EGFR Inhibition by Erlotinib Rescues Desmosome Ultrastructure and Keratin Anchorage and Protects Against Pemphigus Vulgaris IgG-Induced Acantholysis in Human Epidermis.

Pemphigus is a severe blistering disease caused by autoantibodies primarily against the desmosomal cadherins desmoglein (DSG)1 and DSG3 which impair desmosome integrity. Especially for the acute phase, additional treatment options allowing to reduce corticosteroids would fulfill an unmet medical need. Here, we provide evidence that epidermal growth factor receptor (EGFR) inhibition by erlotinib ameliorates pemphigus vulgaris immunoglobulin G (PV-IgG) -induced acantholysis in intact human epidermis. PV-IgG caused phosphorylation of EGFR (Y845) and SRC in human epidermis. In line with that, a phosphotyrosine kinome analysis revealed a robust response associated with EGFR and SRC family kinase signaling in response to PV-IgG but not pemphigus foliaceus autoantibodies. Erlotinib inhibited PV-IgG-induced epidermal blistering and EGFR phosphorylation, loss of desmosomes as well as ultrastructural alterations of desmosome size, plaque symmetry, keratin filament insertion and restored the desmosome midline considered as hallmark of mature desmosomes. Erlotinib enhanced both single molecule DSG3 binding frequency and strength and delayed DSG3 fluorescence recovery supporting that EGFR inhibition increases DSG3 availability and cytoskeletal anchorage. Our data indicate that EGFR is a promising target for pemphigus therapy due to its link to several signaling pathways known to be involved in pemphigus pathogenesis.

Eosinophils, Basophils, and Neutrophils in Bullous Pemphigoid.

Bullous pemphigoid (BP) is an autoimmune blistering skin disease, of which the incidence has increased in recent years. BP is characterized by circulating IgG and IgE autoantibodies against the hemidesmosomal proteins BP180 and BP230. Although autoantibodies trigger inflammatory cascades that lead to blister formation, effector cells and cell-mediated autoimmunity must also be considered as important factors in the pathogenesis of BP. The aim of this review is to outline the current knowledge on the role of eosinophils, basophils, and neutrophils in BP.

Topical Application of the PI3Kß-Selective Small Molecule Inhibitor TGX-221 Is an Effective Treatment Option for Experimental Epidermolysis Bullosa Acquisita.

Class I phosphoinositide 3-kinases (PI3K) have been implemented in pathogenesis of experimental epidermolysis bullosa acquisita (EBA), an autoimmune skin disease caused by type VII collagen (COL7) autoantibodies. Mechanistically, inhibition of specific PI3K isoforms, namely PI3Kß or PI3Kδ, impaired immune complex (IC)-induced neutrophil activation, a key prerequisite for EBA pathogenesis. Data unrelated to EBA showed that neutrophil activation is also modulated by PI3Kα and γ, but their impact on the EBA has, so far, remained elusive. To address this and to identify potential therapeutic targets, we evaluated the impact of a panel of PI3K isoform-selective inhibitors (PI3Ki) on neutrophil function in vitro, and in pre-clinical EBA mouse models. We document that distinctive, and EBA pathogenesis-related activation-induced neutrophil in vitro functions depend on distinctive PI3K isoforms. When mice were treated with the different PI3Ki, selective blockade of PI3Kα (alpelisib), PI3Kγ (AS-604850), or PI3Kß (TGX-221) impaired clinical disease manifestation. When applied topically, only TGX-221 impaired induction of experimental EBA. Ultimately, multiplex kinase activity profiling in the presence of disease-modifying PI3Ki identified unique signatures of different PI3K isoform-selective inhibitors on the kinome of IC-activated human neutrophils. Collectively, we here identify topical PI3Kß inhibition as a potential therapeutic target for the treatment of EBA.

Mast cells promote melanoma colonization of lungs.

