Targeted delivery of a vaccine protein to Langerhans cells in the human skin via the C-type lectin receptor Langerin.

Human skin is a preferred vaccination site as it harbors multiple dendritic cell (DC) subsets, which display distinct C-type lectin receptors (CLR) that recognize pathogens. Antigens can be delivered to CLR by antibodies or ligands to boost antigen-specific immune responses. This concept has been established in mouse models but detailed insights into the functional consequences of antigen delivery to human skin DC in situ are sparse. In this study, we cloned and produced an anti-human Langerin antibody conjugated to the EBV nuclear antigen 1 (EBNA1). We confirmed specific binding of anti-Langerin-EBNA1 to Langerhans cells (LC). This novel LC-based vaccine was then compared to an existing anti-DEC-205-EBNA1 fusion protein by loading LC in epidermal cell suspensions before coculturing them with autologous T cells. After restimulation with EBNA1-peptides, we detected elevated levels of IFN-γ- and TNF-α-positive CD4+ T cells with both vaccines. When we injected the fusion proteins intradermally into human skin explants, emigrated skin DC targeted via DEC-205-induced cytokine production by T cells, whereas the Langerin-based vaccine failed to do so. In summary, we demonstrate that antibody-targeting approaches via the skin are promising vaccination strategies, however, further optimizations of vaccines are required to induce potent immune responses.

Laser-assisted epicutaneous immunization to target human skin dendritic cells.

Dendritic cells (DC) are promising targets for immunotherapy of cancer. Clinically, immunization against cancer antigens by means of the most potent antigen-presenting cells, that is DC, remains an important treatment option in combination with the modern immune checkpoint approaches. Instead of adoptively transferring in vitro monocyte-derived DC, they can also be loaded in situ by antibody-mediated targeting of antigen. Conventionally, these vaccines are delivered by classical intradermal injections. Here, we tested an alternative approach, namely laser-assisted epicutaneous immunization. With an infrared laser ("Precise Laser Epidermal System"/P.L.E.A.S.E.® Laser System), we created micropores in human skin and applied monoclonal antibodies (mAbs) against C-type lectins, for example DEC-205/CD205 and Langerin/CD207. Optimal parameters for formation of pores in epidermis and dermis were determined. We could induce pores of defined depths without enhanced apoptosis around them. Antibodies applied epicutaneously to the laser-porated skin could be detected both in Langerhans cells (LC) in situ in the epidermis and in migratory skin DC subsets from short term human skin explant culture, demonstrating uptake and transport of Langerin and DEC-205 mAbs. Efficacy of targeting was similar between the different laser treatments and pore depths. Thus, laser-assisted epicutaneous immunization may be a valuable alternative to intradermal injection, yet the loading efficacy of DC needs to be further improved.

The versatility of the medial thigh lift for defect coverage in the genito-perineal region.

Soft tissue defects in the genito-perineal region are predominantly because of trauma, infections, neoplasms or iatrogenic causes. As a result of the region's urological, reproductive and gastrointestinal function, defects in this area may cause devastating physical and psychological consequences as well as diminished sexual functioning. The purpose of this study was to examine the efficacy of implementing a medial thigh lift for defect coverage in the perineal region. A retrospective analysis of all medial thigh lift procedures for defect coverage in the genito-perineal region between November 2010 and March 2015 was conducted at three institutions. Ten consecutive patients underwent a medial thigh lift for defect coverage in the genito-perineal region. Nine patients were male, and one was female. The causative factors were Fournier's gangrene in eight patients, one patient had a straddle injury, and one suffered from extramammary Paget's disease. The mean follow-up time was 19·8 months. The average total defect size was 11·1 × 11 cm (length × width). The medial thigh lift procedure is a safe, technically easy and reliable technique with discrete scars. Outstanding aesthetic and functional outcomes result in a high rate of patient satisfaction. Through immediate wound closure, a reduction of recovery time can be achieved.

The John D. Constable International Traveling Fellowship: A Reciprocal Education in Plastic Surgery.

The John D. Constable International Traveling Fellowship has been an integral part of the American Association of Plastic Surgeons since 2006 and has provided an opportunity for international plastic surgeons to work with leaders in American plastic surgery.

