Koji Ono

Nε-(Carboxymethyl)lysine Modification of Elastin Alters Its Biological Properties: Implications for the Accumulation of Abnormal Elastic Fibers in Actinic Elastosis

Accumulation of degenerated elastic fibers in the sun-exposed skin designated as actinic elastosis is a histological hallmark of photodamaged skin. Previous studies have indicated that the elastic fibers of actinic elastosis interact with lysozyme and are modified by N(ɛ)-(carboxymethyl)lysine (CML), one of the major advanced glycation end products (AGEs). We studied here how CML modification of elastin is involved in the pathogenesis of actinic elastosis. The CML-modified insoluble elastin became resistant to neutrophil elastase digestion, which was reversed by treatment with aminoguanidine, a potent inhibitor of AGE formation. In a temperature-dependent aggregation assay, CML-modified elastin rapidly formed self-aggregates, the size of which was larger than unmodified elastin. The elastic fiber sheets prepared from CML-modified α-elastin showed 3D wider diameter, tortuous appearance, and decreased elasticity on tensile tests. The CML-modified α-elastin, but not unmodified α-elastin, was found to bind to lysozyme in vitro, supporting the immunohistochemical findings that the antibodies for lysozyme and CML reacted simultaneously with the elastic fibers of actinic elastosis and UV-irradiated skin. The glycated elastin is likely to cause the accumulation of abnormally aggregated elastic fibers and unusual interaction with lysozyme in actinic elastosis.

Galectin‐7 and actin are components of amyloid deposit of localized cutaneous amyloidosis

The precursor protein of localized cutaneous amyloidosis (LCA) is believed to be cytokeratins on the basis of previous immunohistochemical studies. To identify the candidate amyloid protein biochemically, amyloid proteins were extracted with distilled water from lesional skin of LCA associated with Bowens disease. The proteins were resolved on one‐ or two‐dimensional polyacrylamide gel electrophoresis followed by characterization with immunoblot analysis. The proteins with multiple molecular weights of 50–67 kDa and two proteins with 25 and 35 kDa were identified as keratins, serum amyloid P component and apolipoprotein E, respectively. The unknown 14‐kDa (pI = 7.0) and 42‐kDa (pI = 5.4) proteins reacted with the antibody against galectin‐7 and actin, respectively. The protein with the molecular weight of 14 kDa was identified as galectin‐7 by MALDI‐TOF mass spectrometer. Their electrophoretic mobilities were identical with normal counterparts extracted from cultured normal human keratinocytes. Galectin‐7 and actin were detected by immunoblot assay in the water‐soluble fractions prepared from the lesional skins of two patients with primary LCA. Immunohistochemical studies of tumor‐associated (n = 9) and primary (n = 10) LCA revealed various degrees of positive immunoreactivities with the antibodies for galectin‐7 and F‐actin. Galectin‐7 and actin, which contain considerable amount of β‐sheet structure, may be candidate amyloidogenic proteins of primary and secondary LCA.

Primary cutaneous follicle center lymphoma in a patient with WHIM syndrome.

