Norihisa Ishii

Oculocutaneous Albinism Type 4 Is One of the Most Common Types of Albinism in Japan

Oculocutaneous albinism (OCA) is a complex genetic disease with great clinical heterogeneity. Four different types of OCA have been reported to date (OCA1, OCA2, OCA3, and OCA4). MATP was recently reported in a single Turkish OCA patient as the fourth pathological gene, but no other patients with OCA4 have been reported. Here, we report the mutational profile of OCA4, determined by genetic analysis of the MATP gene in a large Japanese population with OCA. Of 75 unrelated patients that were screened, 18 individuals (24%) were identified as having OCA4; they harbored seven novel mutations, including four missense mutations (P58S, D157N, G188V, and V507L) and three frameshift mutations (S90CGGCCA-->GC, V144insAAGT, and V469delG), showing that MATP is the most frequent locus for tyrosinase-positive OCA in Japanese patients. We discuss the functional melanogenic activity of each mutant allele, judging from the relationship between the phenotypes and genotypes of the patients. This is the first report on a large group of patients with OCA4.

Predominance of type 2 cytokine–producing CD4+ and CD8+ cells in patients with atopic dermatitis

BACKGROUND Recently, increased IL-4 and decreased interferon-gamma (IFN-gamma) production in peripheral blood mononuclear cells (PBMCs) from patients with atopic dermatitis have been reported by several groups. They measured the total amount of each cytokine in culture supernatants in their studies. These studies suggested a predominance of type 2 cytokine-producing cells in atopic dermatitis. However, it is still unclear whether the cytokine imbalance is the result of an imbalance of specific T-cell subsets or of dysregulation of the cytokine-producing ability in T-cell subsets. Here, we examined frequencies of IL-4-, IFN-gamma-, or IL-2-producing CD4+ cells and CD8+ cells in PBMCs from patients with atopic dermatitis at a single cell level by using flow cytometry. METHODS PBMCs from 45 patients with atopic dermatitis and 24 healthy control subjects were stimulated with immobilized anti-CD3 monoclonal antibody for 6 hours in the presence of monensin. Cells were fixed, made permeable, and stained for intracellular cytokines in combination with staining for cell surface markers CD4 and CD8. RESULTS The frequency of IL-4-producing CD4+ cells and CD8+ cells from patients with atopic dermatitis was significantly higher (p < 0.001) than that from healthy control subjects. In contrast, the frequency of IFN-gamma-producing CD4+ cells and CD8+ cells was significantly decreased (p < 0.01) in the patients with atopic dermatitis. The frequency of IL-2-producing cells from patients with atopic dermatitis was comparable to that from healthy control subjects. CONCLUSION Our findings indicate that frequencies of type 2 cytokine-producing cells, not only among CD4+ cells but also among CD8+ cells, were significantly higher in patients with atopic dermatitis than in healthy control subjects.

Adjuvant formulations and delivery systems for DNA vaccines.

DNA vaccines have become a reliable and major means to elicit immune responses in the past decade. We and others have attempted to obtain stronger, more long lasting, and optimized immune responses, subsequent to the pioneering works demonstrating the ability of plasmid DNA to raise specific immune responses. Advances in molecular biology and biotechnology allow the application of various adjuvants, immunologic agents that increase the antigenic response, in DNA vaccines. Adjuvants can be broadly separated into two classes based on their origin-genetic and conventional. Genetic adjuvants are expression vectors of cytokines or other molecules that can modulate immune responses when administered with a vaccine antigen. Conventional adjuvants are chemical compounds that enhance, prolong, or modulate antigen-specific immune responses. The use of an appropriate adjuvant is pivotal in optimizing the response to DNA vaccines. Moreover, DNA vaccines themselves possess their own adjuvant activity because of the presence of unmethylated CpG motifs in particular base contents. The route of inoculation is also a critical factor in determining the outcome of vaccination. It is well known that intramuscular injection preferentially induces Th1-type immunity, whereas particle bombardment by gene gun predominantly induces Th2-type response. This article focuses on providing the detailed procedure to construct genetic adjuvant plasmids and prepare DNA vaccines formulated with conventional adjuvants. We also offer a practical guide for the procedure of intramuscular DNA injection.

