Treatment of giant congenital melanocytic nevi with cultured epithelial autografts: Clinical and histopathological analysis.

INTRODUCTION: Curettage and dermabrasion are effective in the treatment of giant congenital melanocytic nevi (GCMN); however, local infection and hypertrophic scar formation are major issues. Thus, we applied cultured epithelial autografts (CEA) on skin defects after curettage or abrasion of GCMN and assessed the postoperative outcomes. METHODS: Seven nevi lesions of five patients (aged 3 months to 24 years) were treated with CEA after curettage or abrasion with a dermatome or a surgical bar, respectively. We assessed the postoperative outcomes, including CEA take ratio, erosion and/or ulcer formation in the acute phase, hospitalization days, Vancouver scar scale, and color improvement one year after the operation. In addition, a histological evaluation of a skin biopsy was performed over one year after the operation. RESULTS: The CEAs took well on the wound, and the wound surface was mostly epithelized by postoperative day 7 in all cases. While hypertrophic scar formation and slight pigmentation were observed in some lesions, the color was improved in all of the treated lesions. Histopathological examination revealed that the regenerated epidermis had stratified keratinocytes with rete ridges, and the dermal layer without nevus cells regenerated above the remaining dermis layer. CONCLUSIONS: In this study, we found that early epithelialization and regeneration of the dermal layer was achieved after the application of CEA, suggesting that CEA could be an effective option after curettage or abrasion of GCMN.

Evaluation of serum IgE in peach-allergic patients with systemic reaction by using recombinant Pru p 7 (gibberellin-regulated protein)

BACKGROUND: Lipid transfer protein (LTP) is a major fruit allergen. It has, however, recently been revealed that the systemic reaction in peach-allergic patients is related not only to LTP (Pru p 3) but also to gibberellin-regulated protein (Pru p 7). We investigated recombinant Pru p 7 (rPru p 7) for its potential use in worldwide standardization for the diagnosis of peach allergy. METHODS: Natural Pru p 7 (nPru p 7) was purified from peach crude extract using a monoclonal antibody affinity column. Complementary DNA for Pru p 7 was cloned and expressed in Escherichia coli and Pichia pastoris. Serum immunoglobulin (Ig) E in peach-allergic patients was examined by enzyme-linked immunosorbent assay (ELISA) using nPru p 7 and rPru p 7 (E. coli product: erPru p 7 and P. pastoris product: prPru p 7). RESULTS: Peach-allergic patients (n = 27) were diagnosed and categorized into oral reaction (n=10) or systemic reaction (n = 17). The nPru p 7 positivity based on serum IgE levels was 52% in the systemic-reaction group and 0% in the oral-reaction group (P<0.05). In the systemic-reaction group, there was no significant difference in reactivity between nPru p 7 and prPru p 7, but the reactivity of erPru p 7 was significantly lower than those of nPru p 7 and prPru p 7 (P < 0.05). CONCLUSIONS: We found that prPru p 7 exhibited reactivity in ELISA comparable to that of nPru p 7 for the diagnosis of peach allergy with systemic reaction

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Evaluation of serum IgE in peach-allergic patients with systemic reaction by using recombinant Pru p 7 (gibberellin-regulated protein).

BACKGROUND: Lipid transfer protein (LTP) is a major fruit allergen. It has, however, recently been revealed that the systemic reaction in peach-allergic patients is related not only to LTP (Pru p 3) but also to gibberellin-regulated protein (Pru p 7). We investigated recombinant Pru p 7 (rPru p 7) for its potential use in worldwide standardization for the diagnosis of peach allergy. METHODS: Natural Pru p 7 (nPru p 7) was purified from peach crude extract using a monoclonal antibody affinity column. Complementary DNA for Pru p 7 was cloned and expressed in Escherichia coli and Pichia pastoris. Serum immunoglobulin (Ig) E in peach-allergic patients was examined by enzyme-linked immunosorbent assay (ELISA) using nPru p 7 and rPru p 7 (E. coli product: erPru p 7 and P. pastoris product: prPru p 7). RESULTS: Peach-allergic patients (n=27) were diagnosed and categorized into oral reaction (n=10) or systemic reaction (n=17). The nPru p 7 positivity based on serum IgE levels was 52% in the systemic-reaction group and 0% in the oral-reaction group (P<0.05). In the systemic-reaction group, there was no significant difference in reactivity between nPru p 7 and prPru p 7, but the reactivity of erPru p 7 was significantly lower than those of nPru p 7 and prPru p 7 (P<0.05). CONCLUSIONS: We found that prPru p 7 exhibited reactivity in ELISA comparable to that of nPru p 7 for the diagnosis of peach allergy with systemic reaction.

