Rafal P. Krol

Immunological and statistical studies of anti-BP180 antibodies in paraneoplastic pemphigus.

Abbreviations: BMZ, basement membrane zone; BP, bullous pemphigoid; HaCaT, concentrated culture supernatant of HaCaT cells; IB, immunoblotting; IF, immunofluorescence; PNP, paraneoplastic pemphigus; RP, recombinant protein

Homozygous deletion of six genes including corneodesmosin on chromosome 6p21.3 is associated with generalized peeling skin disease

BACKGROUND Peeling skin syndrome (PSS) is a rare autosomal recessive form of ichthyosis showing skin exfoliation. PSS is divided into acral and generalized PSS, and the latter is further classified into non-inflammatory type (PSS type A) and inflammatory type (PSS type B). PSS type B is now called peeling skin disease (PSD). Different loss-of-function mutations in the corneodesmosin (CDSN) gene have been reported to cause PSD. OBJECTIVE The aim of this study was to determine genetic basis of disease in a 14-year-old Japanese patient with PSD. METHODS AND RESULTS Immunohistochemical study showed lack of corneodesmosin (CDSN) in the skin, and standard PCR for genomic DNA failed to amplify CDSN product, suggesting CDSN defect. Multiplex ligation-dependent probe amplification and genomic quantitative real-time PCR analyses detected large homozygous deletion of 59,184bp extending from 40.6kb upstream to 13.2kb downstream of CDSN, which included 6 genes (TCF19, CCHCR1, PSORS1C2, PSORS1C1, CDSN and C6orf15). The continuous gene lost did not result in additional clinical features. Inverted repeats with 85% similarity flanking the deletion breakpoint were considered to mediate the deletion by non-homologous end joining or fork stalling and template switching/microhomology-mediated break-induced replication. Parents were clinically unaffected and were heterozygote carriers of the same deletion, which was absent in 284 ethnically matched control alleles. We also developed simple PCR method, which is useful for detection of this deletion. CONCLUSION Although 5 other genes were also deleted, homozygous deletion of CDSN was considered to be responsible for this PSD.

Human dermal fibroblast migration induced by fibronectin in autocrine and paracrine manners

Although fibronectin (FN) is known as a chemoattractant for human dermal fibroblasts (HDFs), it is unclear whether HDF migration is stimulated by FN produced by HDFs (autocrine manner) or by keratinocytes (paracrine manner). In this study, we investigated HDF migration by Boyden chamber assay using conditioned media from HDFs and HaCaT cells (keratinocyte cell line). Immunoblotting and enzyme‐linked immunosorbent assay revealed that FN existed in both conditioned media. Boyden chamber assay showed both conditioned media stimulated HDF migration, which was inhibited by anti‐FN antibody. Antibodies to both integrin β1and β3 subunits inhibited HDF migration induced by HDF‐conditioned medium almost completely and that by HaCaT cell‐conditioned medium with 50–60%. These results suggested that HDF migration was stimulated by FN in both autocrine and paracrine manners. However, the mechanisms of HDF migration by FN, particularly the role of integrin β1 and β3 subunits, were slightly different between autocrine and paracrine manners.

Mutation study for 9 genes in 23 unrelated patients with autosomal recessive congenital ichthyosis in Japan and Malaysia

