Yasuyuki Amo

Clinical significance of enzyme-linked immunosorbent assay for the detection of circulating anti-BP180 autoantibodies in patients with bullous pemphigoid

The NC16A domain of the 180-kDa bullous pemphigoid antigen (BP180) is the most immunogenic and, probably, pathogenic region in bullous pemphigoid (BP). In the present study, in order to determine whether serum level of circulating anti-BP180 autoantibodies is a valuable serum marker in BP, the immunoreactivity of sera against the NC16A domain of BP180 was measured using enzyme-linked immunosorbent assay (ELISA) in ten patients with BP. Serum levels of anti-BP180 autoantibodies correlated with the clinical course in BP patients, who received various therapeutic agents. The result suggests that this NC16A-ELISA is a useful method for evaluating the clinical course and efficacy of the therapy in patients with BP.

Expression of vascular endothelial growth factor in a human hemangiosarcoma cell line (ISO-HAS).

Abstract Vascular endothelial growth factor (VEGF), in addition to being a specific mitogen of endothelial cells in vitro, is also known to induce angiogenesis in vivo. These functions suggest that VEGF may play an important role in the growth of hemangiosarcomas. Previous studies have demonstrated the expression of VEGF and its receptors, flt-1 or KDR/flk-1, in hemangiosarcomas by immunohistochemical staining and in situ hybridization. In the present study, however, we demonstrated that tumor cells of the hemangiosarcoma cell line ISO-HAS express mRNA of VEGF and its two receptors, flt-1 and KDR, and secrete VEGF protein. VEGF mRNA expression and protein secretion were found to be enhanced by phorbol 12-myristate 13-acetate. In addition, we demonstrated that ISO-HAS cells respond to recombinant human VEGF 165 with a dose-dependent up-regulation of cell proliferation and growth. These results suggest that the VEGF-VEGF receptor system plays a role in proliferation and growth of hemangiosarcoma cells.

Observations on angiopoietin 2 in patients with angiosarcoma

SIR, Vascular remodelling in host tissues surrounding growing tumours is implicated in the successful development of tumour neovasculature. Cooperation between vascular endothelial growth factor (VEGF) and angiopoietins (Angs) is considered to be critical in this context. VEGF is a prime regulator of endothelial cell proliferation, angiogenesis, vasculogenesis and vascular permeability. We have recently established a human angiosarcoma cell line, ISO-HAS. We have previously demonstrated that tumour cells of the angiosarcoma cell line ISO-HAS secrete VEGF-A protein. Tie2 is an endothelium-specific receptor tyrosine kinase known to play a role in tumour angiogenesis. Modulation of Tie2 receptor activity by its Ang ligands is crucial for angiogenesis, blood vessel maturation and integrity of the vascular endothelium. Ang1 and Ang2 are respectively proangiogenic and antiangiogenic owing to their respective agonist and antagonist signalling action through the Tie2 receptor. It has recently been reported that in the presence of endogenous VEGF-A, Ang2 promotes a rapid increase in capillary diameter, remodelling of the basal lamina and proliferation and migration of endothelial cells, and