Proteomic analysis of filaggrin deficiency identifies molecular signatures characteristic of atopic eczema.

BACKGROUND: Atopic eczema (AE) is characterized by skin barrier and immune dysfunction. Null mutations in filaggrin (FLG), a key epidermal barrier protein, strongly predispose to AE; however, the precise role of FLG deficiency in AE pathogenesis remains incompletely understood. OBJECTIVES: We sought to identify global proteomic changes downstream of FLG deficiency in human epidermal living skin-equivalent (LSE) models and validate findings in skin of patients with AE. METHODS: Differentially expressed proteins from paired control (nontargeting control short hairpin RNA [shNT]) and FLG knockdown (FLG knockdown short hairpin RNA [shFLG]) LSEs were identified by means of proteomic analysis (liquid chromatography-mass spectrometry) and Ingenuity Pathway Analysis. Expression of key targets was validated in independent LSE samples (quantitative RT-PCR and Western blotting) and in normal and AE skin biopsy specimens (immunofluorescence). RESULTS: Proteomic analysis identified 17 (P ≤ .05) differentially expressed proteins after FLG knockdown, including kallikrein-7 (KLK7; 2.2-fold), cyclophilin A (PPIA; 0.9-fold), and cofilin-1 (CFL1, 1.3-fold). Differential protein expression was confirmed in shNT/shFLG LSEs; however, only KLK7 was transcriptionally dysregulated. Molecular pathways overrepresented after FLG knockdown included inflammation, protease activity, cell structure, and stress. Furthermore, KLK7 (1.8-fold) and PPIA (0.65-fold) proteins were differentially expressed in lesional biopsy specimens from patients with AE relative to normal skin. CONCLUSIONS: For the first time, we show that loss of FLG in the absence of inflammation is sufficient to alter the expression level of proteins relevant to the pathogenesis of AE. These include proteins regulating inflammatory, proteolytic, and cytoskeletal functions. We identify PPIA as a novel protein with levels that are decreased in clinically active AE skin and show that the characteristic upregulation of KLK7 expression in patients with AE occurs downstream of FLG loss. Importantly, we highlight disconnect between the epidermal proteome and transcriptome, emphasizing the utility of global proteomic studies.

Human IgG1 monoclonal antibody against human collagen 17 noncollagenous 16A domain induces blisters via complement activation in experimental bullous pemphigoid model.

Bullous pemphigoid (BP) is an autoimmune blistering disease caused by IgG autoantibodies targeting the noncollagenous 16A (NC16A) domain of human collagen 17 (hCOL17), which triggers blister formation via complement activation. Previous in vitro analysis demonstrated that IgG1 autoantibodies showed much stronger pathogenic activity than IgG4 autoantibodies; however, the exact pathogenic role of IgG1 autoantibodies has not been fully demonstrated in vivo. We constructed a recombinant IgG1 mAb against hCOL17 NC16A from BP patients. In COL17-humanized mice, this mAb effectively reproduced a BP phenotype that included subepidermal blisters, deposition of IgG1, C1q and C3, neutrophil infiltration, and mast cell degranulation. Subsequently, alanine substitutions at various C1q binding sites were separately introduced to the Fc region of the IgG1 mAb. Among these mutated mAbs, the one that was mutated at the P331 residue completely failed to activate the complement in vitro and drastically lost pathogenic activity in COL17-humanized mice. These findings indicate that P331 is a key residue required for complement activation and that IgG1-dependent complement activation is essential for blister formation in BP. This study is, to our knowledge, the first direct evidence that IgG1 Abs to hCOL17 NC16A can induce blister formation in vivo, and it raises the possibility that IgG1 mAbs with Fc modification may be used to block pathogenic epitopes in autoimmune diseases.

Infantile Gastroesophageal Reflux: Adherence to Treatment Guidelines in the Hospital Setting.

