Yin Jiang

Validation of Nonradioactive Chemiluminescent Immunoassay Methods for the Analysis of Thyroxine and Cortisol in Blood Samples Obtained from Dogs, Cats, and Horses

The performances of a radioimmunoassay method, a chemiluminescent immunoassay method, and a chemiluminescent-enzyme immunoassay method were evaluated for the analysis of cortisol and total thyroxine in blood samples obtained from dogs, cats, horses, and humans (reference samples). The analysis of cortisol in human and animal samples exhibited good precision, linearity, and recovery. The 3 methods gave comparable values for the ACTH-induced increase and the dexamethasone-induced decrease in cortisol concentrations in animal samples. The recoveries of total thyroxine from human samples, analyzed by the 3 methods, were comparable. However, the basal total thyroxine concentrations determined by the chemiluminescent immunoassay method were 30–40% lower than those determined by the radioimmunoassay and the chemiluminescent-enzyme immunoassay methods in animal samples. In both human and animal samples, the plot of thyroxine values obtained by the radioimmunoassay method against those obtained by the chemiluminescent immunoassay method or the chemiluminescent-enzyme immunoassay method was linear. However, although the slope of the radioimmunoassay versus chemiluminescent-enzyme immunoassay curve was close to unity, the slope of the radioimmunoassay versus chemiluminescent immunoassay curve was 0.6. This result suggests that, compared with the radioimmunoassay method, the chemiluminescent immunoassay method underestimated thyroxine values in animal samples but not in human samples. Although all 3 methods yielded comparable changes in thyroxine concentrations in response to thyroid stimulating hormone, they did not yield comparable thyroxine concentrations in response to T3 suppression in dogs and cats.

Detection of Influenza a Virus in Porcine Oral Fluid Samples

Porcine oral fluids have been used for the detection of Porcine reproductive and respiratory syndrome virus and Porcine circovirus-2. The objective of the present study was to determine whether Influenza A virus (FLUAV) is present in porcine oral fluids at detectable levels and to validate a standard FLUAV molecular diagnostic test for porcine oral fluids. Pen-based oral fluid samples were collected on 3, 4, 5, and 6 days postinfection (DPI) from 4 groups of 6 pigs each that were inoculated intratracheally with A/Swine/ Iowa/00239/2004 H1N1 and from 2 untreated or mock-inoculated groups of 6 pigs each that served as negative controls. Individual nasal swabs were also collected from these 36 pigs on 3 and 7 DPI. All oral fluid samples were examined for the presence of FLUAV by matrix gene real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and virus isolation. Nasal swabs were tested initially by virus isolation followed by retest of negative samples with real-time RT-PCR. No oral fluid sample from virus-inoculated pigs was positive by virus isolation, but 15 of 16 positive (94%) oral fluids were positive by real-time RT-PCR. In contrast, virus was isolated from 32 of 48 (67%) nasal swabs collected from virus-inoculated pigs. In addition, 382 of 910 porcine oral fluids collected from pigs in the field between August 1, 2009, and January 31, 2010, were positive by real-time RT-PCR. The results of the present study indicate that pen-based oral fluids provide an easy, effective, and safe collection method for the detection of FLUAV with rapid testing methods such as real-time RT-PCR.

Complete Genome Sequence of Porcine Epidemic Diarrhea Virus Strain USA/Colorado/2013 from the United States

ABSTRACT Porcine epidemic diarrhea virus (PEDV) is newly emerging in the United States. PEDV strain USA/Colorado/2013 (CO/13) was obtained from a 7-day-old piglet with severe diarrhea, and the complete genome was sequenced to further study the PEDV outbreak in the United States.

Rapid detection, complete genome sequencing, and phylogenetic analysis of porcine deltacoronavirus.

In February 2014, porcine deltacoronavirus (PDCoV) was identified in the United States. We developed a PDCoV real-time reverse transcription PCR that identified PDCoV in 30% of samples tested. Four additional PDCoV genomes from the United States were sequenced; these had ≈99%–100% nt similarity to the other US PDCoV strains.

