Toll-like receptor signalling induces the expression of serum amyloid A in epidermal keratinocytes and dermal fibroblasts.

BACKGROUND: Toll-like receptors (TLRs) play critical roles in innate immune response by sensing pathogen- or damage-associated molecular patterns. Epidermal keratinocytes and dermal fibroblasts also produce proinflammatory cytokines and chemokines under stimulation with TLR ligands. Serum amyloid A (SAA) is an essential factor in the pathogenesis of secondary amyloidosis, and also has immunomodulatory functions. SAA are produced mainly by hepatocytes but also by a variety of cells, including immune cells, endothelial cells, synoviocytes, and epidermal keratinocytes. However, SAA expression in human dermal fibroblasts has not been shown to date. AIM: To investigate the effect of TLR ligands on SAA expression in epidermal keratinocytes and dermal fibroblasts. METHODS: We investigated whether TLR ligands induce the expression of SAA in normal human epidermal keratinocytes (NHEKs) and normal human dermal fibroblasts (NHDFs) by real-time quantitative PCR and ELISA. The effect of SAA on its own expression in NHDFs was also studied. RESULTS: SAA expression was induced via nuclear factor-κB by TLR1/2, 3, 5 and 2/6 ligands in NHEKs. In NHDFs, TLR1/2 and TLR2/6 ligands increased SAA expression. SAA further induced its own expression via TLR1/2 and NF-κB in NHDFs, as previously reported for NHEKs. CONCLUSIONS: Our results provide new evidence that the skin's innate immune response contributes to the production of SAA, which might lead to an increased risk of systemic complications such as secondary amyloidosis of recessive dystrophic epidermolysis bullosa.

Long-term efficacy and safety of ixekizumab in Japanese patients with erythrodermic or generalized pustular psoriasis: subgroup analyses of an open-label, phase 3 study (UNCOVER-J).

BACKGROUND: Erythrodermic and generalized pustular psoriasis are rare, difficult to treat forms of psoriasis. In previous reports, we documented 24- and 52-week findings of an open-label, phase 3 trial (UNCOVER-J) of ixekizumab in Japanese patients with erythrodermic or generalized pustular psoriasis; most patients responded to treatment and maintained response through 52 weeks. OBJECTIVE: To assess the long-term (>3 years) efficacy and safety of ixekizumab in Japanese patients with erythrodermic or generalized pustular psoriasis. METHODS: These subgroup analyses were of a partial population of patients from UNCOVER-J (NCT01624233; Sponsored by Eli Lilly and Company), specifically those with erythrodermic psoriasis (N = 8) or generalized pustular psoriasis (N = 5). These patients received 160 mg ixekizumab at Week 0, ixekizumab 80 mg every 2 weeks through Week 12, and ixekizumab 80 mg every 4 weeks thereafter up to Week 244. This regimen is consistent with the regimen approved in Japan for plaque, erythrodermic, and generalized pustular psoriasis and psoriatic arthritis. Efficacy assessments included Global Improvement Score (GIS), Psoriasis Area and Severity Index (PASI), dermal symptoms (for patients with generalized pustular psoriasis), Dermatology Life Quality Index (DLQI) and Itch Numeric Rating Scale (NRS). Safety assessments included treatment-emergent adverse events and adverse events of special interest. RESULTS: Most patients had a GIS of resolved or improved from Week 12 onwards, and all patients had early and sustained improvement in PASI and dermal symptom (generalized pustular psoriasis only) scores. Mean improvements in DLQI and Itch NRS at Week 12 were sustained through Week 244. Ixekizumab was well tolerated over 3 years of treatment in patients with erythrodermic psoriasis or generalized pustular psoriasis, and no new safety concerns were identified. CONCLUSION: These findings suggest that ixekizumab can be an effective long-term treatment option for erythrodermic or generalized pustular psoriasis.

Expression of serine protease inhibitors in epidermal keratinocytes is increased by calcium but not 1,25-dihydroxyvitamin D3 or retinoic acid.

