Makoto Inaoki

Connective tissue growth factor causes persistent proα2(I) collagen gene expression induced by transforming growth factor‐β in a mouse fibrosis model

Skin fibrotic disorders such as systemic sclerosis (SSc) are characterized by an excessive production of extracellular matrix (ECM) and understood to develop under the influence of certain growth factors. Connective tissue growth factor (CTGF) is a cysteine‐rich mitogenic peptide that is implicated in various fibrotic disorders and induced in fibroblasts after activation with transforming growth factor‐β (TGF‐β). To better understand the mechanisms of persistent fibrosis seen in SSc, we previously established an animal model of skin fibrosis induced by exogenous application of growth factors. In this model, TGF‐β transiently induced subcutaneous fibrosis and serial injections of CTGF after TGF‐β caused persistent fibrosis. To further define the mechanisms of skin fibrosis induced by TGF‐β and CTGF in vivo, we investigated in this study, the effects of growth factors on the promoter activity of the proα2 (I) collagen (COL1A2) gene in skin fibrosis. For this purpose, we utilized transgenic reporter mice harboring the −17 kb promoter sequence of the mouse COL1A2 linked to either a firefly luciferase gene or a bacterial β‐galactosidase gene. Serial injections of CTGF after TGF‐β resulted in a sustained elevation of COL1A2 mRNA expression and promoter activity compared with consecutive injection of TGF‐β alone on day 8. We also demonstrated that the number of fibroblasts with activated COL1A2 transcription was increased by serial injections of CTGF after TGF‐β in comparison with the injection of TGF‐β alone. Furthermore, the serial injections recruited mast cells and macrophages. The number of mast cells reached a maximum on day 4 and remained relatively high up to day 8. In contrast to the kinetics of mast cells, the number of macrophages was increased on day 4 and continued to rise during the subsequent consecutive CTGF injections until day 8. These results suggested that CTGF maintains TGF‐β‐induced skin fibrosis by sustaining COL1A2 promoter activation and increasing the number of activated fibroblasts. The infiltrated mast cells and macrophages may also contribute to the maintenance of fibrosis.

Both Th1 and Th2 chemokines are elevated in sera of patients with autoimmune blistering diseases

Although chemokines are critical elements for the selective attraction and activation of various leukocyte subsets in the inflammatory process, there are few findings concerning T helper (Th) 1 or Th2 chemokines in autoimmune blistering disease (ABD). To determine whether serum levels of chemokines that are preferentially chemotactic for Th1 (monokine induced by IFN-γ (MIG/CXCL9)) and Th2 (thymus and activation regulated chemokine (TARC/CCL17) and macrophage derived chemokine (MDC/CCL22)) cells were elevated and whether they correlated with the clinical features in patients with ABD. Serum chemokine levels were examined using ELISA in patients with pemphigus vulgaris (PV, n=19), pemphigus foliaceous (PF, n=14), or bullous pemphigoid (BP, n=27) and normal controls (n=20). Serum MIG levels were significantly higher in patients with PV, PF, or BP than those in the control subjects. Serum levels of TARC and MDC were also significantly elevated in patients with PV, PF, or BP relative to the normal controls. Among the ABD subgroups, the levels of each chemokine tended to be higher in BP patients than in PV patients. Furthermore, serum TARC levels correlated positively with serum IgE levels in patients with ABD. Levels of TARC, MDC, and MIG were significantly decreased after treatment when the skin lesions disappeared in these patients. Furthermore, serum MIG levels correlated positively with serum levels of TARC and MDC in the ABD patients. These results suggest that both a Th1 chemoattractant MIG and Th2 chemoattractants, TARC and MDC, cooperatively play a role in the development of ABD.

Biotin deficiency in an infant fed with amino acid formula.

