Kiichi Kajino

BRCA1 binds c-Myc and inhibits its transcriptional and transforming activity in cells.

c-Myc, a proto-oncogene that is implicated in tumorigenesis, embryonic development and apoptosis, can physically associate with BRCA1. We have found that BRCA1 interacts with c-Myc in yeast, in in vitro assays and in mammalian cells. Endogenous interactions between BRCA1 and c-Myc were also observed. Efficient BRCA1-Myc association requires the intact helix–loop–helix region of c-Myc, a motif involved in Myc–Max dimerization. BRCA1 does not however bind to Max. Our studies revealed that BRCA1 represses Myc-mediated transcription while having no effect on some other transcriptional activities. Furthermore, BRCA1 reverses the phenotype of embryonic fibroblasts transformed by the activation of Myc and Ras, but only minimally affects the transformed phenotype induced by SV40 virus. These data indicate that BRCA1 may function as a tumor suppressor by regulating the behavior of c-Myc and provide a molecular explanation for some of the effects of the BRCA1 gene product.

Intranasal administration of a synthetic peptide vaccine encapsulated in liposome together with an anti-CD40 antibody induces protective immunity against influenza A virus in mice.

Mucosal immunity is critical for protection from viral infections. We attempted to activate mucosal cytotoxic T lymphocytes (CTLs) specific for influenza A virus nucleoprotein (NP) which play an important role in protective immunity. It has been shown that dendritic cells (DCs) activated by signaling via CD40-CD40 ligand (CD40L) interaction are required for the differentiation of naive CD8(+) T cells into antigen-specific CTLs in a non-mucosal environment. We herein inoculated mice intranasally with an anti-CD40 monoclonal antibody (anti-CD40 mAb) and NP366-374 peptide, corresponding to a CTL epitope on NP, encapsulated in liposome (liposomal NP366-374) to induce protective CTL responses against influenza A virus. Intranasal but not subcutaneous immunization with liposomal NP366-374 effectively induced mucosal immunity to reduce virus replication in the lung, suggesting that anti-CD40 mAb also functioned as a mucosal adjuvant. Interestingly, neither MHC class I- nor class II-deficient mice immunized intranasally with these materials were resistant to the infection. Since anti-CD40 mAb was considered to help replace CD4(+) T cells, another help of CD4(+) T cells are presumably required for the induction of CTL activity in the lung. This approach may prove promising for developing vaccines to induce mucosal CTL responses, and seems to highlight differences between mucosal and non-mucosal immunity.

VCP, a weak ATPase involved in multiple cellular events, interacts physically with BRCA1 in the nucleus of living cells.

BRCA1, a breast/ovarian cancer susceptibility gene, undergoes mutations in as many as 50% of familial breast tumors. Recent studies indicate that BRCA1 may be involved in DNA damage repair. Here, we demonstrate that the BRCA1 protein physically associates with valosin-containing protein (VCP), a member of the ATPases associated with a variety of cellular activities (AAA) superfamily. In vitro studies revealed that VCP, via its N- terminal region, binds to amino acid residues 303-625 in the BRCA1 protein. Although found predominantly in the cytoplasm and, less abundantly, in the nucleus, VCP can be translocated from the nucleus after stimulation with epidermal growth factor. Collectively, our results suggest that VCP, by binding to BRCA1, participates in the DNA damage-repair function as an ATP transporter, possibly facilitating the transcription-coupled repair.

Development of Loop-Mediated Isothermal Amplification (LAMP) assays for rapid detection of Ehrlichia ruminantium

BackgroundThe rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater, a potential zoonotic disease of ruminants transmitted by ticks of the genus Amblyomma. The disease is distributed in nearly all of sub-Saharan Africa and some islands of the Caribbean, from where it threatens the American mainland. This report describes the development of two different loop-mediated isothermal amplification (LAMP) assays for sensitive and specific detection of E. ruminantium.ResultsTwo sets of LAMP primers were designed from the pCS20 and sodB genes. The detection limits for each assay were 10 copies for pCS20 and 5 copies for sodB, which is at least 10 times higher than that of the conventional pCS20 PCR assay. DNA amplification was completed within 60 min. The assays detected 16 different isolates of E. ruminantium from geographically distinct countries as well as two attenuated vaccine isolates. No cross-reaction was observed with genetically related Rickettsiales, including zoonotic Ehrlichia species from the USA. LAMP detected more positive samples than conventional PCR but less than real-time PCR, when tested with field samples collected in sub-Saharan countries.ConclusionsDue to its simplicity and specificity, LAMP has the potential for use in resource-poor settings and also for active screening of E. ruminantium in both heartwater-endemic areas and regions that are at risk of contracting the disease.

