Chemical Tattoo Treatment Leading to Systemic Cobalt Hypersensitivity.

An otherwise healthy 36-year-old Caucasian woman, without prior history of atopic dermatitis or eczema, presented to an outside dermatologist with a generalized, severely pruritic eruption involving the entire body except the face. One month previously, she had used a 50% trichloroacetic acid tattoo removal solution on a blue-colored tattoo on the medial aspect of the left ankle. The patient's eruption persisted for 7 months, and after several attempts to slowly taper her prednisone dose, she presented to our institution. On physical examination, there was a 3-cm erythematous, lichenified plaque surrounding the tattoo (Figure). On the trunk and upper regions of the arms, there were scattered, 1- to 2-cm, nummular patches and plaques. Biopsy of a truncal lesion revealed spongiotic pustules with a mixed dermal infiltrate and scattered eosinophils, consistent with subacute spongiotic dermatitis.

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.

MicroRNA-135b Regulates Leucine Zipper Tumor Suppressor 1 in Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin malignancy and it presents a therapeutic challenge in organ transplant recipient patients. Despite the need, there are only a few targeted drug treatment options. Recent studies have revealed a pivotal role played by microRNAs (miRNAs) in multiple cancers, but only a few studies tested their function in cSCC. Here, we analyzed differential expression of 88 cancer related miRNAs in 43 study participants with cSCC; 32 immunocompetent, 11 OTR patients, and 15 non-lesional skin samples by microarray analysis. Of the examined miRNAs, miR-135b was the most upregulated (13.3-fold, 21.5-fold; p=0.0001) in both patient groups. Similarly, the miR-135b expression was also upregulated in three cSCC cell lines when evaluated by quantitative real-time PCR. In functional studies, inhibition of miR-135b by specific anti-miR oligonucleotides resulted in upregulation of its target gene LZTS1 mRNA and protein levels and led to decreased cell motility and invasion of both primary and metastatic cSCC cell lines. In contrast, miR-135b overexpression by synthetic miR-135b mimic induced further down-regulation of LZTS1 mRNA in vitro and increased cancer cell motility and invasiveness. Immunohistochemical evaluation of 67 cSCC tumor tissues demonstrated that miR-135b expression inversely correlated with LZTS1 staining intensity and the tumor grade. These results indicate that miR-135b functions as an oncogene in cSCC and provide new understanding into its pathological role in cSCC progression and invasiveness.

A synthetic superoxide dismutase/catalase mimetic EUK-207 mitigates radiation dermatitis and promotes wound healing in irradiated rat skin.

In the event of a radionuclear attack or nuclear accident, the skin would be the first barrier exposed to radiation, though skin injury can progress over days to years following exposure. Chronic oxidative stress has been implicated as being a potential contributor to the progression of delayed radiation-induced injury to skin and other organs. To examine the causative role of oxidative stress in delayed radiation-induced skin injury, including impaired wound healing, we tested a synthetic superoxide dismutase (SOD)/catalase mimetic, EUK-207, in a rat model of combined skin irradiation and wound injury. Administered systemically, beginning 48 hours after irradiation, EUK-207 mitigated radiation dermatitis, suppressed indicators of tissue oxidative stress, and enhanced wound healing. Evaluation of gene expression in irradiated skin at 30 days after exposure revealed a significant upregulation of several key genes involved in detoxication of reactive oxygen and nitrogen species. This gene expression pattern was primarily reversed by EUK-207 therapy. These results demonstrate that oxidative stress has a critical role in the progression of radiation-induced skin injury, and that the injury can be mitigated by appropriate antioxidant compounds administered 48 hours after exposure.

Two-dimensional dielectric imaging for dermatologic screening: a feasibility study.