stimulates sprouting of new blood vessels in vivo. By contrast, Ang2 promotes endothelial cell death and vessel regression if the activity of endogenous VEGF is inhibited. These observations support a model for regulation of vascularity where VEGF can convert the consequence of Ang2 stimulation from antiangiogenic to proangiogenic. In the present study, we have demonstrated that tumour cells of the angiosarcoma cell line ISO-HAS express mRNA of Ang2 and its receptor, Tie2, and secrete the Ang2 protein, and that serum levels of Ang2 increase with advancing stages of the tumour in patients with angiosarcoma. A human angiosarcoma cell line (ISO-HAS) and a murine phenotypic angiosarcoma cell line (ISO-S1) maintained in our laboratory and normal human endothelial cells (HMvEC) (Morinaga, Yokohama, Japan) were used in this study. ISO-HAS cells were derived from the periauricular metastatic tissue of an 84-year-old Japanese man. ISO-S1 cells were cultured in a complete medium, and ISO-HAS cells were cultured in a complete medium mixed with 50% (v ⁄ v) of the conditioned medium of ISO-S1. Complete medium consisted of high-glucose Dulbecco’s modified Eagle’s medium (Gibco-BRL, Gaithersburg, MD, U.S.A.) supplemented with 15% (v ⁄ v) heat-inactivated fetal calf serum (JRH Biosciences, Lanexa, KS, U.S.A.). HMvEC were cultured in the medium provided by the manufacturer. Polymerase chain reaction (PCR) was performed with primers specific to Ang1, Ang2 and Tie2 (which have been reported by Zhang et al.), and to b-actin as a control. For PCR, 1 lL of cDNA was added to a 25-lL reaction mixture containing 10 mmol L Tris–HCl pH 9Æ0, 50 mmol L KCl, 1Æ5 mmol L MgCl2, 0Æ1% (w ⁄ v) gelatin, 0Æ2 mmol L deoxyribonucleoside triphosphates, 25 pmol L 5¢ and 3¢ oligonucleotide primers, and 2Æ5 U of Taq polymerase (Takara Shuzo Co., Kyoto, Japan). A DNA thermocycler 480 (PerkinElmer Cetus, Norwalk, CT, U.S.A.) was used for one cycle of 95 C for 9 min, followed by 35 cycles of denaturation at 95 C for 30 s, annealing at 58 C for 30 s, and a final cycle of 72 C for 7 min. The PCR product was subjected to electrophoresis in 1Æ5% agarose gel and was visualized by staining with ethidium bromide. We investigated 11 elderly patients (seven men and four women; mean age 75Æ3 years, range 67–84) with definite angiosarcoma of the face and scalp. The serum level of VEGF was measured using a human Ang2 enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, U.S.A.) according to the manufacturer’s protocol. Normal control sera were obtained from 18 healthy volunteers (10 men and eight women; mean age 70Æ3 years, range 62–81). We also analysed Ang2 protein levels by ELISA in the conditioned media of ISO-HAS cells and HMvEC. ISO-HAS cells and HMvEC were treated with trypsin, plated out at a density of 5Æ0 · 10 cells per well in 24-multiwell plates, and allowed to attach overnight. After 24 h, cell-free culture supernatants were removed and assayed for VEGF protein levels by ELISA. We have previously demonstrated that tumour cells of the angiosarcoma cell line ISO-HAS secrete VEGF-A protein. In addition, we have observed serum VEGF protein levels to increase with advancing tumour stage in the patient from whom the ISO-HAS cells were derived. In the present

