Changes in estrogen receptor expression in the chick thymus during late embryonic development.

In chickens, although estrogen receptors (ER) are reported to be associated with the immunological processes, detailed information about the differences in ER expression in the tissues related to the development of lymphocytes is not fully known, especially during the developmental stage. To learn more about this immunological relationship, we used semi-quantitative polymerase chain reaction method to detect the ER expression levels in the thymus tissues of chicks during the developmental stage. Furthermore, ER-expressing cells were detected by immunohistochemistry. The results of this study show that the expression level of ER increased on embryonic day 16 and decreased on day 20. Furthermore, ER expression was significantly higher in male than in female chickens at day 16. The increased expression on day 16 and decreased level on day 20 were also reproduced in the incidence of immunoreactive cells, although there was a 1-day delay in the elevated incidence of the cells. This study revealed the changes in ER expression and the incidence of ER-positive cells in the thymus of chickens during the developmental stage.

Localization of estrogen receptor in the central lymphoid organs of chickens during the late stage of embryogenesis.

Immunological function in chicks is greatly affected by estrogen treatment during embryogenesis, but the mechanism of the estrogen effect is not fully understood. To elucidate the effect of estrogen on immune function, we observed estrogen receptor expression in the thymus and bursa of chick embryos by immunohistochemistry. We compared the distribution of estrogen receptor-positive cells with that of keratin-positive epithelial cells. Intense expression of estrogen receptors was detected in thymic and bursal lymphocytes. In peripheral lymphocytes, ER mRNA was detected by RT-PCR analysis. The results of fluorescence-activated cell sorting analysis indicated that the estrogen receptor was expressed in the cytoplasm of the lymphocytes. Furthermore, intense expression of the estrogen receptor was also confirmed in thymic Hassall's corpuscles, bursal follicle-associated epithelial cells, and the bursal interfollicular epithelium. Our results indicate that estrogen affects the differentiation of thymic and bursal lymphocytes, suggesting that the underlying role for estrogen in immune function.

Effects of estrogen on estrogen receptor expression in the bursal cells of chick embryos and steroidogenic enzymes gene expression in the bursa: relevance of estrogen receptor and estrogen synthesis in the bursa of chick embryos.

Estrogen has been reported to act on B cell genesis in the bursa of Fabricius of chick embryos. In this study, we attempted to demonstrate the hypothesis that B cell genesis is controlled by estrogen receptor (ER) in the bursal cells and steroidogenic enzymes synthesized in the bursa. We previously reported the presence of estrogen receptor α (ERα) in the bursa during the late stage of embryogenesis and an increase in the expression of ERα messenger RNA (mRNA) between the 13th day and 16th day. The number of ER-positive cells was maximal on the 16th day. In the present study, ER-positive cells in the bursa during the late stage of embryogenesis increased 4 h after ß-estradiol treatment on the 14th to 18th day. The concentration of ß-estradiol in the embryonic bursa increased. These results suggest that this stage of embryogenesis is critical in B cell development in the bursa in connection with the effect of estrogen treatment. Our findings also showed that the mRNA expression of five steroidogenic enzymes occurred in the bursa of chick embryos. These results suggest that estrogen is synthesized in the embryonic bursa and estrogen acts on the bursal cells in a paracrine fashion.

Immunomodulatory effects of Alliums and Ipomoea batata extracts on lymphocytes and macrophages functions in White Leghorn chickens: in vitro study.

We previously described that supplementary garlic, onion and purple sweet potato (PSP) enhance humoral immune response in White Leghorn chickens. In the present in vitro study, we investigated the effects of garlic (GE), onion (OE) and PSP (PSPE) extracts on proliferation, interleukin (IL)-2 and interferon (INF)-γ gene expression of stimulated lymphocytes. The effects on microbicidal activity, reactive oxygen species (ROS) and nitric oxide (NO) productions of stimulated peritoneal macrophages were studied as well. The results showed that GE augmented Concanavalin A (ConA)-induced splenocytes (4, 8 and 16µg/mL) and thymocytes (2, 4 and 8µg/mL) proliferations, and gene expression of IL-2 (8 and 16µg/mL) and INF-γ (16µg/mL). None of the examined extracts had mitogenic effect nor stimulated bursacytes response to phorbol 12-myristate 13-acetate (PMA). Macrophages exhibited superior microbicidal activity and ROS production with GE at 4 and 8µg/mL and with OE at 25.6µg/mL. None of the extracts showed stimulatory effects on NO production. The extracts showed concentration-dependent inhibitory effects on all measured parameters at higher concentrations. Taken together, it is likely that garlic has direct stimulatory effects on immune cell functions, whereas the in vitro inhibitory effects of onion and PSP were likely attributed to high flavonoid contents.