In vitro Neurogenesis from Neural Progenitor Cells Isolated from the Hippocampus Region of the Brain of Adult Rats Exposed to Ethanol during Early Development through Their Alcohol-Drinking Mothers

AIMS This study was aimed to determine whether ethanol exposure during early development altered neurogenesis in the brain of adult rats. METHODS Pregnant rats were given either ethanol-mixed or mannose-mixed (for control) rodent liquid diet ad libitum. Ethanol drinking continued during pregnancy and nursing. After weaning, the pups (AC(o): pups from control mothers, AE(o): pups from ethanol exposed mothers) received normal diet and water ad libitum for 11 weeks. Then the rats were anesthetized, their brains were collected and the hippocampal samples were processed for isolation of neural progenitor cells (NPCs). AC(o) NPCs and AE(o) NPCs were sequentially grown in media containing different growth factors that induced proliferation and differentiation. RESULTS AND CONCLUSIONS Neuronal maturation was significantly delayed in ethanol-exposed rats. AC(o) NPCs, up to day 7 of culture, exhibited high beta-catenin-probe binding, an increase in Ca(2+) when exposed to gamma-amino butyric acid (GABA) and lack of response to glutamate (Glu) exposure. beta-Catenin-probe binding and the stimulatory effects of GABA declined thereafter. AC(o) NPCs, at culture day 29, exhibited high beta-catenin-probe binding, lack of response to GABA and elevated Glu-induced increase in Ca(2+i). Cultures of AE(o) NPCs showed an amplified stimulatory effects of GABA, attenuated stimulatory effects of Glu and attenuated the delayed (culture day 29) increase in the expression of Wnt proteins and beta-catenin-probe binding. This suggests a significant alteration in neurogenesis and synapse formation in adult rats exposed to ethanol at early development through their alcohol-drinking mothers.

Complete Genome Sequence of Strain SDCV/USA/Illinois121/2014, a Porcine Deltacoronavirus from the United States

ABSTRACT To investigate the causative agent of swine diarrhea, next-generation sequencing (NGS) was performed on a porcine fecal sample. The NGS reads were assembled, which generated a complete swine Deltacoronavirus genome sequence, that of strain SDCV/USA/Illinois121/2014.

Quantitative chromatographic analysis of inositol phospholipids and related compounds

The metabolism of phospholipids and the mobilization of second messengers such as inositol-1,4,5-trisphosphate, 1,2-diacylglycerol (DAG) and arachidonic acid (AA) from phospholipids is commonly studied by radiolabelling phospholipids with [3H]myo-inositol or [32P]ATP and measuring the incorporation of radioactivity in different phospholipids or their hydrolysis products. However, for the radiolabelling method to accurately reflect changes in the compounds mass, it is essential that the tissue is labelled to isotopic equilibrium which is difficult to achieve. To circumvent the disadvantages of the radiolabelling method, several analytical procedures have been developed for the mass analysis of phospholipids and inositolphosphates (IPs). Quantitation of the mass or the radiolabelling of phospholipids is a complex multi-step procedure that involves quantitative isolation of phospholipids, fractionation of individual phospholipids and either determination of radioactivity in each component or the measurement of their mass. Phospholipids, DAG and AA are extracted from tissue sample with organic solvents such as chloroform-methanol (2:1) containing HCl or formic acid. The extract is separated by TLC, cartridge-column chromatography or HPLC on a reversed-phase column. Phospholipids are quantitated by measuring inorganic phosphate, absorption at 200 nm or mass spectrometry. Inositol phosphates are extracted with perchloric acid or trichloroacetic acid and separated by ion-exchange cartridge-column or HPLC with an ion-exchange column. IPs are quantitated by measuring inorganic phosphate or by using enzymatic reaction, metal-dye coupling, NMR or mass spectrometry.

Lipopolysaccharide (LPS) induced activation of the immune system in control rats and rats chronically exposed to a low level of the organothiophosphate insecticide, acephate

Lipopolysaccharide (LPS), a key inflammatory component of gram-negative bacteria, induces a distinctive pattern of cytokine release that regulates inflammation. An alteration in the LPS response may play a fundamental role in the pathogenesis of a number of inflammatory diseases. Therefore, this study was conducted to determine whether chronic exposure to a low level of acephate (Ace), a commonly used organophosphate insecticide, impaired the LPS response in rats. This study showed that LPS injection in control rats caused (1) a time-dependent increase in blood lymphocyte enumeration and differentiation, and (2) a sequential increase the pro-inflammatory (interleukin-1b (IL1b), tumor necrosis factor-a (TNFa), interferon-g (INTg), and inducible nitric oxide synthase (iNOS)) and anti-inflammatory (interleukin-4 (IL-4), corticotropin-releasing factor (CRF), and blood corticosterone (Cort)) cytokines. The pro-inflammatory cytokines increased after 30 min, while the anti-inflammatory cytokines increased 3 h after LPS injection. An increase in proinflammatory cytokines increased lymphocyte enumeration and differentiation, while the increase in anti-inflammatory cytokines re-established homeostasis. In comparison to the control rats, the Ace-exposed rats exhibited (1) lower levels of IL1b, TNFa and iNOS, (2) higher levels of CRF and Cort, and (3) lower levels of IL-4 in blood and/or brain samples. The abnormal cytokine production may be associated with abnormal phenotypic distribution of B and T cells. Blood IgMhiIgDhi, IgMloIgDlo and CD8+ CD45RA −CCR7+ cells were elevated, while IgMloIgDhi, IgMhiIgDlo, IgMinIgDlo, CD8+ CD45RA +CCR7+ and CD8+ CD45RA− CCR7+ cells were depressed in Ace-exposed rats. Thus, chronic low-level Ace exposure may impair the lineage commitment in lymphocytes, possibly by altering cytokine signaling in the brain.

