R. Shibagaki

Merkel cell carcinoma with partial spontaneous regression: An immunohistochemical, ultrastructural, and TUNEL labeling study

We report a case of Merkel cell carcinoma that partially regressed after biopsy. A 76-year-old woman presented with an 1 month history of a rapidly enlarging nodule on her left cheek. After biopsy, the nodule reduced to almost half the size and was excised 1 month later. The excised specimen showed a dense cluster of lymphocytes and fibrosis around the tumor nests. In addition, lymphocytes showed apposition with tumor cells. An immunohistologically dense, even infiltration of CD4+ and CD8+ cells was found around the tumor nests, and more CD8+ cells than CD4+ cells were seen in the tumor nests. By electron microscopy (EM), apoptosis of tumor cells and lymphocytes was observed. Many apoptotic cells were also detected by in situ nick end-labeling (TUNEL) of DNA-breaks, especially in the marginal area of tumor nests surrounded by dense lymphocytic infiltrates. It seems likely that T-cell immunity, which induces apoptosis of tumor cells, may have been involved in tumor regression.

PUVA-induced lichen planus pemphigoides

A 72‐year‐old woman had suffered from parapsoriasis en plaque (large plaque type) controlled by topically applied psoralen ultraviolet A (PUVA) therapy. The parapsoriasis lesions gradually disappeared, but numerous tiny red papules with pruritus appeared over the forearms and lower legs 120 days after starting PUVA therapy. These papules developed to form violaceous plaques. Histological findings demonstrated the characteristics of lichen planus. Two months later, tense bullae developed on the plaques and on uninvolved skin of the limbs. These were subepidermal, with linear deposits of IgG and C3 along the basement membrane zone (BMZ) in immunofluorescence of peribullous skin, and immunodeposits of type IV collagen along the floor of the bullae. We therefore, diagnosed lichen planus pemphigoides (LPP). Using systemic and topical steroid therapy, the lesions rapidly resolved and there has been no recurrence. This case suggests that the combination of basal cell injuries caused by chronic inflammation and PUVA therapy could expose BMZ components to autoreactive lymphocytes and induce LPP.

Glomeruloid hemangioma in POEMS syndrome shows two different immunophenotypic endothelial cells

The case of a Japanese woman with glomeruloid hemangioma, an initial marker for POEMS syndrome, is reported. Her cutaneous lesions were multiple and consisted of glomeruloid hemangiomas, cherry‐type capillary hemangiomas, and a mixture of both. The specimens of glomeruloid hemangiomas were studied by paraffin section immunohistochemistry with a large panel of antibodies and electron microscopy, respectively. The lesions, whose size ranged from minute foci to large nodules, were composed of anastomosing vascular channels resembling renal glomeruli and had irregular lumina, often featuring capillaries and sinusoid‐like spaces. The vascular channels were lined by a single layer of endothelial cells, which showed two types of cells. The capillary‐type endothelium possessed large vesicular nuclei with open chromatin and large amount of cytoplasm. The sinusoidal endothelium possessed small basal nuclei with dense chromatin as well as scant amount of cytoplasm. The former cells had a characteristic CD31+/CD34+/UEA I +/CD68− phenotype. Some of these cells ultrastructurally showed intracytoplasmic lumen formation. The latter cells had a characteristic CD31+/CD34−/UEA I−/CD68+ phenotype. The present study shows that glomeruloid hemangioma has unique morphologic and immunologic features that differ from the traditional hemangiomas as well as littoral cell angioma of the spleen.

Apoptosis and p53 protein expression increase in the process of burn wound healing in guinea-pig skin

Apoptosis and the expression of p53 protein, an apoptosis‐related protein, in the process of healing of a full‐thickness burn wound in guinea‐pig skin were studied with the terminal deoxynucleotide transferase nick‐end labelling method, electron microscopy, and immunohistochemistry, respectively. Apoptosis was detected in the peripheral zone of heat‐injured skin from 12 h until day 10 after the burn, with the peak occurring on day 2. The peripheral zone of heat‐injured skin showed p53 protein from 12 h through day 2, with the peak occurring on day 2. Apoptosis was also detected in tissues regenerated for covering skin defects. The peak of apoptosis in the regenerated epidermis occurred at days 7–10, when the epidermis was most acanthotic. p53 protein reactivity was also detected in the acanthotic regenerated epidermis, with a peak on day 7. The peak of apoptosis in the granulation and scar tissue took place from day 10 to 14, when the granulation tissue started diminishing, but p53 protein reactivity was not detected there. These findings suggest that apoptosis plays an important part in the elimination of dying and/or dead cells resulting from heat stress, the terminal differentiation of the regenerated epidermis, and the decrease in cellularity during remodelling. The apoptotic process during remodelling may be mediated by some p53‐independent pathway.

Recurrent malignant proliferating trichilemmal tumour: local management with ethanol injection.

