Targeting Melanoma-Associated Fibroblasts (MAFs) with Activated γδ (Vδ2) T Cells: An In Vitro Cytotoxicity Model.

The tumor microenvironment (TME) has gained considerable scientific attention by playing a role in immunosuppression and tumorigenesis. Besides tumor cells, TME is composed of various other cell types, including cancer-associated fibroblasts (CAFs or MAFs when referring to melanoma-derived CAFs) and tumor-infiltrating lymphocytes (TILs), a subpopulation of which is labeled as γδ T cells. Since the current anti-cancer therapies using γδ T cells in various cancers have exhibited mixed treatment responses, to better understand the γδ T cell biology in melanoma, our research group aimed to investigate whether activated γδ T cells are capable of killing MAFs. To answer this question, we set up an in vitro platform using freshly isolated Vδ2-type γδ T cells and cultured MAFs that were biobanked from our melanoma patients. This study proved that the addition of zoledronic acid (1-2.5 µM) to the γδ T cells was necessary to drive MAFs into apoptosis. The MAF cytotoxicity of γδ T cells was further enhanced by using the stimulatory clone 20.1 of anti-BTN3A1 antibody but was reduced when anti-TCR γδ or anti-BTN2A1 antibodies were used. Since the administration of zoledronic acid is safe and tolerable in humans, our results provide further data for future clinical studies on the treatment of melanoma.

Ocular paraneoplastic pemphigoid / Szemészeti érintettséggel járó paraneoplasiás pemphigoid

Paraneoplastic mucous membrane pemphigoid, a rare pemphigoid variant is associated with primary malignancy, and characterised by fulminant progression and frequent ineffectivity of classical systemic immunosuppression. In this paper, the clinical features, diagnostic and therapeutical challenges are presented through three cases. Detailed history and analysis of the immunofluorescent samples help the diagnosis. The therapeutic goal is to prevent the progression with systemic immunosuppressive treatment, which can be contraindicated during the ongoing oncological therapy. In absence of consent in the exact diagnostic criteria and management protocol of this rare condition, consultation with other specialists (ophthalmologist, dermatologist, dentist, ear-nose-throat specialist, immunologist) has high importance in early diagnosis and treatment.

Mesenchymal-Stromal Cell-like Melanoma-Associated Fibroblasts Increase IL-10 Production by Macrophages in a Cyclooxygenase/Indoleamine 2,3-Dioxygenase-Dependent Manner.

Melanoma-associated fibroblasts (MAFs) are integral parts of melanoma, providing a protective network for melanoma cells. The phenotypical and functional similarities between MAFs and mesenchymal stromal cells (MSCs) prompted us to investigate if, similarly to MSCs, MAFs are capable of modulating macrophage functions. Using immunohistochemistry, we showed that MAFs and macrophages are in intimate contact within the tumor stroma. We then demonstrated that MAFs indeed are potent inducers of IL-10 production in various macrophage types in vitro, and this process is greatly augmented by the presence of treatment-naïve and chemotherapy-treated melanoma cells. MAFs derived from thick melanomas appear to be more immunosuppressive than those cultured from thin melanomas. The IL-10 increasing effect is mediated, at least in part, by cyclooxygenase and indoleamine 2,3-dioxygenase. Our data indicate that MAF-induced IL-10 production in macrophages may contribute to melanoma aggressiveness, and targeting the cyclooxygenase and indoleamine 2,3-dioxygenase pathways may abolish MAF-macrophage interactions.

Melanoma-associated fibroblasts impair CD8+ T cell function and modify expression of immune checkpoint regulators via increased arginase activity.

This study shows that melanoma-associated fibroblasts (MAFs) suppress cytotoxic T lymphocyte (CTL) activity and reveals a pivotal role played by arginase in this phenomenon. MAFs and normal dermal fibroblasts (DFs) were isolated from surgically resected melanomas and identified as Melan-A-/gp100-/FAP+ cells. CTLs of healthy blood donors were activated in the presence of MAF- and DF-conditioned media (CM). Markers of successful CTL activation, cytotoxic degranulation, killing activity and immune checkpoint regulation were evaluated by flow cytometry, ELISPOT, and redirected killing assays. Soluble mediators responsible for MAF-mediated effects were identified by ELISA, flow cytometry, inhibitor assays, and knock-in experiments. In the presence of MAF-CM, activated/non-naïve CTLs displayed dysregulated ERK1/2 and NF-κB signaling, impeded CD69 and granzyme B production, impaired killing activity, and upregulated expression of the negative immune checkpoint receptors TIGIT and BTLA. Compared to DFs, MAFs displayed increased amounts of VISTA and HVEM, a known ligand of BTLA on T cells, increased L-arginase activity and CXCL12 release. Transgenic arginase over-expression further increased, while selective arginase inhibition neutralized MAF-induced TIGIT and BTLA expression on CTLs. Our data indicate that MAF interfere with intracellular CTL signaling via soluble mediators leading to CTL anergy and modify immune checkpoint receptor availability via L-arginine depletion.