Human skin dendritic cells can be targeted in situ by intradermal injection of antibodies against lectin receptors.

Skin dendritic cells (DC) express C-type lectin receptors for the recognition of pathogens. Langerhans cells (LC) express the receptor Langerin/CD207, whereas DEC-205/CD205 is mainly expressed by dermal DC, but can also be detected at low levels on LC. In this study, we tested an ex vivo approach for targeting DC in situ with monoclonal antibodies (mAb) against Langerin and DEC-205. The targeting mAb was injected intradermally into human skin biopsies or added to the medium during skin explant culture. Corresponding to the expression patterns of these lectin receptors on skin DC, Langerin mAb was detected merely in LC in the epidermis and DEC-205 mainly in dermal DC in human skin explants, regardless of the application route. Migratory skin DC bound and carried targeting mAb from skin explants according to their lectin receptor expression profiles. In contrast to the very selective transport of Langerin mAb by LC, DEC-205 mAb was more widely distributed on all CD1a(+) skin DC subsets but almost absent in CD14(+) dermal DC. As effective vaccination requires the addition of adjuvant, we co-administered the toll-like receptor (TLR)-3 ligand poly I:C with the mAb. This adjuvant enhanced binding of DEC-205 mAb to all skin DC subsets, whereas Langerin targeting efficacy remained unchanged. Our findings demonstrate that LC can be preferentially targeted by Langerin mAb. In contrast, DEC-205 mAb can be bound by all CD1a(+) skin DC subsets. The efficacy of DEC-205 mAb targeting strategy can be boosted by addition of poly I:C underlining the potential of this combination for immunotherapeutical interventions.

How to avoid pectus bar dislocation in the MIRPE or MOVARPE technique: results of 12 years' experience.

Bar displacement remains the most common complication of the minimally invasive repair of pectus excavatum (MIRPE). To date, no studies show results from 12 years' experience using a bar fixation technique with only absorbable sutures. Our aim is to show how to stabilize the bar using a modified approach for bar fixation. A retrospective review of 68 patients, who underwent MIRPE or the minor open videoendoscopic assisted repair of pectus excavatum, was performed. To stabilize the pectus bar, both wings of the pectus bar were tied to the ribs in 52 patients with circumcostal absorbable sutures using a Deschamps needle under endoscopic survey and in 16 patients with lateral stabilizers. The stability of pectus bar after the operation was assessed by lateral chest X-ray films and classified as being perfect, incomplete, or poor. No complications were observed in the perioperative period with the circumcostal suture technique. Lateral chest X-rays showed an excellent position of the pectus bar in 50 patients, incomplete position in 1, and poor position in another patient. Our technique seems to be effective in preventing bar displacement following pectus excavatum repair. It does not add any significant cost or time to the operation, and it is fairly simple to perform.

Excessive Production of Hydrogen Peroxide in Mitochondria Contributes to Atopic Dermatitis.

Atopic dermatitis (AD) is a complex disease characterized by chronic recurring eczema and pruritus. In addition, patients with AD display increased cutaneous and systemic levels of oxidative damage markers, whose source remains elusive. In this study, we investigated oxidative and mitochondrial stress in AD epidermis. The levels of superoxide dismutase 2 and hydrogen peroxide are augmented in the mitochondria of flaky tail (ft/ft) mouse keratinocytes, which is associated with the inhibition of the glutathione system and catalase. Furthermore, reduced levels of glutathione peroxidase 4 are associated with accumulation of malondialdehyde, 4-hydroxy-2-nonenal, and oxidized phosphatidylcholines in ft/ft epidermis. Cytochrome c is markedly increased in ft/ft epidermis, hence showing mitochondrial stress. Topical application of MitoQ, which is a mitochondrial-targeting antioxidant, to ft/ft mouse skin reduced damage to macromolecules and inflammation and restored epidermal homeostasis. Absence of alteration in the expression of superoxide dismutase 2, catalase, and glutathione peroxidase 4 and limited lipid peroxidation as well as oxidized phosphatidylcholines in the epidermis of Flg-/- mice suggest that FLG deficiency marginally contributes to oxidative stress in ft/ft epidermis. Increased superoxide dismutase 2, lipid peroxidation, and cytochrome c in the epidermis of patients with AD, associated with reduced antioxidant response in primary AD keratinocytes, corroborate mitochondrial dysfunction and lack of cellular adjustment to oxidative stress in AD epidermis.

