T-cell subtype is scarce. Here, we investigated the cytokine influence on skin T cells with focus on IL-9-producing T cells because a better understanding of their biology may identify novel therapeutic approaches. Healthy human skin biopsies were cultured either in the presence of IL-2, IL-4, and TGF-β [T helper (T h )9-promoting condition (T h 9-PC)] or IL-2 and IL-15 [standard condition (SC)]. Paired analysis of enzymatically isolated skin T cells and emigrated T cells after 4 wk of skin culture showed significant alterations of T-cell phenotypes, cytokine production, and IL-9-producing T-cell frequency. RNA sequencing analysis revealed differentially regulated pathways and identified CXCL8 and CXCL13 as top up-regulated genes in T h 9-PC compared with SC. Functionally supernatant of stimulated skin-derived T cells, CXCL8 and CXCL13 increased neutrophil survival. We report that the cytokine environment alters skin-derived T-cell phenotype and functional properties.-Kienzl, P., Polacek, R., Reithofer, M., Reitermaier, R., Hagenbach, P., Tajpara, P., Vierhapper, M., Gschwandtner, M., Mildner, M. Jahn-Schmid, B., Elbe-Bürger, A. The cytokine environment influence on human skin-derived T cells.

Ideal agents for the topical treatment of skin wounds should have antimicrobial efficacy without negative influence on wound healing. Octenidine (OCT) has become a widely used antiseptic in professional wound care, but its influence on several components of the wound healing process remains unclear. In the present study, we have used a superficial wound model using tape stripping on human full-thickness skin ex vivo to investigate the influence of OCT on epidermal Langerhans cells (LCs) and cytokine secretion pattern of skin cells during wound healing in a model without disruption of the normal skin structure. Histological and immunofluorescence studies showed that OCT neither altered human skin architecture nor the viability of skin cells upon 48 hours of culture in unwounded or wounded skin. The epidermis of explants and LCs remained morphologically intact throughout the whole culture period upon OCT treatment. OCT inhibited the upregulation of the maturation marker CD83 on LCs and prevented their emigration in wounded skin. Furthermore, OCT reduced both pro- and anti-inflammatory mediators (IL-8, IL-33, and IL-10), while angiogenesis and growth factor mediators (VEGF and TGF- β 1) remained unchanged in skin explant cultures. Our data provide novel insights into the host response to OCT in the biologically relevant environment of viable human (wounded) skin.

Herpes simplex virus (HSV) infections can cause considerable morbidity. Currently, nucleoside analogues such as acyclovir are widely used for treatment. However, HSV infections resistant to these drugs are a clinical problem among immunocompromised patients. To provide more efficient therapy and to counteract resistance, a different class of antiviral compounds has been developed. Pritelivir, a helicase primase inhibitor, represents a promising candidate for improved therapy. Here, we established an HSV-1 infection model on microneedle-pretreated human skin ex vivo. We identified HSV-1-specific histological changes (e.g., cytopathic effects, multinucleated giant cells), down-regulation of nectin-1, nuclear translocation of NF-κB (p65), interferon regulatory factor 3 (IRF3), and signaling of the IFN-inducible protein MxA. Accordingly, this model was used to test the potency of pritelivir compared with the standard drug acyclovir. We discovered that both drugs had a comparable efficacy for inhibiting HSV-1 replication, suggesting that pritelivir could be an alternative therapeutic agent for patients infected with acyclovir-resistant strains. To our knowledge, we present a previously unreported ex vivo HSV-1 infection model with abdominal human skin to test antiviral drugs, thus bridging the gap between in vitro and in vivo drug screening and providing a valuable preclinical platform for HSV research., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Human skin dermis is composed of the superficial papillary dermis and the reticular dermis in the lower layers, which can easily be distinguished histologically. In vitro analyses of fibroblasts from explant cultures from superficial and lower dermal layers suggest that human skin comprises at least two fibroblast lineages with distinct morphology, expression profiles, and functions. However, while for mouse skin cell surface markers have been identified, allowing the isolation of pure populations of one lineage or the other via FACS, this has not been achieved for human skin fibroblasts. We have now discovered two cell surface markers that discriminate between papillary and reticular fibroblasts. While FAP + CD90 - cells display increased proliferative potential, express PDPN and NTN1, and cannot be differentiated into adipocytes, FAP - CD90 + fibroblasts express high levels of ACTA2, MGP, PPARγ, and CD36 and readily undergo adipogenic differentiation, a hallmark of reticular fibroblasts. Flow cytometric analysis of fibroblasts isolated from superficial and lower layers of human dermis showed that FAP + CD90 - cells are enriched in the papillary dermis. Altogether, functional analysis and expression profiling confirms that FAP + CD90 - cells represent papillary fibroblasts, whereas FAP - CD90 + fibroblasts derive from the reticular lineage. Although papillary and reticular fibroblasts are enriched in the upper or lower dermis, respectively, they are not spatially restricted, and the microenvironment seems to affect their function., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Together with keratinocytes (KCs) and the dense network of Langerhans cells (LCs), the epidermis is an ideal portal for vaccine delivery. Pattern recognition receptor agonists, in particular polyinosinic-polycytidylic acid [p(I:C)], are promising adjuvant candidates for therapeutic vaccination to generate protective T-cell immunity. Here we established an ex vivo skin explant model to study the expression and activation of double-stranded RNA (dsRNA)-sensing pattern recognition receptors in LCs and KCs in human skin. Whereas KCs expressed all known dsRNA sensing receptors at a constitutive and inducible level, LCs exclusively expressed melanoma differentiation-associated protein 5 (MDA5) in untreated skin and freshly isolated cells. Comparative assessments of downstream signaling pathways induced by p(I:C) revealed distinct mitochondrial antiviral-signaling protein, IFN-regulatory factor 3, and NF-κB activation in LCs and KCs. Consequently, p(I:C) treatment of LCs significantly induced IFN-α and IFN-β mRNA expression, while in KCs an up-regulation of IFN-β and TNF-α mRNA was detectable. Stimulation of LCs with specific ligands revealed that not the TLR3- but only the MDA5-specific ligand induced IFN-α2, IFN-β, and TNF-α cytokines, but no IL-6 and -8. In KCs, both ligands induced production of high IL-6 and IL-8 levels, and low IFN-α2 and IFN-β levels, indicating that different dsRNA-sensing receptors and/or downstream signaling pathways are activated in both cell types. Our data suggest that MDA5 may be an attractive adjuvant target for epicutaneous delivery of therapeutic vaccines with the goal to target LCs.-Tajpara, P., Schuster, C., Schön, E., Kienzl, P., Vierhapper, M., Mildner, M., Elbe-Bürger, A. Epicutaneous administration of the pattern recognition receptor agonist polyinosinic-polycytidylic acid activates the MDA5/MAVS pathway in Langerhans cells.

