Hitoki Yamanaka

Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion

The development of technologies for the in vitro amplification of abnormal conformations of prion protein (PrPSc) has generated the potential for sensitive detection of prions. Here we developed a new PrPSc amplification assay, called real-time quaking-induced conversion (RT-QUIC), which allows the detection of ≥1 fg of PrPSc in diluted Creutzfeldt-Jakob disease (CJD) brain homogenate. Moreover, we assessed the technique first in a series of Japanese subjects and then in a blind study of 30 cerebrospinal fluid specimens from Australia, which achieved greater than 80% sensitivity and 100% specificity. These findings indicate the promising enhanced diagnostic capacity of RT-QUIC in the antemortem evaluation of suspected CJD.

Detection of cytokines in bovine colostrum

Colostrum contains factors that are protective for the neonate and may be a source of immunomodulary molecules that positively influence the immune status of the neonate. To confirm that colostrum contains a variety of cytokines with immunomodulatory properties, we established a bovine cytokine specific ELISA and five cytokines (IL-1 beta, IL-6, TNF-alpha, INF-gamma or IL-1 receptor antagonist, IL-1ra) in the whey samples from cows at different stages of lactation were monitored. The expression of cytokine mRNAs (IL-1 beta, IL-6, TNF-alpha and INF-gamma) in the colostral cells was detected by RT-PCR. The concentrations of cytokines in colostrum were significantly higher concentrations than those in the mature milk. A positive correlation was observed between the concentrations of IL-1ra and IL-1 beta in the colostrum samples. In conclusion, colostrum contains high levels of cytokines that could be produced and secreted in the mammary gland and that may have an immunomodulatory activity and influence neonatal immunity.

Concentrations of IL-6 in Serum and Whey from Healthy and Mastitic Cows

Inflammatory cytokines, such as interleukin (IL)-6, have been shown to reflect clinical signs in certain conditions in diseased animals. In this study, we quantified the IL-6 concentrations in the serum and milk whey from 94 dairy cows with acute clinical mastitis and 55 healthy lactating cows. The IL-6 concentrations in serum from mastitic cows were significantly higher on the first day of illness compared to those of normal cows. Higher concentrations of IL-6 were also detected in the whey from mastitic cows, whereas low concentrations of IL-6 were detected in both serum and whey samples from normal cows. IL-6 concentrations in the serum taken at the onset of illness from cows that later required euthanasia were significantly higher than those in samples from cows that later recovered. These results suggest that serum IL-6 concentrations may be of prognostic value in identifying cows with severe mastitis.

A Nasal Interleukin-12 DNA Vaccine Coexpressing Yersinia pestis F1-V Fusion Protein Confers Protection against Pneumonic Plague

ABSTRACT Previous studies have shown that mucosal application of interleukin-12 (IL-12) can stimulate elevated secretory immunoglobulin A (IgA) responses. Since possible exposure to plague is via Yersinia pestis-laden aerosols that results in pneumonic plague, arming both the mucosal and systemic immune systems may offer an added benefit for protective immunity. Two bicistronic plasmids were constructed that encoded the protective plague epitopes, capsular antigen (F1-Ag) and virulence antigen (V-Ag) as a F1-V fusion protein but differed in the amounts of IL-12 produced. When applied nasally, serum IgG and mucosal IgA anti-F1-Ag and anti-V-Ag titers were detectable beginning at week 6 after three weekly doses, and recombinant F1-Ag boosts were required to elevate the F1-Ag-specific antibody (Ab) titers. Following pneumonic challenge, the best efficacy was obtained in mice primed with IL-12(Low)/F1-V vaccine with 80% survival compared to mice immunized with IL-12(Low)/F1, IL-12(Low)/V, or IL-12(Low) vector DNA vaccines. Improved expression of IL-12 resulted in lost efficacy when using the IL-12(High)/F1-V DNA vaccine. Despite differences in the amount of IL-12 produced by the two F1-V DNA vaccines, Ab responses and Th cell responses to F1- and V-Ags were similar. These results show that IL-12 can be used as a molecular adjuvant to enhance protective immunity against pneumonic plague, but in a dose-dependent fashion.

Proinflammatory Cytokines in Bovine Colostrum Potentiate the Mitogenic Response of Peripheral Blood Mononuclear Cells from Newborn Calves through IL-2 and CD25 Expression

Bovine colostrum contains high concentrations of cytokines, and colostral cytokines are considered to be an important factor in stimulation of maturation of the immune system in newborns. In this study, 5 proinflammatory cytokines (IL‐1β, IL‐6, TNF‐α, IFN‐γ and IL‐1 receptor antagonist, IL‐1ra) present in colostrum were tested for their potential to enhance mitogenic response and to elicit expression of IL‐2 mRNA and CD25 in peripheral blood mononuclear cells (PBMC) from newborn calves before being fed colostrum. PBMC were pretreated with each recombinant bovine cytokine for 2 hr before stimulation with concanavalin A (ConA). Pretreatment of PBMC from newborn calves with IL‐1β, TNF‐α or IFN‐γ significantly enhanced the ConA response, whereas IL‐1ra inhibited the response. The degree of enhancement or inhibition of mitogenic response by these cytokines was more pronounced in PBMC from newborn calves than in those from adult cows. Although IL‐2 mRNA expression in ConA‐stimulated PBMC from newborn calves was weaker than that in those from adult cows of ConA‐stimulated controls, the expression levels became comparable after pretreatment with IL‐1β, TNF‐α or IFN‐γ. The CD25 expression in PBMC from newborn calves was also enhanced by pretreatment with IL‐1β, TNF‐α and IFN‐γ. These results suggest that pretreatment of neonatal PBMC with IL‐1β, TNF‐α or IFN‐γ promotes mitogenic response to ConA through up‐regulating the production of IL‐2 and the expression of the mature IL‐2 receptor.