Direct blood dry LAMP: a rapid, stable, and easy diagnostic tool for Human African Trypanosomiasis.

Loop-mediated isothermal amplification (LAMP) is a rapid and sensitive tool used for the diagnosis of a variety of infectious diseases. One of the advantages of this method over the polymerase chain reaction is that DNA amplification occurs at a constant temperature, usually between 60–65°C; therefore, expensive devices are unnecessary for this step. However, LAMP still requires complicated sample preparation steps and a well-equipped laboratory to produce reliable and reproducible results, which limits its use in resource-poor laboratories in most developing countries. In this study, we made several substantial modifications to the technique to carry out on-site diagnosis of Human African Trypanosomiasis (HAT) in remote areas using LAMP. The first essential improvement was that LAMP reagents were dried and stabilized in a single tube by incorporating trehalose as a cryoprotectant to prolong shelf life at ambient temperature. The second technical improvement was achieved by simplifying the sample preparation step so that DNA or RNA could be amplified directly from detergent-lysed blood samples. With these modifications, diagnosis of HAT in local clinics or villages in endemic areas becomes a reality, which could greatly impact on the application of diagnosis not only for HAT but also for other tropical diseases.

Involvement of IL-10 in exhaustion of myeloid dendritic cells and rescue by CD40 stimulation

It has recently been shown that immature dendritic cells (DCs) stimulated by a danger signal undergo transient maturation followed by exhaustion. However, the exact mechanism for this has not been elucidated. In this study, we show that interleukin‐10 (IL‐10) secreted from transiently matured DCs stimulated by danger signals is responsible for this rapid DC exhaustion. Blocking of the autocrine IL‐10 enabled transient mature DCs to maintain the mature phenotype for several days. However, these DCs remained phenotypically unstable because the addition of IL‐10 altered the transient mature DCs to exhausted DCs. More importantly, stimulation of DCs by CD40 protected transient mature DCs from IL‐10‐dependent exhaustion, with the result that mature DCs remained stable in the presence of IL‐10. Furthermore, in vivo administration of stable mature DCs pulsed with ovalbumin protein induced antigen‐specific cytotoxic T lymphocytes (CTLs) effectively, whereas neither exhausted DCs nor transient mature DCs were able to prime a strong antigen‐specific CTL response. These results indicate that DC−T cell engagement via CD40−CD154 is required for stable DC maturation leading to effective CTL induction. Otherwise, DCs stimulated solely by a danger signal are temporarily activated, but then rapidly lose their immune‐activating capacity under the influence of autocrine IL‐10.

Cross-Protective Peptide Vaccine against Influenza A Viruses Developed in HLA-A*2402 Human Immunity Model

Background The virus-specific cytotoxic T lymphocyte (CTL) induction is an important target for the development of a broadly protective human influenza vaccine, since most CTL epitopes are found on internal viral proteins and relatively conserved. In this study, the possibility of developing a strain/subtype-independent human influenza vaccine was explored by taking a bioinformatics approach to establish an immunogenic HLA-A24 restricted CTL epitope screening system in HLA-transgenic mice. Methodology/Principal Findings HLA-A24 restricted CTL epitope peptides derived from internal proteins of the H5N1 highly pathogenic avian influenza A virus were predicted by CTL epitope peptide prediction programs. Of 35 predicted peptides, six peptides exhibited remarkable cytotoxic activity in vivo. More than half of the mice which were subcutaneously vaccinated with the three most immunogenic and highly conserved epitopes among three different influenza A virus subtypes (H1N1, H3N2 and H5N1) survived lethal influenza virus challenge during both effector and memory CTL phases. Furthermore, mice that were intranasally vaccinated with these peptides remained free of clinical signs after lethal virus challenge during the effector phase. Conclusions/Significance This CTL epitope peptide selection system can be used as an effective tool for the development of a cross-protective human influenza vaccine. Furthermore this vaccine strategy can be applicable to the development of all intracellular pathogens vaccines to induce epitope-specific CTL that effectively eliminate infected cells.

