Evaluation of Francisella tularensis ΔpdpC as a candidate live attenuated vaccine against respiratory challenge by a virulent SCHU P9 strain of Francisella tularensis in a C57BL/6J mouse model.

Francisella tularensis, which causes tularemia, is an intracellular gram-negative bacterium. F. tularensis has received significant attention in recent decades because of its history as a biological weapon. Thus, development of novel vaccines against tularemia has been an important goal. The attenuated F. tularensis strain ΔpdpC, in which the pathogenicity determinant protein C gene (pdpC) has been disrupted by TargeTron mutagenesis, was investigated as a potential vaccine candidate for tularemia in the present study. C57BL/6J mice immunized s.c. with 1 × 106 CFUs of ΔpdpC were challenged intranasally with 100× the median lethal dose (LD50 ) of a virulent SCHU P9 strain 21 days post immunization. Protection against this challenge was achieved in 38% of immunized C57BL/6J mice administered 100 LD50 of this strain. Conversely, all unimmunized mice succumbed to death 6 days post challenge. Survival rates were significantly higher in vaccinated than in unimmunized mice. In addition, ΔpdpC was passaged serially in mice to confirm its stable attenuation. Low bacterial loads persisted in mouse spleens during the first to tenth passages. No statistically significant changes in the number of CFUs were observed during in vivo passage of ΔpdpC. The inserted intron sequences for disrupting pdpC were completely maintained even after the tenth passage in mice. Considering the stable attenuation and intron sequences, it is suggested that ΔpdpC is a promising tularemia vaccine candidate.

Tolerance mechanisms of human-residential bifidobacteria against lysozyme.

We previously reported that lysozyme present in breast milk is a selection factor for bifidobacterial colonization in infant human intestines. This study is aimed at examining their underlying mechanisms. Human-residential bifidobacteria (HRB) generally exhibited higher tolerance than non-HRB to lysozymes, except B. bifidum subspecies. To assess the involvement of enzymatic activity of lysozyme, peptidoglycan (PG) was isolated and the degree of O-acetylation (O-Ac) in 19 strains, including both HRB and non-HRB, was determined. Variety in the degree of O-Ac was observed among each of the Bifidobacterium species; however, all purified PGs were found to be tolerant to lysozyme, independent of their O-Ac degree. In addition, De-O-Ac of PGs affected the sensitivity to lysozyme of only B. longum-derived PG. To examine the non-enzymatic antibacterial activity of lysozyme on bifidobacteria, lysozyme was heat-denatured. The HRB and non-HRB strains exhibited similar patterns of susceptibility to intact lysozyme as they did to heat-denatured lysozyme. In addition, strains of B. bifidum (30 strains), which showed various tolerance of lysozyme, also exhibited similar patterns of susceptibility to intact lysozyme as they did to heat-denatured lysozyme. These results suggest that bifidobacteria are resistant to the peptidoglycan-degrading property of lysozyme, and the tolerance to lysozyme among some HRB strains is due to resistance to the non-enzymatic antibacterial activity of lysozyme.

Virulence of representative Japanese Francisella tularensis and immunologic consequences of infection in mice.

Francisella tularensis, which causes tularemia, is widely distributed in the Northern hemisphere. F. tularensis strains isolated in Japan are genetically unique from non-Japanese strains; however, their phenotypic properties have not been well studied. Thus, mice were infected with representative Japanese strains of F. tularensis and their virulence and mouse immune responses to them assessed. Of four representative Japanese strains, the Ebina, Jap and Tsuchiya strains were susceptible to H2 O2 and did not grow well intracellularly. Only Yama strain grew intracellularly and was lethal to mice. Infection with Yama strain resulted in drastic increases in IFN-γ, CD4 and CD8 double-positive T cells and Th1 cells (CD3, CD4 and Tim3-positive cells), and a decrease in the ratio of CD8-positive CD4-negative T cells in mice. C57BL/6J mice that survived infection produced IgM antibodies to LPS and IgG2c antibodies to 43, 19 and 17 kDa proteinase K-sensitive components. These data are valuable for understanding the phenotypic properties of F. tularensis in Japan.

