Lonicera flos and Cnicus japonicus extracts improved egg quality partly by modulating antioxidant status, inflammatory-related cytokines and shell matrix protein expression of oviduct in laying hens.

This study was conducted to investigate the effects of Lonicera flos and Cnicus japonicus extracts (LCE) on the laying performance, egg quality, morphology, antioxidant status, inflammatory-related cytokines, and shell matrix protein expression of oviduct in laying hens. A total of 1,728 Roman Pink laying hens aged 73-wk-old were randomly assigned into 4 groups (18 replicates/group, 24 layers/replicate) fed basal diets supplemented with 0, 300, 500, and 1,000 mg of LCE per kg of diet, respectively. The trial lasted for 11 wk, including 2-wk adjustment period and 9-wk testing period. The results indicated that laying hens fed diets supplemented with LCE linearly increased egg weight, yolk color and shell thickness at wk 78 and albumen height, Haugh unit and shell thickness at wk 83 (P < 0.05). At wk 78, LCE groups linearly affected the hydrogen peroxide content in magnum (P < 0.05) and 300 mg/kg LCE groups had the highest catalase activity in isthmus (P < 0.05). At wk 83, LCE groups linearly reduced (P < 0.05) hydrogen peroxide content in the magnum and isthmus and malondialdehyde content in the uterus whereas increased catalase activity in isthmus (P < 0.05). Furthermore, LCE levels quadratically affected glutathione peroxidase activity in isthmus at wk 83 (P < 0.05). At wk 78, the mRNA expressions of inducible nitric oxide synthase and interferon-γ in isthmus and ovalbumin and ovocleidin-116 in uterus had linear effects in response to LCE levels (P < 0.05) and 1,000 mg/kg LCE group had the lowest mRNA expression of interleukin-6 in magnum (P < 0.05). At wk 83, LCE supplementation linearly decreased the mRNA expression of interleukin-1ß, interferon-γ and tumor necrosis factor-α in magnum and tumor necrosis factor-α and inducible nitric oxide synthase in uterus (P < 0.05). It is concluded that LCE improved egg quality partly by modulating antioxidant status, inflammatory-related cytokines and shell matrix protein expression of oviduct in laying hens.

Effects of salpingitis simulation on the morphology and expression of inflammatory-related genes of oviduct in laying hens.

This study was conducted to simulate salpingitis of laying hens by observing the morphology and expression of inflammatory genes in the oviduct. A total of one hundred twenty 81-wk-old Roman Pink laying hens in good physical condition without the oviduct disease with an average egg production rate of 76% were fed a basal diet for 2 wks and then randomly allocated into 4 groups (6 replicates/group, 5 birds/replicate). The experimental treatments were as follows: 1) Control group (treated with PBS); 2) Organic chemical reagent (OCR) group; 3) Lipopolysaccharide (LPS) group; 4) LPS + OCR group. First, the chickens were kept upside down to make ectropion and exposure of the apertura uterinae; then prepared reagents were poured into the uterine part of the fallopian tube by using the chicken vas deferens (1 mL/layer); finally, the chickens were kept in the inverted position for 5 to 10 min. The fallopian tube samples (the magnum, isthmus, and uterus) were collected after 48 h of treatment. Compared with the control, treatment with LPS+OCR decreased (P < 0.05) the secondary villus length and primary villus area in magnum and villus length in isthmus (P < 0.05). An increase (P < 0.05) of the intervillous space of uterus was observed in LPS + OCR group compared with the control. The expressions of interleukin-6 mRNA of magnum and interferon-γ (IFN-γ) of isthmus in the LPS and LPS+OCR treatments were higher (P < 0.05) than that in control. Compared with the control, treatment with LPS+OCR increased (P < 0.05) the expressions of IFN-γ mRNA of magnum and IFN-γ, tumor necrosis factor-α and inducible nitric oxide synthase mRNA of uterus in laying hens. In conclusion, the results of morphological damage of fallopian tube tissue and increased expression of inflammatory factors in LPS + OCR treatment group suggested that LPS+OCR treatment can provide data basis to establish salpingitis model in laying hens for studying the pathogenesis of it.

Cerium Oxide Nanoparticles: A Potential Medical Countermeasure to Mitigate Radiation-Induced Lung Injury in CBA/J Mice.