CD34+ -derived Langerhans cell-like cells are different from epidermal Langerhans cells in their response to thymic stromal lymphopoietin.

Thymic stromal lymphopoietin (TSLP) endows human blood-derived CD11c(+) dendritic cells (DCs) and Langerhans cells (LCs) obtained from human epidermis with the capacity to induce pro-allergic T cells. In this study, we investigated the effect of TSLP on umbilical cord blood CD34(+) -derived LC-like cells. These cells are often used as model cells for LCs obtained from epidermis. Under the influence of TSLP, both cell types differed in several ways. As defined by CD83, CD80 and CD86, TSLP did not increase maturation of LC-like cells when compared with freshly isolated LCs and epidermal émigrés. Differences were also found in the production of chemokine (C-C motif) ligand (CCL)17. LCs made this chemokine only when primed by TSLP and further stimulated by CD40 ligation. In contrast, LC-like cells released CCL17 in response to CD40 ligation, irrespective of a prior treatment with TSLP. Moreover, the CCL17 levels secreted by LC-like cells were at least five times higher than those from migratory LCs. After maturation with a cytokine cocktail consisting of tumour necrosis factor-α, interleukin (IL)-1ß, IL-6 and prostaglandin (PG)E(2) LC-like cells released IL-12p70 in response to CD40 ligation. Most importantly and in contrast to LC, TSLP-treated LC-like cells did not induce a pro-allergic cytokine pattern in helper T cells. Due to their different cytokine secretion and the different cytokine production they induce in naïve T cells, we conclude that one has to be cautious to take LC-like cells as a paradigm for 'real' LCs from the epidermis.

The internal mammary artery perforator (IMAP) breast-flap harvested from an asymmetric hyperplastic breast for correction of a mild funnel chest deformity.

Pectus excavatum deformity is the most frequent congenital anomaly of the thoracic wall. If the invasive surgical procedures of thoracoplasty are not indicated or the patient refuses them, alternative treatment options should be considered. In such cases, local or distant transposition of autologous tissue could be appropriate. This report presents a selected case of funnel chest deformity and concomitant unilateral breast hyperplasia. Both deformities were corrected simultaneously using a pedicled internal mammary artery perforator (IMAP) flap dissected from the hyperplastic breast. This is a safe, reliable, low-morbidity, one-stage option for adult women that uses an easy-to-harvest flap for simultaneous correction of mild funnel chest deformity and concomitant breast hyperplasia with a single resulting scar.

The questionable benefit of pectus excavatum repair on cardiopulmonary function: a prospective study.

OBJECTIVES: Since the introduction of the minimally invasive technique for repair of pectus excavatum (MIRPE), increasing numbers of patients are presenting for surgery. However, controversy remains regarding cardiopulmonary outcomes of surgical repair. Therefore, the aim of our prospective study was to investigate cardiopulmonary function, at rest and during exercise before surgery, first after MIRPE and then after pectus bar removal. METHODS: Forty-seven patients were enrolled in a prospective, open-label, single-arm, single-centre clinical trial (Impact of Surgical Treatments of Thoracic Deformation on Cardiopulmonary Function) [NCT02163265] between July 2013 and November 2019. All patients underwent a modified MIRPE technique for surgical correction of pectus excavatum (PE), called Minor Open Videoendoscopically Assisted Repair of Pectus Excavatum. The patients underwent pre- and postoperative chest X-ray, three-dimensional volume-rendering computer tomography thorax imaging, cardiopulmonary function tests at rest and during stepwise cycle spiroergometry (sitting and supine position) and Doppler echocardiography. Daily physical activity questionnaires were also completed. RESULTS: The study was completed by 19 patients (15 males, 4 females), aged 13.9-19.6 years at the time of surgery. The surgical patient follow-up was 5.7 ± 7.9 months after pectus bar removal. No significant differences in cardiopulmonary and exercise parameters were seen after placement of the intrathoracic bar, or after pectus bar removal, compared to presurgery. CONCLUSIONS: Our findings indicate that surgical correction of PE does not impair cardiopulmonary function at rest or during exercise. Therefore, no adverse effects on exercise performance should be expected from surgical treatment of PE via the modified MIRPE technique. CLINICAL TRIAL REGISTRATION NUMBER: clinicaltrials.gov [ClinicalTrials.gov number, NCT02163265].