Skin-homing CD8+ T cells preferentially express GPI-anchored peptidase inhibitor 16, an inhibitor of cathepsin K.

This study sought to identify novel CD8+ T cell homing markers by studying acute graft versus host disease (aGvHD), typically involving increased T cell homing to the skin and gut. FACS-sorted skin-homing (CD8ß+ /CLA+ ), gut-homing (CD8ß+ /integrinß7+ ), and reference (CD8ß+ /CLA- /integrinß7- ) T cells were compared in patients affected by cutaneous and/or gastrointestinal aGVHD. Microarray analysis, qPCR, and flow cytometry revealed increased expression of peptidase inhibitor 16 (PI16) in skin-homing CD8+ T cells. Robust association of PI16 with skin homing was confirmed in all types of aGvHD and in healthy controls, too. PI16 was not observed on CLA+ leukocytes other than T cells. Induction of PI16 expression on skin-homing T cells occurred independently of vitamin D3. Among skin-homing T cells, PI16 expression was most pronounced in memory-like CD45RO+ /CD127+ /CD25+ /CD69- /granzyme B- cells. PI16 was confined to the plasma membrane, was GPI-anchored, and was lost upon restimulation of memory CD8+ T cells. Loss of PI16 occurred by downregulation of PI16 transcription, and not by Phospholipase C (PLC)- or Angiotensin-converting enzyme (ACE)-mediated shedding, or by protein recycling. Inhibitor screening and pull-down experiments confirmed that PI16 inhibits cathepsin K, but may not bind to other skin proteases. These data link PI16 to skin-homing CD8+ T cells, and raise the possibility that PI16 may regulate cutaneous cathepsin K.

[Epidermolysis bullosa: oral manifestations and their treatments]. / Az epidermolysis bullosa szájüregi tünetei és annak ellátása.

The aim of this comprehensive article is to provide guidelines for the daily treatment of patients with epidermolysis bullosa, thus contributing to the attainment of their higher quality of life through the improvement of their oral health. Moreover, it is our intention to facilitate the cooperation among Hungarian general practitioners, dermatologists and dentists. Relying on recent research findings of the international literature, we intend to help general practitioners or dermatologists treating epidermolysis bullosa patients on a daily basis by identifying symptoms that require consulting an oral professional on the one hand, and to present the most important prevention strategies and further treatments advised for dentists on the other. Focusing on various aspects of dental treatment, we specify how a dentist can treat the patient without causing additional wounds or pain, and what kinds of therapy are justified by this approach. Orv Hetil. 2017; 158(40): 1577-1583.

[Role of cancer stem cells in the progression and heterogeneity of melanoma]. / Tumorossejtek szerepe a melanoma progressziójában és heterogenitásában.

Over the past decade a rare cell population called cancer stem cells has been identified in both solid tumors and hematologic cancers. These cells are reminiscent of somatic and embryonic stem cells and play a critical role in the initiation and progression of malignancies. As all stem cells, they are able to undergo asymmetric cell division and hence renew themselves and create various other progenies with heterogenous phenotypes. A growing body of literature suggested that stem cell subpopulations contribute significantly to the growth and metastatic properties of melanoma. This review gives a comprehensive overview of the current literature on melanoma stem cells, with a special emphasis on the signaling pathways responsible for the homeostatic growth of melanocytes and the uncontrolled proliferation of melanoma cells. The importance of the local microenvironment are demonstrated through summarizing the role of various cell types, soluble factors and cell adhesion molecules in the progression of melanoma and the creation of treatment resistant cancer cell clones. Last but not least, the models of melanoma progression will be introduced and a variety of cellular markers will be presented that may be used to identify and therapeutically target melanoma. Orv. Hetil., 2016, 157(34), 1339-1348.