Effects of bacterial toxins on endothelial tight junction in vitro: a mechanism-based investigation.

ABSTRACT Lipopolysaccharide (LPS) and lipoteichoic acid (LTA), principal cell wall components of Gram-negative and Gram-positive bacteria, respectively, play a central role in altering the blood-brain barrier and facilitate bacterial infection of the host brain. Despite the significance of bacterial toxins in disease pathogenesis, mechanisms by which toxins impair the barrier are not yet known. This study, using an in vitro cell culture model, showed that LPS and LTA interacted with the endothelial cells and disrupted the tight junction between the cells that increased the barriers permeability. Both toxins increased inducible nitric oxide synthase (iNOS) mRNA that is indicative of an increase in intracellular NO release, disrupted architecture of the tight junction proteins, suppressed zonula occludens-1 (ZO-1) and occludin (OCL) and junctional adhesive molecules (JAM) mRNA levels, and increased tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL-1β) mRNA levels. Anti-CD14 antibodies blocked the increase in TNFα and IL-1β mRNA levels but did not affect either changes in the tight junction or iNOS, ZO-1, OCL, and JAM mRNA levels in endothelial cells and astrocytes. Although both toxins did not cross the endothelial barrier, the abluminal neurons exhibited high inflammatory activity characterized by a sequential increase in TNFα, IL-1β, external receptor kinase (ERK), and RelA-p50 that induced inflammation, followed by an increase in anti-inflammatory/apoptotic factors including IL-10 and cysteine-aspartic acid protease-8 (CASPASE-8), which resolve inflammation and induce apoptosis. Anti-CD14 antibodies in luminal buffer blocked the pro- and anti-inflammatory effects of the toxins in neurons. Thus, the CD14-TLR cascade that participates in the inflammatory effects of toxins may not participate in the toxin-induced barrier disruption in vitro. Since the toxins did not cross the endothelial barrier, induction of inflammation in neurons was due to a release of proinflammatory cytokines in the abluminal fluid.

Anti-Inflammatory Potency of Nano-Formulated Puerarin and Curcumin in Rats Subjected to the Lipopolysaccharide-Induced Inflammation

Puerarin (PU) and curcumin (CU), used commonly in traditional Chinese medicine and Ayurveda, have been shown to possess potent anti-inflammatory, anti-oxidation, and neuro-protective properties. Despite the experimental success of CU and PU in in vitro and animal models, their effectiveness has not yet been demonstrated in clinical trials, possibly because of their poor bioavailability. We hypothesized that gold nanoparticle (AuNP)-formulated PU (PU-AuNP), CU (CU-AuNP), or a combination of PU and CU (PU-CU-AuNP) were a more effective and nontoxic alternative to their bulk (nonformulated) counterparts. To test the hypothesis, bioavailability, therapeutic potency, and toxicity of bulk CU and/or PU were compared with those of their nanotized counterparts in rats subjected to the lipopolysaccharide (LPS)-induced inflammation. This study showed that a 20-mg/kg dose of bulk PU or a mixture of PU and CU did not, while their nanotized counterparts, PU-AuNP, CU-AuNP, or PU-CU-AuNP, effectively suppressed the LPS-induced inflammation and cytotoxicity in rats. In addition, PU-CU-AuNP was more potent than PU-AuNP or CU-AuNP alone. The blank AuNP (bAuNP) at ≤40 mg/kg dose did not cause any adverse effects (blood and brain lactic acid concentrations, kidney function, and neuronal apoptosis were measured) in animals. Therefore, the present observations suggest that a bi-functional AuNP loaded with CU and PU may effectively suppress the LPS-induced inflammation and cytotoxicity provided the following conditions are met: (1) The AuNP dose is at or below the no-effect dose; (2) the nanoparticles release a therapeutic dose of CU and PU in vivo; and (3) the active ingredients are released into the intracellular component of the brain.