IgE sensitivity to Malassezia pachydermatis and mite allergens in dogs with atopic dermatitis.

In this study, dogs with atopic dermatitis were separated into non-food-induced atopic dermatitis (NFIAD) group (n = 15) and food-induced atopic dermatitis (FIAD) group (n = 37) based on an elimination diet test. IgE reactivity for crude Malassezia pachydermatis (M. pachydermatis) and house dust mites (HDM) allergen extracts was investigated in the two groups using fluorometric enzyme-linked immunosorbent assay (ELISA) and intradermal skin test (IDST). Nine (60%) of the 15 dogs in NFIAD group and 6 (16%) of the 37 dogs in FIAD group showed specific IgE for M. pachydermatis (Mann-Whitney U-test, P < 0.01). By immunoblotting analysis, the pooled serum samples from dogs with IgE for M. pachydermatis showed IgE reactivity for 50 kDa protein of M. pachydermatis. Twelve (80%) of the 15 dogs in NFIAD group and 8 (22%) of the 37 dogs in FIAD group showed specific IgE for HDM (Mann-Whitney U-test, P < 0.01). In addition, the dogs in NFIAD group significantly show a positive IDST to M. pachydermatis and HDM extracts compared with the dogs in FIAD group. The results suggest that dogs with NFIAD are at increased risk of becoming sensitized to the normal commensal organism M. pachydermatis compared with dogs with FIAD, perhaps co-sensitization occurred due to an HDM protease antigen's, Der f 1 and/or Der p 1, proteolytic activity related epidermal skin barrier defects. Treatment to limit skin colonization may thus be especially important in NFIAD.

Screening of KHP30-like prophages among Japanese Helicobacter pylori strains, and genetic analysis of a defective KHP30-like prophage sequence integrated in the genome of the H. pylori strain NY40.

We have recently reported the active Helicobacter pylori bacteriophages (phages), KHP30 and KHP40, the genomic DNAs of which exist as episomes in host bacterial strains isolated in Japan (i.e. pseudolysogeny). In this study, we examined the possibility of the lysogeny of active KHP30-like phages in Japanese H. pylori strains, because their genomes contain a putative integrase gene. Only the NY40 strain yielded partial detection of a KHP30-like prophage sequence in PCR among 174 Japanese H. pylori isolates, except for strains producing the above active phages. Next, according to the genomic analysis of the NY40 strain, the KHP30-like prophage sequence was found to be located from ca. 524 to 549 kb in the host chromosome. The attachment sites, attL and attR, in the NY40 genome showed almost the same genomic location and sequence as those detected in a French isolate B38, suggesting that an active parental KHP30-like phage had integrated into the ancestral NY40 genome in a site-specific manner. The prophage found in the NY40 genome was assumed to have been genetically modified, after site-specific integration. These, together with the data in the KHP30-like prophages of other H. pylori genomes, suggest that the lysogenic state of the KHP30-like phages is generally unstable.

IgE reactivity to hen egg white allergens in dogs with cutaneous adverse food reactions.

Dogs with cutaneous adverse food reactions (CAFR) often have specific IgE to food allergens. Egg white, which is majorly composed of ovomucoid, ovalbumin, ovotransferrin, and lysozyme, is a food allergen in dogs. Information of the IgE reactivity to purified egg white allergens supports accurate diagnosis and efficiency treatment in humans. However, to the best of our knowledge, there have been no studies on the IgE reactivity to purified egg white allergens in dogs. Here, we investigated the IgE reactivity to crude and purified allergens of hen egg white in dogs with CAFR. First, when we examined serum samples from 82 dogs with CAFR for specific IgE to crude egg white by ELISA, 9.8% (8/82) of the dogs with CAFR showed the IgE reactivity to crude egg white. We then used sera from the eight dogs with positive IgE reactivity to crude egg white to examine the IgE reactivity to four purified allergens, ovomucoid, ovalbumin, ovotransferrin, and lysozyme, by ELISA. We found that 75% (6/8) of the dogs showed IgE reactivity to both ovomucoid and ovalbumin, and that 37.5% (3/8) of the dogs showed IgE reactivity to ovotransferrin. None (0/8) showed IgE reactivity to lysozyme. Moreover, validating these results, the immunoblot analyses were performed using the sera of the three dogs showing the highest IgE reactivity to crude egg white. Both anti-ovomucoid and anti-ovalbumin IgE were detected in the sera of these dogs, while anti-ovotransferrin IgE was not detected. Considering these, ovomucoid and ovalbumin appears to be the major egg white allergens in dogs with CAFR.