Long-term outcome of pediatric renal transplantation: a single center study in Japan.

Little is known about the risk factors for long-term poor outcome in pediatric renal transplantation. Between 1973 and 2010, 111 renal transplants (92 living donations) were performed in 104 children (56 males, mean age, 12.5 yr) at the Social Insurance Chukyo Hospital, and followed-up for a mean period of 13.6 yr. The patient survival at 1, 5, 10, 15, 20 (living- and deceased-donor transplants), and 30 yr (living-donor transplants only) was 98.1%, 92.8%, 87.8%, 84.9%, 82.6%, and 79.3%. The graft survival at 1, 5, 10, 15, 20, and 30 yr was 92.0%, 77.3%, 58.4%, 50.8%, 38.5%, and 33.3%. The most common cause of graft loss was CAI, AR, death with functioning, recurrent primary disease, ATN, and malignancy. Donor gender, ATN, malignancy/cardiovascular events, and eras affected patient survival. AR and CAI were the risk factors for graft loss. The evolved immunosuppression protocols improved the outcome by reducing AR episodes and ATN but not CAI, suggesting CAI as the major risk factor for graft loss. CAI was correlated with AR episodes, CMV infection, and post-transplant hypertension. Strategies for preventing the risk factors for malignancy/cardiovascular events and CAI, including hypertension/infection, are crucial for better outcomes.

Necl-2/CADM1 interacts with ErbB4 and regulates its activity in GABAergic neurons.

The neuronal network is tightly regulated by a large variety of locally connected GABAergic neurons. Neuregulin1 (Nrg1) and its receptor ErbB4 are master regulators in the morphological and functional development of excitatory synapses in GABAergic neurons. We previously showed that the immunoglobulin-like cell adhesion molecule, nectin-like molecule-2 (Necl-2)/CADM1, interacts with the ErbB3 and ErbB4 receptors, and that the interaction of Necl-2 with ErbB3 inhibits the Nrg1-induced ErbB3/ErbB2 signaling in epithelial cells. Here, we examined the role of the interaction of Necl-2 with ErbB4 in GABAergic neurons. Necl-2 was co-expressed with ErbB4 in parvalbumin-positive GABAergic neurons in the mouse hippocampus and co-localized with ErbB4 at excitatory synapses. Necl-2 knockdown enhanced the Nrg1-induced phosphorylation of ErbB4. Moreover, overexpression of PTPN13, which is a tyrosine phosphatase bound to the cytoplasmic tail of Necl-2, suppressed the Nrg1-induced development of excitatory synapses in GABAergic neurons through the inhibition of ErbB4 activity. These results indicate that Necl-2 interacts with ErbB4 and regulates the development of excitatory synapses via the regulation of ErbB4 activity in GABAergic neurons.

Antigen capture ELISA system for henipaviruses using polyclonal antibodies obtained by DNA immunization.

A novel antigen-capture sandwich ELISA system targeting the glycoproteins of the henipaviruses Nipah virus (NiV) and Hendra virus (HeV) was developed. Utilizing purified polyclonal antibodies derived from NiV glycoprotein-encoding DNA-immunized rabbits, we established a system that can detect the native antigenic structures of the henipavirus surface glycoproteins using simplified and inexpensive methods. The lowest detection limit against live viruses was achieved for NiV Bangladesh strain, 2.5 × 10(4) TCID(50). Considering the recent emergence of genetic variants of henipaviruses and the resultant problems that arise for PCR-based detection, this system could serve as an alternative rapid diagnostic and detection assay.

Age and gender may affect posterior reversible encephalopathy syndrome in renal disease.