Modulatory effects of two levels of dietary Alliums on immune response and certain immunological variables, following immunization, in White Leghorn chickens.

This study aimed at investigating the effects of dietary Allium sativum (garlic, G) and Allium cepa (onion, O) on immune functions in White Leghorn chicken. One-week-old chicks, were fed diets without (control) or with Alliums (GL and OL, 10 g or GH and OH, 30 g/kg diet). Chickens were immunized with Newcastle disease virus (NDV), sheep red blood cells (SRBC) and Brucella abortus (BA). Antibodies, lymphocyte proliferation, and ratios of CD4(+) , CD8(+) and CD4⁻ CD8⁻ lymphocytes were investigated. Histology and weights of the spleen, thymus and bursa (BF), and white blood cell (WBC) counts were studied as well. Alliums at 10 g/kg diet enhanced anti-NDV, anti-SRBC and anti-BA antibody productions, whereas 30 g/kg diet had less stimulatory effects. Histology of the lymphoid organs and proliferation of peripheral blood lymphocytes (PBL) were not influenced. However, splenocyte and thymocyte proliferations were augmented with garlic. Flow cytometry analysis showed reduction in CD4(+) and increase in CD4⁻ CD8⁻ lymphocyte ratios in GH and OH groups. Garlic-supplemented chickens had heavier spleen and thymus, and higher WBC counts, whereas BF weight increased with both Alliums at 30 g/kg diet. These results suggest that dietary Alliums have a potential to enhance the immune functions in White Leghorn chickens.

Gastrointestinal tract disorder in natriuretic peptide receptor B gene mutant mice.

Natriuretic peptide receptor B (NPR-B), which has high affinity for C-type natriuretic peptide (CNP) and synthesizes intracellular cGMP, may be involved in gastrointestinal tract (GIT) regulation. A mutant allele of the NPR-B-encoding gene (Npr2) is responsible for the phenotype of the short-limb dwarfism (SLW) mouse. Homozygosity for this autosomal-recessive gene (slw/slw) leads to dwarfism and death before weaning because of milk retention in the stomach and intestinal distention. To elucidate the relationship between CNP/NPR-B signaling and GIT function, we investigated the association between Npr2 mutation and the GIT phenotype in slw/slw mice. The pylorus and large intestine of the mutants did not respond to CNP stimulation; further, they showed pyloric lumen narrowing with randomly aligned circular muscle cells. Comparison of the cGMP and neuronal marker distribution in GIT tissues confirmed cGMP expression in neuronal tissues. An Auerbach's plexus and submucosal tissues of the mutants didn't express cGMP and expressed Ca(2+). In contrast, those of normal mice (controls) expressed both cGMP and Ca(2+). Sequencing revealed that the causative Npr2 mutation was a 7-base deletion in exon 8, resulting in a frameshift and premature termination codon appearance. Therefore, the GIT phenotype of slw/slw mice is because of a CNP/NPR-B-signaling defect caused by an Npr2 mutation. These results facilitate better understanding of the role of CNP/NPR-B signaling in GIT motility.

In vivo immunomodulatory effects of dietary purple sweet potato after immunization in chicken.