BACKGROUND: In human skin, the serine proteases kallikrein-related peptidase (KLK)5 and KLK7 degrade corneodesmosome proteins, leading to desquamation. Serine protease activity of the skin is tightly regulated by the interplay between such proteases and serine protease inhibitors, including lymphoepithelial Kazal-type related inhibitor (LEKTI), encoded by SPINK5; secretory leucocyte peptidase inhibitor (SLPI); and elafin. Expression of KLK5 and KLK7 is controlled and upregulated by stimulants such as calcium, 1,25-dihydroxyvitamin D3 [1,25(OH)2 VD3 ] and retinoic acid (RA). OBJECTIVES: To understand the effect of calcium, 1,25(OH)2 VD3 and RA on the expression of serine protease inhibitors in epidermal keratinocytes. METHODS: We stimulated normal human epidermal keratinocytes (NHEKs) with high calcium, 1,25(OH)2 VD3 or RA, and then analysed the expression of serine protease inhibitors using quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay and immunocytofluorescence. We also analysed trypsin- and chymotrypsin-like serine protease activities in stimulated NHEKs. RESULTS: High calcium, but not 1,25(OH)2 VD3 or RA, significantly induced the expression of LEKTI, SLPI and elafin at both transcript and protein levels in NHEKs. These inductions were time- and dose-dependent. The activities of trypsin- and chymotrypsin-like serine proteases were significantly up- and downregulated by high calcium, respectively, in NHEKs. CONCLUSIONS: High calcium, but not 1,25(OH)2 VD3 or RA, increases the expression of serine protease inhibitors in epidermal keratinocytes. Our findings contribute to the understanding of the mechanisms by which serine protease activities are regulated by serine proteases and related inhibitors in epidermal keratinocytes.

An unusual clinical presentation of lupus erythematosus tumidus localized on the thigh.

A 44-year-old woman with seronegative polyarthritis presented with a 2-year history of a solitary, bluish-red, oedematous, nonscaly, annular and partially reticulated macule on her right thigh. Histopathological findings revealed perivascular and periadnexal lymphocytic infiltrate in the dermis. Alcian blue and colloidal iron stains highlighted mucinous deposit in the upper and mid dermis. Direct immunofluorescence showed a linear deposit of IgG and C3 along the basement membrane zone. Antinuclear antibody was positive at a titre of 1 : 80, with homogenous and speckled patterns. Except for its unusual localization and lack of photosensitivity, our case had the clinical and histopathological features of lupus erythematosus tumidus. These characteristics were also reminiscent of reticular erythematous mucinosis and erythema annulare centrifugum, both of which are considered to be associated with cutaneous lupus erythematosus (CLE). Hydroxychloroquine 200 mg daily led to improvement of the skin lesion. The unusual clinical presentation of our case emphasizes the heterogeneity of clinical manifestations of CLE.

Epitope analysis of antidesmoglein 1 autoantibodies from patients with pemphigus foliaceus across different activity stages.

BACKGROUND: Pemphigus foliaceus (PF) and pemphigus vulgaris (PV) are closely related, but clinically distinct, autoimmune blistering diseases caused by autoantibodies against desmoglein (Dsg)1 and Dsg3, respectively. Using ethylenediaminetetraacetic acid (EDTA)-treated Dsg3 enzyme-linked immunosorbent assay (ELISA) we have shown that the proportion of anti-Dsg3 antibodies against calcium-dependent epitopes decreased upon shifting to the inactive phase in patients with PV. OBJECTIVES: To analyse the epitope profiles of anti-Dsg1 antibodies across the different activity stages of PF. METHODS: We evaluated five patients with PF who retained high serum levels of anti-Dsg1 antibodies in the inactive phase. Sera were obtained in both the active and inactive phases, and were analysed by EDTA-treated and exfoliative toxin-treated ELISAs. To map the epitopes of anti-Dsg1 antibodies, immunoprecipitation-immunoblotting was performed using a set of Dsg1/Dsg2 domain-swapped molecules. RESULTS: Anti-Dsg1 antibodies against the calcium-dependent epitopes of Dsg1 were the predominant antibodies in both the active and inactive phases. The proportion of anti-Dsg1 antibodies against the calcium-dependent epitopes did not change upon shifting to the inactive phase. The results of immunoprecipitation-immunoblotting showed that most of the anti-Dsg1 antibodies bound to the extracellular domains (EC)1-2 of Dsg1. CONCLUSIONS: In patients with PF, the calcium-dependent epitopes on EC1 and EC2 of Dsg1 contained definitively pathogenic and nonpathogenic epitopes. The disease activity might be differentially controlled by the antibodies between PF and PV depending on the presence or absence of the nonpathogenic epitope.