It remains elusive what factors affect posterior reversible encephalopathy syndrome (PRES). Eleven PRES children, all with acute glomerulonephritis, Alport syndrome, and lupus nephritis, 5 with nephrosis, and 3 renal transplant recipients, were studied. PRES recurred in 1 patient. Neurological symptoms were graded as 1: mild (headache, nausea/vomiting, or tremor), 2: moderate (vision change), and 3: severe (mental dysfunction, cerebellar symptoms, seizures, recurrence of seizures, and coma). Magnetic resonance imaging was graded as 1: subtle change, 2: abnormal large areas, and 3: complete involvement of the regions. The common symptoms were seizures (100%), headache (82%), nausea/vomiting (73%), coma (55%), and vision change (46%). Seizures recurred in 7 (64%). All but one (91%) developed hypertension and 7 (64%) received calcineurin inhibitors (CNI). Edema occurred in 7 (64%) and renal insufficiency/end-stage renal disease (ESRD) in 4 (36%). Seizures recurred frequently in younger patients. Symptoms were severe in girls. Duration or severity of the condition with predisposing factors (hypertension, CNI, nephrosis or renal insufficiency/ERSD) did not make a difference in the symptoms and neuroimaging. Two patients developed chronic epilepsy. Age and gender may affect PRES symptoms. Our results are limited by small sample size and should be determined using larger numbers of patients.

Recurrent posterior reversible encephalopathy syndrome in nephrotic syndrome: case report and review of the literature.

Sequential neuroimaging before and after seizures has not been reported in patients with nephrotic syndrome and recurrent posterior reversible encephalopathy syndrome (PRES). We report a 13-year-old nephrotic female patient with recurrent PRES during and after cessation of cyclosporine A (CyA). She had headache, visual disturbance and acute hypertension, followed by seizures. The brain magnetic resonance imaging (MRI) before seizures revealed a high signal intensity area on fluid-attenuated inversion recovery and diffusion-weighted images and a low signal intensity area on T1-weighted images in bilateral parieto-occipital and left temporal lobes. Cessation of CyA resulted in clinical improvement. The follow-up MRI 2 months after the initial episode showed a complete resolution. Six months later, she had similar symptoms, edema, severe hypoalbuminemia, renal insufficiency, and acute pancreatitis before seizures. The brain MRI after seizures showed similar abnormalities in the same regions, which completely resolved 2 months later. Recurrence of acute severe hypertension, nephrotic state (edema/ hypoalbuminemia), and renal insufficiency may lead to recurrent PRES and thus early treatment of trigger factors, especially of hypertension, is important to reduce the episodes of PRES.

Fasciitis as a common lesion of dermatomyositis, demonstrated early after disease onset by en bloc biopsy combined with magnetic resonance imaging.

OBJECTIVE: To investigate whether fasciitis is histopathologically demonstrable in patients with dermatomyositis (DM), and to analyze the process of inflammatory progression in myopathy accompanying DM. METHODS: STIR or fat-suppressed T2-weighted magnetic resonance imaging (MRI) and en bloc biopsy were performed in 14 patients with newly diagnosed adult-onset DM. The severity of inflammatory cell infiltration around the fascial and intramuscular small blood vessels was evaluated using the total vascular inflammation score (TVIS). RESULTS: In all patients, MRI revealed abnormal hyperintensity in the fascia and in marginal sites of the muscle, predominantly over central sites. En bloc biopsy revealed the presence of fasciitis in most of the patients, as shown by inflammatory infiltrates around the fascial small blood vessels. In those patients who underwent en bloc biopsy earlier than 2 months after the appearance of muscle symptoms, the TVIS of the fascia was significantly higher than the TVIS of the muscle. In contrast, in those patients who underwent en bloc biopsy >2 months after muscle symptom onset, the TVIS of the fascia did not differ significantly from the TVIS of the muscle. CONCLUSION: Fasciitis was histopathologically demonstrated in patients with newly diagnosed adult-onset DM as early as 2 months after the onset of muscle symptoms. These results indicate that fasciitis is a common lesion of DM and suggest that the fascial microvasculature is the primary site of inflammatory cell infiltration in DM. Fasciitis may contribute to muscle symptoms in patients with DM without myositis.

