Timo Buhl

A sensory neuron–expressed IL-31 receptor mediates T helper cell–dependent itch: Involvement of TRPV1 and TRPA1

BACKGROUND Although the cytokine IL-31 has been implicated in inflammatory and lymphoma-associated itch, the cellular basis for its pruritic action is yet unclear. OBJECTIVE We sought to determine whether immune cell-derived IL-31 directly stimulates sensory neurons and to identify the molecular basis of IL-31-induced itch. METHODS We used immunohistochemistry and quantitative real-time PCR to determine IL-31 expression levels in mice and human subjects. Immunohistochemistry, immunofluorescence, quantitative real-time PCR, in vivo pharmacology, Western blotting, single-cell calcium imaging, and electrophysiology were used to examine the distribution, functionality, and cellular basis of the neuronal IL-31 receptor α in mice and human subjects. RESULTS Among all immune and resident skin cells examined, IL-31 was predominantly produced by TH2 and, to a significantly lesser extent, mature dendritic cells. Cutaneous and intrathecal injections of IL-31 evoked intense itch, and its concentrations increased significantly in murine atopy-like dermatitis skin. Both human and mouse dorsal root ganglia neurons express IL-31RA, largely in neurons that coexpress transient receptor potential cation channel vanilloid subtype 1 (TRPV1). IL-31-induced itch was significantly reduced in TRPV1-deficient and transient receptor channel potential cation channel ankyrin subtype 1 (TRPA1)-deficient mice but not in c-kit or proteinase-activated receptor 2 mice. In cultured primary sensory neurons IL-31 triggered Ca(2+) release and extracellular signal-regulated kinase 1/2 phosphorylation, inhibition of which blocked IL-31 signaling in vitro and reduced IL-31-induced scratching in vivo. CONCLUSION IL-31RA is a functional receptor expressed by a small subpopulation of IL-31RA(+)/TRPV1(+)/TRPA1(+) neurons and is a critical neuroimmune link between TH2 cells and sensory nerves for the generation of T cell-mediated itch. Thus targeting neuronal IL-31RA might be effective in the management of TH2-mediated itch, including atopic dermatitis and cutaneous T-cell lymphoma.

Selection of Patients for Long-term Surveillance With Digital Dermoscopy by Assessment of Melanoma Risk Factors

OBJECTIVE To identify patients at increased melanoma risk who benefit from long-term surveillance with digital dermoscopy. DESIGN Prospective, nonrandomized, observational study. SETTING University-based surveillance program. PARTICIPANTS Six hundred eighty-eight patients prospectively categorized into defined melanoma risk groups and followed up (mean, 44.3 months) by clinical examinations, dermoscopy, and, for atypical nevi, sequential digital dermoscopy. MAIN OUTCOME MEASURE Association between patient risk factors and detection of melanomas. RESULTS Odds ratios from a multivariate logistic regression analysis indicated a highly increased melanoma risk for patients with familial atypical mole and multiple melanoma (FAMMM) syndrome, atypical mole syndrome (AMS), or previous melanoma. Each digitally documented atypical lesion (range, 1-17 lesions per patient) denoted a significant 10% increase in melanoma risk. Patients with higher melanoma risk (1) showed a higher percentage of melanomas detected by digital dermoscopy (FAMMM syndrome group, 50%; AMS group, 22%), (2) more often developed multiple melanomas within shorter intervals, and (3) showed a ratio of melanoma to benign results for lesions excised because of dynamic changes of 1:15 (AMS group) or 1:4 (FAMMM syndrome group). Melanomas detected by digital dermoscopy were significantly thinner (0.41 mm in mean Breslow thickness) compared with melanomas detected by other means (0.62 mm; P = .04). CONCLUSIONS We suggest an individualized surveillance plan, with digital dermoscopy performed at follow-up intervals of 3 months for patients with FAMMM syndrome and 6 to 12 months (depending on additional risk factors) for those with AMS. Patients with multiple common nevi and no additional risk factors had no benefit from sequential digital dermoscopy.