Hen's Egg Allergy.

Egg allergy is one of the most frequent food allergies in infants and young children. The prevalence of egg allergy is estimated to be between 1.8 and 2% in children younger than 5 years of age. The reactions are mainly mediated by IgE and partially by non-IgE or are a mix of both types. Egg white contains more than 20 different proteins and glycoproteins. Ovomucoid (Gal d 1), ovalbumin (Gal d 2), conalbumin (ovotransferrin) (Gal d 3) and lysozyme (Gal d 4) have been identified as major allergens in hen's egg. Alpha-livetin (Gal d 5) is thought to be a main egg yolk allergen responsible for bird-egg syndrome. The diagnosis of egg allergy is based on history taking, antigen-specific IgE measurements, such as the skin prick test, in vitro antigen-specific blood IgE tests and histamine release tests, and oral food challenges. The measurements of specific IgE to ovomucoid and its linear epitopes are more useful in the diagnosis of heated egg allergy and in the prediction of prognosis. Currently, the management of egg allergy is essentially minimal elimination based on the correct identification of the causative allergen. Although oral immunotherapy is promising as a tolerance induction protocol, several questions and concerns still remain, predominantly regarding safety.

Hyper IgM syndrome and complement Clq deficiency in an individual with systemic lupus erythematosus-like disease.

Many immunedeficiency syndromes are associated with autoimmune disorders. We here report on a girl with a systemic lupus erythematosus-like disease who suffered from both hyperimmunoglobulin M syndrome (HIGMS) and C1q deficiency. Despite severe central nervous system-lupus like disease, probably due to C1q deficiency, kidney function was relatively spared. IgM autoantibody might play a protective role against lupus-glomerulonephritis.

A convenient and sensitive allergy test: IgE crosslinking-induced luciferase expression in cultured mast cells.

BACKGROUND: For the detection of allergen-specific IgE in sera, solid-phase IgE-binding assays like the CAP test are commonly used. Although such immunochemical methods are very sensitive, they frequently produce false positives. Degranulation of the human IgE receptor (FcεRI)-transfected rat mast cell (RBL) lines seems to be a possible indicator for human IgE, but spontaneous mediator release from these cells in the presence of human sera is not negligible. METHODS: The nuclear factor of activated T-cells (NFAT)-responsive luciferase reporter gene was stably transfected into human FcεRI-expressing RBL-SX38 cells. One established clone (RS-ATL8) was sensitized with 1 : 100 dilution of sera from patients with egg white allergy and then stimulated with purified or a crude extract of egg white allergen. RESULTS: Sensitization with 15 pg/ml IgE was sufficient to detect IgE crosslinking-induced luciferase expression (EXiLE) by anti-IgE stimulation. Allergen-specific EXiLE was elicited by as little as 1 fg/ml of egg white protein without cytotoxicity. There was a good correlation between results with EXiLE and oral food challenge tests on patients with egg allergy (P = 0.001687, Fisher's exact test). The measured values of EXiLE and the CAP test also correlated well (R = 0.9127, Spearman's test). CONCLUSION: The EXiLE test using RS-ATL8 cells is a promising in vitro IgE test to evaluate the biological activity of the binding between IgE and allergens.

Up-regulated cytokine-inducible SH2-containing protein expression in allergen-stimulated T cells from hen's egg-allergic patients.