Longitudinal analysis of antibody profiles against plakins in severe drug eruptions: emphasis on correlation with tissue damage in drug-induced hypersensitivity syndrome and drug reaction with eosinophilia and systemic symptoms.

BACKGROUND: The evidence for severe drug eruption as a trigger for autoimmune disease has recently increased. No information is available on how tissue damage in severe drug eruptions can induce autoimmune responses. OBJECTIVES: To investigate whether the generation of autoantibodies (autoAbs) against plakin family proteins could be the cause or result of tissue damage in patients with severe drug eruptions and whether the generation of autoAbs could be prevented by systemic corticosteroids during the acute stage. METHODS: We retrospectively analysed alterations of serum levels of autoAbs against plakin family proteins in patients with Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) and drug-induced hypersensitivity syndrome (DiHS)/drug reaction with eosinophilia and systemic symptoms (DRESS) during the acute stage and long after resolution over a period of more than 10 years. RESULTS: AutoAbs against plakin family proteins were detected in patients with either SJS/TEN or DiHS/DRESS regardless of the epidermal damage in the acute stage, and were sustained even long after resolution in DiHS/DRESS, indicating that those autoAbs are neither the cause nor the consequence of epidermal damage, at least in DiHS/DRESS. Severe liver damage and noncorticosteroid therapy during the early and acute stages of DiHS/DRESS were associated with the subsequent generation of these autoAbs. CONCLUSIONS: These autoAbs are neither necessarily the cause nor the result of epidermal damage in DiHS/DRESS, because the presence of these autoAbs was not restricted to patients with SJS/TEN but was also observed in those with DiHS/DRESS, which is characterized by lack of epidermal damage. Severe liver damage and/or immune responses that could be prevented by corticosteroids in the acute stage of DiHS/DRESS are among the causal factors contributing to the generation of autoimmune responses.

Survival rates and prognostic factors of Epstein-Barr virus-associated hydroa vacciniforme and hypersensitivity to mosquito bites.

BACKGROUND: Epstein-Barr virus (EBV)-associated T/natural-killer lymphoproliferative disorders form a group of diseases that includes classical and systemic hydroa vacciniforme (HV) and hypersensitivity to mosquito bites (HMB). Patients with systemic HV (sHV) and HMB often have a poor prognosis, although little is known about the prognostic factors. OBJECTIVES: To elucidate the prognostic factors of HV and HMB. METHODS: We studied clinicopathological manifestations, routine laboratory findings, anti-EBV titres, EBV DNA load and EBV-encoded gene expression, including expression of BZLF1, in 50 patients with classical HV (cHV), sHV, HMB only and HMB with HV (HMB + HV), and further analysed 30 patients who were available for follow-up. RESULTS: The median age of disease onset was 5 years (range 1-74). A follow-up study indicated that fatal outcomes were observed in three of eight patients with sHV, two of six patients with HMB only, and two of five patients with HMB + HV. The main causes of death were complications from haematopoietic stem-cell transplantation and multiorgan failure. There were no fatalities among the 11 patients with cHV. Univariate analysis revealed two poor prognostic indicators: (i) onset age > 9 years and (ii) the expression of an EBV-encoded immediate-early gene transcript, BZLF1 mRNA, in the skin lesions (P < 0·001 and P = 0·003, respectively). CONCLUSIONS: No prognostic correlation was observed in EBV-infected lymphocyte subsets, anti-EBV antibody titres or EBV DNA load. Late onset and EBV reactivation are both related to more severe phenotypes of the disease, and thus may predict a poor prognosis.