Analyses of Short-Term Antagonistic Evolution of Pseudomonas aeruginosa Strain PAO1 and Phage KPP22 (Myoviridae Family, PB1-Like Virus Genus).

UNLABELLED: Pseudomonas aeruginosa causes serious intractable infections in humans and animals. Bacteriophage (phage) therapy has been applied to treat P. aeruginosa infections, and phages belonging to the PB1-like virus genus in the Myoviridae family have been used as therapeutic phages. To achieve safer and more effective phage therapy, the use of preadapted phages is proposed. To understand in detail such phage preadaptation, the short-term antagonistic evolution of bacteria and phages should be studied. In this study, the short-term antagonistic evolution of bacteria and PB1-like phage was examined by studying phage-resistant clones of P. aeruginosa strain PAO1 and mutant PB1-like phages that had recovered their infectivity. First, phage KPP22 was isolated and characterized; it was classified as belonging to the PB1-like virus genus in the Myoviridae family. Subsequently, three KPP22-resistant PAO1 clones and three KPP22 mutant phages capable of infecting these clones were isolated in three sets of in vitro experiments. It was shown that the bacterial resistance to phage KPP22 was caused by significant decreases in phage adsorption and that the improved infectivity of KPP22 mutant phages was caused by significant increases in phage adsorption. The KPP22-resistant PAO1 clones and the KPP22 mutant phages were then analyzed genetically. All three KPP22-resistant PAO1 clones, which were deficient for the O5 antigen, had a common nonsense mutation in the wzy gene. All the KPP22 mutant phage genomes showed the same four missense mutations in the open reading frames orf060, orf065, and orf086 The information obtained in this study should be useful for further development of safe and efficient phage therapy. IMPORTANCE: Pseudomonas aeruginosa causes serious intractable infections in humans and animals; bacteriophage (phage) therapy has been utilized to treat P. aeruginosa infections, and phages that belong to the PB1-like virus genus in the family Myoviridae have been used as therapeutic phages. The preadapted phage is trained in advance through the antagonistic evolution of bacteria and phage and is proposed to be used to achieve safer and more effective phage therapy. In this study, to understand the phage preadaptation, the in vitro short-term antagonistic evolution was studied using P. aeruginosa strain PAO1 and the newly isolated PB1-like phage KPP22. Phage KPP22 was characterized, and the molecular framework regarding the phage preadaptation of KPP22 was elucidated. The importance of study of antagonistic evolution of bacteria and phage in phage therapy is discussed.

Cross-reactivity between major IgE core epitopes on Cry j 2 allergen of Japanese cedar pollen and relevant sequences on Cha o 2 allergen of Japanese cypress pollen.

BACKGROUND: Cry j 2 and Cha o 2 are major allergens in Japanese cedar (Cryptomeria japonica; CJ) and Japanese cypress (Chamaecyparis obtusa; CO) pollen, respectively. Here, we assessed the epitopes related to the cross-reactivity between Cry j 2 and Cha o 2 using in vitro analyses. METHODS: Peptides were synthesized based on Cry j 2 sequential epitopes and relevant Cha o 2 amino acid sequences. Four representative monoclonal antibodies (mAbs) against Cry j 2 were used according to their epitope recognitions. Serum samples were collected from 31 patients with CJ pollinosis. To investigate cross-reactivity between Cry j 2 and Cha o 2, ELISA and inhibition ELISA were performed with mAbs and sera from patients with CJ pollinosis. RESULTS: Two of four mAbs had reactivity to both Cry j 2 and Cha o 2. Of these two mAbs, one mAb (T27) recognized the amino acid sequence (169)KVVNGRTV(176) on Cha o 2. This is related to the core epitope (169)KWVNGREI(176) on Cry j 2, which is an important IgE epitope. In addition, we found that these correlative sequences and purified allergens showed cross-reactivity between Cry j 2 and Cha o 2 in IgE of CJ patients. CONCLUSIONS: We demonstrated the importance of (169)KVVNGRTV(176) in Cha o 2 for cross-reactivity with the Cry j 2 epitope (169)KWVNGREI(176), which plays an important role in allergenicity in CJ pollinosis. Our results are useful for the development of safer and more efficient therapeutic strategies for the treatment of CJ and CO pollen allergies.