Editor A 32-year-old Japanese man presented with a 10-year history of nodules on his head. Physical examination revealed multiple dermal and/or subcutaneous nodules of various sizes (Fig. 1a). His past history included pneumonia at 12 and 31 years old. A biopsied specimen showed diffuse cellular infiltrates comprising relatively large centrocyte-like cells with cleaved nuclei and small reactive lymphocytes throughout the entire dermis and subcutaneous tissues (Fig. 1b,c). Tumour cells were positive for CD20, CD79a and bcl-6, but not for CD4, CD10, bcl2 or MUM-1 (Fig. 1d). In addition, a small number of CD21 (+) cells were scattered within the tumour. Predominant expression of j light chain was detected by flow cytometric analysis. Re-activation of Epstein–Barr virus (EBV) was not involved, as demonstrated by negative results for latent membrane protein (LMP)-1 and EBV-encoded small RNAs (EBERs). Positron emission tomography-computed tomography revealed no involvement of visceral organs or lymph nodes. Skin lesions were diagnosed as primary cutaneous follicle center lymphoma (PCFCL; diffuse type). Laboratory tests showed: white blood cell count, 900/lL; neutrophils, 431/lL; lymphocytes, 335/lL; IgG, 394 mg/dL; IgA, 25 mg/dL; IgM, 17 mg/dL and IgE, 5 IU/mL. CD3 T cells and CD19 B cells were 77.2%/lymphocytes and 4.1%/lymphocytes respectively. Ratio of CD4 to CD8 cells was 0.9. Proliferative lymphocyte responses to phytohaemagglutinin and concanavalin A were within normal limits. Results of bone marrow aspiration showed myelokathexis characterized by increased mature neutrophils with hypersegmented nuclei connected by long, thin chromatin filaments and vacuolated cytoplasm. Hypogammaglobulinaemia with myelokathexis, neutropenia, B-cell lymphopenia and a history of pneumonia prompted us to consider the possibility of WHIM syndrome. As a result, genetic analysis of the proband’s genomic DNA revealed a mutation in the CXCR4 gene (c.1000C>T hetero p.Arg334X). This is the most frequently reported mutation (R334X) in WHIM syndrome. Although the patient did not have warts and no family members had symptoms suggestive of the syndrome, a diagnosis of WHIM syndrome was made based on the clinical and genetic

Increase of elastic fibers in lichen sclerosus et atrophicus.

Lichen sclerosus et atrophicus (LSA) is histopathologically characterized by upper dermal hyalinization with vacuolar alteration, whereas no particular microscopic change in the mid to lower dermis has been described. The purpose of this study was to investigate any histopathologic changes involving elastic fibers in the mid to lower dermis in patients with LSA.

In Vitro Amyloidogenic Peptides of Galectin-7 POSSIBLE MECHANISM OF AMYLOIDOGENESIS OF PRIMARY LOCALIZED CUTANEOUS AMYLOIDOSIS

Background: Characterization of amyloid precursor protein and the mechanism of amyloidogenesis in primary localized cutaneous amyloidosis have not been elucidated previously. Results: Galectin-7 fragments containing β-strand peptides are highly amyloidogenic in vitro. Conclusion: Galectin-7 is an amyloid precursor protein in primary localized cutaneous amyloidosis. Significance: We have proposed the possible mechanism of amyloid deposition in primary localized cutaneous amyloidosis. Pathogenesis of primary localized cutaneous amyloidosis (PLCA) is unclear, but pathogenic relationship to keratinocyte apoptosis has been implicated. We have previously identified galectin-7, actin, and cytokeratins as the major constituents of PLCA. Determination of the amyloidogenetic potential of these proteins by thioflavin T (ThT) method demonstrated that galectin-7 molecule incubated at pH 2.0 was capable of binding to the dye, but failed to form amyloid fibrils. When a series of galectin-7 fragments containing β-strand peptides were prepared to compare their amyloidogenesis, Ser31-Gln67 and Arg120-Phe136 were aggregated to form amyloid fibrils at pH 2.0. The rates of aggregation of Ser31-Gln67 and Arg120-Phe136 were dose-dependent with maximal ThT levels after 3 and 48 h, respectively. Their synthetic analogs, Phe33-Lys65 and Leu121-Arg134, which are both putative tryptic peptides, showed comparable amyloidogenesis. The addition of sonicated fibrous form of Ser31-Gln67 or Phe33-Lys65 to monomeric Ser31-Gln67 or Phe33-Lys65 solution, respectively, resulted in an increased rate of aggregation and extension of amyloid fibrils. Amyloidogenic potentials of Ser31-Gln67 and Phe33-Lys65 were inhibited by actin and cytokeratin fragments, whereas those of Arg120-Phe136 and Leu121-Arg134 were enhanced in the presence of Gly84-Arg113, a putative tryptic peptide of galectin-7. Degraded fragments of the galectin-7 molecule produced by limited trypsin digestion, formed amyloid fibrils after incubation at pH 2.0. These results suggest that the tryptic peptides of galectin-7 released at neutral pH, may lead to amyloid fibril formation of PLCA in the intracellular acidified conditions during keratinocyte apoptosis via regulation by the galectin-7 peptide as well as actin and cytokeratins.