Mast cells have been implicated in malignant processes, mainly through clinical correlative studies and by experiments performed using animals lacking mast cells due to defective c-kit signaling. However, mast cell-deficient mouse models based on c-kit defects have recently been questioned for their relevance. Here we addressed the effect of mast cells in a tumor setting by using transgenic Mcpt5-Cre+ R-DTA+ mice, in which the deficiency of mast cells is independent of c-kit defects. Melanoma cells (B16.F10) were administered either subcutaneously or intravenously into Mcpt5-Cre+ R-DTA+ mice or Mcpt5-Cre- R-DTA+ littermate controls, followed by the assessment of formed tumors. In the subcutaneous model, mast cells were abundant in the tumor stroma of control mice but were absent in Mcpt5-Cre+ R-DTA+ mice. However, the absence of mast cells did not affect tumor size. In contrast, after intravenous administration of B16.F10 cells, melanoma colonization of the lungs was markedly reduced in Mcpt5-Cre+ R-DTA+ vs. Mcpt5-Cre- R-DTA+ animals. Decreased melanoma colonization of the lungs in Mcpt5-Cre+ R-DTA+ animals was accompanied by increased inflammatory cell recruitment into the bronchoalveolar lavage fluid, suggesting that mast cells suppress inflammation in this setting. Further, qPCR analysis revealed significant alterations in the expression of Twist and E-cadherin in lungs of Mcpt5-Cre+ R-DTA+ vs. control Mcpt5-Cre- R-DTA+ animals, suggesting an impact of mast cells on epithelial-mesenchymal transition. In conclusion, this study reveals that mast cells promote melanoma colonization of the lung.

ß1 integrin signaling maintains human epithelial progenitor cell survival in situ and controls proliferation, apoptosis and migration of their progeny.

ß1 integrin regulates multiple epithelial cell functions by connecting cells with the extracellular matrix (ECM). While ß1 integrin-mediated signaling in murine epithelial stem cells is well-studied, its role in human adult epithelial progenitor cells (ePCs) in situ remains to be defined. Using microdissected, organ-cultured human scalp hair follicles (HFs) as a clinically relevant model for studying human ePCs within their natural topobiological habitat, ß1 integrin-mediated signaling in ePC biology was explored by ß1 integrin siRNA silencing, specific ß1 integrin-binding antibodies and pharmacological inhibition of integrin-linked kinase (ILK), a key component of the integrin-induced signaling cascade. ß1 integrin knock down reduced keratin 15 (K15) expression as well as the proliferation of outer root sheath keratinocytes (ORSKs). Embedding of HF epithelium into an ECM rich in ß1 integrin ligands that mimic the HF mesenchyme significantly enhanced proliferation and migration of ORSKs, while K15 and CD200 gene and protein expression were inhibited. Employing ECM-embedded ß1 integrin-activating or -inhibiting antibodies allowed to identify functionally distinct human ePC subpopulations in different compartments of the HF epithelium. The ß1 integrin-inhibitory antibody reduced ß1 integrin expression in situ and selectively enhanced proliferation of bulge ePCs, while the ß1 integrin-stimulating antibody decreased hair matrix keratinocyte apoptosis and enhanced transferrin receptor (CD71) immunoreactivity, a marker of transit amplifying cells, but did not affect bulge ePC proliferation. That the putative ILK inhibitor QLT0267 significantly reduced ORSK migration and proliferation and induced massive ORSK apoptosis suggests a key role for ILK in mediating the ß1 integrin effects. Taken together, these findings demonstrate that ePCs in human HFs require ß1 integrin-mediated signaling for survival, adhesion, and migration, and that different human HF ePC subpopulations differ in their response to ß1 integrin signaling. These insights may be exploited for cell-based regenerative medicine strategies that employ human HF-derived ePCs.

KREX2 is not essential for either procyclic or bloodstream form Trypanosoma brucei.