This study was intended to determine the modulatory effects of dietary supplementation of purple sweet potato (Ipomoea batats Poir., PSP) on the immune response of chickens. PSP was included in a basal starter diet by 1% (PSP(L)) or 3% (PSP(H)) and continually fed. Newcastle disease (NDV) vaccine, Brucella abortus (BA) and sheep red blood cells (SRBC) were used for chicken immunization. Antibody titers against these antigens were used to estimate humoral immunity. Concanavalin A (Con A)-induced proliferations of splenocytes, thymocytes and peripheral blood lymphocytes (PBL), ratios of CD4- and CD8-single positive and CD4-CD8-double negative (DN) cells in splenocytes, were both used to indicate cellular immunity. Relative weights of spleen, thymus and bursa and white blood cell (WBC) counts were studied. PSP(H) increased anti-NDV (P < 0.05), anti-BA (P < 0.01) and anti-SRBC titers (P < 0.05) in response to secondary immunization, whereas PSP(L) increased titers of anti-BA (P < 0.05) and anti-SRBC (P < 0.01). Proliferations of splenocytes and thymocytes were augmented with PSP(L) (P < 0.05). PSP(H)-treated chickens had lower (P < 0.05) ratios of CD4-single positive lymphocytes. Proliferation of PBL, weights of lymphoid organs and WBC counts were not affected. These results suggest that dietary PSP supplementation could enhance the immune response after immunization in chickens.

Mobiloization of intracellular calcium ions in chicken and rat lymphocytes induced by T cell mitogens.

Cytosolic Ca(2+) is known to be an important factor in intracellular signaling pathways that regulate several cellular functions. The present study was designed to measure the intracellular concentrations of Ca(2+) ([Ca(2+)](i)) in T cell mitogen-stimulated chicken lymphocytes, and to compare the results with those in rat lymphocytes. [Ca(2+)](i) was increased in the thymocytes, splenocytes and bursacytes of chickens, and in the thymocytes and splenocytes of rats following exposure to the mitogens phytohaemagglutinin (PHA) and concanavalin A (ConA). Increases were greatest in the thymocytes followed by the splenocytes and bursacytes. The PHA-induced changes in the thymocytes and splenocytes were similar in chickens and rats, but the ConA-induced increases were significantly lower in the chickens than rats. Pretreatment with EGTA before the application of PHA and ConA completely suppressed the rise in [Ca(2+)](i) in all the chicken lymphocytes, indicating that the increases that occurred in PHA- and ConA-treated chicken lymphocytes could be entirely attributed to the influx of extracellular Ca(2+). On the other hand, the PHA- and ConA-induced increase in [Ca(2+)](i) in rat lymphocytes was not completely suppressed by EGTA, indicating the recruitment of Ca(2+) from the intracellular Ca(2+) pool. The results suggest species differences in the Ca(2+)-based responses to T cell mitogens between chicken lymphocytes and rat lymphocytes.

Extracellular signal-regulated kinase (ERK) activation in chicken heterophils stimulated with phorbol 12-myristate 13-acetate (PMA), formyl-methionylleucyl-phenylalanine (fMLP) and lipopolysaccharide (LPS).

The signaling pathways leading to the activation of extracellular signal-regulated kinase (ERK) by phorbol 12-myristate 13-acetate (PMA), formyl-methionylleucyl-phenylalanine (fMLP) and lipopolysaccharide (LPS) in chicken heterophils were examined. To determine the mechanism of ERK's activation and its relation with the influx of calcium ions, heterophils were stimulated by PMA, fMLP and LPS. ERK was not activated by fMLP. LPS- and PMA-stimulated activation of ERK, based on Western blotting with antibodies against the phosphorylated form of ERK, was attenuated by the pretreatment of cells with the intracellular calcium chelator BAPTA/AM (1,2-bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid) but not with the extracellular calcium chelator EGTA (glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid). Exposure of cells to the protein kinase C (PKC) inhibitor GF109203X inhibited the LPS- and PMA-stimulated phosphorylation of ERK in a concentration-dependent manner. The LPS-stimulated phosphorylation was inhibited by pretreatment with the phospholipase C (PLC) inhibitor U73122 but not the phosphatidylinositol 3-kinase (PI(3)K) inhibitor LY294002. These results indicate that the LPS-induced phosphorylation of ERK in the chicken heterophils is mediated by PLC, PKC and intracellular calcium, and the PMA-stimulated phosphorylation is dependent on intracellular calcium ion and PKC.

B cell differentiation in the bursa of Fabricius and spleen of embryos and chicks immediately after hatching.