Characterization of Pseudomonas aeruginosa phage KPP21 belonging to family Podoviridae genus N4-like viruses isolated in Japan.

Bacteriophages (phages) belonging to the family Podoviridae genus N4-like viruses have been used as therapeutic agent in phage therapy against Pseudomonas aeruginosa infections. P. aeruginosa phage KPP21 was isolated in Japan, and phylogenetically investigated the phages belonging to this viral genus. Morphological and genetic analyses confirmed that phage KPP21 belongs to the family Podoviridae genus N4-like viruses. Moreover, phylogenetic analyses based on putative DNA polymerase and major virion protein showed that P. aeruginosa phages belonging to the genus N4-like viruses are separated into two lineages and that phage KPP21 is in the same clade as phage LUZ7.

Analysis of conformational and sequential IgE epitopes on the major allergen Cry j 2 of Japanese cedar (Cryptomeria japonica) pollen in humans by using monoclonal antibodies for Cry j 2.

PURPOSE: Japanese cedar (Cryptomeria japonica; CJ) pollinosis is a type I allergy induced by CJ pollen, and Cry j 2 is one of the major allergens in this pollen. In a previous study, we analyzed IgE epitopes on Cry j 2 in humans by using synthetic peptides. The main purpose of this study was to identify B-cell epitopes on Cry j 2 in patients with CJ pollinosis by using monoclonal antibodies (mAbs) for Cry j 2. METHODS: We used ELISA with mAbs for the epitope analysis. Sera samples were collected from 80 patients with CJ pollinosis, and allergenic epitopes for mAbs and human IgE were identified using ELISA with synthetic peptides. The importance of the epitopes for human IgE was analyzed using an inhibition ELISA. RESULTS: Four independent epitopes (epitope #1, #2, #3, and #4) were identified on Cry j 2 with the use of mAbs. Epitope #3 and #4, corresponding to peptides No. 25 and No. 33, respectively, were newly determined as epitopes for mAbs and human IgE. Inhibition ELISA showed that not only epitope #2 (sequential) but epitope #1 (conformational) may play an important role in the CJ pollinosis. CONCLUSIONS: Our results revealed 4 epitopes, including two new ones, on Cry j 2. We also found that inhibition ELISA with appropriate mAbs could be a viable method of evaluating the importance of the conformational and sequential epitopes for human IgE. These results are beneficial for the development of safer and more efficient therapeutic strategies for treating CJ pollinosis.

T-cell receptor repertoires of tumor-infiltrating lymphocytes after hyperthermia using functionalized magnetite nanoparticles.

AIM: Accumulating evidence has indicated that hyperthermia using magnetite nanoparticles induces antitumor immunity. This study investigated the diversity of T-cell receptors (TCRs) in tumor-infiltrating lymphocytes after hyperthermia using magnetite nanoparticles. MATERIALS & METHODS: Functionalized magnetite nanoparticles, N-propionyl-4-S-cysteaminylphenol (NPrCAP)/magnetite, were synthesized by conjugating the melanogenesis substrate NPrCAP with magnetite nanoparticles. NPrCAP/magnetite nanoparticles were injected into B16 melanomas in C57BL/6 mice, which were subjected to an alternating magnetic field for hyperthermia treatment. RESULTS: Enlargement of the tumor-draining lymph nodes was observed after hyperthermia. The TCR repertoire was restricted in tumor-infiltrating lymphocytes, and expansion of Vß11(+) T cells was preferentially found. DNA sequences of the third complementaritydetermining regions revealed the presence of clonally expanded T cells. CONCLUSION: These results indicate that the T-cell response in B16 melanomas after hyperthermia is dominated by T cells directed toward a limited number of epitopes and that epitope-specific T cells frequently use a restricted TCR repertoire.

