Noriaki Nakai

Therapeutic RNA interference of malignant melanoma by electrotransfer of small interfering RNA targeting Mitf

Microphthalmia-associated transcription factor (Mitf) is critically involved in melanin synthesis as well as differentiation of cells of the melanocytic lineage. Some earlier studies suggested that Mitf is also essential in the survival of melanoma cells, but this notion remains controversial. We synthesized short interfering RNA (siRNA) duplexes corresponding to the mitf sequence and transfected them into B16 melanoma. Lipid-mediated transfection in vitro of Mitf-specific siRNA resulted in specific downregulation of Mitf and of the tyrosinase that is a transcriptional target of Mitf. This treatment also remarkably reduced the viability of melanoma cells by inducing apoptosis. To examine the potential feasibility of RNAi therapy against melanoma, B16 cells were subcutaneously injected into syngenic mice and siRNA was transfected into the pre-established tumor by means of electroporation. The Mitf-specific siRNA drastically reduced outgrowth of subcutaneous melanoma, while nonspecific siRNA failed to affect tumor progression. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-based analysis of tumor specimens demonstrated that the tumor cells transfected with Mitf-siRNA effectively underwent apoptosis in vivo. The present results indicate that Mitf plays important roles in melanoma survival. Intratumor electrotransfer of Mitf-specific siRNA may provide a powerful strategy for therapeutic intervention of malignant melanoma.

Vaccination of Japanese patients with advanced melanoma with peptide, tumor lysate or both peptide and tumor lysate-pulsed mature, monocyte-derived dendritic cells

We performed a clinical trial to assess the feasibility and efficacy of immunotherapy with peptides, tumor lysate or both peptides and tumor lysate‐pulsed mature, monocyte‐derived dendritic cells (DC) for advanced malignant melanoma patients that are resistant to conventional therapies. Sixteen patients were enrolled in this trial. All patients received DC vaccines i.d. in the proximal thigh, close to the inguinal lymph nodes, one treatment per week or 2 weeks. Several factors such as clinical findings, computed tomography (CT) images, delayed type hypersensitivity (DTH) response, enzyme‐linked immunosorbent spot (ELISPOT) assay, and immunohistochemistry in primary, metastatic lesions and the DTH site were evaluated. Clinical results through DC vaccination were as follows: in 11 evaluable cases, three stable disease, six progression of disease and two disease‐free from the time of study entry to the completion of one vaccination course. One patient showed reduction of the tumors in the metastases on chest CT during the first and second course of DC vaccination. Ten out of 14 evaluable cases showed positive DTH responses to more than one treatment with melanoma peptides or tumor lysate. Eight out of 13 evaluable cases showed positive immunological responses to more than one treatment with melanoma peptides or tumor lysate in an ELISPOT assay. As for the experiences with toxicity and adverse reactions, autosensitization dermatitis‐like eruptions appeared in five cases during DC vaccination. No severe adverse effects were seen in any of the patients. In our study, the clinical efficacy in prolongation of the patients’ survival was confirmed. At the same time, cancer immunoediting of the tumor was also found. It will be necessary to improve the tumor‐specificity of this therapeutic approach and to analyze the mechanism(s) of tumor escape from immunosurveillance in melanoma.

Immunohistological analysis of peptide-induced delayed-type hypersensitivity in advanced melanoma patients treated with melanoma antigen-pulsed mature monocyte-derived dendritic cell vaccination.