Biotin deficiency is rarely encountered in an infant on weaning from breast and formula feeding. It is characterized by alopecia and scaly, erythematous dermatitis distributed around the body orifices. We report a 5‐month‐old Japanese infant with typical skin lesions who had been diagnosed as a neonate with dyspepsia and fed only an amino acid formula. Serum and urine levels of biotin were below the normal range, but zinc and biotinidase were within normal range. Urinary excretion of 3‐methylcrotonylglycine, 3‐hydroxyisovaleric acid, and methylcitric acid was significantly elevated. Daily oral supplementation with 1 mg of biotin resulted in dramatic improvement of the periorificial dermatitis and hair growth together with a complete disappearance of the organic aciduria. Our case shows that the characteristic skin manifestations are the most important clue to the diagnosis of biotin deficiency and demonstrated that urinary excretion of biotin and organic aciduria, rather than the serum concentration of biotin, are the sensitive indicators for evaluating the patients status of biotin deficiency.

A failure of mucocutaneous lymphangiogenesis may underlie the clinical features of lipoid proteinosis.

Lipoid proteinosis (LiP) (OMIM 247100) is a rare autosomal recessive disease caused by loss of function mutations in the extracellular matrix protein 1 gene, ECM1, on chromosome 1q21. LiP is characterized clinically by hoarseness in early infancy, followed by waxy papules and plaques on the face and body along with pox‐like and acneiform scars. We studied a 20‐year‐old Japanese woman with LiP. She was born of consanguineous parents. Biopsy specimens obtained from a nodule on the elbow were used for histopathology, immunohistology and electron microscopy. Exons 6 and 7 of ECM1 were amplified by polymerase chain reaction (PCR) from genomic DNA from the proband, her parents, her brother and an unrelated person. PCR products were sequenced to detect the mutation. Histopathological examination revealed an irregular mass of calcium beneath deposits of a hyaline material in the dermis. Immunofluorescence double staining showed that the CD31‐positive microvascular density was increased but that staining for the lymphatic‐specific hyaluronan receptor LYVE‐1 was drastically diminished in lesional compared with nonlesional skin of the patient and with normal skin. Electron microscopy revealed marked concentric reduplication of basal laminae not only around blood vessels but also around solitary dermal cells positive for Weibel–Palade bodies scattered in the hyaline material. Sequencing of the PCR products revealed a homozygous frameshift mutation, 507delT, in exon 6. This led to a premature stop codon 23 bp downstream. The results of immunopathological and ultrastructural characterization suggest that a failure of mucocutaneous lymphangiogenesis may underlie the clinical features of LiP. Identification of mutation 507delT in a Japanese patient with LiP further supports the thesis that this mutation represents a recurrent mutation in ECM1 in patients with LiP. To our knowledge, this case represents the first report of calcinosis cutis occurring in LiP.

Inflammatory Stage of Disseminated Superficial Porokeratosis

Disseminated superficial porokeratosis (DSP) is a keratinization disorder characterized by multiple small lesions with a slightly elevated, sharply defined ridge over the whole body. Unusual DSP cases with acute exacerbation of their lesions accompanied by severe pruritus have been reported and designated as “eruptive pruritic papular porokeratosis” or “inflammatory DSP”. Histologically, the pruritic lesions in the majority of these unusual DSP cases had a dense infiltration of eosinophils and lymphocytes in the vicinity of blood vessels in the upper dermis. In this report, we describe an additional case of DSP with a similar clinical course and histopathological findings. A review of the literature showed that the pruritic condition in these unusual DSP cases can be transient and is not necessarily related to spontaneous regression. We propose the term “Inflammatory stage of DSP” for describing this unusual variant of DSP.

Melanocytic nevi clinically simulating melanoma

Melanoma and other benign or malignant pigmented skin tumors can significantly overlap in their clinical and dermoscopical presentations. Thus, pigmented skin lesions may be misdiagnosed in a large number of cases. An extensive review of the published work provides numerous examples of benign lesions mimicking melanoma. Although a number of melanocytic nevi may have been identified as melanomas, information about their clinical appearance is limited. In this report, we present the clinical appearances of two melanocytic nevi on the vulva and the upper extremity that were difficult to diagnose clinically. Detecting melanoma at an early stage is of the utmost importance. However, more attention should be given to the diagnostic accuracy of benign pigmented skin lesions, which otherwise may be diagnosed and treated as melanoma.

