Non-invasive determination of the immune physiological state of reindeer (Rangifer tarandus) in the Greater Khingan Mountains, China.

Immunoglobulin and cortisol levels are good indicators of well-being and living status in animals. In this study, the concentrations of fecal immunoglobulins A ([IgAF]), G ([IgGF]), and M ([IgMF]), and cortisol ([cortisolF]) were examined by enzyme-linked immunosorbent assay in reindeer of the Greater Khingan Mountains of Inner Mongolia, China. [IgAF] was significantly higher than [IgGF] and [IgMF], and [IgGF] was significantly higher than [IgMF] (P < 0.05). Both [IgAF] and [IgGF] were higher in the Adult group than in Aged or Infant groups, and higher in the Young than Infant group (P < 0.05). The four age group [IgMF]s were not significantly different (P > 0.05). [IgAF], [IgGF], and [IgMF] in each age group were higher in females than in males, with a significant difference in the Young group (P < 0.05). The Infant group had the highest [cortisolF], and the Adult group the lowest; [cortisolF] was significantly higher in the Infant group than in other age groups (P < 0.05). In each age group, [cortisolF] was higher in females than males, and there were significant differences among the Infant, Young, and Aged groups (P < 0.05). A significant negative correlation was observed between [cortisolF] and [IgAF] and [IgGF] (P > 0.05). Overall physical condition was better in the Adult and Young groups than in the Aged and Infant groups as determined by the comprehensive analysis of fecal Ig levels in the four age groups, with the Infant group the worst.

Attenuated mRNA expression of lipid metabolism genes in primary hepatocytes following lipopolysaccharide treatment in dairy cows.

The influence of ruminal acidosis on ruminal microbiology and metabolite production has received considerable attention, but little is known regarding the systemic manifestations that arise from ruminal acidosis. Lipopolysaccharide (LPS) is released in the gastrointestinal tract upon ingestion of high-grain or high-fat diets, and it has been implicated in the etiology of multiple energy- and lipid-related metabolic disturbances in ruminants. The liver plays a crucial role in the acute phase response to intruding pathogens. The effect of blood LPS in subacute ruminal acidosis on lipid metabolism in the liver has not been established. In this study, cell cultures were photographed using an inverted microscope. We observed that hepatocytes changed their morphologies from irregular triangle to circular (contraction) shapes; the number of contracted cells increased with the increasing LPS doses. This suggests that LPS can promote cell contraction and take off the wall, ultimately leading to cell apoptosis. With changes in LPS exposure, hepatocyte number also changes. We explored lipid metabolism in the liver using quantitative reverse transcription-polymerase chain reaction to detect the expression of key lipid metabolism enzymes in hepatocytes. We found that Toll-like receptor 4 signaling pathway mediated by LPS could attenuate mRNA expression of fatty acid synthesis genes and increase the expression of fatty acid transport genes in primary hepatocytes following LPS treatment in dairy cows.

Lactoferrin mRNA expression in mouse mammary glands during pregnancy and lactation.

Lactoferrin (Lf) is an iron-binding glycoprotein that is produced by mucosal epithelial cells in mammals. Lf has non-immune natural defense functions and biological functions in addition to and distinct from its role in regulating inflammatory responses. Lf also improved some physiological and immunological parameters. Lf is a biomarker for monitoring medical treatment in inflammatory bowel diseases. Current LF research focuses on iron absorption, antimicrobial activity, and the modulation of iron metabolism during inflammation. No systematic research about Lf expression levels in mouse mammary glands during pregnancy and lactation exists. We investigated Lf mRNA expression levels in mouse mammary glands by collecting samples on days 1, 6, 12, and 18 of pregnancy and lactation (six mice per group). The expression levels of Lf mRNA were measured by semi-quantitative reverse transcription polymerase chain reaction using GAPDH as an internal control. Lf mRNA was not expressed in mammary glands on days 1, 6, and 12 of pregnancy, but it was expressed on day 18 (IOD: integrated optical density; Lf(IOD)/GAPDH(IOD) = 0.46). Lf expression levels were higher during lactation stages than during pregnancy stages, and it stabilized at 0.71-0.73 (Lf(IOD)/GAPDH(IOD)) from day 1 to 12 of lactation; however, the difference was not significant (P > 0.05). At day 18 of lactation, Lf expression began to decline (Lf(IOD)/GAPDH(IOD) = 0.61), but the difference was not significant (P > 0.05). Based on these results, the variation in Lf expression levels during developmental stages may be related to its regulatory role in mouse mammary gland immunity.