A patient with elderly-onset atypical hydroa vacciniforme with an indolent clinical course.

Hydroa vacciniforme (HV) is a rare photodermatosis that mainly affects children and manifests as vesiculopapular eruptions in sun-exposed areas without systemic symptoms. HV-like lymphoma (HVLL) is one of the Epstein-Barr virus (EBV)-associated lymphoproliferative disorders (LPD) of childhood. Its diagnosis is based on monoclonal T-cell proliferation; however, its degree of malignancy is controversial owing to its variable prognosis. Elderly-onset cases of these diseases are extremely rare, and the clinical features remain unknown. It has been shown that late onset is closely associated with a severe phenotype in EBV-associated LPD including atypical HV. Here we describe a case of elderly-onset atypical HV accompanied by T-cell monoclonality, but with a very indolent clinical course. Our patient indicates a possible case with elderly-onset atypical HV manifesting a favourable course, and that T-cell monoclonality and age of onset cannot always predict the disease severity, and highlights the difficulty of prognosis prediction in elderly-onset atypical HV.

Cathelicidin antimicrobial peptide LL-37 augments interferon-ß expression and antiviral activity induced by double-stranded RNA in keratinocytes.

BACKGROUND: Cathelicidin antimicrobial peptide LL-37 has the capacity to kill a wide range of microbes and to modify host immunity. Recently, our group observed that the activation of keratinocytes by LL-37 and DNA greatly increases interferon (IFN)-ß through Toll-like receptor (TLR)9. However, the effect of LL-37 on the induction of IFN-ß through TLR3, a sensor of double-stranded (ds) RNA, in keratinocytes is not well known. OBJECTIVES: To investigate whether LL-37 could affect TLR3 signalling and antiviral activity in normal human epidermal keratinocytes (NHEKs). METHODS: We investigated the production of IFN-ß in NHEKs stimulated with a TLR3 ligand, poly (I:C), in the presence of LL-37. To examine the effect of LL-37 and poly (I:C) on antiviral activity, a virus plaque assay using herpes simplex (HS) virus type-1 was carried out. The uptake of poly (I:C) conjugated with fluorescein isothiocyanate (FITC) into the keratinocytes was observed in the presence of LL-37. Immunostaining for TLR3 and LL-37 was performed using skin samples from HS. RESULTS: LL-37 and poly (I:C) synergistically induced the expression of IFN-ß in NHEKs. Furthermore, co-stimulation with LL-37 and poly (I:C) significantly decreased the viral plaque numbers compared with poly (I:C) or LL-37 alone. LL-37 enhanced the uptake of FITC-conjugated poly (I:C) into cells. Immunohistochemical analysis demonstrated that the expression of TLR3 and LL-37 is upregulated in HS lesions. CONCLUSIONS: Our findings suggest that LL-37 augments the antiviral activity induced by dsRNA in keratinocytes, which may contribute to the innate immune response to cutaneous viral infections such as HS.

New screening methods for probiotics with adhesion properties to sialic acid and sulphate residues in human colonic mucin using the Biacore assay.

