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1.  Role of Pathogenicity Determinant Protein C (PdpC) in Determining the Virulence of the Francisella tularensis Subspecies tularensis SCHU 
PLoS ONE  2014;9(2):e89075.
Francisella tularensis subspecies tularensis, the etiological agent of tularemia, is highly pathogenic to humans and animals. However, the SCHU strain of F. tularensis SCHU P0 maintained by passaging in artificial media has been found to be attenuated. To better understand the molecular mechanisms behind the pathogenicity of F. tularensis SCHU, we attempted to isolate virulent bacteria by serial passages in mice. SCHU P5 obtained after 5th passages in mice remained avirulent, while SCHU P9 obtained after 9th passages was completely virulent in mice. Moreover, SCHU P9 grew more efficiently in J774.1 murine macrophages compared with that in the less pathogenic SCHU P0 and P5. Comparison of the nucleotide sequences of the whole genomes of SCHU P0, P5, and P9 revealed only 1 nucleotide difference among P0, P5 and P9 in 1 of the 2 copies of pathogenicity determinant protein C (pdpC) gene. An adenine residue deletion was observed in the pdpC1 gene of SCHU P0, P5, and P9 and in the pdpC2 gene of SCHU P0, and P5, while P9 was characterized by the wild type pdpC2 gene. Thus, SCHU P0 and P5 expressed only truncated forms of PdpC protein, while SCHU P9 expressed both wild type and truncated versions. To validate the pathogenicity of PdpC, both copies of the pdpC gene in SCHU P9 have been inactivated by Targetron mutagenesis. SCHU P9 mutants with inactivated pdpC gene showed low intracellular growth in J774.1 cells and did not induce severe disease in experimentally infected mice, while virulence of the mutants was restored by complementation with expression of the intact PdpC. These results demonstrate that PdpC is crucial in determining the virulence of F. tularensis SCHU.
PMCID: PMC3928404  PMID: 24558472
2.  Detection of Francisella tularensis-Specific Antibodies in Patients with Tularemia by a Novel Competitive Enzyme-Linked Immunosorbent Assay 
A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R2 = 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species.
PMCID: PMC3535769  PMID: 23114700
3.  Altered specificity of single-chain antibody fragments bound to pandemic H1N1-2009 influenza virus after conversion of the phage-bound to the soluble form 
BMC Research Notes  2012;5:483.
In 2009, a novel influenza A/H1N1 virus (H1N1pdm) quickly spread worldwide and co-circulated with then-existing seasonal H1N1 virus (sH1N1). Distinguishing between these 2 viruses was necessary to better characterize the epidemiological properties of the emergent virus, including transmission patterns, pathogenesis, and anti-influenza drug resistance. This situation prompted us to develop a point-of-care virus differentiation system before entering the 2009–2010 influenza season. Aiming to establish H1N1pdm-specific detection tools rapidly, we employed phage display libraries to select H1N1pdm-specific single-chain variable fragments (scFvs).
Human single-fold scFv libraries (Tomlinson I + J) underwent selection for the ability to bind H1N1pdm virus particles. Three rounds of panning brought 1152 phage-bound scFvs, of which 58 clones reacted with H1N1pdm specifically or preferentially over sH1N1 in an enzyme-linked immunosorbent assay (ELISA). After conversion of the scFvs to soluble form, 7 clones demonstrating high/stable expression were finally obtained. However, all the soluble scFvs except No. 29 were found to have lost their specificity/preference for H1N1pdm in ELISA. The specificity/preference of No. 29 was also confirmed by immunofluorescence assay and immunoprecipitation, and the viral nucleoprotein was identified by ELISA as its target protein. The change in specificity associated with scFv conversion from phage-bound to soluble form could be due to loss of phage scaffold pIII protein, which likely provides structural support for the scFv antigen-binding site. It is also possible that the similar antigenic properties of H1N1pdm and sH1N1 led to the observed alterations in scFv specificity.
Using a phage display library, we obtained 7 soluble scFv clones reactive against H1N1pdm; however, only 1 showed specificity/preference toward H1N1pdm. Our results confirmed that using phage display libraries was highly advantageous for the rapid development of molecules to detect target antigens. However, our results also indicated that this strategy might not have been effective for selecting H1N1pdm-specific antibodies during the 2009 pandemic, where the co-circulating sH1N1 virus shared similar antigenic properties. This suggests that it might be advisable to use a synthetic scFv phage display library by strategically considering the characteristics of target antigens and the potential situations.