BACKGROUND In melanoma patients vaccinated with monocyte-derived melanoma peptide-pulsed dendritic cells (DC), the delayed-type hypersensitivity (DTH) reactions have been examined as a surrogate marker to determine if acquired immunity is induced by DC vaccination. To date, however, only limited information has been reported as for histopathological analyses of DTH. OBJECTIVE To evaluate tumor-specific immunomonitoring histopathologically after DC vaccination in melanoma patients. METHODS Seven patients previously vaccinated with monocyte-derived melanoma peptide-pulsed DCs were challenged with recall antigenic peptide injection in the skin of the forearm. Using immunohistochemical techniques, the presence of immune cells and the expression of CD4, CD8, interleukin (IL)-2, IL-4, IL-10, Foxp3, CD1a, CD1d, and interferon (IFN)-gamma was investigated at the site of injection where a DTH reaction developed. RESULTS Strong DTH reactions from infiltrated erythema to bullae formation were detected in all 7 cases. Biopsies taken from the DTH site revealed heavy infiltration of mononuclear cells and eosinophils in the dermis and subcutaneous tissue. Cells staining positively for CD4, CD8, IL-2, IL-4, Foxp3, CD1d, and IFN-gamma were increased at the site 48h after antigen injection in all cases. Cells positive for IL-10 were never found in any patient. Regulatory T cells appeared 6h after injection and reached their maximum at day 7. CONCLUSIONS The significant induction of CD8(+)T cells as well as both Th1 and Th2-type cells at the site of DTH suggests that effective antigen presentation leading to anti-tumor immune responses has taken place. Inhibitory mechanisms may also develop as the disappearance of the DTH response could be related to an increase in Foxp3+ cells.

Acute Generalized Exanthematous Pustulosis Caused by Dihydrocodeine Phosphate in a Patient With Psoriasis Vulgaris and a Heterozygous IL36RN Mutation

IMPORTANCE Acute generalized exanthematous pustulosis (AGEP) is a rare and severe type of drug eruption. Dihydrocodeine phosphate is a semisynthetic opioid analgesic. Recently, recessive mutations in IL36RN have been identified in generalized pustular psoriasis (GPP). To date, 4 cases of AGEP and IL36RN mutation without previous history of psoriasis vulgaris (PV) have been reported. OBSERVATIONS A woman in her 60s with PV presented with diffuse erythema, nonfollicular pustules, and fever. She had been treated with dextromethorphan hydrobromide hydrate, amoxicillin hydrate, clarithromycin, dihydrocodeine phosphate, tipepidine hibenzate, and tulobuterol tape for a cough and common cold. Based on histopathologic results and a positive result in a drug provocation test with dihydrocodeine phosphate, she was diagnosed with AGEP. A heterozygous IL36RN mutation c.28C>T (p.Arg10X) was also confirmed by mutation analysis. CONCLUSIONS AND RELEVANCE This is the first report of dihydrocodeine phosphate-induced AGEP. In this case, helper T cells, type 17, might have been activated because of morphine and underlying PV, followed by increased production of interleukin (IL) 36. However, because of the IL36RN mutation, IL-36 signaling was uncontrolled, which might have resulted in the occurrence of AGEP. An IL36RN mutation might underlie several different pustular skin eruptions, including AGEP and GPP, and further accumulation of patient data is required.

Immunoregulatory T cells in the peripheral blood of melanoma patients treated with melanoma antigen-pulsed mature monocyte-derived dendritic cell vaccination

BACKGROUND Regulatory T cells (Treg) may inhibit monocyte-derived melanoma antigen-pulsed dendritic cells (DC) vaccination in treatment of melanoma. However, the Treg level in peripheral blood mononuclear cells (PBMCs) following DC vaccination has not been examined in melanoma patients in Japan. OBJECTIVE To evaluate differences in the helper T cell and Treg population and mRNA levels of Treg in pre- and post-DC vaccination PBMCs obtained from melanoma patients. METHODS Levels of intracellular forkhead box protein 3 (Foxp3) mRNA as well as levels of CD4(+)CD25(+)Foxp3(+) and CD4(+)CD25(+) T cells were examined by real-time PCR and flow cytometry using PBMCs from 9 patients who received DC vaccination. RESULTS Eight of the 9 cases and 7 of the 9 cases showed increased populations of CD4(+)CD25(+) T cells and CD4(+)CD25(+)Foxp3(+) T cells, respectively after repeated DC vaccination. Five of 8 cases showed an increase of Foxp3 mRNA after treatment. Four of these 5 cases also had increased CD4(+)CD25(+) and CD4(+)CD25(+)Foxp3(+) T cells, but the fifth case showed a decrease in CD4(+)CD25(+)Foxp3(+) T cells. Three cases showed a decrease of Foxp3 mRNA. One of these 3 cases showed decreased population of CD4(+)CD25(+)Foxp3(+) T cells, but two cases showed increased population of CD4(+)CD25(+)Foxp3(+) T cells. In 3 of 8 cases Foxp3 expression at the cellular (protein) and mRNA level were inconsistent. CONCLUSION Repeated DC vaccination may commonly induce Treg and helper T cells at the cellular level. However, there are a few discrepancies of Treg expression at cellular and mRNA level.