Transcriptional Regulation of the Human TLR9 Gene

To clarify the molecular basis of human TLR9 (hTLR9) gene expression, the activity of the hTLR9 gene promoter was characterized using the human myeloma cell line RPMI 8226. Reporter gene analysis and EMSA demonstrated that hTLR9 gene transcription was regulated via four cis-acting elements, cAMP response element, 5′-PU box, 3′-PU box, and a C/EBP site, that interacted with the CREB1, Ets2, Elf1, Elk1, and C/EBPα transcription factors. Other members of the C/EBP family, such as C/EBPβ, C/EBPδ, and C/EBPε, were also important for TLR9 gene transcription. CpG DNA-mediated suppression of TLR9 gene transcription led to decreased binding of the trans-acting factors to their corresponding cis-acting elements. It appeared that suppression was mediated via c-Jun and NF-κB p65 and that cooperation among CREB1, Ets2, Elf1, Elk1, and C/EBPα culminated in maximal transcription of the TLR9 gene. These findings will help to elucidate the mechanism of TLR9 gene regulation and to provide insight into the process by which TLR9 evolved in the mammalian immune system.

Mycobacterium leprae and Leprosy: A Compendium

Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which was discovered by G.H.A. Hansen in 1873. M. leprae is an exceptional bacterium because of its long generation time and no growth in artificial media. Entire sequencing of the bacterial genome revealed numerous pseudogenes (inactive reading frames with functional counterparts in M. tuberculosis) which might be responsible for the very limited metabolic activity of M. leprae. The clinical demonstration of the disease is determined by the quality of host immune response. Th1‐type immune response helps to kill the bacteria, but hosts are encroached upon when Th2‐type response is predominant. The bacteria have affinity to the peripheral nerves and are likely to cause neuropathy. M. leprae/laminin‐α2 complexes bind to α/β dystroglycan complexes expressed on the Schwann cell surface. WHO recommends a chemotherapy protocol [multidrug therapy (MDT)] which effectively controls the disease and contributes to the global elimination program. Leprosy has been stigmatized throughout history, and recent topics regarding the disease in Japan are also discussed.

TRAF4 acts as a silencer in TLR-mediated signaling through the association with TRAF6 and TRIF.

Toll‐like receptors (TLR) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase play an essential role in intracellular eradication of engulfed pathogens. Here, we demonstrate the physical and functional association between components of the cytosolic NADPH oxidase and TLR‐mediated signaling molecules. Cytosolic components of NADPH oxidase suppressed TLR‐mediated NF‐κB activation as well as IFN‐β promoter activation. We demonstrate that TNF‐associated factor (TRAF) 4 associates with p47phox, a component of cytosolic NADPH oxidase, and physically interacts and functionally counteracts with TRAF6 and Toll‐IL‐1 receptor (TIR) domain‐containing adaptor‐inducing IFN‐β (TRIF) molecules that critically regulate TLR‐mediated signaling. TRAF4 mRNA expression was elicited in RPMI 8226 cells following LPS or CpG DNA treatment. These results suggest that TRAF4 participates in the molecular mechanism underlying silencing of TLR‐mediated signaling through the interaction with molecules harboring phagosome/endosome membrane.

Toll-Like Receptor Adaptor Molecules Enhance DNA-Raised Adaptive Immune Responses against Influenza and Tumors through Activation of Innate Immunity

ABSTRACT Toll-like receptors (TLRs) recognize microbial components and trigger the signaling cascade that activates the innate and adaptive immunity. TLR adaptor molecules play a central role in this cascade; thus, we hypothesized that overexpression of TLR adaptor molecules could mimic infection without any microbial components. Dual-promoter plasmids that carry an antigen and a TLR adaptor molecule such as the Toll-interleukin-1 receptor domain-containing adaptor-inducing beta interferon (TRIF) or myeloid differentiation factor 88 (MyD88) were constructed and administered to mice to determine if these molecules can act as an adjuvant. A DNA vaccine incorporated with the MyD88 genetic adjuvant enhanced antigen-specific humoral immune responses, whereas that with the TRIF genetic adjuvant enhanced cellular immune responses. Incorporating the TRIF genetic adjuvant in a DNA vaccine targeting the influenza HA antigen or the tumor-associated antigen E7 conferred superior protection. These results indicate that TLR adaptor molecules can bridge innate and adaptive immunity and potentiate the effects of DNA vaccines against virus infection and tumors.