BACKGROUND: Although changes in the fine balance of allergen-specific T cells are crucial in the pathogenesis of allergic diseases, their roles in the allergic reaction to hen's eggs (HE) have not yet been fully analysed. OBJECTIVE: Using microarray technology, allergen-stimulated T cells from HE-allergic children were analysed to identify genes that are specifically up-regulated in these cells. METHODS: RNA from CD4(+) CD14(-) cells, fractionated from allergen-stimulated peripheral mononuclear cells, was analysed using a whole-genome microarray and real-time RT-PCR. The protein expression of selected genes was ascertained by flow cytometry. RESULTS: In microarray analyses of allergen-stimulated T cells, 43 genes were up-regulated in HE-allergic children but not in non-HE-allergic children. Among these, up-regulation of three genes, cytokine -inducible SH2-containing protein (CISH), nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor Z (NFKBIZ) and B-cell CLL/lymphoma 2 (BCL2), was confirmed by real-time quantitative RT-PCR. CISH, but not NFKBIZ or BCL2, showed a significantly higher ratio of antigen-stimulated cell transcription over unstimulated cells in HE-allergic than in non-HE-allergic children (P<0.01). Flow-cytometric analysis revealed that the percentage of CD25(+)CISH(+) cells in CD4(+) cells from patients with HE allergy was significantly higher than that in controls (P<0.01). The expression level of CISH was significantly higher in IL-4(+) Th2 cells than in IFN-gamma(+) Th1 cells. CONCLUSION: We noted that CISH expression in allergen-stimulated CD4(+) T cells from HE-allergic patients was significantly increased in both mRNA and protein levels compared with that from non-HE-allergic children.

Allergen-specific helper T cell response in patients with cow's milk allergy: Simultaneous analysis of proliferation and cytokine production by carboxyfluorescein succinimidyl ester dilution assay.

BACKGROUND: The role of antigen-specific T cells in the allergic reaction to cow's milk or in tolerance induction is not yet fully understood. OBJECTIVE: This study was designed to analyse both cow's milk protein (CMP)-specific T cell proliferation and cytokine production simultaneously in children with cow's milk allergy (CMA) in comparison with subjects with various allergic backgrounds. METHODS: Carboxyfluorescein succinimidyl ester was used to detect cow's milk-specific T cells by flow cytometry. The intra-cytoplasmic cytokine production of these antigen-specific T cells was also analysed. RESULTS: Significant differences of both CMP-specific CD4+ cell proliferation and cytokine production between CMA and non-allergic children were observed. While the proliferative responses of children who recently outgrew CMA were not significantly different from those of patients, the patterns of cytokine production were similar to those of non-allergic children. CONCLUSION: These results suggest that the presence of CMP-specific T cell clones per se does not produce CMA, but that the T-helper type 2-skewed pattern of those T cells is associated with adverse reactions. Although it is not possible to distinguish between individual patients with and without CMA on the basis of CFSE assays, these results contribute to the understanding of the pathogenesis and tolerance induction of CMA.

Prospective monitoring of the Epstein-Barr virus DNA by a real-time quantitative polymerase chain reaction after allogenic stem cell transplantation.

Epstein-Barr virus (EBV)-related lymphoproliferative disorder (LPD) is a serious complication of haematopoietic stem cell transplantation (HSCT). To clarify the frequency, natural course and risk factors for LPD, we prospectively monitored 38 allogeneic (allo)-HSCT patients, focusing on the use of anti-thymocyte globulin (ATG). We used a recently developed real-time polymerase chain reaction assay to monitor EBV genome load. The subjects consisted of 19 patients given ATG for conditioning and 19 patients not given ATG. Of the 19 patients given ATG, 47.4% (nine patients) had a significant increase in EBV genome load (10(2.5) copies/microg DNA). Of these nine patients, two developed LPD. Therefore, 10.5% of the patients receiving allo-HSCT with ATG developed LPD. In contrast, none of the 19 patients without ATG had a significantly increased EBV load. The increases in viral load were observed in the second or third month after HSCT. We found that the peak viral loads of LPD patients were > 10(4.0 ) copies/microg DNA. On the other hand, the viral loads of most patients with no symptoms were < 10(2.5) copies/microg DNA. In conclusion, routine monitoring of EBV load during the second and third months after transplantation may benefit patients undergoing HSCT with ATG. We propose that an EBV load > 10(2.5) copies/microg DNA is the reactivation of EBV, and that an EBV load > 10(4.0) copies/microg DNA is indicative of developing LPD.