BACKGROUND: Most mitochondrial mRNAs in Trypanosoma brucei require RNA editing for maturation and translation. The edited RNAs primarily encode proteins of the oxidative phosphorylation system. These parasites undergo extensive changes in energy metabolism between the insect and bloodstream stages which are mirrored by alterations in RNA editing. Two U-specific exonucleases, KREX1 and KREX2, are both present in protein complexes (editosomes) that catalyze RNA editing but the relative roles of each protein are not known. METHODOLOGY/PRINCIPAL FINDINGS: The requirement for KREX2 for RNA editing in vivo was assessed in both procyclic (insect) and bloodstream form parasites by methods that use homologous recombination for gene elimination. These studies resulted in null mutant cells in which both alleles were eliminated. The viability of these cells demonstrates that KREX2 is not essential in either life cycle stage, despite certain defects in RNA editing in vivo. Furthermore, editosomes isolated from KREX2 null cells require KREX1 for in vitro U-specific exonuclease activity. CONCLUSIONS: KREX2 is a U-specific exonuclease that is dispensable for RNA editing in vivo in T. brucei BFs and PFs. This result suggests that the U deletion activity, which is required for RNA editing, is primarily mediated in vivo by KREX1 which is normally found associated with only one type of editosome. The retention of the KREX2 gene implies a non-essential role or a role that is essential in other life cycle stages or conditions.

KREPB6, KREPB7, and KREPB8 are important for editing endonuclease function in Trypanosoma brucei.

Three distinct editosomes are required for the uridine insertion/deletion editing that creates translatable mitochondrial mRNAs in Trypanosoma brucei. They contain KREPB6, KREPB7, or KREPB8 proteins and their respective endonucleases KREN3, KREN2, or KREN1. RNAi knockdowns of KREPB6, KREPB7, and KREPB8 variably affect growth and RNA editing. KREPB6 and KREPB7 knockdowns substantially reduced in vitro insertion site cleavage activity of their respective editosomes, while KREPB8 knockdown did not affect its editosome deletion site cleavage activity despite inhibition of growth and editing. KREPB6, KREPB7, and KREPB8 knockdowns disrupted tagged KREN3, KREN2, or KREN1 editosomes, respectively, to varying degrees, and in the case of KREN1 editosomes, the deletion editing site cleavage activity shifted to a smaller S value. The varying effects correlate with a combination of the relative abundances of the KREPB6-8 proteins and of the different insertion and deletion sites. Tagged KREPB6-8 were physically associated with deletion subcomplexes upon knockdown of the centrally interactive KREPA3 protein, while KREN1-3 endonucleases were associated with insertion subcomplexes. The results indicate that KREPB6-8 occupy similar positions in editosomes and are important for the activity and specificity of their respective endonucleases. This suggests that they contribute to the accurate recognition of the numerous similar but diverse editing site substrates.

The zinc-fingers of KREPA3 are essential for the complete editing of mitochondrial mRNAs in Trypanosoma brucei.

Most mitochondrial mRNAs in trypanosomes undergo uridine insertion/deletion editing that is catalyzed by approximately 20S editosomes. The editosome component KREPA3 is essential for editosome structural integrity and its two zinc finger (ZF) motifs are essential for editing in vivo but not in vitro. KREPA3 function was further explored by examining the consequence of mutation of its N- and C-terminal ZFs (ZF1 and ZF2, respectively). Exclusively expressed myc-tagged KREPA3 with ZF2 mutation resulted in lower KREPA3 abundance and a relative increase in KREPA2 and KREL1 proteins. Detailed analysis of edited RNA products revealed the accumulation of partially edited mRNAs with less insertion editing compared to the partially edited mRNAs found in the cells with wild type KREPA3 expression. Mutation of ZF1 in TAP-tagged KREPA3 also resulted in accumulation of partially edited mRNAs that were shorter and only edited in the 3'-terminal editing region. Mutation of both ZFs essentially eliminated partially edited mRNA. The mutations did not affect gRNA abundance. These data indicate that both ZFs are essential for the progression of editing and perhaps its accuracy, which suggests that KREPA3 plays roles in the editing process via its ZFs interaction with editosome proteins and/or RNA substrates.

An improved, standardised protocol for the isolation, enrichment and targeted neural differentiation of Nestin+ progenitors from adult human dermis.