Molecular and Morphological Characterization of Inflammatory Infiltrate in Rosacea Reveals Activation of Th1/Th17 Pathways

Rosacea is a common chronic inflammatory skin disease of unknown etiology. Our knowledge about an involvement of the adaptive immune system is very limited. We performed detailed transcriptome analysis, quantitative real-time reverse-transcriptase-PCR, and quantitative immunohistochemistry on facial biopsies of rosacea patients, classified according to their clinical subtype. As controls, we used samples from patients with facial lupus erythematosus and healthy controls. Our study shows significant activation of the immune system in all subtypes of rosacea, characterizing erythematotelangiectatic rosacea (ETR) already as a disease with significant influx of proinflammatory cells. The T-cell response is dominated by Th1/Th17-polarized immune cells, as demonstrated by significant upregulation of IFN-γ or IL-17, for example. Chemokine expression patterns support a Th1/Th17 polarization profile of the T-cell response. Macrophages and mast cells are increased in all three subtypes of rosacea, whereas neutrophils reach a maximum in papulopustular rosacea. Our studies also provide evidence for the activation of plasma cells with significant antibody production already in ETR, followed by a crescendo pattern toward phymatous rosacea. In sum, Th1/Th17 polarized inflammation and macrophage infiltration are an underestimated hallmark in all subtypes of rosacea. Therapies directly targeting the Th1/Th17 pathway are promising candidates in the future treatment of this skin disease.

Seven-point checklist for dermatoscopy: Performance during 10 years of prospective surveillance of patients at increased melanoma risk

BACKGROUND The retrospectively developed 7-point checklist is one of the most applicable dermatoscopic algorithms for clinical use. However, until today no prospective data on the diagnostic performance of this algorithm were reported. OBJECTIVE Our aim was to assess the sensitivity, specificity, and diagnostic accuracy of the 7-point checklist in the setting of a prospective long-term study. METHODS Patients at increased melanoma risk (n = 688) were screened at regular intervals by naked-eye examination, the dermatoscopic 7-point checklist, and digital dermatoscopy follow-up (10-year study interval). RESULTS We detected 127 melanomas including 50 melanomas in situ. The mean Breslow thickness of invasive melanomas was 0.57 mm. A total of 79 melanomas displayed the 7-point checklist melanoma threshold of 3 or more points (62% sensitivity, compared with 78%-95% in retrospective settings). In all, 48 melanomas scored fewer than 3 points and were excised because of complementary information (eg, lesional history, dynamic changes detected by digital dermatoscopy). The specificity of the 7-point checklist was 97% (compared with 65%-87% in retrospective settings). Regression patterns, atypical vascular patterns, and radial streaming were associated with the highest relative risk for melanoma (odds ratio 3.26, 95% confidence interval 2.05-5.16; odds ratio 3.04, 95% confidence interval 1.70-5.46; odds ratio 2.91, 95% confidence interval 1.64-5.15; P < .0003, respectively). Melanomas thicker than 0.5 mm exhibited significantly more regression patterns and atypical vascular patterns (P < .02). The malignant versus benign ratio for all excised lesions was 1:8.6 (127 melanomas, 1092 nonmelanomas). LIMITATIONS Calculation of the specificity was a limitation. True negative lesions were defined by a score less than 3 points and either the histopathological diagnosis of nonmelanoma or the absence of dynamic changes during digital dermatoscopy follow-up (nonexcised, nonsuspicious, no change). CONCLUSIONS The 7-point checklist for dermatoscopy was less sensitive but highly specific in this prospective clinical setting. Complementary information clearly increased the sensitivity. Regression patterns or radial streaming in nevi of patients at high risk should raise a higher melanoma suspicion than might be concluded from retrospective studies.

Neural peptidase endothelin-converting enzyme 1 regulates endothelin 1–induced pruritus

In humans, pruritus (itch) is a common but poorly understood symptom in numerous skin and systemic diseases. Endothelin 1 (ET-1) evokes histamine-independent pruritus in mammals through activation of its cognate G protein-coupled receptor endothelin A receptor (ETAR). Here, we have identified neural endothelin-converting enzyme 1 (ECE-1) as a key regulator of ET-1-induced pruritus and neural signaling of itch. We show here that ETAR, ET-1, and ECE-1 are expressed and colocalize in murine dorsal root ganglia (DRG) neurons and human skin nerves. In murine DRG neurons, ET-1 induced internalization of ETAR within ECE-1-containing endosomes. ECE-1 inhibition slowed ETAR recycling yet prolonged ET-1-induced activation of ERK1/2, but not p38. In a murine itch model, ET-1-induced scratching behavior was substantially augmented by pharmacological ECE-1 inhibition and abrogated by treatment with an ERK1/2 inhibitor. Using iontophoresis, we demonstrated that ET-1 is a potent, partially histamine-independent pruritogen in humans. Immunohistochemical evaluation of skin from prurigo nodularis patients confirmed an upregulation of the ET-1/ETAR/ECE-1/ERK1/2 axis in patients with chronic itch. Together, our data identify the neural peptidase ECE-1 as a negative regulator of itch on sensory nerves by directly regulating ET-1-induced pruritus in humans and mice. Furthermore, these results implicate the ET-1/ECE-1/ERK1/2 pathway as a therapeutic target to treat pruritus in humans.