Impact of surgical treatment of pectus carinatum on cardiopulmonary function: a prospective study.

OBJECTIVES: The frequency of sternochondroplasty in cases of pectus carinatum (PC) has increased due to greater surgeon experience and modified surgical techniques. PC deformity does not usually cause cardiopulmonary malfunction or impairment. However, whether cardiopulmonary function changes after surgical repair remains a matter of controversy. The aim of our prospective study was to determine if surgery changes preoperative cardiopulmonary function. METHODS: Nineteen patients (16 males, 3 females) were enrolled in a prospective, open-label, single-arm, single-centre clinical trial (Impact of Surgical Treatments of Thoracic Deformation on Cardiopulmonary Function) (NCT02163265) between July 2013 and January 2017. All patients underwent PC repair via a modified Ravitch procedure and wore a lightweight, patient-controlled chest brace for 8 weeks postoperatively (the Innsbruck protocol). The average follow-up surgical examination was 8.3 months after surgery. In all enrolled patients, before surgery and not before 6 months postoperatively chest X-ray, 3-dimensional volume-rendered computed tomography thorax imaging, cardiopulmonary function tests with stepwise cycle spiroergometry (sitting and supine position) and Doppler echocardiography were performed; questionnaires about daily physical activity were also completed. RESULTS: Fourteen patients (aged 16.3 ± 2.6 years at study entry) completed the study. Changes in submaximal and peak power output were not detected during sitting, or when in the supine position. Also, no clinically relevant postoperative changes in spirometry or echocardiography were noted. CONCLUSIONS: Our findings confirm that surgical correction of PC does not impair cardiopulmonary function at rest or during physical exercise. CLINICAL REGISTRATION NUMBER: clinicaltrials.gov NCT02163265.

Specific Protein Antigen Delivery to Human Langerhans Cells in Intact Skin.

Immune modulating therapies and vaccines are in high demand, not least to the recent global spread of SARS-CoV2. To achieve efficient activation of the immune system, professional antigen presenting cells have proven to be key coordinators of such responses. Especially targeted approaches, actively directing antigens to specialized dendritic cells, promise to be more effective and accompanied by reduced payload due to less off-target effects. Although antibody and glycan-based targeting of receptors on dendritic cells have been employed, these are often expensive and time-consuming to manufacture or lack sufficient specificity. Thus, we applied a small-molecule ligand that specifically binds Langerin, a hallmark receptor on Langerhans cells, conjugated to a model protein antigen. Via microneedle injection, this construct was intradermally administered into intact human skin explants, selectively loading Langerhans cells in the epidermis. The ligand-mediated cellular uptake outpaces protein degradation resulting in intact antigen delivery. Due to the pivotal role of Langerhans cells in induction of immune responses, this approach of antigen-targeting of tissue-resident immune cells offers a novel way to deliver highly effective vaccines with minimally invasive administration.

Peroxisomal Fatty Acid Oxidation and Glycolysis Are Triggered in Mouse Models of Lesional Atopic Dermatitis.

Alterations of the lipid profile of the stratum corneum have an important role in the pathogenesis of atopic dermatitis (AD) because they contribute to epidermal barrier impairment. However, they have not previously been envisioned as a cellular response to altered metabolic requirements in AD epidermis. In this study, we report that the lipid composition in the epidermis of flaky tail, that is, ft/ft mice mimics that of human lesional AD (ADL) epidermis, both showing a shift toward shorter lipid species. The amounts of C24 and C26 free fatty acids and C24 and C26 ceramides-oxidized exclusively in peroxisomes-were reduced in the epidermis of ft/ft mice despite increased lipid synthesis, similar to that seen in human ADL edpidermis. Increased ACOX1 protein and activity in granular keratinocytes of ft/ft epidermis, altered lipid profile in human epidermal equivalents overexpressing ACOX1, and increased ACOX1 immunostaining in skin biopsies from patients with ADL suggest that peroxisomal ß-oxidation significantly contributes to lipid signature in ADL epidermis. Moreover, we show that increased anaerobic glycolysis in ft/ft mouse epidermis is essential for keratinocyte proliferation and adenosine triphosphate synthesis but does not contribute to local inflammation. Thus, this work evidenced a metabolic shift toward enhanced peroxisomal ß-oxidation and anaerobic glycolysis in ADL epidermis.