Human skin-derived Nestin+ cells serve as a convenient source for autologous, adult, pluripotent progenitor cells that offer new therapeutic possibilities in cell-based regenerative medicine. However, the isolation of human Nestin+ cells has tended to be of very low efficiency and to produce highly variable cell yields. Here we report a standardised protocol that facilitates the isolation and enrichment of Nestin+ progenitor cells from enzymatically digested adult human scalp dermis. The use of distinct media like Dulbecco's modified Eagle medium supplemented with foetal bovine serum or, alternatively, serum-free, supplemented neural stem cell medium greatly affected cell morphology, proliferation and differentiation (e.g. towards a neural versus mesenchymal phenotype). Finally, Nestin+ cells were isolated from a heterogeneous dermis-derived progenitor cell population, which proliferates within clones or floating microspheres under defined serum-free culture conditions. Supplementation of the medium with epidermal growth factor and basic fibroblast growth factor as well as coating with fibronectin allowed the highest enrichment level of Nestin+ progenitors and differentiation towards neural fate. These methodological advances should greatly facilitate the isolation, culture and targeted differentiation of primary, adult human scalp skin dermis-derived Nestin+ cells.

Nestin in human skin: exclusive expression in intramesenchymal skin compartments and regulation by leptin.

Cutaneous nestin+ cells are of substantial interest in regenerative medicine. However, the location of nestin+ cells in situ remains controversial. We therefore sought to determine their location in female human scalp skin, using stringently controlled immunohistochemical techniques, Western blot analysis, and in situ hybridization and complementing those techniques with relative and quantitative reverse transcriptase-PCR of enzymatically digested or laser-capture microdissected human hair follicle (HF) compartments. We show here that the immunoreactivity (IR) patterns obtained with anti-nestin antibodies are highly dependent on the tissue-fixation and immunohistochemical methods used. NESTIN mRNA could not be detected within HF-associated epithelial cells in situ or in RNA extracts of the microdissected HF epithelium. Instead, NESTIN transcripts were found only in intramesenchymal skin compartments. Individual cells showing both, specific nestin IR and NESTIN mRNA were detectable in the connective-tissue sheaths of human HFs, sebaceous and sweat glands. Moreover, stimulation of organ-cultured human scalp skin with the adipokine leptin increased the number of nestin+ cells in these intramesenchymal skin locations, whereas no specific nestin IR could be induced by leptin within the HF epithelium, including the bulge. Therefore, nestin expression at the gene and protein levels in human scalp skin is restricted to the periappendage mesenchyme and can be stimulated by leptin.

Differential functions of two editosome exoUases in Trypanosoma brucei.

Mitochondrial RNAs in trypanosomes are edited by the insertion and deletion of uridine (U) nucleotides to form translatable mRNAs. Editing is catalyzed by three distinct editosomes that contain two related U-specific exonucleases (exoUases), KREX1 and KREX2, with the former present exclusively in KREN1 editosomes and the latter present in all editosomes. We show here that repression of KREX1 expression leads to a concomitant reduction of KREN1 in approximately 20S editosomes, whereas KREX2 repression results in reductions of KREPA2 and KREL1 in approximately 20S editosomes. Knockdown of KREX1 results in reduced cell viability, reduction of some edited RNA in vivo, and a significant reduction in deletion but not insertion endonuclease activity in vitro. In contrast, KREX2 knockdown does not affect cell growth or editing in vivo but results in modest reductions of both insertion and deletion endonuclease activities and a significant reduction of U removal in vitro. Simultaneous knockdown of both proteins leads to a more severe inhibition of cell growth and editing in vivo and an additive effect on endonuclease cleavage in vitro. Taken together, these results indicate that both KREX1 and KREX2 are important for retention of other proteins in editosomes, and suggest that the reduction in cell viability upon KREX1 knockdown is likely a consequence of KREN1 loss. Furthermore, although KREX2 appears dispensable for cell growth, the increased inhibition of editing and parasite viability upon knockdown of both KREX1 and KREX2 together suggests that both proteins have roles in editing.

Basic fibroblast growth factor: a potential new therapeutic tool for the treatment of hypertrophic and keloid scars.