Dominant-negative Effects of the N-terminal Half of Prion Protein on Neurotoxicity of Prion Protein-like Protein/Doppel in Mice

Prion protein-like protein/doppel is neurotoxic, causing ataxia and Purkinje cell degeneration in mice, whereas prion protein antagonizes doppel-induced neurodegeneration. Doppel is homologous to the C-terminal half of prion protein but lacks the amino acid sequences corresponding to the N-terminal half of prion protein. We show here that transgenic mice expressing a fusion protein consisting of the N-terminal half, corresponding to residues 1-124, of prion protein and doppel in neurons failed to develop any neurological signs for up to 730 days in a background devoid of prion protein. In addition, the fusion protein prolonged the onset of ataxia in mice expressing exogenous doppel. These results suggested that the N-terminal part of prion protein has a neuroprotective potential acting both cis and trans on doppel. We also show that prion protein lacking the pre-octapeptide repeat (Δ25-50) or octapeptide repeat (Δ51-90) region alone could not impair the antagonistic function against doppel.

An IL-12 DNA vaccine co-expressing Yersinia pestis antigens protects against pneumonic plague

Pneumonic plague remains problematic in endemic areas, and because it can be readily transmitted and has high mortality, the development of efficacious vaccines is warranted. To test whether stimulation of cell-mediated immunity with IL-12 will improve protective immunity against plague, we constructed two IL-12 DNA vaccines using a bicistronic plasmid encoding the protective plague epitopes, capsular (F1) antigen and virulence antigen (V-Ag) as F1-V fusion protein and V-Ag only, respectively. When applied intramuscularly, antibody responses to F1- and V-Ag were detectable beginning at week 6 after 3 weekly doses, and F1-Ag protein boosts were required to induce elevated Ab responses. These Ab responses were supported by mixed Th cell responses, and the IL-12/V-Ag DNA vaccine showed greater cell-mediated immune bias than IL-12/F1-V DNA vaccine. Following pneumonic challenge, both IL-12 DNA vaccines showed similar efficacy despite differences in Th cells simulated. These results show that IL-12 can be used as a molecular adjuvant to enhance protective immunity against pneumonic plague.

Oral administration of IL-1β enhanced the proliferation of lymphocytes and the O2− production of neutrophil in newborn calf

Recently, we demonstrated the presence of IL-1 beta in the colostral whey from dairy cows. Here, authors examined oral transmission of colostral IL-1 beta and its immunological effects on the neonatal calves. Biotin-labeled recombinant bovine (rb) IL-1 beta was administered orally to newborn calves and monitored in the serum. The results disclosed the passive transfer of colostral cytokines via the oral route, and a potent increase in white blood cell (WBC) count was observed in all calves administered with rbIL-1 beta. Oral administration of IL-1 beta significantly increased the proliferation of peripheral blood mononuclear cells (PBMCs) stimulated with concanavalin A, and the O(2)(-) production of stimulates neutrophils in newborn calves. These results suggest that the oral administration of IL-1 beta has an immunostimulatory activity in the newborn calf.

Antigenic mimicry-mediated anti-prion effects induced by bacterial enzyme succinylarginine dihydrolase in mice

Prions, the causative agents of prion diseases, are immunologically tolerated because their major component, prion protein (PrP), is a host-encoded molecule. Therefore, no effective prion vaccines have been developed. We previously showed that heterologous bovine and sheep PrP immunizations of mice overcame tolerance by an antigenic mimicry mechanism to efficiently induce anti-PrP auto-antibodies (Abs), significantly prolonging incubation times in mice subsequently infected with the mouse-adapted Fukuoka-1 prion. These results prompted us to investigate if non-mammal derived molecules able to antigenically mimic anti-prion epitopes, could act as prion vaccines. We show here that immunization of mice with recombinant succinylarginine dihydrolase, a bacterial enzyme with a peptide sequence similar to an anti-prion epitope, induced anti-PrP auto-Abs with anti-prion activity and significantly retarded survival times of the mice subsequently infected with Fukuoka-1 prions. These results might open a way for development of a new type of antigenic mimicry-based prion vaccine.

A Parenteral DNA vaccine protects against pneumonic plague

The chemokine, lymphotactin (LTN), was tested as a molecular adjuvant using bicistronic DNA vaccines encoding the protective Yersinia capsular (F1) antigen and virulence antigen (V-Ag) as a F1-V fusion protein. The LTN-encoding F1-V or V-Ag vaccines were given by the intranasal (i.n.) or intramuscular (i.m.) routes, and although serum IgG and mucosal IgA antibodies (Abs) were induced, F1-Ag boosts were required for robust anti-F1-Ag Abs. Optimal efficacy against pneumonic plague was obtained in mice i.m.-, not i.n.-immunized with these DNA vaccines. These vaccines stimulated elevated Ag-specific Ab-forming cells and mixed Th cell responses, with Th17 cells markedly enhanced by i.m. immunization. These results show that LTN can be used as a molecular adjuvant to enhance protective immunity against plague.