Clinical Characterization and Treatment Response of Folliculitis Decalvans Lichen Planopilaris Phenotypic Spectrum: A Unicentre Retrospective Series of 31 Patients.

Folliculitis decalvans and lichen planopilaris phenotypic spectrum has been described as a form of cicatricial alopecia. The aim of this study is to describe the clinical and trichoscopic features and therapeutic management of this condition in a series of patients. A retrospective observational unicentre study was designed including patients with folliculitis decalvans and lichen planopilaris phenotypic spectrum confirmed with biopsy. A total of 31 patients (20 females) were included. The most common presentation was an isolated plaque of alopecia (61.3%) in the vertex. Trichoscopy revealed hair tufting with perifollicular white scaling in all cases. The duration of the condition was the only factor associated with large plaques (grade III) of alopecia (p = 0.026). The mean time to transition from the classic presentation of folliculitis decalvans to folliculitis decalvans and lichen planopilaris phenotypic spectrum was 5.2 years. The most frequently used treatments were topical steroids (80.6%), intralesional steroids (64.5%) and topical antibiotics (32.3%). Nine clinical relapses were detected after a mean time of 18 months (range 12-23 months). Folliculitis decalvans and lichen planopilaris phenotypic spectrum is an infrequent, but probably underdiagnosed, cicatricial alopecia. Treatment with anti-inflammatory drugs used for lichen planopilaris may be an adequate approach.

Genotyping of the rs1800440 Polymorphism in CYP1B1 Gene and the rs9258883 Polymorphism in HLA-B Gene in a Spanish Cohort of 223 Patients with Frontal Fibrosing Alopecia.

The pathogenesis of frontal fibrosing alopecia has been linked to specific genetic variants. CYP1B1 codes for a component of the cytochrome p450 machinery that is involved in the metabolism of xenobiotic oestrogens. The study of the prevalence of polymorphisms in this gene may help to understand their role in the development of frontal fibrosing alopecia. The aim of this study is to describe the frequency of genetic variations in the alleles HLA-B*07:02 and CYP1B1 in patients with frontal fibrosing alopecia. A cross-sectional study was designed to evaluate blood samples from patients with frontal fibrosing alopecia who attended the Dermatology Department at University Hospital Ramón y Cajal (Madrid, Spain), in search of the polymorphisms rs9258883 and rs1800440 in the alleles HLA-B*07:02 and CYP1B1, respectively. A total of 223 patients were included in the study. Among the 83.8% of patients who carried the rs9258883 polymorphism in HLA-B*07:02, 58.7% were heterozygous for this variant and it was not present in 14.8% of the cases. The majority of patients with frontal fibrosing alopecia lacked the protective rs1800440 polymorphism in CYP1B1 (75.2%). This suggests a relevant role of this variant in development of frontal fibrosing alopecia. The genetic approach to this condition might influence patient prognosis and therapy options.

CRISPR/Cas9-engineering of HMC-1.2 cells renders a human mast cell line with a single D816V-KIT mutation: An improved preclinical model for research on mastocytosis.

The HMC-1.2 human mast cell (huMC) line is often employed in the study of attributes of neoplastic huMCs as found in patients with mastocytosis and their sensitivity to interventional drugs in vitro and in vivo. HMC-1.2 cells express constitutively active KIT, an essential growth factor receptor for huMC survival and function, due to the presence of two oncogenic mutations (D816V and V560G). However, systemic mastocytosis is commonly associated with a single D816V-KIT mutation. The functional consequences of the coexisting KIT mutations in HMC-1.2 cells are unknown. We used CRISPR/Cas9-engineering to reverse the V560G mutation in HMC-1.2 cells, resulting in a subline (HMC-1.3) with a single mono-allelic D816V-KIT variant. Transcriptome analyses predicted reduced activity in pathways involved in survival, cell-to-cell adhesion, and neoplasia in HMC-1.3 compared to HMC-1.2 cells, with differences in expression of molecular components and cell surface markers. Consistently, subcutaneous inoculation of HMC-1.3 into mice produced significantly smaller tumors than HMC-1.2 cells, and in colony assays, HMC-1.3 formed less numerous and smaller colonies than HMC-1.2 cells. However, in liquid culture conditions, the growth of HMC-1.2 and HMC-1.3 cells was comparable. Phosphorylation levels of ERK1/2, AKT and STAT5, representing pathways associated with constitutive oncogenic KIT signaling, were also similar between HMC-1.2 and HMC-1.3 cells. Despite these similarities in liquid culture, survival of HMC-1.3 cells was diminished in response to various pharmacological inhibitors, including tyrosine kinase inhibitors used clinically for treatment of advanced systemic mastocytosis, and JAK2 and BCL2 inhibitors, making HMC-1.3 more susceptible to these drugs than HMC-1.2 cells. Our study thus reveals that the additional V560G-KIT oncogenic variant in HMC-1.2 cells modifies transcriptional programs induced by D816V-KIT, confers a survival advantage, alters sensitivity to interventional drugs, and increases the tumorigenicity, suggesting that engineered huMCs with a single D816V-KIT variant may represent an improved preclinical model for mastocytosis.