Heat-inducible transgene expression with transcriptional amplification mediated by a transactivator.

PURPOSE: Control of therapeutic gene expression in tumours is a major goal of gene therapy research, as it can restrict cytotoxic gene expression in cancer cells. In addition, the combination of hyperthermia with gene therapy through the application of heat-inducible vectors can result in considerable improvements in therapeutic efficiency. In this study, to combine heat-inducibility with high-level transgene expression, we developed a heat-inducible transgene expression system with transcriptional amplification mediated by a tetracycline-responsive transactivator. MATERIALS AND METHODS: A hybrid promoter was generated by placing the heat shock protein (HSP) 70B' promoter under the tetracycline-repressor responsive element sequence, and a reporter/therapeutic gene expression plasmid was constructed by placing a reporter/therapeutic gene under the control of this hybrid promoter. RESULTS: When the transactivator expression plasmid harbouring an expression cassette of the tetracycline-responsive transactivator gene was co-transfected with a reporter gene expression plasmid, the reporter gene expression was controlled by heat treatment. With this system, high levels of heat-induced transgene expression were observed compared to that from the HSP promoter alone without the transactivator. Evaluation of in vitro therapeutic effects using cancer cell lines revealed that therapeutic gene expression effectively caused cell death in a greater percentage of the cells. CONCLUSION: These findings indicate that this strategy improves the efficacy of cancer gene therapy.

IgE reactivity to a Cry j 3, an allergen of Japanese cedar (Cryptomeria japonica) pollen in dogs with canine atopic dermatitis.

The present study investigated IgE reactivity to a new Cryptomeria japonica pollen allergen (Cry j 3) in dogs with atopic dermatitis by using a fluorometric ELISA. Serum samples from 15 dogs that showed IgE sensitivity to crude C. japonica pollen allergen by ELISA were tested for specific IgE to each allergen, individually. All 15 dogs had anti-Cry j 1 IgE, 6 (40%) had anti-Cry j 2 IgE, and 11 (73%) had anti-Cry j 3 IgE. Further, we found that these anti-Cry j 3 IgE reacted to Cry j 3 with immunoblotting analysis. These findings indicate that Cry j 3 may be a major allergen in dogs.

Heat-inducible transgene expression system incorporating a positive feedback loop of transcriptional amplification for hyperthermia-induced gene therapy.

One of the major goals of gene therapy is to regulate the expression of therapeutic genes in desired cells or tissues. For this purpose, heat-inducible vectors have been exploited for cancer gene therapy combined with hyperthermia, which can result in considerable improvement of therapeutic effects. In the present study, we constructed a novel heat-inducible gene expression system incorporating a transactivation system with a positive feedback loop of transcriptional amplification. The target gene expression mediated by the transactivator under the control of a heat shock protein 70B' promoter is enhanced by self-promoted transactivator gene expression. This expression system showed tight control of target gene expression together with high-level expression; enhanced expression of the reporter gene was observed in transfected cells upon heat treatment, while negligible gene expression was detected in non-heated cells. When a therapeutic gene was used as the target gene, a considerable cytotoxic effect was observed after heat treatment of cancer cells transfected with the plasmids. The heat-induced transgene expression system is a promising new approach for the development of both a safe and effective vector for hyperthermia-based cancer gene therapy.

Impact of the mobilization regimen and the harvesting technique on the granulocyte yield in healthy donors for granulocyte transfusion therapy.