Notch-Mediated Generation of Monocyte-Derived Langerhans Cells: Phenotype and Function.

Langerhans cells (LCs) in the skin are a first line of defense against pathogens but also play an essential role in skin homeostasis. Their exclusive expression of the C-type lectin receptor Langerin makes them prominent candidates for immunotherapy. For vaccine testing, an easily accessible cell platform would be desirable as an alternative to the time-consuming purification of LCs from human skin. Here, we present such a model and demonstrate that monocytes in the presence of GM-CSF, TGF-ß1, and the Notch ligand DLL4 differentiate within 3 days into CD1a+Langerin+cells containing Birbeck granules. RNA sequencing of these monocyte-derived LCs (moLCs) confirmed gene expression of LC-related molecules, pattern recognition receptors, and enhanced expression of genes involved in the antigen-presenting machinery. On the protein level, moLCs showed low expression of costimulatory molecules but prominent expression of C-type lectin receptors. MoLCs can be matured, secrete IL-12p70 and TNF-α, and stimulate proliferation and cytokine production in allogeneic CD4+ and CD8+ T cells. In regard to vaccine testing, a recently characterized glycomimetic Langerin ligand conjugated to liposomes demonstrated specific and fast internalization into moLCs. Hence, these short-term in vitro‒generated moLCs represent an interesting tool to screen LC-based vaccines in the future.

Reconstruction of large head and neck deformities: experience with free gracilis muscle and myocutaneous flaps.

Microvascular free flaps continue to revolutionize coverage options in head and neck reconstruction. The authors describe their experience with the gracilis free flap and the myocutaneous gracilis free flap with reconstruction of head and neck defects. Eleven patients underwent 12 free tissue transfer to the head and neck region. The reconstruction was performed with the transverse myocutaneous gracilis (TMG) flap (n = 7) and the gracilis muscle flap with skin graft (n = 5). The average patient age was 63.4 years (range, 17-82 years). The indications for this procedure were tumor and haemangioma resections. The average patient follow-up was 20.7 months (range, 1 month-5.7 years). Total flap survival was 100%. There were no partial flap losses. Primary wound healing occurred in all cases. Recipient site morbidities included one hematoma. In our experience for reconstruction of moderate volume and surface area defects, muscle flaps with skin graft provide a better color match and skin texture relative to myocutaneous or fasciocutaneous flaps. The gracilis muscle free flap is not widely used for head and neck reconstruction but has the potential to give good results. As a filling substance for large cavities, the transverse myocutaneus gracilis flap has many advantages including reliable vascular anatomy, relatively great plasticity and a concealed donor area.

Aesthetic outcomes after surgical repair of pectus excavatum in females: Differences between patients and professional evaluators.

BACKGROUND: Pectus excavatum is less common in females than in males, and it often aggravates a coexistent breast asymmetry. We conducted a study comparing female patients' versus medical professionals' evaluation of pectus excavatum repair to assess differences in aesthetic outcome ratings. Moreover, we evaluated the influence of surgical correction on patients' self-perception. METHODS: Of 30 female patients who were initially screened, 18 patients (mean age, 20 years) who underwent bar removal after surgical correction of pectus excavatum deformity participated in the survey (60%). They completed a questionnaire rating their appearance before and after surgery and responded to a psychological questionnaire about the changes that they had experienced. The mean interval between pectus bar removal and evaluation was 28 months. Standardized preoperative and postoperative patient photographs were evaluated using the same questionnaire by a panel of medical professionals and students (n=24) and the results were compared. RESULTS: Patients rated their preoperative deformity as more severe than the other evaluators, revealing the significant impact of the deformity on patients' self-perception. Postoperatively, patient and professional evaluations were much better than before and were very similar. The psychological evaluation showed a clear improvement in well-being. The ratings of the medical professionals were not influenced by their degree of medical education. CONCLUSIONS: Surgical correction of pectus excavatum in female patients positively influences body perception and psychological well-being. It should therefore not be considered as a merely aesthetic correction, but as an important procedure to restore a patient's self-perception.