Research Advances in Mast Cell Biology and Their Translation Into Novel Therapies for Anaphylaxis.

Anaphylaxis is an acute, potentially life-threatening systemic allergic reaction for which there are no known reliable preventative therapies. Its primary cell mediator, the mast cell, has several pathophysiologic roles and functions in IgE-mediated reactions that continue to be poorly understood. Recent advances in the understanding of allergic mechanisms have identified novel targets for inhibiting mast cell function and activation. The prevention of anaphylaxis is within reach with new drugs that could modulate immune tolerance, mast cell proliferation and differentiation, and IgE regulation and production. Several US Food and Drug Administration-approved drugs for chronic urticaria, mastocytosis, and cancer are also being repurposed to prevent anaphylaxis. New therapeutics have not only shown promise in potential efficacy for preventing IgE-mediated reactions, but in some cases, they are able to inform us about mast cell mechanisms in vivo. This review summarizes the most recent advances in the treatment of anaphylaxis that have arisen from new pharmacologic tools and our current understanding of mast cell biology.

Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

Activating mutations in the receptor KIT promote the dysregulated proliferation of human mast cells (huMCs). The resulting neoplastic huMCs secrete extracellular vesicles (EVs) that can transfer oncogenic KIT among other cargo into recipient cells. Despite potential contributions to diseases, KIT-containing EVs have not been thoroughly investigated. Here, we isolated and characterized KIT-EV subpopulations released by neoplastic huMCs using an immunocapture approach that selectively isolates EVs containing KIT in its proper topology. Immunocapture of EVs on KIT antibody-coated electron microscopy (EM) affinity grids allowed to assess the morphology and size of KIT-EVs. Immunoblot analysis demonstrated KIT-EVs have a distinct protein profile from KIT-depleted EVs, contain exosome and microvesicle markers, and are separated into these subtypes by ultracentrifugation. Cell treatment with sphingomyelinase inhibitors shifted the protein content among KIT-EV subtypes, suggesting different biogenesis routes. Proteomic analysis revealed huMC KIT-EVs are enriched in proteins involved in signalling, immune responses, and cell migration, suggesting diverse biological functions, and indicated neoplastic huMCs disseminate KIT via shuttling in heterogeneous microvesicle- and exosome-like EVs. Further, selective KIT-immunocapture will enable the enrichment of specific huMC-derived EVs from complex human biosamples and facilitate an understanding of their in vivo functions and potential to serve as biomarkers of specific biological pathologies.

Effect of non-ionic surfactant in the solvothermal synthesis of anatase TiO2 nanoplates with a high percentage of exposed {001} facets and its role in the photocatalytic degradation of methylene blue dye.

The synthesis of anatase TiO2 nanoparticles with controlled morphology and increased {001} facets exposed without the presence of fluorine-derived substances is a challenge. Herein, we report a highly effective approach to fabricate anatase TiO2 nanoplates with exposed {001} facets and their exploitation as robust photocatalytic materials for dye remediation. These materials were synthesized under controlled hydrolysis and condensation reactions, using titanium (IV) n-butoxide in an ethanolic solution, with acetic and sulfuric acids, by a solvothermal method at 190 °C with or without the presence of the non-ionic surfactant Triton® X-100 and then characterized. During TiO2 crystal synthesis, the effect of a non-ionic surfactant on the TiO2 particle growth was investigated. Our results demonstrate that the proposed method can synthesize pure and crystalline anatase TiO2 square nanoplates that form nanostructured spheres with high surface area, uniformly sized mesopores, and exposed {001} facets. The presence of non-ionic surfactant increased the exposed {001} facets percentage of the formed nanoplates from 69 to 80%, decreased the crystallite thickness, but unaffected its crystalline phase and band gap energy. The kinetic constants (Ka e Kb) for the synthesized TiO2 anatase nanoplates are considerably higher than the commercial TiO2 anatase constant (Kc). The synthesized photocatalysts show higher efficiency in the photocatalytic removal of methylene blue (MB) than commercial TiO2 (for t = 120 min).