Numerous tissue niches in the human body, such as skin, are now recognized to harbour adult stem cells. In this study, we analyze multipotent human dermis-derived progenitor cell populations, isolated and propagated from mechanically and enzymatically processed adult scalp skin. The populations encompass Nestin-positive and -negative cells, which may serve as a convenient and abundant source for various therapeutic applications in regenerative medicine. Here, we show that these cultures exhibit a strong tendency to differentiate into mesodermal derivatives, particularly myofibroblasts, when maintained in media containing serum. Since undesired and excessive myofibroblast formation is a frequent postsurgical complication, we sought culture conditions that would prevent myofibroblast formation. In particular, we analyzed the effect of growth factors, such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and platelet-derived growth factor AB (PDGF AB). Our results demonstrate that bFGF is a potent inhibitor of mesodermal differentiation, whereas PDFG AB favours myofibroblast formation and up-regulates expression of TGFbeta receptors I and II. This interesting discovery may help in the prevention and treatment of tissue fibrosis and in particular in the eradication of hypertrophic and keloid scars.

The KREPA3 zinc finger motifs and OB-fold domain are essential for RNA editing and survival of Trypanosoma brucei.

Three types of editosomes, each with an identical core containing six related KREPA proteins, catalyze the U insertion and deletion RNA editing of mitochondrial mRNAs in trypanosomes. Repression of expression of one of these, KREPA3 (also known as TbMP42), shows that it is essential for growth and in vivo editing in both procyclic (PF) and bloodstream (BF) life cycle stages of Trypanosoma brucei. RNA interference knockdown results in editosome disruption and altered in vitro editing in PFs, while repression by regulatable double knockout results in almost complete loss of editosomes in BFs. Mutational analysis shows that the KREPA3 zinc fingers and OB-fold domain are each essential for growth and in vivo editing. Nevertheless, KREPA3 with mutated zinc fingers incorporates into editosomes that catalyze in vitro editing and thus is not essential for editosome integrity, although stability is affected. In contrast, the OB-fold domain is essential for editosome integrity. Overall, KREPA3, especially its OB-fold, functions in editosome integrity, and its zinc fingers are essential for editing in vivo but not for the central catalytic steps. KREPA3 may function in editosome organization and/or RNA positioning.

Pandemic human viruses cause decline of endangered great apes.

Commercial hunting and habitat loss are major drivers of the rapid decline of great apes [1]. Ecotourism and research have been widely promoted as a means of providing alternative value for apes and their habitats [2]. However, close contact between humans and habituated apes during ape tourism and research has raised concerns that disease transmission risks might outweigh benefits [3-7]. To date only bacterial and parasitic infections of typically low virulence have been shown to move from humans to wild apes [8, 9]. Here, we present the first direct evidence of virus transmission from humans to wild apes. Tissue samples from habituated chimpanzees that died during three respiratory-disease outbreaks at our research site, Côte d'Ivoire, contained two common human paramyxoviruses. Viral strains sampled from chimpanzees were closely related to strains circulating in contemporaneous, worldwide human epidemics. Twenty-four years of mortality data from observed chimpanzees reveal that such respiratory outbreaks could have a long history. In contrast, survey data show that research presence has had a strong positive effect in suppressing poaching around the research site. These observations illustrate the challenge of maximizing the benefit of research and tourism to great apes while minimizing the negative side effects.

KREPA6 is an RNA-binding protein essential for editosome integrity and survival of Trypanosoma brucei.

Most mitochondrial mRNAs in kinetoplastid protozoa require post-transcriptional RNA editing that inserts and deletes uridylates, a process that is catalyzed by multiprotein editosomes. KREPA6 is the smallest of six editosome proteins that have predicted oligonucleotide-binding (OB) folds. Inactivation of KREPA6 expression results in disruption and ultimate loss of approximately 20S editosomes and inhibition of procyclic form cell growth. Gel shift studies show that recombinant KREPA6 binds RNA, but not DNA, with a preference for oligo-(U) whether on the 3' end of gRNA or as a (UU)(12) homopolymer. Thus, KREPA6 is essential for the structural integrity and presence of approximately 20S editosomes and for cell viability. It functions in RNA binding perhaps primarily through the gRNA 3' oligo(U) tail. The significance of these findings to key steps in editing is discussed.

An essential role of KREPB4 in RNA editing and structural integrity of the editosome in Trypanosoma brucei.