Phylogenetic typing of Bacillus anthracis isolated in Japan by multiple locus variable-number tandem repeats and the comprehensive single nucleotide polymorphism.

Twelve strains of Bacillus anthracis isolated in Japan were subjected to multiple locus variable-number tandem repeats analysis using 25 marker loci (MLVA25). The results showed that Japanese strains could be divided into two distinct genetic clusters, A3a and A3b. By using newly devised comprehensive single nucleotide polymorphisms (SNPs) analysis, Japanese strains were also divided into two groups. The results obtained by the MLVA25 and plasmids SNP analysis well coincided, indicating that both methods were highly sensitive to discriminate B. anthracis strains. These results suggested that MLVA25 had sufficient discrimination power to identify B. anthracis at the strain level, and that MLVA25 as well as comprehensive SNPs analysis could facilitate further studies of B. anthracis strains including Japanese and other Asian strains.

Virulence of Francisella tularensis Subspecies holarctica Biovar japonica and Phenotypic Change during Serial Passages on Artificial Media.

Francisella tularensis (F. tularensis) is the etiological agent of the zoonotic disease tularemia. F. tularensis subspecies holarctica biovar japonica has rarely been isolated in Japan and is considered to have moderate virulence, although the biological properties of fresh isolates have not been analyzed in detail. Here, we analyzed the virulence of two strains of F. tularensis subspecies holarctica biovar japonica (NVF1 and KU-1) and their phenotypic stability during serial passages in Eugon chocolate agar (ECA) and Chamberlain's chemically defined medium (CDM) based agar (CDMA). C57BL/6 mice intradermally inoculated with 101 colony-forming units of NVF1 or KU-1 died within 9 days, with a median time to death of 7.5 and 7 days, respectively. Both NVF1 and KU-1 strains passaged on ECA 10 times had comparable virulence prior to passaging, whereas strains passaged on ECA 20 times and on CDMA 50 times were attenuated. Attenuated strains had decreased viability in 0.01% H2O2 and lower intracellular growth rates, suggesting both properties are important for F. tularensis virulence. Additionally, passage on ECA of the KU-1 strains altered lipopolysaccharide antigenicity and bacterial susceptibility to ß-lactam antibiotics. Our data demonstrate F. tularensis strain virulence in Japan and contribute to understanding phenotypic differences between natural and laboratory environments.

Interaction and localization of Necl-5 and PDGF receptor beta at the leading edges of moving NIH3T3 cells: Implications for directional cell movement.

It was previously shown that platelet-derived growth factor (PDGF) receptor physically and functionally interacts with integrin alpha(v)beta(3), effectively inducing cell movement. We previously showed that Necl-5, originally identified as a poliovirus receptor, interacts with integrin alpha(v)beta(3) and enhances its clustering and the formation of focal complexes at the leading edges of moving cells, resulting in an enhancement of cell movement. We showed here that Necl-5 additionally interacts with PDGF receptor in NIH3T3 cells and regulates the interaction between PDGF receptor and integrin alpha(v)beta(3), effectively inducing directional cell movement. PDGF receptor co-localized with Necl-5 and integrin alpha(v)beta(3) at peripheral ruffles over lamellipodia, which were formed at the leading edges of moving cells in response to PDGF, but not at the focal complexes under these ruffles, whereas Necl-5 and integrin alpha(v)beta(3) co-localized at these focal complexes. The clustering of these three molecules at peripheral ruffles required the activation of integrin alpha(v)beta(3) by vitronectin and the PDGF-induced activation of the small G protein Rac and subsequent re-organization of the actin cytoskeleton. These results indicate a key role of Necl-5 in directional cell movement by physically and functionally interacting with both integrin alpha(v)beta(3) and PDGF receptor.