PMCID: PMC3492028  PMID: 22943792
Influenza; Pandemic; Diagnosis; Single-chain variable fragment (scFv); Altered specificity
4.  Pleiotropic role of the Sco1/SenC family copper chaperone in the physiology of Streptomyces 
Microbial biotechnology  2012;5(4):477-488.
Antibiotic production and cell differentiation in Streptomyces is stimulated by micromolar levels of Cu2+. Here, we knocked out the Sco1/SenC family copper chaperone (ScoC) encoded in the conserved gene cluster ‘sco’ (the S treptomycescopper utilization) in Streptomyces coelicolor A3(2) and S. griseus. It is known that the Sco1/SenC family incorporates Cu2+ into the active centre of cytochrome oxidase (cox). The knockout caused a marked delay in antibiotic production and aerial mycelium formation on solid medium, temporal pH decline in glucose‐containing liquid medium, and significant reduction of cox activity in S. coelicolor. The scoC mutant produced two‐ to threefold higher cellular mass of the wild type exhibiting a marked cox activity in liquid medium supplied with 10 µM CuSO4, suggesting that ScoC is involved in not only the construction but also the deactivation of cox. The scoC mutant was defective in the monoamine oxidase activity responsible for cell aggregation and sedimentation. These features were similarly observed with regard to the scoC mutant of S. griseus. The scoC mutant of S. griseus was also defective in the extracellular activity oxidizing N,N′‐dimethyl‐p‐phenylenediamine sulfate. Addition of 10 µM CuSO4 repressed the activity of the conserved promoter preceding scoA and caused phenylalanine auxotrophy in some Streptomyces spp. probably because of the repression of pheA; pheA encodes prephenate dehydratase, which is located at the 3′ terminus of the putative operon structure. Overall, the evidence indicates that Sco is crucial for the utilization of copper under a low‐copper condition and for the activation of the multiple Cu2+‐containing oxidases that play divergent roles in the complex physiology of Streptomyces.
PMCID: PMC3815325  PMID: 22117562
5.  Autoimmunity to citrullinated type II collagen in rheumatoid arthritis 
Modern Rheumatology  2006;16(5):276-281.
The production of autoantibodies to citrullinated type II collagen and the citrullination of type II collagen were analyzed in rheumatoid arthritis. Autoantibodies to citrullinated type II collagen were detected in 78.5% of serum samples from 130 rheumatoid arthritis patients. Autoantibodies to native noncitrullinated type II collagen were detected in 14.6% of serum samples, all of which were positive for anti-citrullinated type II collagen antibodies. Serum samples were also positive for anti-citrullinated type II collagen antibodies in 1 of 31 systemic lupus erythematosus patients and 2 of 55 patients with osteoarthritis of the knee. In contrast, sera samples from 24 systemic sclerosis patients, 21 dermatomyositis/polymyositis patients, 21 ankylosing spondylitis patients, and 18 psoriatic arthritis patients were all negative for anti-citrullinated type II collagen antibodies. Anti-citrullinated type II collagen antibodies and fragments of citrullinated type II collagen were found in the synovial fluid obtained from affected knee joints of 15 rheumatoid arthritis patients. Moreover, anti-citrullinated type II collagen antibodies were isolated from the synovium of affected knee joints in 8 rheumatoid arthritis patients using antigen/antibody immunocomplex dissociation buffer but not by using standard buffers. These findings indicate that autoantibodies that react with citrullinated type II collagen are specifically produced and that immunocomplexes composed of fragments of citrullinated type II collagen and autoantibodies are deposited in the inflamed articular synovium in rheumatoid arthritis patients. Assaying for the presence of anti-citrullinated type II collagen antibodies may therefore be useful for diagnosing rheumatoid arthritis, and the deposition of these immunocomplexes in the articular synovium may be involved in pathogenesis.