Clinical and virologic characteristics of chronic active Epstein-Barr virus infection.

Thirty patients with chronic active Epstein-Barr virus (CAEBV) infection were analyzed. The study group included 18 male and 12 female patients, ranging in age from 5 to 31 years with a mean age of 14.2 years. Not all patients had high titers of EBV-specific antibodies, but all patients had high viral loads in their peripheral blood (more than 10(2.5) copies/microg DNA). Fifty percent of the patients displayed chromosomal aberrations, and 79% had monoclonality of EBV. Patients were divided into 2 clinically distinct groups, based on whether the predominantly infected cells in their peripheral blood were T cells or natural killer (NK) cells. Over a 68-month period of observation, 10 patients died from hepatic failure, malignant lymphoma, or other causes. Patients with T-cell CAEBV had a shorter survival time than those with NK-cell type of disease.

Vidarabine therapy for severe chronic active Epstein-Barr virus infection.

PURPOSE: Severe chronic active Epstein-Barr virus infection (SCAEBV) is an intractable disease with a poor prognosis, and a definitive treatment has not been established. We administered vidarabine to patients with natural killer (NK) cell-type SCAEBV and evaluated clinical and virologic effects. PATIENTS AND METHODS: Four patients with SCAEBV were enrolled in this study. These patients had various symptoms, including fever, chronic hepatitis, hepatosplenomegaly, and hypersensitivity to mosquito bites. All patients had increased numbers of NK cells in their peripheral blood, and most of these were infected with EBV. Viral load was measured by in situ hybridization and quantitative polymerase chain reaction (PCR). RESULTS: The patients all responded to the therapy, and their symptoms improved. After the therapy, the number of NK cells in their peripheral blood decreased. In two patients who were closely monitored, the viral load measured by in situ hybridization and quantitative PCR decreased in parallel with the symptomatic improvement. After discontinuing this drug, the patient's symptoms returned and the Epstein-Barr virus load increased again. CONCLUSION: These results indicate that vidarabine therapy is a therapeutic choice to control SCAEBV, although its effect may be transient.

Impaired cytotoxic T lymphocyte response to Epstein-Barr virus-infected NK cells in patients with severe chronic active EBV infection.

Clinical evidence of a relationship between severe chronic active Epstein-Barr virus (EBV) infection and clonal expansion of EBV-infected T or NK cells has been accumulated. In order to clarify pathogenesis of EBV-infected cell proliferation in patients with severe chronic active EBV infection, cytotoxic T lymphocyte (CTL) responses of two patients against B-lymphoblastoid cell lines (B-LCL) and EBV-infected NK cells were examined in comparison with those of HLA-identical healthy siblings. Unexpectedly, patients' CTL activities induced by mixed culture with autologous B-LCLs were markedly reduced, although uncontrolled EBV-related B-cell proliferations have never been experienced. In contrast, limiting dilution analysis demonstrated that B-LCL-specific CTL precursor (CTLp) frequencies of patients were comparable to those of their healthy sisters. The existence of normal levels of B-LCL-specific T cell responses was confirmed by flow-cytometric analysis of IFN-gamma-producing T cells after stimulation with B-LCLs. Infected NK-cell-specific CTLp frequencies of the patients were at undetectable levels despite their expression of latent membrane protein (LMP) 1, suggesting mechanisms to escape immunologic surveillance. In the patients' HLA-identical healthy sisters, infected NK-cell-specific CTLps were detected, and infected NK-cell-specific CTL clones could be established. From these findings, two treatment options for severe chronic active EBV infection are offered for consideration: adoptive transfer of in vitro-cultured CTL, and bone marrow transplantation from HLA-identical donors.

Characterization of the gammac chain among 27 unrelated Japanese patients with X-linked severe combined immunodeficiency (X-SCID).