Ecthyma gangrenosum without pseudomonas septicemia in a kidney transplant recipient

We report a case of ecthyma gangrenosum (EG) without septicemia in a renal transplant recipient who presented with a 1‐month history of painful ulcers, vesicles and bullae on the face and extremities. Histopathological findings revealed subepidermal bullae covered by a necrotic epidermis containing an infiltrate of a moderate number of lymphocytes, neutrophils and necrotic collagen. Many dilated and congested capillaries were also present due to thrombi beneath the bullae, with alteration of collagen fibers through the superficial to middle dermis with some infiltrate. A culture from the ulcers revealed the presence of Pseudomonas aeruginosa and methicillin‐resistant Staphylococcus aureus, whereas the results of repeated blood cultures were negative. The ulcers were completely cured by early appropriate i.v. antibiotic therapy with granulocyte colony‐stimulating factor, without progression to EG with septicemia. An immunocompromised state due to immunosuppressive drugs, in addition to diabetes mellitus, hypogammaglobulinemia and hypoproteinemia, may have caused the EG and herpes zoster may have exacerbated the condition.

Mycosis fungoides palmaris et plantaris successfully treated with radiotherapy: Case report and mini‐review of the published work

Mycosis fungoides palmaris et plantaris (MFPP) is a rare variant of cutaneous T‐cell lymphoma limited to the palms and soles that is not widely recognized because of its uncommon occurrence. We report a 73‐year‐old Japanese man who presented with an erosion on the left dorsal hand, a reddish tumor on the right palm, and hyperkeratotic erythematous plaques on the right sole. Skin biopsy showed histological features of mycosis fungoides (MF) with invasion into the deeper layers of skin. There was no visceral or lymph node invasion. We diagnosed this case as MFPP. External beam radiotherapy (EBRT) was performed to treat the hand lesions. Combination treatment with topical steroids and topical psoralen plus ultraviolet light therapy was performed to treat the right sole lesion, but was ineffective. Therefore, sequential EBRT was performed. Complete remission of all lesions was obtained. This is the first report of MFPP with a locally advanced tumor for which the efficacy of radiotherapy is described in detail. MFPP lesions occur on the dorsal aspect of hand or foot, and here we propose a classification of MFPP as hand and foot MF. The pathogenesis of MFPP is still unclear and further accumulation of data is required.

Mitf silencing cooperates with IL-12 gene transfer to inhibit melanoma in mice

Malignant melanoma is a malignant neoplasm originating from the melanocyte lineage. Microphthalmia-associated transcription factor (Mitf) is crucially involved in the melanin synthesis as well as proliferation and survival of melanocyte and melanoma. We previously showed that short interfering RNA (siRNA) that is specific for the Mitf gene (Mitf-siRNA) significantly inhibited growth of B16 melanoma after electro-transfected in vivo into preestablished tumor in mice. Here we assessed efficacy of electroporation-mediated co-transfection of Mitf-siRNA and IL-12 gene in the treatment of murine melanoma. As results, the tumor growth was more strongly inhibited by intratumor co-transfection with Mitf-siRNA and IL-12-encoding plasmid DNA than by transfection with either of the molecules alone. The co-transfection induced intratumor infiltration of CD4+ and CD8+ T cells, and hampered neoangiogenesis in the tumor. The findings suggest that the RNAi/cytokine gene combination therapy by means of electroporation may become a novel and efficacious therapeutic modality to treat neoplasms including melanoma.