Effects of lipopolysaccharide on the stearoyl-coenzyme A desaturase mRNA level in bovine primary hepatic cells.

This study aimed to compare the effects of lipopolysaccharide (LPS) on stearoyl-coenzyme A desaturase (SCD) gene expression in mouse primary hepatic cells. To obtain sufficient total RNA, primary hepatic cells were plated on 6-cm diameter-type collagen 1-coated dishes (1 x 106 cells per dish). The test was divided into 6 groups with 6 replications per group. The 6 groups were treated with the following volumes of LPS (0.1 mg/mL): 0, 1, 1.5, 2, 4, and 8 µL. The cells were cultured for 24 h, and the total RNA was extracted from samples. Reverse transcription polymerase chain reaction was used to analyze SCD mRNA levels. With increasing LPS amounts, the SCD mRNA expression first decreased and then increased slightly; the expression was the lowest in the 2-µL LPS condition. The SCD mRNA levels from the 4- and 8-µL LPS conditions were slightly higher than that from the 2-µL LPS condition, but the difference was not significant (P > 0.05). The SCD mRNA level from the 2-µL LPS condition was obviously lower than that from the 0-, 1-, and 1.5-µL LPS condition, and the differences were significant (P < 0.05), and the SCD mRNA levels from the 0-, 1-, and 1.5-µL LPS conditions were not significantly different (P > 0.05). The SCD mRNA levels from the 4- and 8-µL LPS conditions were obviously lower than those from the 0- and 1-µL LPS conditions, and the differences were significant (P < 0.05).

Toll-like receptor 3 polymorphism is not associated with neovascular age-related macular degeneration and polypoidal choroidal vasculopathy in the Chinese.

Toll-like receptor 3 (TLR3) variants in mainland northern Chinese patients with polypoidal choroidal vasculopathy (PCV) and neovascular age-related macular degeneration (nAMD) were investigated. The complete genes of TLR3, including all exons and the promoter region, were assessed using direct sequencing technology of 284 unrelated mainland northern Chinese individuals: 96 nAMD patients, 92 PCV patients, and 96 controls. Six single nucleotide polymorphisms were identified: rs5743303, rs5743305, rs5743312, rs3775291, rs3775290, and rs6830345. The distribution of TLR3 genotypes for nAMD and PCV was not significantly different compared with normal controls. This study indicates that the TLR3 gene polymorphism is not associated with nAMD and PCV in northern Chinese patients.

Dendritic cells transfected with hepatocellular carcinoma (HCC) total RNA induce specific immune responses against HCC in vitro and in vivo.

BACKGROUND: Immunotherapy is an effective method for preventing metastasis and recurrence of carcinoma. Hepatocellular carcinoma (HCC) is a common malignancy with a high rate of recurrence, and has not successfully been introduced to immunotherapy. METHODS: Peripheral blood mononuclear cells were isolated from whole blood of HCC patients and stimulated to transform into dendritic cells (DCs). These DCs were then transfected with RNA extracted from HepG-2 hepatoma cells to induce expression of specific antigens. RESULTS: The transfected DCs stimulated T lymphocytes to produce cytotoxic T lymphocytes, which specifically attacked HepG-2 cells. Injection of T lymphocytes from HCC patients and transfected DCs into severe combined immunodeficiency mice limited the growth of HepG-2 tumors. CONCLUSION: A specific immune response against hepatoma can be generated in vivo by administering DCs transfected with RNA from a specific tumor. This method may have therapeutic application in humans to reduce recurrence of HCC.

Isolation and identification of bovine primary hepatocytes.