Two cases of erythema exsudativum multiforme associated with Chlamydia pneumoniae infection

We report two cases of erythema exsudativum multiforme (EEM) that we concluded were caused by infections with Chlamydia pneumoniae. High titers of IgG antibody for Chlamydia pneumoniae were shown in the sera of both cases. One case showed the classical symptoms of pneumonia together with radiological changes in the chest; the other case did not show these symptoms. To the best of our knowledge, only three cases of erythema multiforme associated with Chlamydia pneumoniae infection have been reported.

Verruciform Xanthoma results from epidermal apoptosis with galectin‐7 overexpression

Editor Verruciform xanthoma (VX) is a rare histopathological entity first described by Shafer in 1971. The pathological hallmark of VX is verrucous epithelial hyperplasia of squamous epithelium with aggregates of lipid-laden macrophages in the submucosa or papillary dermis. The aetiology of VX remains unknown. We report here two cases of VX with aetiological insight of VX which had not been reported previously. A 79-year-old Japanese man had noticed multiple skin papules gradually increasing in size and number on the scrotum for 1 year. Examination revealed that firm yellowish or reddish papules ranged from 2 to 5 mm in diameter were scattered around a pedunculated glossy red nodule (case 1) (Fig. 1). A 66-year-old Japanese man had noticed two asymptomatic skin papules on the scrotum for 1 year. Examination revealed two adjacent firm keratotic papules with 4 mm in diameter the colour of which was slightly yellowish. He had been on haemodialysis for chronic renal failure (case 2). Biopsy taken from the skin papules of two cases of VX showed epidermal proliferation with epidermolytic hyperkeratosis or verrucous epithelial hyperplasia of squamous epithelium with aggregates of lipid-laden macrophages in papillary dermis (Fig. 2a, b, and c). Yellowish papules surrounding a pedunculated glossy red nodule were diagnosed as epidermolytic acanthoma (EA) (case 1) (Fig. 2a). Thickened cornified layer seemed to imply sudden epidermal apoptosis (case 2) (Fig. 2b). Granular degeneration of EA as well as foam cells in the papillary dermis of VX showed positive reaction against anti-adipophilin antibody (monoclonal, PROGEN Biotecnik, Heidelberg) (Fig. 2d and 2e). Foam cells (macrophages) as well as verrucous epithelium were also positively stained with antibodies against cytokeratin AE1 ⁄ AE3 (Dako), galectin-7 (monoclonal, R&D Systems, Inc.) and HMGCS1 (polyclonal, Sigma, St Louis, MO, USA) (Fig. 2f, g and h). Regarding VX, Zegarelli et al. suggested that injury caused by a local irritation or trauma may induce epidermal degeneration with subsequent ‘lipid incontinence’ that is scavenged by dermal histiocytes. There is a reported case of VX surrounded by three isolated EA. In this report, histopathological examination showed granular degeneration in the pedunculated lesion of the VX nodule free from the foam cells. Granular degeneration was also seen in EA red papules surrounding the VX. Requena et al. presented a case of VX having numerous acantholytic cells in the upper layers of the epithelium. Taking together these cases and ours, it is reasonable to consider that acantholytic cells and granular degeneration may be related to the coalescence of lipid-laden foam cells in the papillary dermis. There have been several reported cases of disseminated EA induced by sun exposure or PUVA therapy. UV and epidermolysis led us to investigate whether galectin-7 participates in the pathogenesis of EA and VX. Galectin-7 is thought to function in stratified epithelial tissue response to environmental injuries, such as wound healing or UV light. Galectin-7 mRNA and protein have been shown to be rapidly induced in skin keratinocytes by UV light exposure and to take part in UV-induced apoptosis. Expression of galectin-7 was also reported to render culture cells more sensitive to apoptosis induced by a variety of stimuli in addition to UVB. Microarray analysis revealed that galectin-7 transfected cell expressed 9 times more HMG-CoA synthase 1 (HMGCS1) mRNA than untreated cells. Recently, we discovered that galectin-7 interacts with HMGCS1 while inducing its expression (unpublished data). HMGCS1 is a key rate-limiting enzyme, preceding HMG-CoA reductase, in the pathway for endogenous cholesterol synthesis. Therefore, some stimuli accompanied with galectin-7 overexpression are considered to induce cholesterol synthesis, epidermal apoptosis, and subsequent lipid incontinence via HMGCS1. This explains well the possible aetiological process of VX.