Decreased fibrinolytic potential and morphological changes of fibrin structure in dermatitis herpetiformis.

BACKGROUND: Recently, high prevalence of cryofibrinogenaemia has been observed in plasma of untreated dermatitis herpetiformis (DH) patients, and the pathological IgA and TG3 deposits in the papillary dermis were found to co-localize with fibrin and fibrinogen. OBJECTIVE: To study the fibrinolytic potential in plasma of untreated, dapsone and or/gluten-free diet treated DH patients as well as the in vitro effect of dapsone on the fibrinolytic profile. METHOD: Plasma samples of 23 DH patients, 19 healthy subjects and 5 pemphigus vulgaris patients were investigated by a turbidimetric-clot lysis assay. Out of them 5 DH plasma samples representing different fibrinolytic parameters, and 3 healthy controls were selected for parallel fibrin clot preparation. The clot fibrin structure was examined by scanning electron microscopy (SEM), and the diameters of 900 fibrin fibres were determined in each clot. RESULTS: A significantly prolonged clot lysis time was detected in untreated DH patients. The turbidity values of DH plasma clots indicated an altered fibrin structure that was also confirmed by SEM: significantly thicker fibrin fibers were observed in untreated, TG3 antibody positive DH patients compared to healthy controls, whereas the fiber diameters of dapsone-treated patients were similar or thinner than the control values. In line with the structural changes of fibrin, the fibrinolytic profile of 5 DH patients under dapsone treatment approached the control values. CONCLUSION: This study revealed that the fibrinolytic potential was impaired in the plasma of untreated DH patients, whereas dapsone corrected the fibrinolytic defect. These data suggest a pathogenic role for plasma-derived factors in the development of skin symptoms and add a new aspect to the long-known beneficial, symptomatic effect of dapsone in active DH.

Characterization of desmoglein-3 epitope region peptides as synthetic antigens: analysis of their in vitro T cell stimulating efficacy, cytotoxicity, stability, and their conformational features.

Desmoglein-3 (Dsg3) adhesion protein is the main target of autoantibodies and autoreactive T cells in Pemphigus vulgaris (PV) autoimmune skin disorder. Several mapping studies of Dsg3 T cell epitope regions were performed, and based on those data, we designed and synthesized four peptide series corresponding to Dsg3 T cell epitope regions. Each peptide series consists of a 17mer full-length peptide (Dsg3/189-205, Dsg3/206-222, Dsg3/342-358, and Dsg3/761-777) and its N-terminally truncated derivatives, resulting in 15 peptides altogether. The peptides were prepared on solid phase and were chemically characterized. In order to establish a structure-activity relationship, the solution conformation of the synthetic peptides has been investigated using electronic circular dichroism spectroscopy. The in vitro T cell stimulating efficacy of the peptides has been determined on peripheral blood mononuclear cells isolated from whole blood of PV patients and also from healthy donors. After 20 h of stimulation, the interferon (IFN)-γ content of the supernatants was measured by enzyme-linked immunosorbent assay. In the in vitro conditions, peptides were stable and non-cytotoxic. The in vitro IFN-γ production profile of healthy donors and PV patients, induced by peptides as synthetic antigens, was markedly different. The most unambiguous differences were observed after stimulation with 17mer peptide Dsg3/342-358, and three truncated derivatives from two other peptide series, namely, peptides Dsg3/192-205, Dsg3/763-777, and Dsg3/764-777. Comparative analysis of in vitro activity and the capability of oligopeptides to form ordered or unordered secondary structure showed that peptides bearing high solvent sensibility and backbone flexibility were the most capable to distinguish between healthy and PV donors.

Evidence for a role of autoantibodies to heat shock protein 60, 70, and 90 in patients with dermatitis herpetiformis.