We report a 59‐year‐old woman who exhibited a recurrent malignant proliferating trichilemmal tumour on the scalp for 15 years. The tumour was recalcitrant to conventional treatments such as chemotherapy, radiation or hyperthermia and we performed intratumoral ethanol injection as an alternative means of reducing tumour mass and obtaining haemostasis. Biopsy specimens obtained after the ethanol injection revealed oedema, haemorrhage in the dermis and degeneration of the tumour cells, showing vacuolization with pyknotic nuclei. For cases of recurrent skin tumours and for patients in poor clinical condition, intratumoral ethanol injection is likely to be a therapeutic alternative to surgery or other conventional treatments.

Verruciform xanthoma in association with a vulval fibroepithelial polyp

Summary We report the clinical, light microscopic, immunohistochemical and ultrastructural features of a verruciform xanthoma that developed in association with a vulval fibroepithelial polyp. To our knowledge, this is the first time that this association has been reported. Immunohistochemical findings confirmed that the xanthoma cells were of a monocyte/macrophage lineage. In addition to typical histological characteristics, prominent vascular ectasias were detected in the deep dermis at the periphery of this lesion. The ectasias may play a part in pathogenesis.

Giant combined dermatofibroma.

Sir, Epidermal grafting with suction blisters is used in treatment of stable vitiligo. Previous reports have shown various complications including postinflammatory hyperpigmentation, peripheral hypopigmentation and hypertrophic scarring. However, the risk of infection has not been reported to date. We report an unusual case of verruca vulgaris that appeared after epidermal grafting. It seems likely that virus particles might have been transferred from the operator, who had verruca vulgaris on his hand, to the patient during the surgical procedure. A 12-year-old girl with localized stable vitiligo was treated by epidermal grafting. Blisters on the recipient site formed within 24 h after three freeze±thaw cycles with liquid nitrogen. Blisters on the donor site were made by suction on the inner portion of the thigh. After approximately 3 h of suction at 200 mmHg, large unilocular bullae appeared. After removal of the blisters at the recipient site, the epidermal sheets were grafted to the denuded recipient site and held in place. Two weeks after grafting, once weekly systemic psoralen-ultraviolet A treatment was started. Almost complete repigmentation was observed 3 months after grafting. Four months after grafting, the patient noticed two verrucous plaques in the grafted site (Fig. 1). She denied warts on any other body sites. There was no history of similar lesions in her family or close friends. The operator, who wore gloves, had a verrucous papule on his hand during the surgery. Skin biopsy of two different lesions, on the patients back and the operators hand, demonstrated histological features of verruca vulgaris. Human papillomavirus (HPV) typing was not performed. Although epidermal grafting appears to be an effective and safe method for the treatment of vitiligo, various complications have been reported, of which some are associated with the application of liquid nitrogen, such as postinflammatory hyperpigmentation, hypertrophic scarring, peripheral hypopigmentation and uneven pigmentation. Koebner phenomenon and recurrence have also been considered complications of epidermal grafting. The possibility of transmission of virus from patient to patient or from patient to doctor has been reported. One study showed that virus may survive on a cotton swab dipped into liquid nitrogen and suggested that virus transmission from patient to patient may occur via this route. Charles and Sire reported the possibility of transmission of papovavirus indirectly by cotton-tipped applicators which had been used earlier to treat verruca in other patients. In our case, the same liquid nitrogen and cotton-tipped applicators were not used for multiple patients. Bergbrant et al. reported that there is a risk of contamination of the operator by HPV DNA, during both carbon dioxide laser and electrocoagulation treatment. Once an individual has been infected, new warts may develop in sites of inoculation over a period of weeks to months. After experimental HPV inoculation, it requires from 2 to 9 months for a verruca to become clinically apparent. This observation implies a relatively long period of subclinical infection. In our patient, the verruca appeared 4 months after grafting. It is unclear whether the verruca vulgaris resulted from direct contact with the operators hand during the surgical procedure, or with another person after grafting. However, there are several possible mechanisms of viral transmission from the operator to the patient: the operator may have palpated the lesion with his bare hand immediately after cryosurgery to evaluate the effectiveness of the freezing, or transmission may have occurred during application of a dressing after surgery. We suggest that the risk of transmission of infection from doctor to patient should be considered as a new complication of epidermal grafting.

Detection of programmed cell death in anagen hair follicles of guinea pig skin by labeling of nick ends of fragmented DNA