X-linked severe combined immunodeficiency (X-SCID) is a rare fatal disease that is caused by mutations in the gene encoding the gammac chain. In this study, 27 unrelated Japanese patients with X-SCID were examined in terms of their genetic mutations and surface expression of the gammac chain. Among 25 patients examined, excluding two patients with large deletions, 23 different mutations were identified in the IL2RG gene, including 10 novel mutations. One patient bearing an extracellular mutation and all three of the patients bearing intracellular mutations after exon 7 expressed the gammac chain on the cell surface. Overall, 84% of patients lacked surface expression of the gammac chain leading to a diagnosis of X-SCID.

Longitudinal dynamics of Epstein-Barr virus-specific cytotoxic T lymphocytes during posttransplant lymphoproliferative disorder.

Epstein-Barr virus (EBV)-associated lymphoproliferative disorder (LPD) is a serious complication after allogeneic bone marrow transplantation (BMT). Dynamics of EBV-specific cytotoxic T lymphocytes (CTL), which are important in controlling EBV during the LPD, have not been fully elucidated. A patient with Wiskot-Aldrich's syndrome was diagnosed as suffering from LPD on day 47 after BMT. Fluorescence-activated cell sorter (FACS) analysis for interferon-gamma production revealed that >70% of the patient's CD8(+) T cells were EBV specific. The patient's lymphocytes were directly cytotoxic to donor-derived EBV-positive lymphoblastoid cells, which was blocked by an anti-class I antibody. EBV-specific CD8(+) T cell counts declined in parallel with EBV genome load, and full recovery of LPD was obtained with relaxation of immunosuppressive drugs. The results illustrate longitudinal dynamics of EBV-specific CTL during the posttransplant LPD; they also illustrate the advantages of using FACS analysis for EBV-specific CTL to make decisions about treatment.

Quantitative analysis of cytomegalovirus load using a real-time PCR assay.

A novel real-time PCR assay system was developed to quantify the cytomegalovirus (CMV) genome load. The real-time PCR assay could detect from 6 to over 10(6) copies of CMV-DNA with a wide linear range. The virus load of immunocompromised patients with symptomatic CMV infections was quantified and compared to that of asymptomatic ones. In symptomatic patients, all 17 peripheral blood leukocytes were positive for CMV DNA, and its mean value was 10(3.3) copies/10(6) cells. On the other hand, only 9 of 38 samples (24%) were positive in the asymptomatic patients, and its mean titer was lower (10(2.0) copies/10(6) cells) than that of the symptomatic group (P = 0.002). In plasma, the virus genome was detected in 13 out of 17 samples from symptomatic patients (76%), and its mean value was 10(4.0) copies/ml. In contrast, for the asymptomatic group, only one out of 36 samples were positive (3%). Finally, this system was used to monitor two patients with CMV infections serially. The CMV DNA copy number changed with their clinical symptoms and anti-CMV therapy, and virtually paralleled the result of the pp65 antigenemia assay in both cases. In one patient with the cord blood transplantation, however, the CMV DNA became positive faster than the antigenemia assay. These results indicate that this assay is sensitive and useful for estimating the CMV genome load not only in peripheral blood leukocytes but also in plasma. It can be very helpful for diagnosing CMV-related diseases and monitoring the virus load in patients with CMV infections.

Successful bone marrow transplantation in a child with X-linked hyper-IgM syndrome.

We report a case of an 11-year-old boy who underwent successful bone marrow transplantation for X-linked hyper-IgM syndrome (XHIM). The donor was an HLA-matched brother. The patient was conditioned with busulfan, cyclophosphamide and anti-thymocyte globulin. He received 4.7 x 10(8) marrow cells per kg from the donor. Prophylaxis against graft-versus-host disease consisted of cyclosporine and short-term methotrexate. The clinical course after the bone marrow transplantation was uneventful, and 12 months after transplantation the patient was doing well with no need for therapy. We examined expression of the CD40 ligand (CD40L) on the patient's activated T lymphocytes and in vitro production of immunoglobulins by his lymphocytes. Although expression of CD40L was totally absent before the bone marrow transplant, subnormal expression appeared after the transplantation. In vitro production of IgG and IgA also was improved by the transplant. Based on our experience bone marrow transplantation appears to be a reasonable therapeutic option for patients with XHIM if HLA-matched family donors are available.