AIMS: To determine the relationship between adhesive ability of probiotics and acidic residues in human colonic mucin, we developed a new screening method using Biacore to evaluate adherence of bacteria before and after sialic acid or sulphate residues were blocked or removed from mucin. METHODS AND RESULTS: Ten strains of lactobacilli and three strains of bifidobacteria isolated from human faeces were evaluated for their adhesive properties to soluble human colonic mucin (sHCM) using the Biacore binding assay. Three strains (Lactobacillus strain ME-522, Lact. gasseri ME-527 and Bifidobacterium bifidum MCC1092) showing significant adherence were selected. Decreased binding activities were observed after removing sialic acid of sHCM using sialidase. However, after removing the sulphate residue using sulphatase, the adhesion of ME-527 decreased; whereas the remaining two strains had increased adhesion. The adhesion of three probiotics significantly decreased after the sulphate residue was blocked by elution with barium chloride. CONCLUSIONS: A new evaluation method using the Biacore assay was developed to observe binding properties to the acidic residues of sHCM. Results indicated that there was a strong relationship between probiotic adhesion and acidic residues of sHCM. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report showing a screening method that quantitatively measures the binding between bacteria and acidic residues in sHCM using the Biacore binding assay; and provides a new method for the selection of probiotics in the future.

Detection of antibodies against the non-calcium-dependent epitopes of desmoglein 3 in pemphigus vulgaris and their pathogenic significance.

BACKGROUND: Antidesmoglein (anti-Dsg) 3 serum antibody titres are usually correlated with the disease activity of pemphigus vulgaris (PV), but some patients retain high titres even in remission. OBJECTIVES: The aim of our study was to determine whether anti-Dsg3 antibodies in PV sera recognized calcium (Ca(2+) )-dependent or non-Ca(2+) -dependent epitopes, and to evaluate their pathogenicity. METHODS: Dsg3 baculoprotein-coated enzyme-linked immunosorbent assay (ELISA) plates were treated with 0.5 mmol L(-1) ethylenediaminetetraacetic acid (EDTA). The binding ability of anti-Dsg3 monoclonal antibodies (mAbs) was analysed. Eight of the 83 patients with PV who were screened had elevated Dsg3 ELISA index values > 00 in remission. The binding ability of these PV sera was analysed. We evaluated the pathogenicity of anti-Dsg3 serum antibodies against the non-Ca(2+) -dependent epitopes using a dissociation assay. RESULTS: The reactivity of pathogenic anti-Dsg3 mAbs against the Ca(2+) -dependent epitopes diminished markedly in the EDTA-treated ELISA, whereas no such reduction was observed in mAbs against the non-Ca(2+) -dependent epitopes. The sera of all the patients contained antibodies against both Ca(2+) -dependent and non-Ca(2+) -dependent epitopes. In six out of the eight patients, the ratio of antibodies against Ca(2+) -dependent to non-Ca(2+) -dependent epitopes decreased in remission. EDTA-treated Dsg3 baculoproteins adsorbed anti-Dsg3 serum antibodies against the non-Ca(2+) -dependent epitopes, but the remnant PV antibodies retained the ability to induce acantholysis in the dissociation assay. CONCLUSIONS: We have established an assay to measure indirectly the titres of anti-Dsg3 serum antibodies against the Ca(2+) -dependent epitopes, based on the differences between EDTA-untreated and EDTA-treated ELISA index values, as a routine laboratory test to reflect the pathogenic anti-Dsg3 serum antibody titres more accurately.

Rapid propidium monoazide PCR assay for the exclusive detection of viable Enterobacteriaceae cells in pasteurized milk.