OBJECTIVES: Recent guidelines defined and differentiated the management of gastroesophageal reflux (GER) and gastroesophageal reflux disease (GERD). The guidelines recommend against using empiric acid suppression therapy for infantile GER. The primary objective of this study was to assess inpatient guideline adherence regarding management of infantile GER through the perspective of pharmacists. Secondary objectives included assessing pharmacist comfort level with differentiation between GER and GERD, observing current trends in practice relating to the primary objective, and determining the availability of institution-specific guidelines that address the management of infantile GER. METHODS: An institutional review board-approved, national, online survey of pharmacists with inpatient pediatric experience was conducted. Pediatric pharmacy membership directories were used to create the listserv of eligible pharmacists. The 2009 NASPGHAN/ESPGHAN (North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition/European Society for Pediatric Gastroenterology, Hepatology, and Nutrition) Pediatric Gastroesophageal Reflux Clinical Practice Guideline was used to develop the survey and to define both GER and GERD. Demographic data was also collected regarding the institutional setting and pharmacists responding. RESULTS: The overall response rate was 14.8% (n = 149). Although 29.7% of pharmacists stated empiric acid suppression trials were not used for infantile GER at their institution, 44.6% responded that these trials are initiated 1 to 2 times per week in their hospitals. In addition, 19.6% responded that these empiric trials were initiated 3 to 5 times per week. A smaller percentage of responders reported even higher frequencies per week at their institutions. CONCLUSIONS: From the results of the survey, infants continue to receive empiric acid suppression trials for GER in the inpatient setting, which is not adherent to the current guideline recommendation.

Vitiligo autoantigen VIT75 is identified as lamin A in vitiligo by serological proteome analysis based on mass spectrometry.

VIT75 is a 75-kDa melanocyte membrane antigen (Ag) that had been observed, but not identified until now. Its immunopathogenic role in vitiligo remains unknown. In this study, serological proteome analysis based on mass spectrometry was employed to identify VIT75. Three disparate 75-, 60-, and 45-kDa proteins on two-dimensional (2D) gel were, respectively, identified as lamin A, tyrosinase-related protein 1, and melanin-concentrating hormone receptor 1. The latter two proteins are well-known Ags. Immunoreactivity analysis showed that the 75-kDa protein displayed on the 2D gel was recognized by human anti-lamin A IgG. Antibody (Ab) reactivity to lamin A was positive in 28.6% of patients' sera. Only 3.1% healthy sera reacted with the lamin A. A total of 91.7% of the positive sera was from the active non-segmental vitiligo (NSV). The positive rate and mean titer of anti-lamin A Ab are higher for NSV with autoimmune disease than for NSV without autoimmune disease. These data demonstrate that VIT75 is lamin A. To our knowledge, this is a previously unreported vitiligo Ag. Anti-lamin A Ab may be a potential marker of NSV with autoimmune disease. The study indicates that the targets of autoantibodies in vitiligo patients can be revealed by serological proteome analysis.

Current advances in gene therapy for the treatment of genodermatoses.

Gene therapy provides the possibility of long term treatment for the severest of congenital disorders. In this review we will examine the recent advances in gene therapy for genodermatoses. Congenital diseases of the skin exhibit a wide range of severity and underlying causes and there are many possible therapeutic avenues. Gene therapy approaches can follow three paths-in vivo, ex vivo and fetal gene therapy, though the later is currently theoretical only it can provide potential results for even the most severe congenital diseases. All approaches utilize the many different vector systems available, including viral and the emerging use of non- viral integrating vectors. In addition, the use of RNAi based techniques to prevent dominant mutant protein expression has been explored as a therapy for specific dominant disorders such as keratin mutation disorders. Progress has been rapid in the past few years with some initial successful clinical trials reported. However, there are still some issues surrounding long term expression, transgene sustainability and safety issues that need to be addressed to further shift from experimental to clinically therapeutic applications. With the continuing development, merger and refinement of existing techniques there is an ever increasing likelihood of gene therapies becoming available for the more severe genodermatoses within the next decade or shortly thereafter.