Evaluation of Echinococcus multilocularis tetraspanins as vaccine candidates against primary alveolar echinococcosis.

Echinococcus multilocularis causes an important zoonotic cestode disease. The metacestode stage proliferates in the liver of intermediate hosts including human and rodents and forms multiple cysts. Recently, members of a transmembrane protein tetraspanin (TSP) family have been used as vaccines against schistosomosis, or as diagnostic antigens for cysticercosis. In this study, seven tetraspanins of E. multilocularis, designated as TSP1 to TSP7, were evaluated for their protective potential against primary alveolar echinococcosis. The large extracellular loop (LEL) region of these tetraspanins was cloned from a full-length enriched cDNA library of E. multilocularis metacestodes and expressed in Escherichia coli as a fusion protein with thioredoxin. Recombinant TSPs were applied as vaccines against an E. multilocularis primary experimental infection in BALB/c mice. Cyst lesions in the livers of vaccinated and non-vaccinated mice were counted. The cyst lesion reduction rates induced by the seven tetraspanins in vaccinated vis-à-vis non-vaccinated mice were: 87.9%, 65.8%, 85.1%, 66.9%, 73.7%, 72.9% and 37.6%. Vaccination conferred protective rates to mice ranging from 0% (TSP5, 6, 7) to maximally 33% (TSP1, 3). The results indicated that recombinant tetraspanins have varying protective effects against primary alveolar echinococcosis and could be used in vaccine development.

The use of Loop-mediated Isothermal Amplification (LAMP) to detect the re-emerging Human African Trypanosomiasis (HAT) in the Luangwa and Zambezi valleys

BackgroundLoop-mediated isothermal amplification (LAMP) is a novel strategy which amplifies DNA with high sensitivity and rapidity under isothermal conditions. In the present study, the performance of the repetitive insertion mobile element (RIME)-LAMP and human serum resistance-associated gene (SRA)-LAMP assays were evaluated using clinical specimens obtained from four male patients from Luangwa and Zambezi valleys in Zambia and Zimbabwe, respectively.FindingsThe cases reported in this preliminary communication were all first diagnosed by microscopy, through passive surveillance, and confirmed by both RIME-LAMP and SRA-LAMP. A good correlation between microscopy and LAMP was observed and contributed to staging and successful treatment of patient. RIME-LAMP and SRA-LAMP complimented each other well in all the cases.ConclusionsBoth RIME-LAMP and SRA-LAMP were able to detect Trypanosoma brucei rhodesiense DNA in patient blood and CSF and hence confirmed HAT in the parasitaemic patients. Our study indicates that the LAMP technique is a potential tool for HAT diagnosis, staging and may be useful for making therapeutic decisions. However, no statistically significant conclusion may be drawn due to the limited sample size used in the present study. It is thus imperative to conduct a detailed study to further evaluate the potential of LAMP as a bedside diagnostic test for HAT.

Relationship of p215BRCA1 to tyrosine kinase signaling pathways and the cell cycle in normal and transformed cells.

We have analysed the relationship of the products of two genes, neu and BRCA1, known to be important in human breast cancer. Highly specific antibodies that recognized both the rodent and human form of the BRCA1 gene product (Mr 215 kDa, p215BRCA1) were developed to facilitate these efforts. p215BRCA1 was identified as a tyrosine phosphorylated protein primarily localized in the nucleus of several breast cancer cell lines. In transformed murine and human cells, levels of p215BRCA1 tyrosine phosphorylation were inversely correlated with the activity of the erbB family receptor-tyrosine-kinases and with the transformed growth features of these cells. Regulation of p215BRCA1 tyrosine phosphorylation was also related to events in the cell cycle. Increased levels of p215BRCA1 phosphotyrosine content were observed in NIH3T3 cells arrested at the G2/M transition. These findings indicate that the products of BRCA1, neu, and erbB breast cancer genes participate in a common or shared signaling pathway important in cell growth and its regulation.