Pasteurized milk is a complex food and contains numerous PCR inhibitors and can often contain high levels of dead Enterobacteriaceae cells, depending on the condition of food sanitation. Usually, propidium monoazide (PMA) or ethidium monoazide PCR techniques decrease the number of dead bacteria by up to 3.5 log to the associated dead bacteria with no treatment. However, this difference could be insufficient to completely inhibit DNA amplification in the PCR from 10(6) cells of dead Enterobacteriaceae bacteria/mL, potentially contaminated in pasteurized milk. Actually, such potentially high levels of dead Enterobacteriaceae cells in milk has prevented milk researchers from applying PMA- or ethidium monoazide PCR to the assay of viable Enterobacteriaceae cells in milk. We, therefore, developed a rapid PMA real-time PCR whose minimum levels of detection were 1.5 log cfu/PCR for Cronobacter muytjensii and Escherichia coli, and 2.5 log cfu/PCR for Salmonella enteritidis without DNA purification in milk matrices. The PMA real-time PCR allowed us to specifically detect viable Enterobacteriaceae cells (5-10 cfu/mL) in pasteurized milk (20 mL) within 7.5h of total testing time, following the hygienic guidelines for pasteurized milk in the United States and European Union. The long DNA amplification (mainly 2,451 bp) of the 16S-23S rRNA gene was completely suppressed in highly contaminated dead Enterobacteriaceae cells (7.5 log cfu of Cronobacter muytjensii) in 20 mL of pasteurized milk by 23-µM PMA treatment. Although the contamination of the PCR reaction with 5% milk usually causes great inhibition, our method led to the successful elongation of PCR from viable Enterobacteriaceae cells still in the pasteurized milk matrices finally corresponding to 2 to 4 mL of milk PCR inhibitors without a DNA purification step. To comply with current customer demands for chilled pasteurized milk at the most excellent possible quality, our new technique could enable laboratory persons in a factory to conduct rapid milk coliform testing before shipping from a factory.

Granulysin-producing cytotoxic T cells in the mucocutaneous lesions of Behçet disease: a distinct inflammatory response from erythema nodosum.

BACKGROUND: Cytotoxic T lymphocytes (CTLs) have been recognized as an important effector cell in Behçet disease (BD). Granulysin is a cytolytic granule protein expressed by CTLs and natural killer cells. AIM: To evaluate the involvement of granulysin-producing T cells in the pathogenesis of BD. METHODS: Using immunohistochemistry, lymphocyte subsets expressing granulysin were investigated in mucocutaneous lesions of BD. Serum granulysin levels were assayed by ELISA. RESULTS: Granulysin-positive cells were seen in specimens from oral ulcers, genital ulcers and acne-like eruptions, but not erythema nodosum-like lesions. Both CD4+ and CD8+ T cells expressed granulysin. Serum granulysin levels did not correlate with disease activity in BD. CONCLUSION: Immune reactions mediated by granulysin-positive CTLs may play an important role in the pathogenesis of acne-like eruptions, oral ulcers and genital ulcers in BD.

Novel multiplex polymerase chain reaction primer set for identification of Lactococcus species.

AIMS: The gram-positive bacterial genus Lactococcus has been taxonomically classified into seven species (Lactococcus lactis, Lactococcus garvieae, Lactococcus piscium, Lactococcus plantarum, Lactococcus raffinolactis, Lactococcus chungangensis and Lactococcus fujiensis). This study aimed to develop a novel multiplex polymerase chain reaction (PCR) primer set for the identification of the seven lactococcal species, as well as to differentiate the two industrially important dairy subspecies, L. lactis subsp. lactis and L. lactis subsp. cremoris. METHODS AND RESULTS: A multiplex PCR primer set was designed based on the nucleotide sequences of the 16S rRNA gene of the seven lactococcal species. The specificity of the established one-step multiplex PCR scheme was verified using more than 200 bacterial strains, in which a complete sequence match was confirmed by partial sequencing of their 16S rRNA gene. CONCLUSIONS: The one-step multiplex PCR enables the identification and speciation of bacterial strains belonging to the genus Lactococcus and the differentiation of strains of L. lactis subsp. lactis and L. lactis subsp. cremoris. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides an efficient method for identification of lactococcal strains of industrial importance.

Improved viability of bifidobacteria in fermented milk by cocultivation with Lactococcus lactis subspecies lactis.