A Case of Ductal Apocrine Carcinoma in the Left Axilla with Tubular Apocrine Adenoma in the Right Axilla

Introduction Apocrine adenoma is considered to be a benign lesion, and its relationship to invasive apocrine carcinoma remains unclear. A few follow-up studies have suggested that apocrine adenoma may be a predictor for the subsequent development of carcinoma (1–3). This report provides a description of a rare case of ductal apocrine carcinoma in the left axilla with tubular apocrine adenoma in the right axilla.

A case of solitary indeterminate cell histiocytosis

Introduction Indeterminate cell histiocytosis (ICH), a disorder of cells of the histiocytic family, has been infrequently reported in the literature (1). Although ICH usually presents as numerous papules or nodules, there have been a few reports of solitary lesions (2, 3). Levisohn et al. (2) reported an ICH patient presenting with a single regressing nodule composed of indeterminate cells. We describe herein an additional case of solitary ICH that showed spontaneous regression.

Serum levels of vascular endothelial growth factor in a haemangiosarcoma patient with a newly typed p53 gene point mutation

Sir, Molluscum contagiosum (MC) is a poxvirus skin infection that often complicates the course of patients with acquired or iatrogenic immunosuppression. Both the clinical and histological features of disease in these cases may be atypical. We report the unique clinical and histological features of a case of fulminant MC infection with concomitant leukaemia cutis in a patient with relapse of chronic myeloid leukaemia (CML) after allogeneic bone marrow transplantation (BMT). A 49-year-old Chinese woman underwent BMT (conditioning: busulphan, cyclophosphamide, total body irradiation) from an HLA identical sister for Ph positive CML in accelerated phase. She engrafted uneventfully with no chronic graft-vs.-host disease (GVHD). Serial bone marrow reassessments, up to 18 months post-BMT, were negative for residual disease by polymerase chain reaction and cytogenetics. At 36 months, she was found to have haematological relapse of CML, with cytogenetic subclonal evolution. She was treated with hydroxyurea and donor lymphocyte infusions (DLI) (6 ́1 10 kg cells infused in three doses) to enhance the graft-vs.-leukaemia (GVL) effect. There was good control of the peripheral cell counts and no evidence of GVHD. A repeat marrow biopsy at 40 months showed suppression of the abnormal clone to 3% of analysed metaphases. Unfortunately, at 46 months the disease progressed again with increased neutrophil counts (52 10 L), and leukaemia cutis documented by skin biopsy (Figs 1a, 1b). This was treated with combination chemotherapy (cytosine arabinoside 150 mg 5, thioguanine 160 mg 5) resulting in normalization of cell count and resolution of all skin lesions. A second course of DLI (4 ́2 10 kg cells) was administered at 49 months post-BMT. Three weeks after DLI, however, the patient presented with a dense eruption of erythematous papular lesions over the entire face, upper limbs and upper trunk. Photography of the lesions was refused. A biopsy of one lesion showed lobules of abnormal epidermal cells with cytoplasm packed with eosinophilic viral inclusion bodies (Fig. 2a). Electron microscopy showed numerous intracytoplasmic poxvirus particles (240 95 nm in size) within the abnormal epidermal cells, consistent with MC (Fig. 2b). In addition, an infiltrate of promyelocytes and immature myeloid blast cells was seen, consistent with recurrent leukaemia cutis. She died 1 week later of an intracranial haemorrhage, probably related to intracerebral disease. The use of DLI is the treatment of choice for relapse of CML after BMT. The main side-effects are profound marrow and immune suppression, with or without acute and chronic GVHD. Reactivation of dormant DNA viruses like cytomegalovirus, varicella zoster and hepatitis B viruses are potential complications of immunosuppression caused by DLI. This is the first report of severe MC complicating DLI. In immunosuppressed hosts, due to the fulminant replication of the poxvirus, the clinical and pathological features of MC can be highly variable, and aggressive treatment is often needed. Viral particles have even been found in the histologically normal skin epidermis adjacent to MC lesions in patients infected with HIV. There have been two previous reports of skin changes in MC mimicking haematological malignancy involving the skin. In our case, the clinical setting and pathological features are highly supportive of a genuine double pathology. It is recognized that post-BMT patients have an increased incidence of leukaemic involvement of extramedullary sites, including the skin. The incidence may be even higher in patients salvaged with DLI, due to a weaker GVL effect outside the marrow. The precise colocalization of MC replication and leukaemic infiltration in the skin lesions may have been due to a high concentration of chemotactic

Dermatitis herpetiformis in a Japanese patient with anaplastic large cell lymphoma.

We report a 73‐year‐old Japanese man with dermatitis herpetiformis which developed after diagnosis of anaplastic large cell lymphoma. The patient suffered fever, sweating, shivering, and multiple enlarged cervical lymph nodes. The diagnosis of anaplastic large cell lymphoma was confirmed by the histologic features of a biopsied cervical lymph node. The patient underwent combination chemotherapy. However, one month after the initial therapy, pruritic erythematous skin lesions with peripheral vesicles appeared on his buttocks. A skin biopsy showed subepidermal blister formation associated with polymorphonuclear and mononuclear cell infiltrates. Direct immunofluorescence examination of the area adjacent to the lesion showed granular deposits of IgA at the dermoepidermal junction. While it is well‐known that dermatitis herpetiformis can develop into lymphoma, there have been only a few reports of its appearance after a diagnosis of lymphoma. This case suggests that dermatitis herpetiformis may be induced by anaplastic large cell lymphoma.

CD40 ligation inhibits trichilemmoma cell proliferation and induces IL-6 production

CD40 is a member of the tumor necrosis factor receptor superfamily expressed by B cells, monocytes, dendritic cells, epithelial cells, and hematopoietic progenitor cells. Recently, CD40 has been reported to also be expressed on human epidermal cells. We have elucidated the function of CD40 on epidermal tumor cells and have found that trichilemmoma (KTL-1) cells constitutively express CD40 and respond to CD40 ligation by anti-CD40 mAb (EA-5) with a significant decrease in proliferation. We were also able to demonstrate that KTL-1 cells respond to CD40 ligation by EA-5 with the up-regulation of interleukin-6 (IL-6) mRNA expression. Together, the results suggest that CD40 on KTL-1 cells may function to regulate their proliferation associated with the induction of IL-6 production.