BACKGROUND: Granulocyte mobilization and harvesting, the two major phases of granulocyte collection, have not been standardized. STUDY DESIGN AND METHODS: The data on 123 granulocyte collections were retrospectively investigated for the effect of the mobilization regimen and the harvesting technique. After a single subcutaneous dose (600 µg) of granulocyte-colony-stimulating factor (G-CSF) with (n = 68) or without (n = 40) 8 mg of orally administered dexamethasone, 108 granulocyte donors underwent granulocyte collections. Moreover, 15 peripheral blood stem cell (PBSC) donors who had received 400 µg/m2 or 10 µg/kg G-CSF for 5 days underwent granulocyte collections on the day after the last PBSC collections (PBSC-GTX donors). Granulocyte harvesting was performed by leukapheresis with (n = 108) or without (n = 15) using high-molecular-weight hydroxyethyl starch (HES). RESULTS: Granulocyte donors who received mobilization with G-CSF plus dexamethasone produced significantly higher granulocyte yields than those who received G-CSF alone (7.2 × 10(10) ± 2.0 × 10(10) vs. 5.7 × 10(10) ± 1.7 × 10(10) , p = 0.006). PBSC-GTX donors produced a remarkably high granulocyte yield (9.7 × 10(10) ± 2.3 × 10(10) ). The use of HES was associated with better granulocyte collection efficiency (42 ± 7.8% vs. 10 ± 9.1%, p < 0.0001). CONCLUSION: G-CSF plus dexamethasone produces higher granulocyte yields than G-CSF alone. Granulocyte collection from PBSC donors appears to be a rational strategy, since it produces high granulocyte yields when the related patients are at a high risk for infection and reduces difficulties in finding granulocyte donors. HES should be used in apheresis procedures.

Small cell osteosarcoma successfully treated by high-dose ifosfamide and methotrexate, combined with carboplatin and pirarubicin.

Small cell osteosarcoma (SCO) is the most rare subtype of osteosarcoma and has a poor prognosis. An 11-year-old boy presented with 2-month history of painful tumefaction in the lower leg. Imaging analysis demonstrated a mixture of osteolytic and osteosclerotic lesions in the proximal tibia and extraskeletal area. Histology of the open biopsy showed small round cells producing mucous matrix. Based on these findings, SCO was suspected. The patient received three cycles of neoadjuvant chemotherapy using high-dose ifosfamide, high-dose methotrexate, pirarubicin and carboplatin. Wide-margin resection was performed followed by tibial lengthening using the Ilizarov method and two cycles of adjuvant chemotherapy with the same drugs as for neoadjuvant chemotherapy. Histology of the resected specimen showed that almost all tumor cells were necrotized. Neither recurrence nor metastasis was found after 4 years. Our experience suggests that neoadjuvant chemotherapy, such as the one used here, would be exceedingly effective for SCO without serious non-hematological toxicities.

CHIP-dependent termination of MEKK2 regulates temporal ERK activation required for proper hyperosmotic response.

The extracellular signal-regulated kinase (ERK) pathway is an important signalling pathway that regulates a large number of cellular processes, including proliferation, differentiation and gene expression. Hyperosmotic stress activates the ERK pathway, whereas little is known about the regulatory mechanisms and physiological functions of ERK activation in hyperosmotic response. Here, we show that MAPK/ERK kinase kinase 2 (MEKK2), a member of the MAPKKK family, mediated the specific and transient activation of ERK, which was required for the induction of aquaporin 1 (AQP1) and AQP5 gene expression in response to hyperosmotic stress. Moreover, we identified the E3 ubiquitin ligase carboxyl terminus of Hsc70-interacting protein (CHIP) as a binding partner of MEKK2. Depletion of CHIP by small-interference RNA or gene targeting attenuated the degradation of MEKK2 and prolonged the ERK activity. Interestingly, hyperosmolality-induced gene expression of AQP1 and AQP5 was suppressed by CHIP depletion and was reversed by inhibition of the prolonged phase of ERK activity. These findings show that transient activation of the ERK pathway, which depends not only on MEKK2 activation, but also on CHIP-dependent MEKK2 degradation, is crucial for proper gene expression in hyperosmotic stress response.

Callosal and diffuse white matter lesions with restricted water diffusion in hemophagocytic syndrome.

We present magnetic resonance (MR) imaging findings of the brain of a 6-year-old girl with fatal hemophagocytic syndrome (HPS); diffusion-weighted imaging shows abnormal intensity in the white matter and entire corpus callosum. HPS is a rare disorder that affects the mononuclear phagocyte system and not uncommonly involves the central nervous system. Various MR imaging findings of HPS have been reported, but restricted water diffusion throughout the entire corpus callosum lesion and lesions of the white matter have not been reported.