Evaluation of survival in Japanese stage IV melanoma patients treated with melanoma antigen-pulsed mature monocyte-derived dendritic cells

Dear Editor, Metastatic melanoma responds poorly to conventional treatments such as chemotherapy and radiation. Nestle et al. first showed the clinical efficacy of dendritic cell (DC) vaccination in advanced melanoma patients in 1998, and subsequently many similar studies have been performed worldwide. However, evaluation of survival using this approach has not been reported in Japan. We have previously showed an induction of in vitro and in vivo antitumor immunity against melanoma using DC vaccination, but we did not report a survival rate because the study was in progress. Herein, we discuss the clinical efficacy of the approach based on a difference in survival periods between patients with melanoma treated with DC vaccination and those receiving conventional treatment historically as non-randomized control. Twenty stage IV melanoma patients (five men and 15 women; aged 33–77 years; mean age, 57.1 years) who were unresponsive to conventional therapies were enrolled in the study for 5 years. The entry criteria, indication of the patients, procedures and evaluation have been described in our previous report. The patients all had stage IV disease, including 13 with lung metastases, 13 with nodal metastases, four with liver metastases, four with bone metastases, two with brain metastases, and one with other visceral metastases. Previous treatment included both surgery and chemotherapy in 16 patients (80.0%), chemotherapy only in three patients (15.0%) and surgery only in one patient (5.0%). The backgrounds of the patients are summarized in Table 1. The results for patients receiving DC vaccination are summarized in Table 2. Fourteen patients

Allergic contact dermatitis due to epsilon-aminocaproic acid: a case report and mini-review of the published work.

Dear Editor, Epsilon-aminocaproic acid (EAA) is a fibrinolysin that has been used to control various hemorrhagic disorders worldwide. In Japan, EAA is also contained in household products and drugs on the market, including eye drops, soap and toothpaste. Here, we report a case of allergic contact dermatitis (ACD) due to EAA and describe the characteristics of this condition based on a review of published cases. A 78-year-old Japanese woman visited our department for diagnosis of pruritic erythema and mild lichenification on the periorbital areas (Fig. 1a). She reported that she had used sodium hyaluronate 0.1% eye drops (Hyalein; Santen Pharmaceutical, Osaka, Japan), sodium chloride 0.4% eye drops (Softsantear; Santen Pharmaceutical) and fluorometholone 0.1% eye drops (Flumetholone; Santen Pharmaceutical) for treatment of allergic keratoconjunctivitis for 4 years. A month before her first visit to our department, she noticed the eruption. On the basis of her history, ACD due to these three eye drops was suspected. The eye drops were stopped and the eruption disappeared in 5 days with p.o. administration of fexofenadine hydrochloride 120 mg/day and topical application of hydrocortisone butyrate. Patch and scratch-patch tests were performed using pure samples of the three eye drops according to the International Contact Dermatitis Research Group guidelines. All patch tests were negative. However, positive scratch-patch test reactions (1+) to sodium hyaluronate 0.1% eye drops were found on days 2 and 3. The sodium hyaluronate 0.1% eye drop is a viscous solution consisting of sodium hyaluronate, EAA, edetate sodium and benzalkonium chloride. Patch and scratchpatch tests were performed individually for the four components (0.1% sodium hyaluronate, 0.2% EAA, 0.01% edetate sodium, 0.003% benzalkonium chloride). Distilled water was used as a vehicle. Positive patch test reactions (1+) to EAA on days 2 and 3 and a positive scratch-patch test reaction (1+) to EAA on day 3 were obtained (Fig. 1b). The erythemas faded but they still remained on day 7. The patch and scratch-patch test reactions to the other three components were negative. Therefore, we diagnosed this case as ACD due to EAA. Including our case, there have been 16 reports of ACD due to EAA in the English-language published work (Table 1). These patients (two male and 14 females) were aged 43–79 years old. Eye drops were the most common cause (n = 13, 81.3%). Therefore, the skin around the eyes and cheek was the most frequently affected site. In all seven evaluable cases, the eruptions were erythematous types with or without scales, edema or lichenification. In four out of five evaluable cases, eruption onset occurred between 7 months and 14 years after the start of exposure to EAA, suggesting that sensitization occurred after long-term exposure to EAA. In all six evaluable cases, topical corticosteroids were effective for treatment of the eruption. In all seven evaluable cases, positive patch test results were seen at concentrations of 0.1–30.0% in petrolatum or aqua, suggesting that a positive reaction to EAA may occur even at a relatively low concentration and 1% of EAA in aqua may be recommended for patch testing. From these results, we suggest that long-term use of drugs and household products that contain EAA may cause ACD, even if the concentration of EAA is low. Thus, inquiries into medical and life history and skin tests are crucial for identification of ACD due to EAA. The present case illustrates the importance of medical practitioners paying attention to products containing EAA.