The poor survival of probiotic bacteria in commercial yogurts may limit their potential to exert health benefits in humans. The objective was to improve the survival of bifidobacteria in fermented milk. Cocultivation with some strains of Lactococcus lactis ssp. lactis improved the survival of bifidobacteria in fermented milk during refrigerated storage. Studies on one strain, Lc. lactis ssp. lactis MCC866, showed that the concentrations of dissolved oxygen were kept lower in the cocultivated fermented milk during storage compared with monocultured Bifidobacterium longum BB536 or samples cocultured with another noneffective Lc. lactis ssp. lactis strain. Degradation of genomic DNA was suppressed in the cocultivating system with Lc. lactis ssp. lactis MCC866. Several genes that participated in protection from active oxygen species (e.g., genes coding for alkyl hydroperoxide reductase and Fe(2+) transport system) were expressed at higher levels during refrigerated storage in Lc. lactis ssp. lactis MCC 866 compared with another noneffective Lc. lactis ssp. lactis strain. Concentration of free iron ion was also lower in supernatants of fermented milk cocultivated with B. longum BB536 and Lc. lactis ssp. lactis MCC866. These results suggest that Lc. lactis ssp. lactis MCC 866 is potentially superior in reducing oxygen damage and consequently improves the survival of bifidobacteria in the cocultivating system. This cocultivation system is of industrial interest for producing fermented milk containing viable bifidobacteria with long shelf life.

Prognostic factors for mature natural killer (NK) cell neoplasms: aggressive NK cell leukemia and extranodal NK cell lymphoma, nasal type.

BACKGROUND: Patients with natural killer (NK) cell neoplasms, aggressive NK cell leukemia (ANKL) and extranodal NK cell lymphoma, nasal type (ENKL), have poor outcome. Both diseases show a spectrum and the boundary of them remains unclear. The purpose of this study is to draw a prognostic model of total NK cell neoplasms. PATIENTS AND METHODS: We retrospectively analyzed 172 patients (22 with ANKL and 150 with ENKL). The ENKLs consisted of 123 nasal and 27 extranasal (16 cutaneous, 9 hepatosplenic, 1 intestinal and 1 nodal) lymphomas. RESULTS: Complete remission rate for ENKL was 73% in stage I, but 15% in stage IV, which was consistent with that for ANKL (18%). The prognosis of ENKL was better than that of ANKL (median survival 10 versus 1.9 months, P < 0.0001) but was comparable when restricted to stage IV cases (4.0 months, P = 0.16). Multivariate analysis showed that four factors (non-nasal type, stage, performance status and numbers of extranodal involvement) were significant prognostic factors. Using these four variables, an NK prognostic index was successfully constructed. Four-year overall survival of patients with zero, one, two and three or four adverse factors were 55%, 33%, 15% and 6%, respectively. CONCLUSION: The current prognostic model successfully stratified patients with NK cell neoplasms with different outcomes.

New approach to use ethidium bromide monoazide as an analytical tool.

AIMS: Ethidium bromide monoazide (EMA) has been determined to cause delay in DNA amplification from dead bacteria at real-time PCR. However, there is concern that the increasing EMA concentration to suppress amplification from high number of dead bacteria also affects live bacteria. The aim is to disclose a novel application of EMA for food hygienic test. METHODS AND RESULTS: We performed a low-dose double EMA treatment. Live or heat-dead Enterobacter sakazakii (reclassified as Cronobacter spp.) in 10% powdered infant formula (PIF) solution was subjected to a treatment with 20 µg ml(-1) of EMA followed by a treatment with 10 µg ml(-1) of EMA without washing, and direct real-time PCR. We observed that DNA amplification from 10(7) cells ml(-1) of dead Ent. sakazakii was completely suppressed within 50 cycles of PCR, whereas 10(2) -10(3) cells ml(-1) of viable cells could be detected. When a 3-h enrichment step in liquid medium was included after the first EMA treatment, live Ent. sakazakii could be detected at initial levels of 10(0) -10(2) cells ml(-1) . We compared the low-dose double-treated EMA-PCR with the culture method using 80 samples of PIF, and completely correlative results were obtained for both methods. CONCLUSIONS: We concluded that the newly developed low-dose double-treated EMA-PCR is a very effective tool for live Ent. sakazakii detection in PIF. SIGNIFICANCE AND IMPACT OF THE STUDY: We focused on the specific nature of photoreactive compound that residual EMA is cancelled by irradiation. We were successful in treating bacteria with EMA in gradient concentration to increase live and dead distinction ability.