Current status of leprosy: Epidemiology, basic science and clinical perspectives

Leprosy has affected humans for millennia and remains an important health problem worldwide, as evidenced by nearly 250 000 new cases detected every year. It is a chronic infectious disorder, caused by Mycobacterium leprae, that primarily affects the skin and peripheral nerves. Recent advances in basic science have improved our knowledge of the disease. Variation in the cellular immune response is the basis of a range of clinical manifestations. The introduction of multidrug therapy has significantly contributed to a decrease in the prevalence of the disease. However, leprosy control activities, including monitoring and prevention programs, must be maintained.

Mycolactone Is Responsible for the Painlessness of Mycobacterium ulcerans Infection (Buruli Ulcer) in a Murine Study

ABSTRACT Buruli ulcer is a chronic skin disease caused by Mycobacterium ulcerans, which produces a toxic lipid mycolactone. Despite the extensive necrosis and tissue damage, the lesions are painless. This absence of pain prevents patients from seeking early treatment and, as a result, many patients experience severe sequelae, including limb amputation. We have reported that mice inoculated with M. ulcerans show loss of pain sensation and nerve degeneration. However, the molecules responsible for the nerve damage have not been identified. In order to clarify whether mycolactone alone can induce nerve damage, mycolactone A/B was injected to footpads of BALB/c mice. A total of 100 μg of mycolactone induced footpad swelling, redness, and erosion. The von Frey sensory test showed hyperesthesia on day 7, recovery on day 21, and hypoesthesia on day 28. Histologically, the footpads showed epidermal erosion, moderate stromal edema, and moderate neutrophilic infiltration up to day 14, which gradually resolved. Nerve bundles showed intraneural hemorrhage, neutrophilic infiltration, and loss of Schwann cell nuclei on days 7 and 14. Ultrastructurally, vacuolar change of myelin started on day 14 and gradually subsided by day 42, but the density of myelinated fibers remained low. This study demonstrated that initial hyperesthesia is followed by sensory recovery and final hypoesthesia. Our present study suggests that mycolactone directly damages nerves and is responsible for the absence of pain characteristic of Buruli ulcer. Furthermore, mice injected with 200 μg of mycolactone showed pulmonary hemorrhage. This is the first study to demonstrate the systemic effects of mycolactone.

Whole-Genome Tiling Array Analysis of Mycobacterium leprae RNA Reveals High Expression of Pseudogenes and Noncoding Regions

Whole-genome sequence analysis of Mycobacterium leprae has revealed a limited number of protein-coding genes, with half of the genome composed of pseudogenes and noncoding regions. We previously showed that some M. leprae pseudogenes are transcribed at high levels and that their expression levels change following infection. In order to clarify the RNA expression profile of the M. leprae genome, a tiling array in which overlapping 60-mer probes cover the entire 3.3-Mbp genome was designed. The array was hybridized with M. leprae RNA from the SHR/NCrj-rnu nude rat, and the results were compared to results from an open reading frame array and confirmed by reverse transcription-PCR. RNA expression was detected from genes, pseudogenes, and noncoding regions. The signal intensities obtained from noncoding regions were higher than those from pseudogenes. Expressed noncoding regions include the M. leprae unique repetitive sequence RLEP and other sequences without any homology to known functional noncoding RNAs. Although the biological functions of RNA transcribed from M. leprae pseudogenes and noncoding regions are not known, RNA expression analysis will provide insights into the bacteriological significance of the species. In addition, our study suggests that M. leprae will be a useful model organism for the study of the molecular mechanism underlying the creation of pseudogenes and the role of microRNAs derived from noncoding regions.