Cerium oxide nanoparticles (CNPs) have a unique surface regenerative property and can efficiently control reactive oxygen/nitrogen species. To determine whether treatment with CNPs can mitigate the delayed effects of lung injury after acute radiation exposure, CBA/J mice were exposed to 15 Gy whole-thorax radiation. The animals were either treated with nanoparticles, CNP-18 and CNP-ME, delivered by intraperitoneal injection twice weekly for 4 weeks starting 2 h postirradiation or received radiation treatment alone. At the study's end point of 160 days, 90% of the irradiated mice treated with high-dose (10 µM) CNP-18 survived, compared to 10% of mice in the radiation-alone (P < 0.0001) and 30% in the low-dose (100 nM) CNP-18. Both low- and high-dose CNP-ME-treated irradiated mice showed increased survival rates of 40% compared to 10% in the radiation-alone group. Multiple lung functional parameters recorded by flow-ventilated whole-body plethysmography demonstrated that high-dose CNP-18 treatment had a significant radioprotective effect on lethal dose radiation-induced lung injury. Lung histology revealed a significant decrease (P < 0.0001) in structural damage and collagen deposition in mice treated with high-dose CNP-18 compared to the irradiated-alone mice. In addition, significant reductions in inflammatory response (P < 0.01) and vascular damage (P < 0.01) were observed in the high-dose CNP-18-treated group compared to irradiated-alone mice. Together, the findings from this preclinical efficacy study clearly demonstrate that CNPs have both clinically and histologically significant mitigating and protective effects on lethal dose radiation-induced lung injury.

Linkage and association study of late-onset Alzheimer disease families linked to 9p21.3.

A chromosomal locus for late-onset Alzheimer disease (LOAD) has previously been mapped to 9p21.3. The most significant results were reported in a sample of autopsy-confirmed families. Linkage to this locus has been independently confirmed in AD families from a consanguineous Israeli-Arab community. In the present study we analyzed an expanded clinical sample of 674 late-onset AD families, independently ascertained by three different consortia. Sample subsets were stratified by site and autopsy-confirmation. Linkage analysis of a dense array of SNPs across the chromosomal locus revealed the most significant results in the 166 autopsy-confirmed families of the NIMH sample. Peak HLOD scores of 4.95 at D9S741 and 2.81 at the nearby SNP rs2772677 were obtained in a dominant model. The linked region included the cyclin-dependent kinase inhibitor 2A gene (CDKN2A), which has been suggested as an AD candidate gene. By re-sequencing all exons in the vicinity of CDKN2A in 48 AD cases, we identified and genotyped four novel SNPs, including a non-synonymous, a synonymous, and two variations located in untranslated RNA sequences. Family-based allelic and genotypic association analysis yielded significant results in CDKN2A (rs11515: PDT p = 0.003, genotype-PDT p = 0.014). We conclude that CDKN2A is a promising new candidate gene potentially contributing to AD susceptibility on chromosome 9p.

Interaction between the alpha-T catenin gene (VR22) and APOE in Alzheimer's disease.

BACKGROUND: APOE is the only gene that has been consistently replicated as a risk factor for late onset Alzheimer's disease. Several recent studies have identified linkage to chromosome 10 for both risk and age of onset, suggesting that this region harbours genes that influence the development of the disease. A recent study reported association between single nucleotide polymorphisms (SNPs) in the VR22 gene (CTNNA3) on chromosome 10 and plasma levels of Abeta42, an endophenotype related to Alzheimer's disease. OBJECTIVE: To assess whether polymorphisms in the VR22 gene are associated with Alzheimer's disease in a large sample of Alzheimer's disease families and an independent set of unrelated cases and controls. RESULTS: Several SNPs showed association in either the family based or case-control analyses (p<0.05). The most consistent findings were with SNP6, for which there was significant evidence of association in both the families and the unrelated cases and controls. Furthermore, there was evidence of significant interaction between APOE-4 and two of the VR22 SNPs, with the strongest evidence of association being concentrated in individuals carrying APOE-4. CONCLUSIONS: This study suggests that VR22 or a nearby gene influences susceptibility to Alzheimer's disease, and the effect is dependent on APOE status.

Defective neuronal sprouting by human apolipoprotein E4 is a gain-of-negative function.

