Noritoshi Kitamoto

Cross-Reactivity among Several Recombinant Calicivirus Virus-Like Particles (VLPs) with Monoclonal Antibodies Obtained from Mice Immunized Orally with One Type of VLP

ABSTRACT Human caliciviruses (HuCVs) are classified into the Norwalk-like viruses (NLV) and Sapporo-like viruses (SLV) as genera within the family Caliciviridae. The NLV genus is further classified into genogroups I and II, based on sequence similarities. To study the antigenic determinants on the HuCV capsid protein and develop new diagnostic tools for field samples, we established and characterized monoclonal antibodies (MAbs) against baculovirus-expressed recombinant HuCV virus-like particles (VLPs). Hybrid clones producing MAbs were obtained from cultures of PAI myeloma cells fused with spleen or mesenteric lymph node cells from mice immunized orally with either a single type of recombinant Norwalk virus (rNV), Kashiwa 47 virus (rKAV), Snow Mountain agent (rSMA), or Sapporo virus (rSV) VLP or with mixtures of two types of VLPs from different genogroups. Twenty MAbs, obtained as mouse ascites, were characterized and classified into six groups according to their enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB) cross-reactivity patterns to VLPs. Five groups of MAbs reacted by both WB and ELISA and were classified as follows: common cross-reactive MAbs for four genogroup I and six genogroup II VLPs (group A), genogroup I-specific MAbs (group B), genogroup II-specific MAbs (group C), and strain-specific MAbs (groups D and E). One MAb group (group F) reacted only by ELISA. The group A MAbs, which showed broad cross-reactivity with VLPs of both NLV genogroups, were obtained from mice immunized orally with a single type of VLP (either rNV or rKAV). Two MAbs, which were obtained from mice immunized with rSV, reacted with rSV but not with any NLV VLP. These are the first MAbs to be reported for any SLV. These strain-, genogroup-, and genus-reactive MAbs will be useful tools for further study of the antigenic and structural topography of the HuCV virion and for diagnostic assays for HuCVs.

Identification of Genogroup I and Genogroup II Broadly Reactive Epitopes on the Norovirus Capsid

ABSTRACT Norwalk virus, a member of the family Caliciviridae, is an important cause of acute epidemic nonbacterial gastroenteritis. Norwalk and related viruses are classified in a separate genus of Caliciviridae called Norovirus, which is comprised of at least three genogroups based on sequence differences. Many of the currently available immunologic reagents used to study these viruses are type specific, which limits the identification of antigenically distinct viruses in detection assays. Identification of type-specific and cross-reactive epitopes is essential for designing broadly cross-reactive diagnostic assays and dissecting the immune response to calicivirus infection. To address this, we have mapped the epitopes on the norovirus capsid protein for both a genogroup I-cross-reactive monoclonal antibody and a genogroup II-cross-reactive monoclonal antibody by use of norovirus deletion and point mutants. The epitopes for both monoclonal antibodies mapped to the C-terminal P1 subdomain of the capsid protein. Although the genogroup I-cross-reactive monoclonal antibody was previously believed to recognize a linear epitope, our results indicate that a conformational component of the epitope explains the monoclonal antibodys genogroup specificity. Identification of the epitopes for these monoclonal antibodies is of significance, as they are components in a commercially available norovirus-diagnostic enzyme-linked immunosorbent assay.

The hydrogen peroxide/copper ion system, but not other metal-catalyzed oxidation systems, produces protein-bound dityrosine

Dityrosine formation leads to the cross-linking of proteins intra- or intermolecularly. The formation of dityrosine in lens proteins oxidized by metal-catalyzed oxidation (MCO) systems was estimated by chemical and immunochemical methods. Among the four MCO systems examined (H(2)O(2)/Cu, H(2)O(2)/Fe-ethylenediaminetetraacetic acid (Fe-EDTA), ascorbate/Cu, ascorbate/Fe-EDTA), the treatment with H(2)O(2)/Cu preferentially caused dityrosine formation in the lens proteins. The success of oxidative protein modification with all the MCO systems was confirmed by carbonyl formation estimated using 2,4-dinitrophenylhydrazine. The loss of tyrosine by the MCO systems was partly due to the formation of protein-bound 3,4-dihydroxyphenylalanine. The formation of dityrosine specific to H(2)O(2)/Cu was confirmed by using poly-(Glu, Ala, Tyr) and N-acetyl-tyrosine as a substrate. The dissolved oxygen concentration in the H(2)O(2)/Cu system hardly affected the amount of dityrosine formation, suggesting that dityrosine generation by the H(2)O(2)/Cu system is independent of oxygen concentration. Moreover, the combination of copper ion with H(2)O(2) is the most effective system for dityrosine formation among various metal ions examined. The addition of reducing agents, glutathione or ascorbic acid, into the H(2)O(2)/Cu system suppressed the generation of the dityrosine moiety, suggesting effective quench of tyrosyl radicals by the reducing agents.