Autosomal recessive congenital ichthyosis (ARCI) is heterogeneous group of ichthyosis and consists of Harlequin ichthyosis (HI, MIM #242500), lamellar ichthyosis (LI, MIM #242304) and congenital ichthyosiform erythroderma (CIE, MIM #242100). Harlequin ichthyosis shows the severest phenotype and used to be almost fatal at birth [1]. Even milder types of ARCI severely influence quality of life of patients. To date, mutations in 9 causative genes have been reported in ARCI, including ATPbinding cassette, subfamily a, member 12 (ABCA12) [2,3], transglutaminase 1 (TGM1) [2,3], arachidonate lipoxygenase 3 [2,3], arachidonate 12-lipoxygenase, 12R type (ALOX12B) [2,3], NIPA-like domain containing 4 [2,3], cytochrome P450, family 4, subfamily F, polypeptide 22 [2,3], lipase, family member N [2,3], patatin-like phospholipase domain-containing 1 [3] and ceramide synthase 3 [4]. However, mutations in these genes have been found in about 78% of ARCI patients [2]. In this study, we performed genetic analyses in 23 unrelated patients with ARCI. Sixteen out of 23 patients were from Japan, and 7 patients from Malaysia. Clinically and histopathologically, 3 patients were diagnosed as HI, 2 patients as LI, and 18 patients as CIE. All experiments in this study were approved by Medical Ethical Committee of Kurume University School of Medicine, and conducted according to Declaration of Helsinki Principles. Written informed consent was obtained from each individual. Mutation and polymorphism analyses of 9 causative genes were performed, basically according to the methods described previously [5]. Briefly, genomic DNA was extracted from peripheral blood and PCR-direct sequencing was performed for all exons and their flanking intron boundaries in all 9 genes. Primer sequences for all 9 genes are shown in Supplemental data (Table S1). Primers which we designed using online Primer3 plus tool are indicated by red-colored letters (Table S1) [6]. We detected mutations in 17 (74%) of 23 patients. Mutations on both alleles were detected in 14 of 17 patients, while only one mutation could be detected in 3 patients. Ten (44%) of 17 patients showed mutations in ABCA12, 6 (26%) patients in TGM1, and one (4%) patient in ALOX12B (Fig. 1A). Eleven unreported mutations in ARCI were identified in this study and 4 out of 11 were missense mutations, 2 were in ABCA12 and

A compound synonymous mutation c.474G>A with p.Arg578X mutation in SPINK5 causes splicing disorder and mild phenotype in Netherton syndrome.

A compound synonymous mutation c.474G>A with p.Arg578X mutation in SPINK5 causes splicing disorder and mild phenotype in Netherton syndrome SanaeNumata, Kwesi Teye, Rafal P. Krol, Yuki Okamatsu, KeikoHashikawa,MitsuhiroMatsuda, Paola Fortugno, Giovanni Di Zenzo, Daniele Castiglia, Giovanna Zambruno, Takahiro Hamada and Takashi Hashimoto Department of Dermatology, Kurume University School of Medicine, and Kurume University Institute of Cutaneous Cell Biology, Fukuoka, Japan; Department of Pediatrics and Child Health, Kurume University School of Medicine, Fukuoka, Japan; Laboratory of Molecular and Cell Biology, Istituto Dermopatico dell’Immacolata IDI-IRCCS, Rome, Italy Correspondence: Takashi Hashimoto, Department of Dermatology, Kurume University School of Medicine, and Kurume University Institute of Cutaneous Cell Biology, 67 Asahimachi, Kurume, Fukuoka 830-0011, Japan, Tel.:/Fax: +81-942-31-7853, e-mail: hashimot@med.kurume-u.ac.jp

Ten cases of severe oral lichen planus showing granular C3 deposition in oral mucosal basement membrane zone.

BackgroundOral lichen planus (OLP) may show depositions of immunoglobulins and complement components in oral mucosal basement membrane zone (BMZ) in direct immunofluorescence, although these finding are not frequently seen.ObjectiveWe collected and examined ten cases of severe OLP showing granular C3 deposition in BMZ.Materials and MethodsIn addition to clinical, histopathological and direct immunofluorescence assessments, we performed various immuneserological tests, including indirect immunofluorescence of normal human skin and 1M NaCl-split skin, immunoblotting of normal human epidermal and dermal extracts, recombinant proteins of BP180 NC16a and C-terminal domains, concentrated culture supernatant of HaCaT cells and purified human laminin-332, and enzyme-linked immunosorbent assays for BP230 and BP180.ResultsDirect immunofluorescence showed C3 deposition in BMZ exclusively of granular pattern in 7 cases and of both granular and linear patterns in 3 cases. The 10 cases showed no positive reactivity for either IgG or IgA antibodies in any immunoserological tests. Detailed analyses of clinical, histopathological and immunological findings revealed striking female prevalence, although other parameters were in general characteristic of OLP.ConclusionsGranular C3 deposition in oral BMZ may be one of the characteristic features of severe OLP, although mechanisms for C3 deposition and its pathogenic role in OLP are currently unknown.