Effect of cellular cholesterol depletion on rabies virus infection.

Although there are several reports on candidates for rabies virus (RABV) receptor, possible roles played by these receptor candidates in determination of highly neurotropic nature of RABV have not been well understood. Since these candidate receptors for RABV were reported to be frequently associated with cholesterol-rich microdomains characterized by lipid rafts and caveolae structures, we attempted to determine whether the disturbance of microdomains caused by the cholesterol depletion showed any effects on RABV infection. When the cellular cholesterol was depleted by methyl-beta-cyclodextrin (MBCD) treatment, increase in RABV adsorption and infection, but not multiplication rather than suppression was observed in both BHK-21 and HEp-2 cells. These effects exerted by MBCD treatment on RABV infection could be reversed by cholesterol reconstitution. These results suggest that RABV enters BHK-21 or HEp-2 cells through ports of entry other than those located on cholesterol-rich microdomains and raise the possibility that RABV uses different mechanisms to enter the non-neuronal cells.

Bloodmeal identification and detection of avian malaria parasite from mosquitoes (Diptera: Culicidae) inhabiting coastal areas of Tokyo Bay, Japan.

Bloodmeal identification and the detection of avian malaria parasite from mosquitoes (Diptera: Culicidae) were carried out by polymerase chain reaction-based methods for field samples collected in coastal areas of Tokyo Bay, Japan, from April to October 2007. The following seven mosquito species were collected: Aedes albopictus (Skuse), Culex pipiens pallens Coquillett, Culex pipiens form molestus Forskal, Culex tritaeniorhynchus Giles, Culex inatomii Kamimura & Wada, Culex bitaeniorhynchus Giles, and Lutzia vorax Edwards. Forty blood-fed mosquitoes were collected and 95% of bloodmeals of Cx. pipiens pallens were avian-derived, whereas only mammalian bloodmeals were identified for Ae. albopictus. Plasmodium DNA was amplified from 65% (15/23) of blood-fed Cx. pipiens pallens and unfed females of Cx. pipiens pallens and Cx. pipiens form molestus with a minimum infection rate of 29.9 and 13.5, respectively. One unfed female of Lt. vorax was also positive for Plasmodium parasites. Five genetically distinct lineages of Plasmodium were identified, with 0.21 to 5.86% sequence divergence. Rinshi-8, the most prevalent lineage at our study site, was identical to the published sequence of Plasmodium relictum-P5.

Survival of avian H5N1 influenza A viruses in Calliphora nigribarbis (Diptera: Calliphoridae).

In a previous study, the highly pathogenic avian influenza (HPAI) H5N1 viruses were isolated from blow flies collected at the Tamba Town of Kyoto prefecture during the outbreak period in March 2004. In this study, we carried out virus exposure experiments to investigate whether the H5N1 virus would survive in a blow fly, Calliphora nigribarbis. The virus exposure experiments showed that the H5N1 influenza virus was isolated from the crop and intestine of C. nigribarbis for at least 24 h, and the viruses remained viable with titers ranging from 0.5 to 4.63 TCID50. This result suggests that C. nigribarbis could possibly transport the H5N1 virus over a distance of 2 km, which is the distance they can migrate within 24 h.

Rapid detection of rabies virus by reverse transcription loop-mediated isothermal amplification.

In this study, reverse transcription loop-mediated isothermal amplification (RT-LAMP) was established which can detect 10(3) copies of viral RNA corresponding to approximately 5 fg of RNA. RT-LAMP with the Phil primer set designed according to the nucleotide sequences obtained from a Kyoto patient who contracted rabies in the Philippines was able to amplify all 16 street viral sequences derived from the Philippines. The specificity of RT-LAMP products was easily confirmed by digestion with RsaI restriction enzyme. The reaction of RT-LAMP could be completed within 1 h and could be conducted under isothermal conditions using a conventional water bath or heat blocks, indicating that RT-LAMP is ideal for the diagnosis of rabies in developing countries. Although further study is required to establish more universal RT-LAMP primers applicable to viruses from other regions or countries, the fast, easy, simple, sensitive and specific RT-LAMP method established here might be useful for rabies diagnosis and can facilitate studies of rabies epidemiology where rabies is enzootic, particularly in developing countries.