Covalent modification of lysine residues by allyl isothiocyanate in physiological conditions: plausible transformation of isothiocyanate from thiol to amine.

We investigated the reactivity of allyl isothiocyanate (AITC) with amino groups under physiological conditions. First, the chemical reaction of AITC with bovine serum albumin (BSA) was investigated. When BSA was incubated with AITC in a phosphate buffer (pH 7.4), the loss of Lys residues was observed. Second, the Lys residue N(alpha)-benzoyl-glycyl-L-lysine (BGK) was reacted with AITC in the buffer, and a novel peak was detected using high performance liquid chromatography (HPLC). The peak was purified and identified as AITC-modified BGK with a N(epsilon)-thiocarbamoyl linkage. However, a thiol residue is known to be a predominant target of an isothiocyanate (ITC). Although AITC may react with a thiol moiety in vivo, a thiocarbamoyl linkage between ITC and thiol is unstable, and an AITC molecule may be regenerated. To prove the plausible transformation of ITC from thiol to amine, synthetic AITC-conjugated N(alpha)-acetyl-L-cysteine (NAC) was incubated with BGK at 37 degrees C in physiological buffer, and the generation of AITC-Lys was analyzed. The loss of the AITC-NAC adduct corresponded to the formation of the AITC-BGK adduct. Furthermore, using a novel monoclonal antibody (A4C7mAb) specific for AITC-Lys, we found that the AITC-Lys residue was generated from the reaction between AITC-NAC and BSA. Although AITC preferentially reacts with thiol rather than with Lys, AITC can be liberated from thiols and can then react with amino groups. The ITC-Lys adduct may be a useful marker for ITC target molecules.

Scavenger Receptor Collectin Placenta 1 (CL-P1) Predominantly Mediates Zymosan Phagocytosis by Human Vascular Endothelial Cells

Collectin placenta 1 (CL-P1), a recently discovered scavenger receptor, mediates the uptake of oxidized low density lipoprotein and microbes. In this study, we investigated CL-P1-mediated binding and ingestion of yeast-derived zymosan bioparticles using Chinese hamster ovary (CHO) cells stably expressing human CL-P1 (CHO/CL-P1) and human vascular endothelial cells constitutively expressed CL-P1. The uptake of zymosan by CHO/CL-P1 was dependent upon the level of CL-P1 expressed on the membrane and was inhibited by cytochalasin D and wortmannin. The binding of zymosan was also inhibited by ligands of other scavenger receptors such as poly(I) and dextran sulfate. Real time reverse transcription-PCR analyses showed that other scavenger receptors, namely LOX-1, Stabilin-2, or macrophage receptor with collagenous structure (MARCO), were not expressed in human umbilical vein endothelial cells isolated from different individuals. Nonopsonic zymosan ingestion was inhibited in three primary cultured vascular endothelial cells, including different human umbilical vein endothelial cells from nine individuals treated with CL-P1 small interfering RNAs, although small interfering RNAs of other scavenger receptors had no effect on zymosan uptake in these cells. Furthermore, we confirmed that CL-P1 is expressed in human and murine vascular endothelial layers. Our results demonstrated that CL-P1 predominantly mediated phagocytosis for fungi in vascular endothelia.

Detection of immunoreactive antigen, with a monoclonal antibody to measles virus, in tissue from a patient with Crohn's disease.

Using immunofluorescence (IF), we investigated reactive antigens present in Crohns disease patients with monoclonal antibodies derived from cells infected with measles virus, but not with the subacute sclerosing panencephalitis virus. During immunoblotting, one monoclonal antibody (mAb 86) reacted with a polypeptide with a molecular weight of 36 000 dalton (M; matrix protein) in measles virus-infected cells. This monoclonal antibody displayed a positive reaction only with tissues from patients with Crohns disease by the IF test. It did not react with samples from patients with other chronic inflammatory bowel diseases, such as ulcerative colitis. Other monoclonal antibodies to the measles virus protein, and monoclonal antibodies toHerpes simplex virus type 1 did not react with the same tissue samples. The role of measles virus infection and/or a viral antigen (possibly the M protein) as a causative agent in Crohns disease poses a challenging avenue for further research.

Quantification of Modified Tyrosines in Healthy and Diabetic Human Urine using Liquid Chromatography/Tandem Mass Spectrometry.