CD40 ligation during dendritic cell maturation reduces cell death and prevents interleukin-10-induced regression to macrophage-like monocytes.

Abstract:  Dendritic cells (DCs) have become popular candidates in cancer vaccination because of their crucial role in inducing T‐cell responses. However, clinical studies greatly differ in their protocols for generating DCs and the efficacy in treating established tumors needs to be improved. We systematically analyzed DCs maturated by five different protocols for surface markers, the alloproliferative T‐cell response, the DC survival after cytokine deprivation, the stability of surface markers under the influence of interleukin‐10 (IL‐10) and the DC cytokine secretion pattern. Monocyte‐derived DCs were maturated by CD40‐ligand (CD40‐L), unmethylated cytosine–guanosine dinucleotides‐oligodinucleotides (CpG‐ODN), an inflammatory cytokine cocktail (ICC), a combination of ICC and CD40‐L, or ICC, CD40‐L and CpG‐ODN. A high co‐expression of DC maturation and costimulation markers was found after treatment with ICC plus CD40‐L (69.3 ± 9.6% CD83/CD80 double positive staining) and correlated with a significantly increased cell survival, a high expression of the antiapoptotic factor bcl‐XL, a stable CD83high/CD14low expression under the influence of IL‐10, and a strong alloproliferative T‐cell response. In conclusion, our data support the use of maturation protocols containing ICC plus CD40‐L in order to generate highly mature, phenotypically stable, cell‐death resistant, and T‐cell stimulatory DCs for clinical application in cancer patients.

Association of Patient Risk Factors and Frequency of Nevus-Associated Cutaneous Melanomas

IMPORTANCE The reported frequencies of associations between primary cutaneous melanomas and melanocytic nevi vary widely between 4% and 72%. However, earlier histopathologic studies were limited by their retrospective design and did not assess the influence of important patient-related risk factors. OBJECTIVES To identify the frequency of nevus-associated melanomas and correlate patient- and melanoma-related factors. DESIGN, SETTING, AND PARTICIPANTS A prospective, single-center, observational study with systematic documentation of melanoma risk factors, clinical and dermoscopic criteria of excised lesions, and results of histopathologic examination was conducted at a university-based dermatology clinic. Participants included 832 patients at high risk for developing melanoma. Evaluation was performed at regular intervals between April 1, 1997, and May 31, 2012, and data analysis was conducted between September 1, 2012, and December 31, 2013. MAIN OUTCOMES AND MEASURES Assessment of the frequency of nevus-associated melanoma and the influence of patient- and melanoma-related factors on their manifestation. RESULTS During the study, 190 melanomas (81 [42.6%] in situ and 109 [57.4%] invasive) were diagnosed in 113 of the 832 patients (13.6%); there were 42 women (37.2%) and 71 men (62.8%). The median (SD) Breslow thickness of invasive melanomas was 0.42 (0.43) mm. Histopathologic examination revealed remnants of melanocytic nevi in 103 melanomas (54.2%). Most nevus-associated melanomas were found on the trunk (67 [65.1%]); however, statistical significance for the localization was not present (P = .06). In univariate analyses, reported as odds ratios (95% CIs), nevus-associated melanomas were found significantly more frequently in patients of lower melanoma risk (risk group 1 [>50 common and/or ≤ 3 atypical nevi], 2.75 [1.14-6.64]; P = .02), with more than 100 nevi (1.63 [1.02-3.60]; P = .04), or with the diagnosis of in situ melanoma (14.01 [6.14-31.96]; P < .001). In contrast, nevus-associated melanomas were found significantly less frequently in patients with 1 or more previous melanomas (0.28 [0.21-0.83]; P = .005). All other factors (eg, age, skin type, hair color, and melanoma thickness) showed no significant influence on the manifestation of nevus-associated melanomas. These observations were confirmed in a separate analysis including all 109 invasive melanomas. Multivariate regression analysis identified 3 independent patient-related factors (high nevus count, low risk for melanoma, and female sex) and 1 melanoma-related factor (in situ melanoma) to be indicative of a significantly increased probability of nevus-associated melanomas. CONCLUSIONS AND RELEVANCE In this prospective study of a high-risk patient cohort, 54.2% of primary melanomas were associated with melanocytic nevi. Patients with many nevi and without previous melanomas or traits of familial atypical mole and multiple melanoma syndrome had a higher frequency of nevus-associated melanomas. These patients could thus benefit from sequential digital dermoscopy in addition to total-body photography.