Programmed cell death (PCD) is a selective process of physiological cell deletion [1]. PCD is an important event in the morphogenesis of fetal skin and maintenance of adult epidermal homeostasis [2]. Morpholigically, PCD is known as apoptosis [3]. Its gross features, identified by electron microscopy, include nuclear chromatin condensation, compactness of cytoplasmatic organelles, and the appearance of pedunculated protuberances on the cell surface [4, 5]. Terminal differentiation of the keratinocyte may be a specialized form of apoptosis [2]. For practical purposes, it is useful to distinguish apoptosis from terminal differentiation. While terminal differentiation of keratinocytes generates functionally important products such as keratinpacked corneocytes and an epidermal barrier, apoptosis causes the selective deletion of individual cells [6, 7]. Furthermore, emigration to, and elimination in, the dermis is a possible deletion mechanism in apoptosis [6]. It is difficult to distinguish in situ cells undergoing PCD by light microscopy [5]. Recently a more sensitive method for identification of PCD via in situ labeling of nuclear DNA fragmentation was developed by Gavrieli et al. [8]. We applied this method to study PCD in normal guinea pig skin. We found PCD in terminal differentiating cells of both the medulla and outer root sheath in anagen hair follicles. Three-week-old male Hartley guinea pigs (body weight 200–250 g) (Shimizu Jitsukenzairyo, Kyoto) were used in this experiment. They were housed for a preliminary period in an air-conditioned room and were fed on a standard diet before the study. Normal skin specimens were obtained from their thigh after being anesthetized with i.p. injection of sodium pentobarbital (30 mg/kg). These specimens were processed for routine light microscopy. As previously reported [9], the terminal deoxynucleotidyltransferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) method of Gavrieli et al. [8] was used with the following modifications. After deparaffinization and rehydration, microtomed sections were incubated with a 20 μg/ml solution of proteinase K (Sigma Chemical, St Louis, Mo.) for 15 min at room temperature to strip away nuclear proteins. After being washed twice with distilled water for 2 min, slides were covered with 2% hydrogen peroxide for 5 min at room temperature to inactivate endogenous peroxidases. Slides were then water-rinsed and immersed with TDT buffer [30 mM tromethamine (Trizma) base, pH 7.2, 140 mM sodium carcodylate, 1 mM cobalt chloride]. A TDT buffer solution containing 0.3 U/μl TdT (Sigma) and 0.04 nmol/μl biotin-dUTP (Boehringer Mannheim, Germany) was added to cover the section, which was then incubated in a humid atmosphere for 60 min at 37°C. The reaction was terminated by transferring the slides to TB buffer (300 mM sodium chloride, 30 mM sodium citrate) for 15 min at room temperature. Sections were rinsed with distilled water, covered with 2% aqueous solution of bovine serum albumin for 10 min at room temperature, rinsed with distilled water, and immersed with phosphatebuffered saline (PBS) for 5 min. Sections were then covered with extra-avidin peroxidase (Sigma), diluted 1 :500 in water, incubated for 30 min at 37°C, washed with distilled water, immersed with PBS for 5 min, and stained with a solution containing 0.02% 3,3′-diaminobenzine tetrahydrochloride and 0.02% H2O2 in 0.05 M Tris buffer, pH 7.6, for 10 min at room temperature. Several additional controls were processed to verify the specificity of the TUNEL reaction. As positive controls, sections were treated with 5000 U/ml DNAase I (Worthington Biochemical Freehold, N.J.) in DN buffer (30 mM Tris-HCl pH 7.2, 140 mM sodium cacodylate, 4 mM magnesium chloride, 0.1 mM dithiothreitol) for 5 min Saburo Kishimoto · Makoto Nagata · Hideya Takenaka · Ryo Shibagaki · Hirokazu Yasuno

Follicular lupus erythematosus: a new cutaneous manifestation of systemic lupus erythematosus

Summary We report the clinical, histopathological and immunological features of follicular erythema and petechiae in a 30‐year‐old Japanese woman with systemic lupus erythematosus (SLE). Histology showed this eruption to constitute a cutaneous manifestation of SLE. To our knowledge, this is the first reported case of follicular erythema and petechiae in association with SLE. Accordingly, we propose that this rare eruption be termed ‘follicular lupus erythematosus’.

Cell death in early burn wounds in guinea pig skin

yasUne Deparbnent of Dermatology, Kyoto Prefectural University of Mediclnc, Kyoto, Japan. In a preliminary study, we induced second and third degree bum wounds in guinea pig skins with various heat-stresses. Second and third degree bum wounds were made with heat-stress of 43°C for 5 minor 180% for 3 set respectively. The wounds were removed 1, 3, 6,12, and 48 hrs after burn. The specimens were processed for TUNEL staining or transmission electron microscopy. TUNELpositivc cells were uniformly dishibuted in a second degree bum wound, but narrowly distribukd to the periphery of the central portion of a third degree burn wound. Positive cells were first found in the granular cell layers of the epidermis at 3 hrs, and then found in the granular and basal cell layers at 6 hrs. Positive cells developed in the whole epidennal cells at 12 hrs and in the deep dermis and follicles at 48 hrs after burn. Electron microscopic findings demonstrated the characteristics for apoptosis or necrosis in TLJNBL-positive epidermis. Furthermore, endothelial cell death was first found in the capillaries of upper dermis beneath the vesicle and then developed to the deep perifollicular capillaries with time in spite of intact follicular keratinocytes. The present findings demonstrate that TUNEL-positive cells show cell death by apptosis or necrosis and that burn wounds are nxtde first with direct heat-stress and then with local ischemia.