The quantification of urinary oxidized tyrosines, dityrosine (DiY), nitrotyrosine (NY), bromotyrosine (BrY), and dibromotyrosine (DiBrY), was accomplished by quadruple liquid chromatography-tandem mass spectrometry (LC/MS/MS). The sample was partially purified by solid phase extraction, and was then applied to the LC/MS/MS using multiple-reaction monitoring (MRM) methods. The analysis for the DiY quantification was done first. The residual samples were further butylated with n-butanol/HCl, and the other modified tyrosines were then quantified with isotopic dilution methods. MRM peaks of the modified tyrosines (DiY, NY, BrY, and DiBrY) from human urine were measured and the elution times coincided with the authentic and isotopic standards. The amounts of modified tyrosines in healthy human urine (n = 23) were 8.8 ± 0.6 (DiY), 1.4 ± 0.4 (NY), 3.8 ± 0.3 (BrY), and 0.7 ± 0.1 (DiBrY) µmol/mol of creatinine, respectively. A comparison of the modified tyrosines with urinary 8-oxo-deoxyguanosine, pentosidine, and Nε-(hexanoyl)lysine was also performed. Almost all products, except for NY, showed good correlations with each other. The amounts of the modified tyrosines (NY, BrY, and DiBrY) in the diabetic urine were higher than those in the urine from healthy people.

Follicular dendritic cell of the knock-in mouse provides a new bioassay for human prions

Infectious prion diseases initiate infection within lymphoid organs where prion infectivity accumulates during the early stages of peripheral infection. In a mouse-adapted prion infection, an abnormal isoform (PrP(Sc)) of prion protein (PrP) accumulates in follicular dendritic cells within lymphoid organs. Human prions, however, did not cause an accumulation of PrP(Sc) in the wild type mice. Here, we report that knock-in mouse expressing humanized chimeric PrP demonstrated PrP(Sc) accumulations in follicular dendritic cells following human prion infections, including variant Creutzfeldt-Jakob disease. The accumulated PrP(Sc) consisted of recombinant PrP, but not of the inoculated human PrP. These accumulations were detectable in the spleens of all mice examined 30 days post-inoculation. Infectivity of the spleen was also evident. Conversion of humanized PrP in the spleen provides a rapid and sensitive bioassay method to uncover the infectivity of human prions. This model should facilitate the prevention of infectious prion diseases.

Comparison of human blood concentrations of collectin kidney 1 and mannan-binding lectin.

Mannan-binding lectin (MBL) was first discovered as a collectin in animal blood, and was shown to have such unique characteristics as a collage-like domain and a carbohydrate recognition domain. We recently identified human collectin kidney 1 (CL-K1, COLEC11) from a human kidney cDNA library. To quantitate the CL-K1 concentration in blood, we developed several polyclonal and monoclonal antibodies using recombinant human CL-K1 in CHO cells and the CL-K1 fragment in Escherichia coli. Using these antibodies, we established a sandwich enzyme-linked immunosorbent assay (ELISA) system. The concentration of CL-K1 in human plasma was 0.34 ± 0.13 µg/ml and that in MBL was 1.72 ± 1.51 µg/ml. Concentrations of MBL are often low due to its single nucleotide polymorphisms (SNPs) which seem to be related to an opsonic defect. However, no low concentrations of CL-K1 were observed on testing over two hundred blood samples. We also found that the blood concentration of CL-K1 was not dependent on gender or age and did not correlate completely with that of MBL. The ELISA system developed in this study will be useful for elucidating the physiological and pathophysiological role of CL-K1 in humans.

Latency of herpesvirus of turkey and Marek's disease virus genomes in a chicken T-lymphoblastoid cell line.

The properties of latent herpesvirus of turkey (HVT) and Mareks disease virus (MDV) genomes have been studied in virus-non-producer MDCC-BO1(T) cells, a T-lymphoblastoid cell line derived from spleen cells of an HVT-vaccinated chicken. The numbers of the two virus genomes in BO1(T) cells remained stable at 1.6 to 1.8 HVT genome equivalents/cell and 3.4 to 3.8 MDV genome equivalents/cell throughout a number of passages and were not decreased by the presence of phosphonoacetic acid in the culture. When the culture temperature of the MDV-producer MDCC-MSB1 cell line was shifted from 41 to 37 degrees C, the cells cultured at 37 degrees C contained about five times as many virus genomes as those cultured at 41 degrees C. In contrast, the numbers of the two virus genomes in BO1(T) cells were not increased by culture at 37 degrees C. RNA extracted from BO1(T) whole cells and from the polyribosomal fraction hybridized to both MDV and HVT DNAs, indicating the expression of both latent virus genomes. Digestion of cell nuclei with micrococcal nuclease revealed that both latent HVT and MDV genomes possess a nucleosomal structure. Closed circular MDV DNA was demonstrated in BO1(T) by isopycnic centrifugation of DNA in ethidium-bromide-CsCl gradients.