Deposition of elafin in the involved vascular wall of neutrophil-mediated cutaneous vasculitis.

Neutrophil elastase plays an important role in skin inflammation induced by neutrophil infiltration. Elafin is an inducible elastase inhibitor expressed by keratinocytes, and is known to be involved in pathogenesis of neutrophilic skin disorders such as psoriasis.

Accumulation of C-reactive protein in basal keratinocytes of normal skins

BACKGROUNDnC-reactive protein (CRP) is a prototypic acute phase protein which increases dramatically in the blood during the first 48h of tissue inflammation and has been recognized as a risk factor for atherosclerosis. CRP interacts with a variety of proteins.nnnOBJECTIVEnTo know the role of accumulated CRP in the skin.nnnMETHODSnInteraction of CRP with basal keratinocytes was studied using immunohistochemical method and keratinocyte culture system.nnnRESULTSnWe found an immunohistochemical deposition of CRP on the basal keratinocyte membrane in some normal human skins (23 out of 46 skins). When added to cultured keratinocytes, heat-denatured but not native CRP was found to adhere to keratinocyte cell membrane after 1h, then internalized into cytoplasm after 24h. The heat-denatured CRP recognized at least four keratinocyte polypeptides with the molecular weights of 56, 42, 32 and 24kDa. Ligand binding assays suggested that multiple populations of receptor-ligand interactions were involved in the binding between CRP and keratinocyte. Cultured dermal microvascular endothelial cells were found to express CRP of which expression was greatly induced by interleukin-1β (IL-1β) treatment, suggesting that the deposited CRP in the basal keratinocytes can be derived from local dermal microvasculatures as well as from systemic circulation (serum). Treatment of cultured keratinocytes with heat-denatured CRP induced interleukin-8 (IL-8) expression, a potent leukocyte chemotactic cytokine. CRP in the medium (liquid phase) and CRP-coated dishes (solid phase) both inhibited the adhesion of keratinocytes in culture.nnnCONCLUSIONnAccumulation of CRP may regulate the skin inflammation and keratinocyte proliferation by modulating keratinocyte cytokine expression and adhesion to substrate.

Erythema induratum of Bazin with anti‐phospholipid antibodies

An 86-year-old woman presented with a 1-month history of nodules and ulcers on the feet. Physical examination revealed multiple, small, dark-red, subcutaneous nodules and/ or ulcers on the peripheral regions of feet (Figures 1a, b). In addition, subcutaneous skin-colored or slightly reddish nodules approximately 1 cm in diameter were identified on the anterior sites of crura (Figure 1c). Ankle brachial pressure index (ABI) was 1.22 on the right and 1.15 on the left. Skin perfusion pressure of the dorsal and plantar aspects of the feet were 58–93 mmHg, suggesting that involvement of largeand medium-sized arteries of the lower extremities was unlikely. Past history included hypertension, schizophrenia, and Alzheimer-type dementia. Histopathological examinations of subcutaneous nodules showed massive infiltration of inflammatory cells into the lower dermis and subcutaneous tissues (Figure 2a). Cellular infiltrates mainly comprised histiocytes and lymphocytes with occasional formation of epithelioid-cell granulomas and Langhans-type multinuclear giant cells surrounding extensive eosinophilic necrosis in the center (Figures 2b, c). Some large veins in the lower dermis were occluded, and Clinical Letter