BACKGROUND/PURPOSE: The diagnosis of skin neoplasia can be very challenging, given the low sensitivity and specificity of traditional methods of diagnosis which are based on visual appearance. Techniques which are based on the dielectric properties of cells can improve the diagnostic accuracy of screening techniques; as an example, point-contact coaxial probes for dielectric measurement can improve diagnostic accuracy. Unfortunately, these probes are not well suited for two-dimensional spatial imaging of the skin surface, given that they must be manually scanned over the skin surface. METHODS/RESULTS: An electronic scanning probe was developed and fabricated to simulate an open-ended coaxial probe suitable for two-dimensional dielectric imaging of human skin in real time. A clinical study was undertaken to demonstrate proof-of-concept for the instrumentation. A select group of normal healthy subjects as well as a subject with diagnosed squamous cell carcinoma participated in this study. The electronic scanning probe was found to be a potentially useful tool for providing two-dimensional images from diseased skin. CONCLUSION: The electronic scanning probe used for the present study addresses existing limitations with current coaxial probes. Measurements of healthy and diseased areas of skin are provided to illustrate the feasibility of the approach.

Sequential intramolecular epitope spreading of humoral responses to human BPAG2 in a transgenic model.

Bullous pemphigoid (BP) is a subepidermal autoimmune disease characterized by a humoral response to an epidermal basement membrane (BM) component, BP antigen 2 (BPAG2). BP patients have IgG autoantibodies against an immunodominant BPAG2 extracellular domain termed NC16A as well as additional epitopes located both in the intracellular and extracellular domains (ICD and ECD, respectively) of this autoantigen. To study the evolution of humoral responses to BPAG2, sequential serum samples obtained from C57BL/6Ncr mice grafted with otherwise syngeneic skin from transgenic mice expressing human BPAG2 (hBPAG2) in epidermal BM were studied for IgG reactivity to seven ECD and ICD hBPAG2 epitopes. All grafted mice developed specific IgG against hBPAG2 ECD and ICD epitopes. In seven of eight mice, anti-hBPAG2 IgG was initially directed against ECD epitopes; in six mice, humoral responses subsequently targeted additional ECD and ICD BPAG2 epitopes. In contrast to IgG specific for ECD epitopes, IgG against ICD epitopes was present at lower levels, detectable for shorter periods, and non-complement fixing. Interestingly, the appearance of IgG directed against ICD epitopes correlated with the development of graft loss in this experimental model. These studies provide a comprehensive and prospective characterization of the evolution of humoral immune responses to hBPAG2 in vivo.

Transient anti-CD40L co-stimulation blockade prevents immune responses against human bullous pemphigoid antigen 2: implications for gene therapy.

Skin grafts from mice expressing human bullous pemphigoid antigen 2 (hBPAG2) in epidermal basement membrane elicit hBPAG2-specific IgG and graft loss in wild-type (Wt) recipients. Graft loss was dependent on CD4+ T cells and correlated with the production and tissue deposition of hBPAG2-specific IgG. To explore the role of CD40/CD40 ligand (CD40L) interaction in this model, Wt mice grafted with transgenic (Tg) skin were treated with hamster anti-CD40L mAb MR1. In contrast to grafted Wt mice treated with equivalent doses of control IgG, 22 of 23 MR1-treated Wt mice did not develop hBPAG2-specific IgG or graft loss for >or=60 days. MR1-treated mice also accepted a second Tg skin graft without durable production of hBPAG2-specific IgG or graft loss. Moreover, splenocytes and enriched CD4+ T cells from MR1-treated graft recipients transferred un- or hyporesponsiveness to hBPAG2 to other mice and demonstrated a dominant tolerant effect over cotransferred naive splenocytes following adoptive transfer to Rag2-/- mice. Successful inhibition of hBPAG2-specific IgG production and Tg graft loss following CD40:CD40L co-stimulatory blockade in this model provides opportunities to study mechanisms of peripheral tolerance and generate antigen-specific regulatory CD4+ cells-issues of relevance to patients with pemphigoid as well as individuals undergoing gene replacement therapy for epidermolyis bullosa.

Bullous pemphigoid and related subepidermal autoimmune blistering diseases.

The pemphigoid group of autoimmune blistering diseases includes distinct entities (bullous pemphigoid, mucous membrane pemphigoid, pemphigoid gestationis, linear IgA dermatosis and lichen planus pemphigoides) that are characterized by relatively consistent clinical, histologic and immunopathologic findings. Patients with these disorders have antibasement membrane autoantibodies that often display pathogenic (blister-forming) activity following passive transfer to experimental animals. Interestingly, such autoantibodies target important structural proteins that promote adhesion of epidermis to epidermal basement membrane in human skin. Autoimmune blistering diseases are characterized by substantial morbidity (for example pruritus, pain, disfigurement) and in some instances mortality. Treatment with systemic immunosuppressives has reduced morbidity and mortality in patients with these diseases.