Anti‐early endosome antigen 1 autoantibodies were detected in a pemphigus‐like patient but not in the majority of pemphigus diseases

Although the major autoantigens in classic pemphigus are desmogleins, sera from various types of pemphigus react with a number of other molecules, including desmocollins and plakin proteins. However, other novel pemphigus‐related autoantigens remain to be identified. In this study, immunoblotting for serum from an atypical autoimmune bullous disease patient identified an unknown 175 kDa protein. Subsequent studies using two‐dimensional gel electrophoresis, immunoblotting and mass‐spectrometry identified the 175 kDa protein as early endosome antigen 1 (EEA1). This finding was confirmed by subsequent immunological studies, including indirect immunofluorescence of skin and cultured keratinocytes, two‐dimensional gel electrophoresis and immunoblotting with anti‐EEA1 polyclonal antibody, and preabsorption with EEA1 recombinant protein. Finally, we developed a novel BIOCHIP assay using full‐length EEA1 recombinant protein to detect anti‐EEA1 antibodies. However, none of 35 sera from various types of pemphigus showed anti‐EEA1 antibodies in the BIOCHIP assay, with the exception of the serum from the index case. In addition, various findings in the index case did not suggest pathogenic role of anti‐EEA1 autoantibodies. Therefore, although we successfully identified the 175 kDa protein reacted by a serum of an atypical pemphigus‐like patient as EEA1, novel BIOCHIP study for other pemphigus sera indicated that EEA1 is not a common and pathogenic autoantigen in pemphigus.

Towards understanding mechanisms of autoimmune bullous skin diseases

Autoimmune bullous skin diseases are characterized by pathogenic autoantibodies targeting distinct adhesion molecules of the skin. Their clinical features are usually heterogeneous, and pathomechanism is believed to involve complex interactions between genetic, immunological, and environmental factors. Environmental causes are still poorly identified, and genetic components are probably multifactorial, with small penetrance.[1] The detailed mechanism of autoimmunity development is however still one of the big enigmas of immunology. Endemic forms of pemphigus foliaceus present unique opportunity to study the pathology of autoimmunity development. They occur in well-defined restricted geographical regions and prevalence in local population is usually very high. Thus, they constitute an excellent model allowing detailed analysis of interactions between clinical, epidemiological, immunological, and environmental aspects of the disease. Endemic pemphigus foliaceus is known for over a 100 years, since prototype was described in rural areas of Brazil. Recently, a new type of endemic pemphigus foliaceus was found in El Bagre, Colombia. The local population was extensively studied by Abreu Velez et al., who performed prospective fieldwork study for over 10 years. Several publications resulting from this study helped to elucidate many aspects of autoimmune bullous skin diseases, including autoantibody characterization, autoantigen identification, genetic factors, and exposure to environmental factors such as mercury, metalloids, and trace elements.[2] Induction of acantholysis and blister formation in pemphigus-type autoimmune bullous skin diseases is a complicated process triggered by interaction of autoantibodies with desmosomal target molecules. Several mechanisms, including various signaling pathways, were suggested for development of acantholysis by experimental evidences.[3] The article by Abreu Velez et al.,[4] published in this issue of North American Journal of Medical Sciences, describes a pilot study, the aim of which was further clarification of blister formation mechanism. The authors studied lesional skin from patients with endemic pemphigus and others autoimmune bullous diseases to clarify the involvement of novel signaling pathway, namely the presence of phosphorylated form of ribosomal protein S6.[4] Previous attempts to clarify mechanism of acantholysis in immunobullous diseases suggested involvement of mammalian target of rapamycin (mTOR). This kinase protein has emerged as important regulator of cell size and protein synthesis. One of the downstream effectors of mTOR is ribosomal protein S6 kinase, primary substrate of which is ribosomal protein S6. Phosphorylation of ribosomal protein S6 was shown to be an important event in signaling pathways, which are involved in global protein synthesis, control of translation, cell size, cell proliferation, and glucose homeostasis.[5] Autoimmune bullous skin diseases are still life-threatening dermatological conditions. Because of their heterogeneity, precise diagnosis is very important. Constant efforts to elucidate pathomechanisms of diseases and to develop new therapies are needed to improve care quality for our patients.