Collagen XVII participates in keratinocyte adhesion to collagen IV, and in p38MAPK-dependent migration and cell signaling.

Collagen XVII (COL17) participates in keratinocyte adhesion and possibly migration, as COL17 defects disrupt keratinocyte-basal lamina adhesion and underlie the disease non-Herlitz junctional epidermolysis bullosa. Using small interference RNA (siRNA) to knock down COL17 expression in HaCaT cells, we assessed cell characteristics, including adhesion, migration, and signaling. Control and siRNA-transfected keratinocytes showed no difference in adhesion on plastic dishes after incubation for 8 hours in serum-free keratinocyte-growth medium; however, when grown on collagen IV alone or BD matrigel (containing collagen IV and laminin isoforms), COL17-deficient cells showed significantly reduced adhesion compared with controls (P<0.01), and mitogen-activated protein kinase (MAPK)/ERK kinase (MEK)1/2 and MAPK showed reduced phosphorylation. Furthermore, COL17-deficient HaCaT cells plated on plastic exhibited reduced motility that was p38MAPK-dependent (after addition of the p38MAPK inhibitor SB203580). Together, these results suggest that COL17 has significantly wider signaling roles than were previously thought, including the involvement of COL17 in keratinocyte adhesion to collagen IV, in p38MAPK-dependent cell migration, and multiple cell signaling events pertaining to MEK1/2 phosphorylation.

Periplakin-dependent re-organisation of keratin cytoskeleton and loss of collective migration in keratin-8-downregulated epithelial sheets.

Collective migration of epithelial sheets requires maintenance of cell-cell junctions and co-ordination of the movement of the migrating front. We have investigated the role of keratin intermediate filaments and periplakin, a cytoskeletal linker protein, in the migration of simple epithelial cells. Scratch wounding induces bundling of keratins into a cable of tightly packed filaments adjacent to the free wound edge. Keratin re-organisation is preceded by a re-distribution of periplakin away from the free wound edge. Periplakin participates with dynamic changes in the keratin cytoskeleton via its C-terminal linker domain that co-localises with okadaic-acid-treated keratin granules. Stable expression of the periplakin C-terminal domain increases keratin bundling and Ser431 keratin phosphorylation at wound edge resulting in a delay in wound closure. Ablation of periplakin by siRNA inhibits keratin cable formation and impairs wound closure. Knockdown of keratin 8 with siRNA results in (1) a loss of desmoplakin localisation at cell borders, (2) a failure of MCF-7 epithelial sheets to migrate as a collective unit and (3) accelerated wound closure in vimentin-positive HeLa and Panc-1 cell lines. Thus, keratin 8 is required for the maintenance of epithelial integrity during migration and periplakin participates in the re-organisation of keratins in migrating cells.

Altered aggregation properties of mutant gamma-crystallins cause inherited cataract.

Protein inclusions are associated with a diverse group of human diseases ranging from localized neurological disorders through to systemic non-neuropathic diseases. Here, we present evidence that the formation of intranuclear inclusions is a key event in cataract formation involving altered gamma-crystallins that are un likely to adopt their native fold. In three different inherited murine cataracts involving this type of gamma-crystallin mutation, large inclusions containing the altered gamma-crystallins were found in the nuclei of the primary lens fibre cells. Their formation preceded not only the first gross morphological changes in the lens, but also the first signs of cataract. The inclusions contained filamentous material that could be stained with the amyloid-detecting dye, Congo red. In vitro, recombinant mutant gammaB-crystallin readily formed amyloid fibrils under physiological buffer conditions, unlike wild-type protein. These data suggest that this type of cataract is caused by a mechanism involving the nuclear targeting and deposition of amyloid-like inclusions. The mutant gamma-crystallins initially disrupt nuclear function, but then this progresses to a full cataract phenotype.