The apolipoprotein E (apoE) epsilon 4 allele (apoE4) is a major risk factor for neurodegenerative conditions, including Alzheimer's disease (AD). A role for apoE in regeneration of synaptic circuitry after neural injury has been shown in several in vitro studies in which apoE3 supports neuronal sprouting better than apoE4. We evaluated sprouting in an in vitro mouse organotypic hippocampal slice culture system derived from transgenic mice expressing apoE3 or apoE4, in which apoE-dependent granule cell mossy fiber sprouting in the presence of apoE4 is only 51% of the level of apoE3. Sprouting supported by apoE4 had a dose response opposite that by supported by apoE3: although increasing E3 expression increased sprouting, increasing E4 expression decreased sprouting, suggesting that the defect in E4 in supporting neuronal sprouting is a gain-of-negative activity. These results may have important pharmacogenomic implications for AD therapies that modulate apoE expression levels.

Human apolipoprotein E isoform-specific differences in neuronal sprouting in organotypic hippocampal culture.

The apolipoprotein E (ApoE) epsilon4 allele is a major risk factor for neurodegenerative conditions, including Alzheimer's disease. A role for ApoE is implicated in regeneration of synaptic circuitry after neural injury. In the in vitro mouse organotypic hippocampal slice culture system, we previously showed that cultures derived from ApoE-knockout mice are defective in mossy fiber sprouting into the dentate gyrus molecular layer. This sprouting defect was rescued in cultures from transgenic mice expressing ApoE3 under the control of the human promoter and in ApoE-knockout cultures treated with ApoE3-conditioned media. Although the ApoE3 transgene fully restored sprouting, ApoE4 restored sprouting to only 58% of ApoE3 levels. These data indicate that ApoE isoform-specific effects on neuroregeneration may contribute to its genetic risk for Alzheimer's disease.

Sialylated human apolipoprotein E (apoEs) is preferentially associated with neuron-enriched cultures from APOE transgenic mice.

Mice transgenic for human APOE2, E3, and E4 alleles express native 34-kDa human apoE and two sialylated apoE isoproteins with approximate molecular weights of 37 kDa (apoEs) and 39 kDa (apoEs2) in brain. These multiple apoE/apoEs/apoEs2 band patterns on Western blot are also observed in human brain, but are not seen in wild-type mouse brain. Both the 37-kDa apoEs and 39-kDa apoEs2 are coprecipitated with native 34-kDa apoE by antibody to human apoE. Neuraminidase digestion eliminates the 37- and 39-kDa forms and results in a downward shift in the bands to the position of the 34-kDa native form. These sialylated apoE isoproteins are found preferentially associated with neurons and contribute significantly (50-60%) to the total neuronal apoE in neuronal cultures from transgenic mice, while only 5-10% of total apoE is sialylated in cultures enriched in glial cells. In situ hybridization and immunocytochemistry demonstrate apoE mRNA and apoE immunoreactivity are predominantly located in cell soma of neurons, not in neuronal processes.

Specific regional transcription of apolipoprotein E in human brain neurons.

In central nervous system injury and disease, apolipoprotein E (APOE, gene; apoE, protein) might be involved in neuronal injury and death indirectly through extracellular effects and/or more directly through intracellular effects on neuronal metabolism. Although intracellular effects could clearly be mediated by neuronal uptake of extracellular apoE, recent experiments in injury models in normal rodents and in mice transgenic for the human APOE gene suggest the additional possibility of intraneuronal synthesis. To examine whether APOE might be synthesized by human neurons, we performed in situ hybridization on paraffin-embedded and frozen brain sections from three nondemented controls and five Alzheimer's disease (AD) patients using digoxigenin-labeled antisense and sense cRNA probes to human APOE. Using the antisense APOE probes, we found the expected strong hybridization signal in glial cells as well as a generally fainter signal in selected neurons in cerebral cortex and hippocampus. In hippocampus, many APOE mRNA-containing neurons were observed in sectors CA1 to CA4 and the granule cell layer of the dentate gyrus. In these regions, APOE mRNA containing neurons could be observed adjacent to nonhybridizing neurons of the same cell class. APOE mRNA transcription in neurons is regionally specific. In cerebellar cortex, APOE mRNA was seen only in Bergmann glial cells and scattered astrocytes but not in Purkinje cells or granule cell neurons. ApoE immunocytochemical localization in semi-adjacent sections supported the selectivity of APOE transcription. These results demonstrate the expected result that APOE mRNA is transcribed and expressed in glial cells in human brain. The important new finding is that APOE mRNA is also transcribed and expressed in many neurons in frontal cortex and human hippocampus but not in neurons of cerebellar cortex from the same brains. This regionally specific human APOE gene expression suggests that synthesis of apoE might play a role in regional vulnerability of neurons in AD. These results also provide a direct anatomical context for hypotheses proposing a role for apoE isoforms on neuronal cytoskeletal stability and metabolism.

