The development of technologies for the in vitro amplification of the abnormal conformers of prion protein (PrP Sc ) has generated the potential for a novel diagnostic assay for prion disease. Previously, we developed a new PrP Sc amplification assay designated quaking-induced conversion (QUIC), which involves intermittent, automated shaking of the substrate, soluble recombinant PrP. We further improved the rapidity and practicality of this method by combining it with thioflavin T fluorescence to monitor the amyloid fibril formation. This assay, termed "real-time QUIC (RT-QUIC)", allows within 48 h, the detection of ≥1 fg of PrP Sc 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 ante-mortem evaluation of suspected CJD. Definitive ante-mortem confirmation of CJD requires the detection of PrP Sc in patient biopsy specimens, the practice of which is discouraged because it is both invasive and poses risks to health care personnel. Recently, however, in vitro PrP Sc amplification techniques, including protein misfolding cyclic amplification (PMCA) 5-7 , the amyloid seeding assay 8 , as well as QUIC have been reported to enable the direct and highly sensitive detection of PrP Sc in various tissues, including cerebrospinal fluid (CSF). QUIC assays involve the use of soluble recombinant PrP (rPrP-sen) as a substrate, which is seeded with PrP Sc , and then subjected to intermittent automated shaking. This technique can be performed more easily than the PMCA, which requires repeated sonication. Previous studies have demonstrated that QUIC assays correctly discriminated between normal and scrapie-infected CSF samples in both hamster and sheep prion disease models 9,10 . However, ultrasensitive PrP Sc detection in CSF from CJD subjects has not yet been accomplished. Accordingly, we further refined the QUIC 5 assay to improve its sensitivity and practicability, and then applied the technique in a blind pilot study to detect PrP Sc in CJD-CSF specimens.Given that a correlation between protease-resistant rPrP aggregate (rPrP-res) levels and thioflavin T (ThT) fluorescence had been shown previously 7 , we sought to determine the relative kinetics of rPrP-res formation by monitoring levels of ThT fluorescence in the QUIC assay. This was intended to minimize the time needed to detect rPrP-res. We first tested whether PrP Sc -dependent rPrP-res (rPrP-res (Sc) ) formation could be induced using a microplate reader with intermittent shaking. Human rPrP-sen (rHuPrP-sen) and a 10 -7 dilution of CJD (molecular subtype MM1) brain homogenate (BH) were used as the substrate and "seed", respectively. We conducted QUIC reactions at various concentrations (0, 0.25, 0.5 and 1.0 M) of guanidine-HCl (GdnHCl), because it h...

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ABSTRACT. Concentrations of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) were determined in serum and whey samples from cattle with naturally occurring coliform mastitis for two weeks after onset using bovine INF-γ-and TNF-α-specific ELISA. In serum and whey samples from healthy cows, IFN-γ was almost undetectable and TNF-α was detected at low levels. At the onset of illness, INF-γ in sera and whey and TNF-α in whey from the mastitic cows were significantly higher than their respective values in healthy cows. Concentrations of IFN-γ and TNF-α in whey from mastitic cattle decreased significantly as the cows recovered. KEY WORDS: bovine mastitis, interferon-γ, tumor necrosis factor-α.

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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 (⌬25-50) or octapeptide repeat (⌬51-90) region alone could not impair the antagonistic function against doppel.

ABSTRACT. To obtain basic information on the state of proinflammatory cytokines in newborn calves, we determined the kinetics of 5 cytokines (IL-1β, IL-6, TNF-α, IFN-γ and IL-1 receptor antagonist) in sera of newborns during the first 4 weeks of life. At birth, none of the 5 cytokines were detected in almost all serum samples, but the cytokines became detectable within 12 hr after being fed colostram. The mean concentrations of the cytokines reached peak levels by 24 hr and then gradually decreased and became undetectable by 4 weeks after birth. Cytokine mRNA expressions in peripheral blood mononuclear cells of newborns were observed without reference to the cytokine concentrations in sera. Serum cytokines detected in newborn calves are probably colostral origin. KEY WORDS: calf-serum, colostrum, cytokine.J. Vet. Med. Sci. 65 (7): [813][814][815][816] 2003 Resistance to infection is determined by the host immune state. The immune state of neonatal calves is largely dependent on the colostrum they are fed. Immunoglobulin, one of resistant factors present in colostrum, is known to play a protective role. The presence of several cytokines in bovine colostrum has been shown by several investigators [4][5][6]15]. In addition, oral administration of recombinant bovine (rb) IL-1β, one of the colostral cytokines, to neonatal calves resulted in the appearance of the cytokine in circulation and the activation of circulating lymphocytes and neutrophils [7]. Furthermore, and TNF-α and IFN-γ [17], which are also present in bovine colostrum [6], have been reported to potentiate immunological functions. Therefore, cytokines transferred to neonates via colostrum are considered to contribute to maturation of neonatal immune functions.To obtain basic information on the state of cytokines in neonatal calves, we measured the levels of five cytokines (IL-1β, IL-6, TNF-α, IFN-γ and IL-1 receptor antagonist, IL-1ra) in serum samples and expression of these cytokine mRNAs in peripheral blood mononuclear cells (PBMC) obtained from neonatal calves after they had been fed colostrum.Specimens were collected from Holstein Friesian cattle that were kept at the dairy farm of Rakuno Gakuen University. Blood was obtained by cervical vessel puncture using a sterile vacuum syringe from healthy calves at birth before being fed colostrum (n=21) and at 12 hr (hr) (n=19), 1 day (n=21), 2 days (n=18), 3 days (n=18), 5 days (n=17), 7 days (n=16), 14 days (n=13), 21 days (n=11) and 28 days (n=11) after birth. Newborn calves were fed about 2 l of colostrum from respective dams (n=10) or pools (n=11) within 2 hr after birth and then fed colostrum from respective dams two times a day by 5 days after birth. The other 4 calves, as controls, fed formula as substitute for colostrum for 3 days and their blood were collected during this period. Sera were stored at -30°C until use. PBMC from newborn calves (n=10) were prepared from heparinized peripheral blood of healthy calves at birth to 28 days after birth as described previously [21].The concentrations of...

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Colostrum contains various agents to compensate for insufficiency of nutrient and immune components in newborns, such as immunoglobulins, lactoferrin, and vitamins (25). In addition, colostrum feeding is considered to enhance T cell-mediated responses and to improve B cell immunity in the immature immune systems in newborns (21,22). Our previous study showed the presence of high concentrations of IL-1β, IL-6, TNF-α, IFN-γ and IL-1 receptor antagonist (ra) in bovine colostrum (13). It is likely that colostral cytokines are transferred to newborn calves (12,14,33). These cytokines, except for IL-1ra, potentiate immunological functions (4,8,23,29,31). Therefore, colostral cytokines are considered to play important roles in development of immunologic functions of neonates (2, 27).Indeed, our previous study demonstrated transfer of orally administered recombinant bovine (rb) IL-1β into serum of neonatal calves and activation of circulating lymphocytes and neutrophils by this cytokine (14).In the present study, to determine the effects of colostral cytokines on neonatal immune functions, we investigated the effect of each of the cytokines mentioned above on the blastogenic activity toward concanavalin A (ConA) of PBMC obtained from newborn calves before the calves had been fed colostrum. In addition, we also examined the expression of IL-2 mRNA and the expression of IL-2 receptor α chain, i.e., CD25, in these PBMC, since IL-2 has been shown to be crucial for the clonal expansion of T cells and growth of B cells and since α chains are expressed on activated cells Proinflammatory Cytokines in Bovine Colostrum