Human bullous pemphigoid antigen 2 transgenic skin elicits specific IgG in wild-type mice.

Bullous pemphigoid antigen 2 (BPAG2) is targeted by autoantibodies in patients with bullous pemphigoid (BP), and absent in patients with one type of epidermolysis bullosa (OMIM #226650). A keratin 14 promoter construct was used to produce transgenic (Tg) mice appropriately expressing human BPAG2 (hBPAG2) in murine epidermal basement membrane (BM). Grafts of Tg skin placed on gender-matched, syngeneic wild type (Wt) or major histocompatibility complex I (MHC I)-/- mice elicited IgG that bound human epidermal BM and BPAG2. Production of such IgG in grafted mice was prompt (detectable within 16+/-2 days), robust (titer > or = 1,280), durable (present > or = 380 days), and correlated with the involution and loss of Tg skin grafts. MHC II-/- mice grafted with Tg skin did not develop anti-hBPAG2 IgG or graft loss indicating that MHC II:CD4+ T cell interactions were crucial for these responses. Tg skin grafts on Wt mice developed neutrophil-rich infiltrates, dermal edema, subepidermal blisters, and deposits of immunoreactants in epidermal BM. This model shows fidelity to alterations seen in patients with BP, has relevance to immune responses that may arise in patients with epidermolysis bullosa following BPAG2 gene replacement, and can be used to identify interventions that may block production of IgG against proteins in epidermal BM.

CD200, a "no danger" signal for hair follicles.

The "danger model" of immune recognition proposes that the immune system does not differentiate between self and non-self when deciding whether to mount a response, but instead, discerns between that which is dangerous or not dangerous to the host. Danger signals incite inflammatory responses, which can lead to the induction of tissue-specific autoimmunity. Immunosuppressive molecules expressed on selected cells have the potential to regulate tissue-specific inflammation, and consequently, autoimmunity. Recent studies have revealed that CD200, a potent immunoregulatory protein, is expressed on Langerhans cells (LCs) and keratinocytes (KCs) in mouse epidermis. CD200 expression is concentrated on KCs comprising the outer root sheath (ORS) of murine hair follicles (HF). Skin deficient in CD200 is highly susceptible to HF-associated inflammation and immune-mediated alopecia. In this concept review, the results of recent studies on CD200 and its inhibitory receptor, CD200R, are summarized and integrated to yield a model whereby CD200-CD200R interaction attenuates perifollicular inflammation, prevents HF-specific autoimmunity and may protect epidermal stem cells from autoimmune destruction. Further elucidation of the CD200-CD200R signaling pathway in cutaneous tissues may advance understanding of how immune homeostasis is established and maintained in the skin.

Expression of CD200 on epithelial cells of the murine hair follicle: a role in tissue-specific immune tolerance?

CD200 (OX-2) is a transmembrane glycoprotein that transmits an immunoregulatory signal through the CD200 receptor (CD200R) to attenuate inflammatory reactions and promote immune tolerance. CD200 expression in the skin has not been described previously. We now report that freshly isolated cells of the murine epidermis contain a subpopulation of major histocompatibility complex (MHC) class II-negative, CD3-negative keratinocytes that are CD200-positive. CD200 expression was accentuated in keratinocytes comprising the outer root sheath of the murine hair follicle (HF). When syngeneic skin grafts were exchanged between gender-matched wild-type (WT) and CD200-deficient C57BL/6 mice, significant perifollicular and intrafollicular inflammation was observed, eventually leading to the destruction of virtually all HF (alopecia) without significant loss of the CD200-negative grafts. Minimal and transient inflammation was observed in WT grafts, which persisted long term with hair. There was a 2-fold increase in graft-infiltrating T cells in CD200-deficient skin at 14 d. Alopecia and skin lesions were induced in CD200-deficient hosts by adoptive transfer of splenocytes from WT mice previously grafted with CD200-negative skin, but not from mice grafted with WT skin. Collectively, these results suggest that the expression of CD200 in follicular epithelium attenuates inflammatory reactions and may play a role in maintaining immune tolerance to HF-associated autoantigens.