Heat shock proteins (Hsp) are highly conserved immunomodulatory molecules upregulated when cells are exposed to stressful stimuli, such as inflammation. Their involvement in various autoimmune diseases, including autoimmune bullous diseases and celiac disease, has been increasingly recognized. To further study the role of Hsp in autoimmune bullous diseases, we have investigated for the first time the humoral autoimmune response to Hsp40, Hsp60, Hsp70, and Hsp90 in patients with dermatitis herpetiformis (DH; n = 26), bullous pemphigoid (BP; n = 23), and pemphigus vulgaris (PV; n = 16), the first representing a cutaneous manifestation of celiac disease. While in patients with active BP and PV, serum levels of autoantibodies against these Hsp did not differ from the corresponding age- and gender-matched healthy controls (n = 9-14); circulating autoantibodies against Hsp60, Hsp70, and Hsp90 were found to be increased at the active disease stage of DH. Further analysis of this latter patient subgroup showed that these anti-Hsp autoantibodies decreased in parallel with serum autoantibodies against epidermal and tissue transglutaminase during remission of skin lesions following a gluten-free diet, revealing significantly positive correlations. Although further studies on larger groups of patients will be needed to confirm the present data, our results support the notion that autoantibodies against Hsp60, Hsp70, and Hsp90 deserve attention in the study of the mechanisms that promote the development and maintenance of DH and possibly also the underlying celiac disease as well as potential novel disease biomarkers.

Rapid systemic and local treatments with the antibacterial peptide dimer A3-APO and its monomeric metabolite eliminate bacteria and reduce inflammation in intradermal lesions infected with Propionibacterium acnes and meticillin-resistant Staphylococcus aureus.

When administered intramuscularly, the designer antibacterial peptide dimer A3-APO is highly efficacious in mouse models of Acinetobacter baumannii and Staphylococcus aureus burn infections. Here we compared the efficacy of A3-APO and its monomeric metabolite in mouse models of S. aureus and Propionibacterium acnes intradermal infections following administration as intramuscular (i.m.) or topical treatments. In the animal models, either (i) the ears of CD-1 mice were infected with P. acnes or (ii) S. aureus was injected into burn wounds inflicted to the back. A3-APO or the monomer were injected intramuscularly at 5 mg/kg one to three times or were applied three times as 1% local treatment in phosphate-buffered saline or Vaseline(®). Despite being inactive against the strains in vitro, in vivo the skin conditions of the mice were dramatically improved upon peptide treatment regardless of dosing frequency, administration mode or drug valency. In the P. acnes study, A3-APO statistically significantly reduced ear thickness and ear bacterial counts. The amount of ear connective tissue and epithelial macrophages correlated with therapeutic success. Bacterial load in the lesions was more representative of physical improvement than ear dimensions. In the S. aureus model, both peptides eliminated wound bacteria from >10(7) CFU/mg to almost background levels, with monomer treatment being somewhat more successful. In conclusion, A3-APO and its monomeric metabolite very efficiently ameliorate resistant aerobic and anaerobic intradermal infections, but the protection is apparently not due to direct bacterial killing. Immunostimulatory and anti-inflammatory actions are likely involved. Nevertheless, topical and i.m. administrations are equally effective.

The mapping of linear B-cell epitope regions in the extracellular parts of the desmoglein 1 and 3 proteins: recognition of immobilized peptides by pemphigus patients' serum autoantibodies.

Desmosomal transmembrane glycoproteins desmogleins (Dsg) 1 and 3 are targets of life-threatening autoimmune blistering disorders such as Pemphigus vulgaris (PV) and Pemphigus foliaceus (PF). In these diseases, pemphigus autoantibodies are produced against Dsg1 and Dsg3 proteins. The autoantibodies bind to these transmembrane elements leading to a loss of desmosomal cell-cell adhesion and clinically, to the presence of blisters and erosions. Identification, characterization, and detailed analysis of the binding sites of autoantibodies have an outstanding importance in understanding the immunopathology of the disease and also in the design of novel diagnostics. Here, we describe the localization of the B-cell epitope regions of Dsg1 and Dsg3 proteins' extracellular parts recognized by IgG-type serum autoantibodies of patients with PV and PF. In our study, overlapping pentadecapeptides were synthesized on hydroxypropyl methacrylate pins based on the results of in silico predictions. To detect the interaction between the serum autoantibodies and the immobilized synthetic peptides, modified Enzyme Linked Immunosorbent Assay (ELISA) was performed with pin-attached peptides testing the serum samples of ten patients and four healthy donors. We identified five possible epitope regions (aa86-110, aa196-220, aa226-250, aa326-340, and aa486-520) within the extracellular part of the Dsg1 and four possible epitope regions (aa64-78, aa330-344, aa375-399, and aa446-460) within that of the Dsg3 protein sequence using these methods. Our data showed that serum autoantibodies of patients, previously identified as Dsg1 and Dsg3 positive, are able to recognize continuous linear epitope regions of both Dsg1 and Dsg3 proteins using pin-bound overlapping peptides in modified ELISAs.