The liver is a unique organ that is endowed with a plethora of specialized functions. Most of its functional traits are controlled by hepatocytes. Primary hepatocytes have been used widely in in vitro models to understand the biological processes occurring in the liver. There are a number of methods used to separate hepatocytes, but the cell activity and purity are much lower in this condition. On the basis of previous research, in this study, the two-step collagenase perfusion technique was used for isolating hepatocytes. The key proteins of hepatocytes, cytokeratin-18 (CK-18) and albumin (ALB), were used to identify cells, and their contents were evaluated by immunohistochemistry and Western blotting. The results showed that the isolated hepatocytes comprised more than 96% of the corresponding protein volume stability. Therefore, this method was demonstrated to be reliable for identifying hepatocytes.

IL-8 mRNA expression in the mouse mammary glands during pregnancy and lactation.

Interkeukin-8 (IL-8) is an important inflammatory mediator. It is an angiogenic factor associated with inflammation and carcinogenesis. To date, research on IL-8 has been limited to its role as an indicator of inflammation. There has been no systematic research concerning IL-8 expression levels in the mouse mammary gland during pregnancy and lactation. Mouse mammary gland samples were collected on days 1, 6, 12, 18 of pregnancy and of lactation (6 mice per group). The expression levels of IL-8 mRNA were measured by semi-quantitative RT-PCR, with GAPDH as an internal control. IL-8 mRNA was highly expressed on day 1 of pregnancy in the mouse mammary glands (IL-8(IOD)/GAPDH(IOD) = 1.68), and then suddenly declined; it reached 0.74 and 0.71 on days 6 and 12 of pregnancy. On day 18 of pregnancy, it started to increase (IL-8(IOD)/GAPDH(IOD) = 1.02). However, the expression levels of IL-8 mRNA were not significant during pregnancy. During lactation, IL-8 expression level was lower than during pregnancy, but it stabilized at 0.32-0.41 (IL-8(IOD)/GAPDH(IOD)) from day 1 to day 18 of lactation, although the difference was not significant. We suggest that the changes in IL-8 expression level during development is related to its regulatory role in mouse mammary gland immunity.

Isolation and characterization of nucleotide-binding site and C-terminal leucine-rich repeat-resistance gene candidates in bananas.

Commercial banana varieties are highly susceptible to fungal pathogens, as well as bacterial pathogens, nematodes, viruses, and insect pests. The largest known family of plant resistance genes encodes proteins with nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. Conserved motifs in such genes in diverse plant species offer a means for the isolation of candidate genes in banana that may be involved in plant defense. Six degenerate PCR primers were designed to target NBS and additional domains were tested on commercial banana species Musa acuminata subsp malaccensis and the Musa AAB Group propagated in vitro and plants maintained in a greenhouse. Total DNA was isolated by a modified CTAB extraction technique. Four resistance gene analogs were amplified and deposited in GenBank and assigned numbers HQ199833-HQ199836. The predicted amino acid sequences compared to the amino acid sequences of known resistance genes (MRGL1, MRGL2, MRGL3, and MRGL4) revealed significant sequence similarity. The presence of consensus domains, namely kinase-1a, kinase-2 and hydrophobic domain, provided evidence that the cloned sequences belong to the typical non-Toll/interleukin-1 receptor-like domain NBS-LRR gene family.

Leptin mRNA expression in the rat mammary gland at different activation stages.

Leptin is expressed in various tissues, suggesting that this protein is effective not only at the central nervous system level, but also peripherically. Recent studies have shown leptin production by other tissues, including the placenta, stomach, and mammary tissues, but there is no information available concerning expression levels of leptin in the rat mammary gland at different activation stages. We used semi-quantitative RT-PCR to investigate leptin mRNA expression levels in the rat mammary gland at different activity stages. Rat mammary gland samples were collected from virgin females and on days 6, 12, 18 of pregnancy and of lactation (six rats per group). The expression levels of leptin mRNA were measured by semi-quantitative RT-PCR, with ß-actin as an internal control. Leptin mRNA was highly expressed in virgin rat mammary glands (leptin(IOD)/ß-actin(IOD) = 1.60). It decreased gradually during pregnancy, being lowest at 18 days of pregnancy, when the levels were significantly lower than in virgin mammary tissue. Leptin mRNA increased slightly during lactation, but the difference was not significant. By day 18 of lactation, expression levels of leptin mRNA reached the same values as in virgin mammary tissue (leptin(IOD)/ß-actin(IOD) = 1.65). Based on these results, we suggest that leptin has an important regulation role in rat mammary gland activation.