Soluble proteins and haptens on bone marrow-derived dendritic cells are presented to host CD4 T cells in an MHC-restricted manner.

Because of their potent antigen-presenting capacity, dendritic cells (DC) have been used extensively in immunotherapy protocols. Our purpose was to functionally characterize mouse bone marrow-derived DC (BMDC) in vitro (in protein antigen- and hapten-specific assays) and in vivo (injecting soluble protein- and hapten-pulsed DC) to determine their suitability for the generation of T(h) cell responses. Furthermore, we determined whether there is cross-presentation on MHC class II molecules during in vivo protein and hapten sensitization. Co-culture of protein-pulsed [with hen egg lysozyme (HEL) or with pigeon cytochrome c (CYT)] DC with T cells from HEL- or CYT- sensitized mice induced antigen-specific T cell proliferation, but compared to cultured Langerhans cells (LC), BMDC required higher protein antigen-pulsing concentrations (100 microg and 1 mg/ml). In contrast, at low protein concentrations (10 microg/ml), BMDC stimulated an HEL-specific hybridoma very efficiently. Using an in vitro T cell proliferation assay and in vivo delayed-type hypersensitivity and contact sensitivity assays, we found that protein- and hapten-pulsed BMDC were able to sensitize syngeneic but not allogeneic hosts. Furthermore, if we injected BALB/c- and C57BL/6-derived HEL-pulsed BMDC into F1 mice, specific secondary proliferation of primed T cells occurred only when antigen-pulsed stimulator cells syngeneic to the injected BMDC were used. Using this model system we found that soluble proteins and haptens are presented by injected BMDC to host T cells in an MHC-restricted manner in vivo.

Fluorine-18 labeled mouse bone marrow-derived dendritic cells can be detected in vivo by high resolution projection imaging.

Immunization with ex vivo generated dendritic cells has become a focus for many clinical applications. The optimal site of injection and the migration pattern of these cells remain to be elucidated. We therefore developed a novel method for labeling mouse bone marrow-derived dendritic cells (BMDC) with the positron emitting radioisotope F-18 using N-succinimidyl-4-[F-18]fluorobenzoate, which covalently binds to the lysine residues of cell surface proteins. When we determined the stability of F-18 labeled BMDC, we found that at 4 h only 44+/-10% of the initial cell-bound activity was retained at 37 degrees C, whereas considerably more (91+/-3%) was retained at 4 degrees C. Labeled cells did not exhibit any significant alteration in cell viability or phenotype as determined by trypan blue exclusion and FACS analysis 24 h after radiolabeling. Furthermore, F-18-labeled BMDC stimulated allogeneic T cells in a mixed leukocyte reaction as potently as did sham-treated BMDC and migrated towards secondary lymphoid tissue chemokine (SLC) in a chemotaxis assay in vitro with the same efficiency as sham-treated BMDC. Migration of F-18-labeled BMDC was studied after footpad injection by (1) ex vivo counting of dissected tissues using a gamma counter and (2) in vivo by imaging mice with PiPET, a 2-mm resolution positron projection imager. After 4 h, the ratio between measured activity in draining vs. contralateral (D/C) lymph nodes (LN) was 166+/-96 (n=7) in the case of live cell injections, whereas if we injected heat-killed F-18-labeled BMDC or F-18-labeled macrophages the D/C ratios were 17+/-2 (n=2) and 14+/-4 (n=2), respectively. Injection of cell-free activity in the form of F-18-labeled 4-fluorobenzoic acid resulted in a D/C ratio of 7+/-2 (n=3), suggesting that the activity measured in the draining lymph node was associated with migrated F-18-labeled BMDC. When F-18-labeled live cells were injected into the footpad, 0.18+/-0.04% (n=7) of footpad activity was found in the draining LN within 4 h, whereas none was found in the contralateral LN. Quantitative assessment of cell migration by PET projection imaging of mice confirmed the ex-vivo counting results. These studies indicate that PET imaging offers a new approach for in vivo studies of dendritic cell biodistribution and migration.