A Specific, Glycomimetic Langerin Ligand for Human Langerhans Cell Targeting.

Langerhans cells are a subset of dendritic cells residing in the epidermis of the human skin. As such, they are key mediators of immune regulation and have emerged as prime targets for novel transcutaneous cancer vaccines. Importantly, the induction of protective T cell immunity by these vaccines requires the efficient and specific delivery of both tumor-associated antigens and adjuvants. Langerhans cells uniquely express Langerin (CD207), an endocytic C-type lectin receptor. Here, we report the discovery of a specific, glycomimetic Langerin ligand employing a heparin-inspired design strategy and structural characterization by NMR spectroscopy and molecular docking. The conjugation of this glycomimetic to liposomes enabled the specific and efficient targeting of Langerhans cells in the human skin. We further demonstrate the doxorubicin-mediated killing of a Langerin+ monocyte cell line, highlighting its therapeutic and diagnostic potential in Langerhans cell histiocytosis, caused by the abnormal proliferation of Langerin+ myeloid progenitor cells. Overall, our delivery platform provides superior versatility over antibody-based approaches and novel modalities to overcome current limitations of dendritic cell-targeted immuno- and chemotherapy.

The effect on cardiopulmonary function after thoracoplasty in pectus carinatum: a systematic literature review.

OBJECTIVES: Creating an aesthetically appealing result using thoracoplasty, especially when correcting extensive deformities, but only causing low morbidity, is challenging. The frequency of thoracoplasties in cases of pectus carinatum (PC) has increased due to improved experience and modified surgical techniques, resulting in low morbidity and low complication rates. The indications for surgical treatment are still controversial and, in most cases, remain aesthetic or psychological rather than physiological. However, whether cardiopulmonary function changes after surgical repair remains a matter of controversy. We sought to investigate and shed light on published knowledge regarding this question. METHODS: We searched MEDLINE and PubMed databases, using various defined search phrases and inclusion criteria, to identify articles on pre- and postoperative cardiopulmonary evaluation and outcomes. RESULTS: Six studies met the inclusion criteria: 5 studies evaluated patients with PC for cardiopulmonary outcomes after chest wall surgery and 1 did so following conservative compression treatment. In these studies, surgical and conservative correction of PC did not reduce absolute lung volumes and spirometric measurements and consequently had no pathogenic effect on cardiopulmonary function. CONCLUSIONS: The results of this systematic review suggest that surgical correction of PC has no symptomatic pathogenic effect on cardiopulmonary function. The results, however, revealed both heterogeneity in the examinations used and inconsistent methods within each study. Further prospective trials with a stronger methodological design are necessary to objectively confirm that surgical correction of PC does not impair cardiopulmonary function.

Epidermal Overexpression of Xenobiotic Receptor PXR Impairs the Epidermal Barrier and Triggers Th2 Immune Response.

The skin is in daily contact with environmental pollutants, but the long-term effects of such exposure remain underinvestigated. Many of these toxins bind and activate the pregnane X receptor (PXR), a ligand-activated transcription factor that regulates genes central to xenobiotic metabolism. The objective of this work was to investigate the effect of constitutive activation of PXR in the basal layer of the skin to mimic repeated skin exposure to noxious molecules. We designed a transgenic mouse model that overexpresses the human PXR gene linked to the herpes simplex VP16 domain under the control of the keratin 14 promoter. We show that transgenic mice display increased transepidermal water loss and elevated skin pH, abnormal stratum corneum lipids, focal epidermal hyperplasia, activated keratinocytes expressing more thymic stromal lymphopoietin, a T helper type 2/T helper type 17 skin immune response, and increased serum IgE. Furthermore, the cutaneous barrier dysfunction precedes development of the T helper type 2/T helper type 17 inflammation in transgenic mice, thereby mirroring the time course of atopic dermatitis development in humans. Moreover, further experiments suggest increased PXR signaling in the skin of patients with atopic dermatitis when compared with healthy skin. Thus, PXR activation by environmental pollutants may compromise epidermal barrier function and favor an immune response resembling atopic dermatitis.