"Stripe" transcription factors provide accessibility to co-binding partners in mammalian genomes.

Regulatory elements activate promoters by recruiting transcription factors (TFs) to specific motifs. Notably, TF-DNA interactions often depend on cooperativity with colocalized partners, suggesting an underlying cis-regulatory syntax. To explore TF cooperativity in mammals, we analyze ∼500 mouse and human primary cells by combining an atlas of TF motifs, footprints, ChIP-seq, transcriptomes, and accessibility. We uncover two TF groups that colocalize with most expressed factors, forming stripes in hierarchical clustering maps. The first group includes lineage-determining factors that occupy DNA elements broadly, consistent with their key role in tissue-specific transcription. The second one, dubbed universal stripe factors (USFs), comprises ∼30 SP, KLF, EGR, and ZBTB family members that recognize overlapping GC-rich sequences in all tissues analyzed. Knockouts and single-molecule tracking reveal that USFs impart accessibility to colocalized partners and increase their residence time. Mammalian cells have thus evolved a TF superfamily with overlapping DNA binding that facilitate chromatin accessibility.

A Critical Function for the Transcription Factors GLI1 and GLI2 in the Proliferation and Survival of Human Mast Cells.

Mast cell hyperactivity and accumulation in tissues are associated with allergy and other mast cell-related disorders. However, the molecular pathways regulating mast cell survival in homeostasis and disease are not completely understood. As glioma-associated oncogene (GLI) proteins are involved in both tissue homeostasis and in the hematopoietic system by regulating cell fate decisions, we sought to investigate the role for GLI proteins in the control of proliferation and survival of human mast cells. GLI1 transcripts were present in primary human mast cells and mast cell lines harboring or not activating mutations in the tyrosine kinase receptor KIT (HMC-1.1 and HMC-1.2, and LAD2 cells, respectively), while GLI2 transcripts were only present in HMC-1.1 and HMC-1.2 cells, suggesting a role for oncogenic KIT signaling in the regulation of GLI2. Reduction in GLI activity by small molecule inhibitors, or by shRNA-mediated knockdown of GLI1 or GLI2, led to increases in apoptotic cell death in both cultured human and murine mast cells, and reduced the number of peritoneal mast cells in mice. Although GLI proteins are typically activated via the hedgehog pathway, steady-state activation of GLI in mast cells occurred primarily via non-canonical pathways. Apoptosis induced by GLI silencing was associated with a downregulation in the expression of KIT and of genes that influence p53 stability and function including USP48, which promotes p53 degradation; and iASPP, which inhibits p53-induced transcription, thus leading to the induction of p53-regulated apoptotic genes. Furthermore, we found that GLI silencing inhibited the proliferation of neoplastic mast cell lines, an effect that was more pronounced in rapidly growing cells. Our findings support the conclusion that GLI1/2 transcription factors are critical regulators of mast cell survival and that their inhibition leads to a significant reduction in the number of mast cells in vitro and in vivo, even in cells with constitutively active KIT variants. This knowledge can potentially be applicable to reducing mast cell burden in mast cell-related diseases.

Targeting KIT by frameshifting mRNA transcripts as a therapeutic strategy for aggressive mast cell neoplasms.

Activating mutations in c-KIT are associated with the mast cell (MC) clonal disorders cutaneous mastocytosis and systemic mastocytosis and its variants, including aggressive systemic mastocytosis, MC leukemia, and MC sarcoma. Currently, therapies inhibiting KIT signaling are a leading strategy to treat MC proliferative disorders. However, these approaches may have off-target effects, and in some patients, complete remission or improved survival time cannot be achieved. These limitations led us to develop an approach using chemically stable exon skipping oligonucleotides (ESOs) that induce exon skipping of precursor (pre-)mRNA to alter gene splicing and introduce a frameshift into mature KIT mRNA transcripts. The result of this alternate approach results in marked downregulation of KIT expression, diminished KIT signaling, inhibition of MC proliferation, and rapid induction of apoptosis in neoplastic HMC-1.2 MCs. We demonstrate that in vivo administration of KIT targeting ESOs significantly inhibits tumor growth and systemic organ infiltration using both an allograft mastocytosis model and a humanized xenograft MC tumor model. We propose that our innovative approach, which employs well-tolerated, chemically stable oligonucleotides to target KIT expression through unconventional pathways, has potential as a KIT-targeted therapeutic alone, or in combination with agents that target KIT signaling, in the treatment of KIT-associated malignancies.