Characterization of Epstein-Barr virus (EBV)-infected natural killer (NK) cell proliferation in patients with severe mosquito allergy; establishment of an IL-2-dependent NK-like cell line.

The clinical evidence of a relationship between severe hypersensitivity to mosquito bite (HMB) and clonal expansion of EBV-infected NK cells has been accumulated. In order to clarify the mechanism of EBV-induced NK cell proliferation and its relationship with high incidence of leukaemias or lymphomas in HMB patients, we studied clonally expanded NK cells from three HMB patients and succeeded in establishing an EBV-infected NK-like cell line designated KAI3. Immunoblotting and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses revealed that KAI3 cells as well as infected NK cells exhibited an EBV latent infection type II, where EBV gene expression was limited to EBNA 1 and LMP1. As KAI3 was established by culture with IL-2, IL-2 responsiveness of peripheral blood NK cells from patients was examined. The results represented markedly augmented IL-2-induced IL-2R alpha expression in NK cells. This characteristic property may contribute to the persistent expansion of infected NK cells. However, KAI3 cells as well as the NK cells from patients were not protected from apoptosis induced by either an anti-Fas antibody or NK-sensitive K562 cells. Preserved sensitivity to apoptosis might explain the relatively regulated NK cell numbers in the peripheral blood of the patients. To our knowledge, KAI3 is the first reported NK-like cell line established from patients of severe chronic active EBV infection (SCAEBV) before the onset of leukaemias or lymphomas. KAI3 cells will contribute to the study of EBV persistency in the NK cell environment and its relationship with high incidence of leukaemias or lymphomas in HMB patients.

Bilateral middle ear squamous cell carcinoma and clinical review of an additional 5 cases of middle ear carcinomas.

We reported a retrospective review of the clinical records for a 64 year old male patient with bilateral middle ear squamous cell carcinoma (MESCC), and for the five other patients with MESCC treated at our institution during the last 20 years. The patient with bilateral MESCC has survived and remained tumor free for more than 1.5 years after extended radical resection of the secondary tumor combined with intra-arterial and systemic chemotherapy, radiotherapy and immunotherapy. Four patients with unilateral MESCC were treated with multidisciplinary treatment (induction chemotherapy, surgery and radiotherapy), and the remaining patient was treated with radiotherapy and mastoidectomy. Five of the six patients are alive with no evidence of disease. The patient treated with radiotherapy and radical mastoidectomy died of local recurrence 3 years after diagnosis. We suggest that MESCC should be considered when refractory granulation, long-standing otorrhea, otalgia and facial paralysis are observed. Multidisciplinary treatment, including intra-arterial chemotherapy and en bloc resection of the temporal tumor is useful for the treatment of MESCC and will improve the prognosis of patients with this disease.

Necrotizing toxoplasmic encephalitis in a child with the X-linked hyper-IgM syndrome.

UNLABELLED: We report on a 9-year-old boy with the hyper-IgM syndrome who presented with rapid impairment of consciousness. The brain CT scan showed multiple round lucencies, and the brain histology revealed necrotizing toxoplasmic encephalitis. This patient, whose CD40/CD40 ligand system was impaired, indicates the importance of this system for defence against toxoplasmic infection. CONCLUSION: Although disseminated toxoplasmosis is a rare complication of the hyper-IgM syndrome, it must be included in the differential diagnosis of infections.

Successful polymerase chain reaction-based diagnosis of fungal meningitis in a patient with chronic granulomatous disease.

Meningitis is not a common complication of chronic granulomatous disease (CGD). Here, we present details of a 3-year-old boy with X-linked CGD, who suffered from fungal meningitis. While 19 samplings using conventional cerebrospinal fluid (CSF) cultures failed to detect any organisms, fungal DNA was identified in the CSF by a new polymerase chain reaction (PCR)-based method. The patient recovered without any sequelae after treatment with a combination of antifungal agents, interferon-gamma and granulocyte infusions. This case report demonstrates that fungal meningitis must be included in the differential diagnosis of infections in CGD patients and that the PCR-based detection of fungal DNA is a powerful tool for diagnosis.