RTN1 is an endoplasmic reticulum-associated protein that was initially identified in neuronal tissues. Here we show that the main isoform RTN1A is a marker for dendritic cells. In the skin, HLA-DR + CD1a high CD207 + CD11c weak Langerhans cells were the only cells in the epidermis, and HLA-DR + CD11c + dendritic cells were the main cells in the dermis, expressing this protein. RTN1A + dendritic cells were also found in gingiva, trachea, tonsil, thymus, and peripheral blood. During differentiation of MUTZ-3 cells into Langerhans cells, expression of RTN1A mRNA and protein preceded established Langerhans cell markers CD1a and CD207, and RTN1A protein partially co-localized with the endoplasmic reticulum marker protein disulfide isomerase. In line with this observation, we found that RTN1A was expressed by around 80% of Langerhans cell precursors in human embryonic skin. Our findings show that RTN1A is a marker for cells of the dendritic lineage, including Langerhans cells and dermal dendritic cells. This unexpected finding will serve as a starting point for the elucidation of the, until now, elusive functional roles of RTN1A in both the immune and the nervous system., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: The value of imaging the deep branch of the ulnar nerve (DBUN) over its entire course has not been clarified. Therefore, this study evaluates the feasibility of visualizing the DBUN from its origin to the most distal point., Methods: We performed high-resolution ultrasound (HRUS) with high-frequency probes (18-22 MHZ), HRUS-guided ink marking, and consecutive dissection in 8 fresh cadaver hands. In both hands of 10 healthy volunteers (n = 20), the cross-sectional area (CSA) was measured at 2 different locations (R1 and R2)., Results: The DBUN was clearly visible in all anatomical specimens and in healthy volunteers. Dissection confirmed HRUS findings in all anatomical specimens. The mean CSA was 1.8 ± 0.5 mm 2 at R1 and 1.6 ± 0.4 mm 2 at R2., Discussion: This study confirms that the DBUN can be reliably visualized over its entire course with HRUS in anatomical specimens and in healthy volunteers. Muscle Nerve 56: 1101-1107, 2017., (© 2017 Wiley Periodicals, Inc.)

Purpose and Hypothesis: Although sex- and gender-specific analyses have been gaining more attention during the last years they have rarely been performed in orthopaedic literature. The primary purpose of this study was to investigate whether for injuries of the UCL the specific location of the rupture is influenced by sex. A secondary study question addressed the sex-independent effect of trauma intensity on the rupture site of the UCL., Methods: This study is a retrospective analysis of all patients with either a proximal or distal bony avulsion or with a mid-substance tear or ligament avulsion of the UCL treated surgically between 1992 and 2015 at two level-I trauma centres. Trauma mechanisms leading to the UCL injury were classified into the following categories: (1) blunt trauma (i.e., strains), (2) low-velocity injuries (e.g., fall from standing height, assaults), and (3) high-velocity injuries (e.g., sports injuries, motor vehicle accidents). After reviewing the surgical records, patients were divided into three groups, depending upon the ligament rupture site: (1) mid-substance tears, (2) proximal ligament or bony avulsions and (3) distal ligament or bony avulsions. Dependencies between the specific rupture site and the explanatory variables (sex, age, and trauma intensity) were evaluated using χ2 test and logistic regression analysis., Results: In total, 1582 patients (1094 males, 488 females) met the inclusion criteria. Mean age was 41 years (range: 9-90 years). Taking into account the effects of sex on trauma intensity (p<0.001) and of trauma intensity on rupture site (p<0.001), mid-substance tears occurred more frequently in women, whereas men were more prone to distal ligament or bony avulsions (p<0.001). In other words, sex and rupture site correlated due to the effects of sex on trauma intensity and of trauma intensity on rupture site, but taking into account those effects there still was a significant effect of sex on rupture site., Conclusions: The results of this study demonstrate that with regression analysis both sex and trauma intensity allow to predict rupture site in UCL injuries.