Eosinophilic Fasciitis associated with Mycoplasma arginini infection.

Eosinophilic fasciitis (EF) with generalized sclerodermiform skin lesions developed over a 19-month period in a previously healthy 23-year-old man. Although we confirmed EF by skin histology and laboratory tests, the recurrent fevers and the clinical observation of sclerotic prepuce with urethritis indicated further bacteriological analysis by conventional microbiological and DNA-based tests. Urethra cultures were positive for an arginine-hydrolyzing mycoplasma and Ureaplasma urealyticum. The patient also had serum IgM antibodies to Mycoplasma pneumoniae using enzyme-linked immunosorbent assay (ELISA)-based qualitative detection. Mycoplasma arginini was isolated from two independent venous blood serum samples and was identified by conventional microbiological tests and sequencing of the 16S rRNA and rpoB genes (GenBank sequence accession numbers HM179555 and HM179556, respectively). M. arginini genomic DNA also was detected by species-specific PCR in the skin lesion biopsy sample. Treatment with corticosteroids and long-term courses of selected antibiotics led to remission of skin symptoms and normalization of laboratory values. This report provides the first evidence of EF associated with mycoplasma infection and the second report of human infection with M. arginini and therefore suggests that this mycoplasma infection might have contributed to the pathogenesis of the disease.

Searching for foreign antigens as possible triggering factors of autoimmunity: Torque Teno virus DNA prevalence is elevated in sera of patients with bullous pemphigoid.

OBJECTIVE: The Torque Teno virus (TTV), a member of virus genus Anellovirus has been shown to be commonly present in humans, yet without detectable pathogenicity. Recent studies imply that TTV may contribute to provoke autoimmune progresses in systemic lupus erythematosus and idiopathic inflammatory myopathies. We aimed to study the presence of TTV in a group of patients with autoimmune bullous diseases with a further goal to identify long-lasting foreign antigen, such as TTV as possible triggers of skin-specific autoimmunity. PATIENTS AND METHODS: We performed in silico research to study similarities between known TTV sequences and antigens of bullous pemphigoid (BP), pemphigus vulgaris (PV) and dermatitis herpetiformis (DH). Basic Local Alignment Search Tool results showed matching regions for the major BP antigens BP180 and BP230, PV antigen desmoglein 3 and DH antigen transglutaminase 3 and disclosed overlapping, antigen-predicted sequences only for BP180 regions. We also assessed the prevalence of TTV in these disorders and compared them with the results from two healthy blood donor groups (group 1: sex- and age-matched for the general bullous group, n = 95; group 2: sex- and age-matched for BP, n = 50). Furthermore, we assayed lymphocytes from four TTV DNA and BP180 NC16A blot-positive BP patients and three controls in a standard lymphocyte transformation test with a TTV peptide from the conserved ORF(Open Reading Frame)1/N22 region. RESULTS: We found that the detection rate of TTV was comparable with that in healthy controls in the group of PV (19/33); whereas detection rates in DH showed a slight, but not significant tendency for elevation (17/20). Contrary, the TTV prevalence in BP patients was significantly elevated (group 1: 36/40 vs group 2: 31/50, P < 0.032). Lymphocytes from all four virus-positive BP patients heavily reacted to TTV peptide while two of the three healthy controls have shown not to recognize the viral sequences. Only the TTV carrier healthy control had a minor reaction at lowest peptide concentration. The combined in silico, polymerse chain reaction and in vitro cell assay data of the present study indicate that a TTV persistence may contribute to the pathogenesis of BP.

[Radical vulvectomy to treat severe vulvar acne inversa]. / Szeméremtesten megjelenõ acne inversa sikeres kezelése radikális vulvectomia útján.

UNLABELLED: A rare case, when radical vulvectomy had to be done to treat a benign skin disorder is presented. PATIENT AND METHOD: A 56-year-old white woman suffered from severe vulvar acne inversa. The systemic treatments, the incisions and drainages were not successful. The only solution was the radical excision of the seriously damaged vulva, with a satisfactory cosmetic and functional result. The pathology, the diagnosis and the treatment of the disease are also discussed. CONCLUSION: The authors put emphasis on the importance of the interdisciplinary collaboration.