[Hepatic arterial infusion of liposomal amphotericin B for multiple fungal abscesses in the liver of a patient with chronic granulomatous disease].

A 26-year-old man with chronic granulomatous disease complicated by multiple liver abscess was admitted to our hospital for hepatic resection and allogeneic bone marrow transplantation (BMT) from an HLA-matched sibling. We diagnosed the patient with Aspergillus liver abscesses based on computed tomographic findings, elevated serum levels of beta-D-glucan, positive test for galactomannan antigen, and the findings of laboratory cultures. Since the liver abscess could not be treated by drainage and administration of antifungals, we resected the posterior segments of the liver, which contained the abscess (S1, S6). However, abscess recurred in the remaining part of the liver 1 month later. The patient received allogeneic BMT from an HLA-matched sibling. During BMT, we continuously administered liposomal amphotericin B (L-AMB) via the hepatic artery (25 mg/day) to treat the liver abscess. There were no adverse effects during hepatic arterial infusion of L-AMB, and the liver abscess disappeared after BMT. These results suggest that hepatic arterial infusion of L-AMB is effective in treating fungal abscess in the liver.

Possible contribution of DNase gamma to immunoglobulin V gene diversification.

Somatic hypermutation (SHM) diversifies the rearranged immunoglobulin variable (V) region gene in B cells, contributing to affinity maturation of antibodies. It is believed that SHM is generated either by direct replication or by error-prone repair systems resolving V region DNA lesions caused directly or indirectly by cytidine deaminase AID. In accord with a part of these mechanisms, it was reported that SHM is associated with staggered double-strand DNA breaks (DSBs) occurring in the rearranged V regions. However, endonucleases responsible for the DSBs remain elusive. Here we show that DNase gamma, a member of DNase I family endonucleases, contributes to the generation of SHM including point mutation, and nucleotide insertion and deletion in chicken DT40 B cell line. DNase gamma also contributes to the generation of staggered DSBs in the rearranged V region. These results raise a possibility that DNase gamma is involved in the V gene mutation machinery.

Artificial lymph nodes induce potent secondary immune responses in naive and immunodeficient mice.

We previously demonstrated that artificial lymph nodes (aLNs) could be generated in mice by the implantation of stromal cell-embedded biocompatible scaffolds into their renal subcapsular spaces. T and B cell domains that form in aLNs have immune response functions similar to those of follicles of normal lymphoid tissue. In the present study, we show that the aLNs were transplantable to normal as well as SCID mice, where they efficiently induced secondary immune responses. Antigen-specific secondary responses were strongly induced in aLNs even 4 weeks after their transplantation. The antigen-specific antibody responses in lymphocyte-deficient SCID mice receiving transplanted aLNs were substantial. The cells from the aLNs migrated to the SCID mouse spleen and BM, where they expanded to generate large numbers of antigen-specific antibody-forming cells. Secondary responses were maintained over time after immunization (i.e., antigen challenge), indicating that aLNs can support the development of memory B cells and long-lived plasma cells. Memory CD4(+) T cells were enriched in the aLNs and spleens of aLN-transplanted SCID mice. Our results indicate that aLNs support strong antigen-specific secondary antibody responses in immunodeficient mice and suggest the possibility of future clinical applications.

Involvement of DNase gamma in the resected double-strand DNA breaks in immunoglobulin genes.

Somatic hypermutation (SHM) of immunoglobulin variable (V) region genes occurs in the germinal center (GC) B cells during immune responses, depending on activation-induced cytidine deaminase (AID). SHM is associated with resected double-strand DNA breaks (DSBs) which were shown to occur specifically in rearranged V regions in the GC B cells and CD40-stimulated B cells expressing AID. So far, endonucleases responsible for the DSBs have not been identified. Here we show that DNase gamma, a member of DNase I family of endonucleases, is expressed in GC B cells and CD40-stimulated B cells. Overexpression of DNase gamma in the mutation-competent Ramos B-cell line resulted in a marked increase in the resected but not blunt DSBs in the V region. Conversely, a selective DNase gamma inhibitor, DR396, suppressed the generation of the resected DSBs. These results suggest that DNase gamma is involved in the generation of resected DSBs associated with SHM.