Purpose: To evaluate in a prospective study the possibility of visualization and diagnostic assessment of the recurrent motor branch (RMB) of the median nerve with high-resolution ultrasound (HRUS)., Materials and Methods: HRUS with high-frequency probes (18-22 MhZ) was used to locate the RMB in eight fresh cadaveric hands. To verify correct identification, ink-marking and consecutive dissection were performed. Measurement of the RMB maximum transverse-diameter, an evaluation of the origin from the median nerve and its course in relation to the transverse carpal ligament, was performed in both hands of ten healthy volunteers (n = 20). Cases referred for HRUS examinations for suspected RMB lesions were also assessed., Results: The RMB was clearly visible in all anatomical specimens and all volunteers. Dissection confirmed HRUS findings in all anatomical specimens. Mean RMB diameter in volunteers was 0.7 mm ± 0.1 (range, 0.6-1). The RMB originated from the radial aspect in 11 (55%), central aspect in eight (40%) and ulnar aspect in one (5%) hand. Nineteen (95%) extraligamentous courses and one (5%) subligamentous course were detected. Three patients with visible RMB abnormalities on HRUS were identified., Conclusion: HRUS is able to reliably visualize the RMB, its variations and pathologies., Key Points: • Ultrasound allows visualization of the recurrent motor branch of the median nerve. • Ultrasound may help clinicians to assess patients with recurrent motor branch pathologies. • Patient management may become more appropriate and targeted therapy could be improved.

Background: The latissimus dorsi muscle flap represents a valuable option in breast reconstruction but can result in postoperative twitching and retraction, discomfort, arm movement limitations, and breast deformation. These complications can be avoided by denervation of the thoracodorsal nerve; however, the optimal method of nerve management is unknown. This study presents the authors' experience with the outcomes of latissimus dorsi flaps for breast reconstruction in the light of thoracodorsal nerve management strategies., Methods: The authors retrospectively collected data from 74 patients who underwent partial or total breast reconstruction with a latissimus dorsi flap alone or with an implant between January of 1999 and October of 2011. Follow-up data were collected at 12 and 24 months postoperatively., Results: In 56 patients (75.7 percent), the latissimus dorsi muscle was denervated at the time of surgery, whereas the thoracodorsal nerve remained intact in 18 patients (24.3 percent). No partial or total flap loss was observed. At 12 and 24 months' follow-up, all patients with an intact thoracodorsal nerve showed twitching of the muscle, and 50 percent and 67.9 percent, respectively, of the denervated patients showed twitching (p < 0.001). No patient had twitching if more than 4 cm of nerve was excised at 12 or 24 months postoperatively, and the length of nerve resection was predictive of the presence of twitching., Conclusion: Denervation of the latissimus dorsi is a safe and reliable procedure that should be performed at the time of breast reconstruction and should include more than 4 cm to achieve a nontwitching breast with a stable volume and shape.

Background: Treating large and extensive pilonidal sinus disease is a challenging task. Long-term reports on flaps suitable for coverage of large, wide, local-excision defects are sparse. We prospectively evaluated data with a minimum 1-year follow-up of the use of a single-sided, innervated, superior gluteal artery perforator flap., Methods: Twenty-one patients (1 woman, 20 men) with a median age of 26 years (min - max = 18 - 46) were included in the study period from September 2005 to April 2010. We recorded flap size, major and minor complications, hospital length of stay, impairment in activities of daily living, pain, aesthetic outcomes, and sensibility in the gluteal region (PSSD, pressure-specified sensory device) at 6 and 12 months postoperatively., Results: The mean defect size (±SD) was 13.0 ± 1.9 × 8.6 ± 1.3 × 5.5 ± 1.2 cm (height × width × depth), and median length of hospital stay was 9 days (range = 7-11). Only two patients developed minor wound-healing complications. Visual analog pain scales significantly improved, with no pain detectable at 12 months postoperatively (p < 0.0001). The aesthetic appearance of the results was good in the majority of patients (61.9-85.7 %). PSSD showed gradual normalization, with retained sensibility in the flap area over 12 months postoperatively (p = 0.0232). During the median 36-month (range = 20-60) follow-up, we have not observed any recurrence in the operated area., Conclusions: The innervated superior gluteal artery perforator flap is a useful technique for covering large and recurrent pilonidal sinus defects following wide local excision and represents an excellent tool in the surgical armamentarium for achieving long-lasting outcomes in this young group of patients.