Integrating static and dynamic features of melanoma: The DynaMel algorithm

BACKGROUND Sequential digital dermatoscopy identifies dynamic changes in melanocytic lesions. However, no algorithm exists that systematically weights dynamic changes regarding their association with melanoma. OBJECTIVE We sought to identify relevant dynamic changes and to integrate these into a novel diagnostic algorithm. METHODS During follow-up (mean 44.28 months) of 688 patients at high risk, 675 pigmented lesions with prospectively documented dynamic changes were excised. The association between specific changes and melanoma was assessed. RESULTS We detected 61 melanomas (38 invasive, median thickness 0.42 mm) with dynamic changes. Multivariate logistic regression analyses revealed a significant association between the diagnosis of melanoma and 5 dynamic criteria. According to the observed odds ratios we defined two dynamic major criteria (2 points each: asymmetric-multifocal enlargement and architectural change) and 3 dynamic minor criteria (1 point each: focal increase in pigmentation, focal decrease in pigmentation, and overall decrease in pigmentation when not accompanied by a lighter pigmentation of the adjacent skin). The DynaMel score was generated by addition of dynamic and 7-point checklist scores with a threshold for excision of 3 or more points. Including information about dynamic changes increased the sensitivity of the 7-point checklist from 47.5% (29 of 61 melanomas detected) to 77.1% (47 of 61 melanomas detected). The specificity slightly decreased from 99.0% to 98.1%. LIMITATIONS Before broad application the DynaMel algorithm needs to be validated using data from a different prospective study. CONCLUSIONS The DynaMel algorithm integrates a scoring system for dynamic dermatoscopic changes into the 7-point checklist for dermatoscopy and thereby increased the sensitivity of melanoma detection.

Intracellular delivery of major histocompatibility complex class I-binding epitopes: dendritic cells loaded and matured with cationic peptide/poly(I:C) complexes efficiently activate T cells.

Abstract:  Based on their role for the induction of T‐cell responses, dendritic cells (DCs) are popular candidates in cancer vaccine development. We established a novel single‐step intracellular delivery of peptide/poly(I:C) complexes for antigen loading and Toll‐like receptor‐3 (TLR3)‐mediated maturation of human DCs using a cell‐penetrating peptide (tat49–57: RKKRRQRRR) as delivery vector. Towards this end, a cationic tat‐sequence was fused with an antigenic, major histocompatibility complex (MHC) class I‐binding melanoma epitope (Melan‐A/Mart‐1 sequence: ELAGIGILTV) and then mixed with negatively charged poly(I:C) dsRNA to form peptide/nucleic acid complexes. Flow cytometry and confocal laser scanning microscopy confirmed intracellular localization of TLR3 in monocyte‐derived immature DCs (iDCs). Peptide/poly(I:C) complexes were readily internalized by iDCs without negatively affecting cell viability. They induced DC maturation and secretion of bioactive interleukin (IL)‐12p70. When peptide/poly(I:C) complex‐loaded DCs were used for autologous T cell stimulation, epitope‐specific interferon‐gamma secretion was quantitatively superior in comparison to peptide‐loaded DCs matured by a cytokine cocktail, as detected by enzyme‐linked immunospot assays. Thus, complexes of cationic antigenic peptides and poly(I:C) might be of great utility for a TLR3‐mediated DC maturation and intracellular peptide targeting in a single step. Resulting DCs induce a strong expansion/activation of antigen‐specific T cells in the context of an IL‐12p70 secretion.

Melanoma arising in segmental nevus spilus: Detection by sequential digital dermatoscopy

BACKGROUND Nevus spilus (NS) defines a café-au-lait macule with superimposed maculopapular speckles. Although the café-au-lait macule often presents at birth, the darker pigmented speckles increase in number and size during a period of several years. The need for close follow-up of patients with NS is underlined by reports of several cases of cutaneous melanoma developing within such lesions. METHODS We followed up 4 adult patients (3 male, one female; mean age 38 years) with unilateral segmental NS of the thoracic or abdominal region. The NS was present at birth in all 4 patients. Follow-up by sequential digital dermatoscopy and digital overview images was scheduled every 6 to 12 months. RESULTS During surveillance (mean follow-up time, 8.15 years), 3 melanocytic lesions were excised. In one patient focal enlargement prompted excision of two dysplastic compound nevi. In another patient new black dots and focal peripheral hyperpigmentation were detected by comparison with previous images. Histologic analysis confirmed the diagnosis of invasive melanoma (Breslow thickness, 0.6 mm). LIMITATIONS This observational clinical study included a small number of patients. Sequential digital dermatoscopy of large NS in children may lead to unnecessary excisions because of physiologic changes. CONCLUSION We suggest close follow-up of patients with segmental NS whenever complete excision is not possible. In adults, follow-up by digital dermatoscopy and excision of lesions with dynamic changes may assist in the early detection of melanoma.