Cicatricial pemphigoid with widespread bullous eruption

Dear Editor, Cicatricial pemphigoid (CP), recently referred to as mucous membrane pemphigoid, is an autoimmune subepidermal blistering disease associated with scarring of the affected mucosa, especially the conjunctiva. Skin lesions appear in 25–35% of patients, and the number and the area of the cutaneous lesions are usually small. To date, only six cases of CP with mucosal scarring and widespread bullous eruption have been reported. We report a case of CP presenting as widespread cutaneous bullous lesions with immunological features similar to those of bullous pemphigoid (BP). A 53-year-old Japanese man presented in June 2003 with a 3-month history of painful erosions of the gingiva, lower eyelids and trunk. A month later, he developed hoarseness and pruritic tense blisters on his trunk and extremities (Fig. 1). Physical examination revealed erosions of the gingiva and the tarsal conjunctivae and symblepharon of the right eye. Scarring of the oral mucosa was not found. A biopsy specimen of the trunk showed subepidermal bullae. Direct immunofluorescence revealed deposition of IgG and C3 along the basement membrane zone (BMZ). Indirect immunofluorescence using normal human skin as a substrate demonstrated antibody titers of 1:160 and 1:40 for immunoglobulin (Ig)G anti-BMZ and IgA anti-BMZ, respectively. Indirect immunofluorescence using 1.0 mol/L sodium chloridesplit skin as a substrate showed that both IgG and IgA antibodies bound to the epidermal side of the split skin. Immunoblotting with extracts of ethylene diamine tetra acetate (EDTA)-separated normal human epidermis revealed that IgG antibodies reacted with BP180 and BP230. IgG antibodies also reacted with the fusion protein of the NC16a domain of BP180, but did not react with the fusion protein of the carboxyl-terminus of BP180 extracellular domain (GST-BP-915). IgA antibodies weakly reacted with the NC16a domain of BP180 but not with the carboxyl-terminus of BP180. Enzymelinked immunosorbent assay (ELISA) using recombinant protein of the NC16a domain of BP180 (Medical and Biological Laboratories, Nagoya, Japan) demonstrated IgG antibodies against NC16a at an index value of 116 (cutoff value = 9). The patient was treated with 40 mg of prednisolone orally, and erosions on his conjunctiva resolved quickly. One month later, lesions on the gingiva, trunk and extremities improved without scarring. Symblepharon and scarring of the tarsal conjunctiva of the right eye became apparent at 5 months (Fig. 2). Prednisolone was gradually tapered. Thereafter, a small number of blisters on the gingiva and the hard palate and erythematous patches on the trunk occasionally appeared, but neither blisters nor erosions occurred in the ocular conjunctiva. Indirect immunofluorescence was negative for IgG and IgA autoantibodies in September 2003 and at subsequent analyses. The BP180 ELISA index was 44 in September 2003 and 70 in November 2004. He remains on prednisolone 5 mg daily.

Two Cases of Fabry's Disease: A Hemizygote with a Point Mutation in the α‐Galactosidase A Gene and His Relative

A 34‐year‐old Japanese male had leg pain, edema of the legs, hypohidrosis, whorl‐like opacities of the bilateral cornea, bilateral subcapsular cataracts, and chest discomfort on exercise. He had no characteristic angiokeratomas but did have telangiectases. The electrocardiogram revealed high voltage. The echocardiogram revealed mild mitral regurgitation. The α‐galactosidase A activity in cultured lymphoblasts was deficient (0.5 nmol/h/mg protein). Electron microscopic examination of the skin revealed lamellar cytoplasmic inclusions in the endothelial cells, pericytes, and fibroblasts. He had a G—A transition at nucleotide 982 in the coding sequence of the α‐galactosidase A gene which resulted in a glycine to arginine amino acid substitution at residue 328. His uncle also had leg pain, edema of the legs, hypohidrosis, and chest pain on exercise. He had no characteristic angiokeratomas but did have telangiectases. Cardiovascular examination revealed hypertrophic cardiomyopathy and stenoses of coronary arteries. Electron microscopic examination of the skin revealed lamellar cytoplasmic inclusions in the endothelial cells, pericytes, and fibroblasts.