RNA editing in the sleeping sickness parasite Trypanosoma brucei remodels mitochondrial transcripts by the addition and deletion of uridylates as specified by guide RNAs. Editing is catalyzed by at least three distinct approximately 20S multiprotein editosomes, all of which contain KREPB4, a protein with RNase III and Pumilio motifs. RNAi repression of KREPB4 expression in procyclic forms (PFs) strongly inhibited growth and in vivo RNA editing, greatly diminished the abundance of 20S editosomes, reduced cellular levels of editosome proteins, and generated approximately 5-10S editosome subcomplexes. Editing TUTase, exoUase, and RNA ligase activities were largely shifted from approximately 20S to approximately 5-10S fractions of cellular lysates. Insertion and deletion endonuclease activities in approximately 20S fractions were strongly reduced upon KREPB4 repression but were not detected in the 5-10S subcomplex fraction. Abundance of MRP1 and RBP16 proteins, which appear to be involved in RNA processing but are not components of the 20S editosome, was unaltered upon KREPB4 repression. These data suggest that KREPB4 is important for the structural integrity of approximately 20S editosomes, editing endonuclease activity, and the viability of PF T. brucei cells.

KREPA4, an RNA binding protein essential for editosome integrity and survival of Trypanosoma brucei.

The 20S editosome, a multiprotein complex, catalyzes the editing of most mitochondrial mRNAs in trypanosomatids by uridylate insertion and deletion. RNAi mediated inactivation of expression of KREPA4 (previously TbMP24), a component of the 20S editosome, in procyclic form Trypanosoma brucei resulted in inhibition of cell growth, loss of RNA editing, and disappearance of 20S editosomes. Levels of MRP1 and REAP-1 proteins, which may have roles in editing but are not editosome components, were unaffected. Tagged KREPA4 protein is incorporated into 20S editosomes in vivo with no preference for either insertion or deletion subcomplexes. Consistent with its S1-like motif, recombinant KREPA4 protein binds synthetic gRNA with a preference for the 3' oligo (U) tail. These data suggest that KREPA4 is an RNA binding protein that may be specific for the gRNA Utail and also is important for 20S editosome stability.

Compositionally and functionally distinct editosomes in Trypanosoma brucei.

Uridylate insertion/deletion RNA editing in Trypanosoma brucei mitochondria is catalyzed by a multiprotein complex, the approximately 20S editosome. Editosomes purified via three related tagged RNase III proteins, KREN1 (KREPB1/TbMP90), KREPB2 (TbMP67), and KREN2 (KREPB3/TbMP61), had very similar but nonidentical protein compositions, and only the tagged member of these three RNase III proteins was identified in each respective complex. Three new editosome proteins were also identified in these complexes. Each tagged complex catalyzed both precleaved insertion and deletion editing in vitro. However, KREN1 complexes cleaved deletion but not insertion editing sites in vitro, and, conversely, KREN2 complexes cleaved insertion but not deletion editing sites. These specific nuclease activities were abolished by mutations in the putative RNase III catalytic domain of the respective proteins. Thus editosomes appear to be heterogeneous in composition with KREN1 complexes catalyzing cleavage of deletion sites and KREN2 complexes cleaving insertion sites while both can catalyze the U addition, U removal, and ligation steps of editing.

A deletion site editing endonuclease in Trypanosoma brucei.

RNA editing in Trypanosoma brucei inserts and deletes uridines in mitochondrial mRNAs by a series of enzymatic steps that are catalyzed by a multiprotein complex, the editosome. KREPB1 and two related editosome proteins KREPB2 and KREPB3 contain motifs that suggest endonuclease and RNA/protein interaction functions. Repression of KREPB1 expression in procyclic forms by RNAi inhibited growth, in vivo editing, and in vitro endoribonucleolytic cleavage of deletion substrates. However, cleavage of insertion substrates and the exoUase, TUTase, and ligase catalytic activities of editing were retained by 20S editosomes. Repression of expression of an ectopic KREPB1 allele in bloodstream forms lacking both endogenous alleles or exclusive expression of KREPB1 with point mutations in the putative RNase III catalytic domain also blocked growth, in vivo editing, and abolished cleavage of deletion substrates, without affecting the other editing steps. These data indicate that KREPB1 is an endoribonuclease that is specific for RNA editing deletion sites.