PMCID: PMC2780673  PMID: 17039307
Autoantibody; Citrullination; Collagen type II; Rheumatoid arthritis (RA)
6.  Elevation of Bombina variegata peptide 8 in mice with collagen-induced arthritis 
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).
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.
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.
Bv8 was elevated in the synovium and bone marrow of CIA mice, suggesting that Bv8 plays an important role in the pathogenesis of arthritis.
PMCID: PMC2687415  PMID: 19405944
7.  Preparation of Monoclonal Antibodies for Detection and Identification of Francisella tularensis▿  
Monoclonal antibodies (MAbs) against Francisella tularensis were obtained. Three MAbs specifically reacted with F. tularensis, while four MAbs reacted with other members of the genus Francisella as well. Fluorescent isothiocyanate-conjugated MAbs unequivocally stained bacterial cells in specimens from experimentally infected mice. Two MAbs agglutinated F. tularensis antigen in the agglutination tests. These MAbs should improve methods for detection and identification of F. tularensis.
PMCID: PMC1797713  PMID: 17121981
8.  Detection of B Virus Antibody in Monkey Sera Using Glycoprotein D Expressed in Mammalian Cells 
Journal of Clinical Microbiology  2001;39(9):3025-3030.
The gene encoding glycoprotein D (gD) of the monkey B virus (Cercopithecine herpesvirus 1) was cloned into a mammalian expression vector, pcDNA3.1(−), and the recombinant plasmid DNA was transfected into COS7 cells. The expression of gD in transfected COS7 cells was detected by indirect immunofluorescence assay or radioimmunoprecipitation analysis (RIPA). Although the expressed gD protein was revealed to react well with sera from monkeys naturally infected with B virus by RIPA, some sera showed reduced reactivity when analyzed by the Western blotting (WB) method. Some sera also showed relatively high background when the WB was performed using gD expressed from recombinant plasmid. The mutant gD protein lacking the transmembrane domain (TM) and cytoplasmic tail (CT) was next expressed in COS7 cells. The mutant protein was secreted into culture medium without apparent loss of the antigenicity. Using the secretory form of the gD protein as antigen in dot blot analysis, sera from B virus-infected monkeys were shown to react with the mutant protein without nonspecific reaction. Since the recombinant gD or its derivative lacking TM and CT could be expressed in mammalian cells with proper antigenicity, these antigens appeared to be useful for serological detection of B virus infection in monkeys.
PMCID: PMC88291  PMID: 11526123
9.  Identification of a Novel GC-Rich 113-Nucleotide Region To Complete the Circular, Single-Stranded DNA Genome of TT Virus, the First Human Circovirus 
Journal of Virology  1999;73(5):3582-3586.
The sequence data (H. Okamoto et al., Hepatol. Res. 10:1–16, 1998) of a newly discovered single-stranded DNA virus, TT virus (TTV), showed that it did not have the terminal structure typical of a parvovirus. Elucidation of the complete genome structure was necessary to understand the nature of TTV. We obtained a 1.0-kb amplified product from serum samples of four TTV carriers by an inverted, nested long PCR targeted for nucleotides (nt) 3025 to 3739 and 1 to 216 of TTV. The sequence of a clone obtained from serum sample TA278 was compared with those registered in GenBank. The complete circular TTV genome contained a novel sequence of 113 nt (nt 3740 to 3852 [=0]) in between the known 3′- and 5′-end arms, forming a 117-nt GC-rich stretch (GC content, 90.6% at nt 3736 to 3852). We found a 36-nt stretch (nt 3816 to 3851) with an 80.6% similarity to chicken anemia virus (CAV) (nt 2237 to 2272 of M55918), a vertebrate circovirus. A putative SP-1 site was located at nt 3834 to 3839, followed by a TATA box at nt 85 to 90, the first initiation codon of a putative VP2 at nt 107 to 109, the termination codon of a putative VP1 at nt 2899 to 2901, and a poly(A) signal at nt 3073 to 3078. The arrangement was similar to that of CAV. Furthermore, several AP-2 and ATF/CREB binding sites and an NF-κB site were arranged around the GC-rich region in both TTV and CAV. The data suggested that TTV is circular and similar to CAV in its genomic organization, implying that TTV is the first human circovirus.
PMCID: PMC104131  PMID: 10196248

Results 1-9 (9)