Demonstration and implications of IL-3 upregulation of CD25 expression on human mast cells.

BACKGROUND: CD25+ human mast cells (huMCs) have been reported in patients with monoclonal mast cell diseases and in rare association with inflammation. However, the regulation of CD25 expression on huMCs and the possible biologic consequences remain poorly understood. OBJECTIVE: We sought to identify conditions that would upregulate CD25 expression on huMCs and to explore possible functional implications. METHODS: huMCs were cultured from peripheral blood progenitor cells over 6 to 8 weeks. Expression of CD25 was determined by fluorescence-activated cell sorting and soluble CD25 by ELISA. Signal transducer and activator of transcription 5 (STAT5) phosphorylation induced by IL-2 in huMCs, regulatory T (Treg) cells, or in cocultured huMCs and Treg cells was examined by fluorescence-activated cell sorting. RESULTS: Addition of IL-3 to CD34+ progenitors at the initiation of huMC cultures in the presence of stem cell factor and IL-6 upregulated the expression of CD25 in developing huMCs and resulted in shedding of soluble CD25 into the media. Removal of IL-3 after the first week of culture did not affect subsequent expression of CD25. Furthermore, addition of IL-3 14 days after the initiation of the culture did not induce significant CD25 expression. Treatment with anti-IL-3 antibody or the Janus kinase inhibitor tofacitinib blocked IL-3-induced CD25 upregulation. Binding of IL-2 to CD25+ huMCs did not induce STAT5 phosphorylation. However, coincubation of Treg cells with CD25+ huMCs pretreated with IL-2 was sufficient to result in STAT5 phosphorylation in Treg cells. CONCLUSIONS: IL-3 promotes CD25 expression and shedding by huMCs. Although CD25+ huMCs do not respond to IL-2, they bind IL-2 and may act as a reservoir of IL-2 to then activate lymphocytes.

MYO1F Regulates IgE and MRGPRX2-Dependent Mast Cell Exocytosis.

The activation and degranulation of mast cells is critical in the pathogenesis of allergic inflammation and modulation of inflammation. Recently, we demonstrated that the unconventional long-tailed myosin, MYO1F, localizes with cortical F-actin and mediates adhesion and migration of mast cells. In this study, we show that knockdown of MYO1F by short hairpin RNA reduces human mast cell degranulation induced by both IgE crosslinking and by stimulation of the Mas-related G protein-coupled receptor X2 (MRGPRX2), which has been associated with allergic and pseudoallergic drug reactions, respectively. Defective degranulation was accompanied by a reduced reassembly of the cortical actin ring after activation but reversed by inhibition of actin polymerization. Our data show that MYO1F is required for full Cdc42 GTPase activation, a critical step in exocytosis. Furthermore, MYO1F knockdown resulted in less granule localization in the cell membrane and fewer fissioned mitochondria along with deficient mitochondria translocation to exocytic sites. Consistent with that, AKT and DRP1 phosphorylation are diminished in MYO1F knockdown cells. Altogether, our data point to MYO1F as an important regulator of mast cell degranulation by contributing to the dynamics of the cortical actin ring and the distribution of both the secretory granules and mitochondria.

Mastocytosis-derived extracellular vesicles deliver miR-23a and miR-30a into pre-osteoblasts and prevent osteoblastogenesis and bone formation.

Osteoporosis and other manifestations of bone disease are frequent in patients with systemic mastocytosis (SM) in association with the presence of mast cell infiltrates in bone marrow, although the mechanisms behind bone disease remain poorly understood. We find that extracellular vesicles (EVs) released by neoplastic mast cells and present in the serum of patients with SM (SM-EVs) block osteoblast differentiation and mineralization in culture, and when injected into mice diminish the expression of osteoblast markers, and trabecular bone volume and microarchitecture. We demonstrate that miRNA-30a and miRNA-23a, increased in SM-EVs and neoplastic mast cell-derived EVs, attenuate osteoblast maturation by suppressing expression of RUNX2 and SMAD1/5, essential drivers of osteogenesis. Thus, SM-EVs carry and deliver miRNAs that epigenetically interfere with bone formation and can contribute to bone mass reduction in SM. These findings also suggest possibilities for novel approaches to the management of bone disease in mast cell proliferative disorders.