Elevation of Bombina variegata peptide 8 in mice with collagen-induced arthritis.

BACKGROUND: Bombina variegate peptide 8 (Bv8) is a small protein secreted by frog skin. Recently it has been shown to contribute to tumor angiogenesis in mouse model. The purpose of this study was to investigate Bv8 in mice with type II collagen-induced arthritis (CIA). METHODS: We induced CIA in male DBA/1J mice. The severity of arthritis was evaluated based on an arthritis score. RNA was extracted from the joint, and examined for Bv8 mRNA expression by RT-PCR and real-time RT-PCR. Synovial tissue and bone marrow were immunohistochemically examined using anti-Bv8 antibody. RESULTS: The level of Bv8 mRNA expression in the joint was below the detection limit in the control group, but was elevated in the CIA group, and was correlated with the arthritis score. In addition, an increase in Bv8-positive cells was observed in the synovium and bone marrow in the CIA group. CONCLUSION: Bv8 was elevated in the synovium and bone marrow of CIA mice, suggesting that Bv8 plays an important role in the pathogenesis of arthritis.

Prevalence of Leptospira spp. in the kidneys of wild boars and deer in Japan.

We surveyed the prevalence of Leptospira spp. from 2005 to 2008 in wild boars and deer in Japan using polymerase chain reaction. Leptospiral flaB was detected in the kidneys of wild boars (positive ratio, 15.2%; 22 of 145) from 9 prefectures and a deer (1.1%; 1 of 94) from 1 prefecture in Japan. There was no annual change in the prevalence of positive animals during the investigation period (chi-squared test, p=0.94) or in the prevalence in male and female wild boars in the 2007 to 2008 season (Fisher's exact test, P=0.45). The Leptospira species harbored by these animals were deduced to be L. interrogans (from 22 animals) and L. borgpetersenii (from 1 animal).

Evaluating the contact rate between companion dogs during dog walking and the practices towards potential cases of rabies among dog owners in Japan.

This study aimed to examine the habits of dog walking in Japan using an internet survey of insured dog owners. About 96.4% of the respondents (n = 1,151) reported that they would take their dogs for a walk and they most frequently walk their dogs once or twice a day (75.9%) for 30 min to 1 hr (83.1%). The probability of a companion dog having contact with other dogs during dog walking was estimated to be 0.83 (95% confidence interval [CI]: 0.81-0.85), and the associated daily contact rate was estimated using log-normal distribution with a mean of 2.73 (95% CI: 2.42-3.11) and a standard deviation (SD) of 6.39 (95% CI: 5.18-7.84). Multiple linear regression revealed that the contact rate is mainly influenced by the social behaviour of the owner and to a lesser degree by his/her demographic characteristics including the area of residence, the breed size of dog and the age of the owner. In addition, ten Likert items measured on a 5-point scale were designed to assess the practices towards potential cases of rabies among dog owners. The respondents (n = 972) achieved a mean score of 2.99 (out of a full score of 4) with a SD of 0.90 in responding to situations related to dog bite incidents and injury from stray cat scratches during dog walking. They achieved a higher score in responding to situations related to sighting a stray or wild animal during dog walking and situations related to non-specific clinical signs of rabies and bite injuries from stray dogs or wild animals during dog walking with a mean of 3.70 (SD = 0.58) and 3.84 (SD = 0.34), respectively. The level of best practice was also proved to be significantly associated with the demographic characteristics of the dog owner.