Regionally specific neuronal expression of human APOE gene in transgenic mice.

Apolipoprotein E (APOE, gene; apoE, protein) is a susceptibility gene for late-onset Alzheimer's disease (AD). To examine whether neurons can synthesize apoE, we performed in situ hybridization on brain tissue of transgenic mice carrying genomic constructs for the three major human APOE alleles. We find human APOE mRNA in glial cells of cerebellum, striatum and cerebral cortex and also in neurons of cerebral cortex, corresponding to apoE localization in humans. Synthesis of apoE by neurons implies that models of AD may need to consider intrinsic apoE production in addition to uptake. Inclusion of human regulatory sequences may result in more realistic transgenic models of human disease.

Cis-acting human ApoE tissue expression element is associated with human pattern of intraneuronal ApoE in transgenic mice.

Apolipoprotein E polymorphic variants (ApoE-epsilon2, epsilon3, and epsilon4) are associated with the age of onset distribution and risk of Alzheimer disease. The question of whether ApoE is expressed at a comparatively low level in human neurons compared to astrocytes, or whether ApoE enters neuronal cytoplasm via altered endosomal metabolism is important to understanding potential pathogenic roles for ApoE as a susceptibility gene in Alzheimer disease. ApoE deficient ("knock-out") mice received large human genomic DNA fragment transgenes for each of the three common apoE alleles. All transgenic mice demonstrated glial/astrocytic (normal rodent pattern), as well as cortical intraneuronal ApoE immunoreactivity with all three human isoforms and at multiple ApoE human allele doses (Xu et al. (32)). To test whether ApoE intraneuronal immunoreactivity was due to ApoE gene sequences between mouse and human, we examined another set of mice constructed using targeted replacement so that the human ApoE gene was placed under mouse gene promoters. Current analyses show that targeted replacement recombinant mice show normal rodent glial expression pattern, but no ApoE neuronal immunoreactivity through six months of age compared to large human genomic DNA fragment transgenic mice, which show neuronal content of ApoE throughout adult life. We conclude that cis-acting DNA sequences, rather than the specific sequence of the ApoE gene, may be responsible for low levels of transcription activity in cortical neurons.

Human apolipoprotein E2, E3, and E4 isoform-specific transgenic mice: human-like pattern of glial and neuronal immunoreactivity in central nervous system not observed in wild-type mice.

Apolipoprotein E (apoE) and its three major alleles (APOE2, E3, and E4) have been implicated in Alzheimer's disease and other neurological disorders. Little is known of the role apoE plays in normal brain function and pathology. To create a model to study apoE in brain, we have generated APOE transgenic mice using microinjection of allele-specific human genomic fragments to establish founders which were then bred to APOE knockout mice lacking a functional mouse apoE protein. This allows the study of apoE without interference from the endogenous mouse APOE gene. Results demonstrate that transgenic lines have been established that transcribe and express apoE appropriately in brain, liver, and other tissues. High cholesterol levels found in APOE knockout mice are substantially corrected in the APOE transgenic lines. ApoE immunoreactivity has been detected in glial cells and selected classes of neurons in all three isoform-specific transgenics. This pattern of immunoreactivity is similar to that observed in nonhuman primates and man, and contrasts with the strictly glial staining pattern of normal rodents.

Human endopeptidase (THOP1) is localized on chromosome 19 within the linkage region for the late-onset alzheimer disease AD2 locus.

A cDNA encoding the rat endopeptidase 24.15 was used to determine the chromosomal localization of the respective human gene. Hybridization to DNA from human-rodent somatic cell hybrids assigned the human gene to chromosome 19. Fluorescence in situ hybridization on human metaphase chromosomes localized the human endopeptidase 24.15 to 19q13.3.