Flow cytometric analysis and immunohistochemical observation were used to qualitatively and quantitatively clarify the nature of B cell differentiation in the bursa of Fabricius of chick embryos and to determine the timing of antibody class switching in chicken spleens based on positivity of IgM and IgG on and in the cells. In the bursa, the sIgM-positive cell population formed from the 12(th) to 15(th) day of embryogenesis. The proportion of sIgM-high expressing (sIgM(high)) cells was lower among bursacytes than splenocytes of hatched chicks, suggesting that the sIgM(high) bursacytes are to be released to peripheral sites. The proportion of sIgM(high) cells was higher at 0 days old than at any other examined stage of development. Colonization of the spleen by B cells occurred between the 18(th) day of embryogenesis and 0 days old. Antibody class switching was thought to start in the spleen between 1 and 2 weeks of age, because IgG-positive cells were present in the spleen of 2-week-old chicks, but not 0-day-old or 1-week-old chicks.

Effects of dietary administration of plant-derived anthocyanin-rich colors to spontaneously hypertensive rats.

Anthocyanins have beneficial effects such as free radical scavenging activity. We investigated the effects of continuous administration of colors from purple corn (PCC), purple sweet potato (PSC) and red radish (RRC) to spontaneously hypertensive rats (SHR). These are rich in anthocyanins. Animals were fed with diets containing PCC, PSC or RRC (1 mass% of diets) for 15 wk. While the body weight and the daily food intake of administered rats were not different from those of the non-administered control rats through the experimental period, the blood pressure and the heart rate of SHR administered each color decreased as compared to the control group from the early stage of administration. These results suggest that plant-derived colors containing anthocyanins have anti-hypertensive effects on hypertensive animals.

Characterization of the pharmacology of YM-198313 on volume-regulated anion channels.

Activation of the volume-regulated anion channels (VRAC) is considered to be involved in arrhythmia, but it has not yet been fully elucidated because of the lack of its high affinitive and selective compounds. A newly synthesized compound, YM-198313 (sodium 4-({[2-(methylthio)benzyl]amino}-5-[(1-phenylethyl)thio]isothiazol-3-olate), strongly inhibited VRAC in HeLa cells with an IC50 of 3.03+/-0.05 microM. However, YM-198313 weakly affected both the Ca2+-activated Cl- channels in HTC cells and the cAMP-activated Cl- channels in T84 cells, demonstrating that this compound is selective for VRAC among Cl- channels. At 10 microM, YM-198313 almost completely (100+/-7.8%) inhibited the VRAC current in guinea pig atrial myocytes. However, at the same concentration, YM-198313 showed little inhibitory effect on the cardiac cation currents in ventricular myocytes. We believe that YM-198313 is a potent and selective VRAC inhibitor, therefore, it should be use to clarify the role VRAC plays in arrhythmia.

Enhancement of norepinephrine-induced transient contraction in aortic smooth muscle of diabetic mice.

Changes in norepinephrine-induced transient contractions in Ca2+-deficient solution were investigated in the aortic smooth muscles of diabetic ALS (alloxan-induced diabetes susceptible) mice. The transient contractions in diabetic mice were significantly larger than those in normal mice. The longer incubation of the muscle preparations in Ca2+-deficient solution made the transient contractions smaller, probably due to the leakage and decrease in norepinephrine-releasable stored Ca2+. The rate of this reduction in contraction was slower in diabetic mice. These results suggest that the leakage of intracellular stored Ca2+ caused by extracellular Ca2+ deficiency is attenuated in diabetic mice, contributing to enhanced norepinephrine-induced transient contractions.

Glucosyl hesperidin improves serum cholesterol composition and inhibits hypertrophy in vasculature.

Long-term administration of hesperidin (HES) or glucosyl hesperidin (GHES), a water-soluble analogue of HES, brings about an antihypertensive effect on spontaneously hypertensive rats (SHR). In the present study, we investigated the effects of long-term administration of HES and GHES (corresponding to 30 mg/d/kg body weight) on serum lipid concentration and morphology of vasculature. Serum HDL cholesterol increased in both SHR and Wistar-Kyoto rats (WKY) fed a HES- or GHES-containing diet for 25 wk. Simultaneously, GHES administration reduced the vascular diameter and media-intimal cross-sectional area of the abdominal aorta in SHR. These results suggest that HES as well as GHES improves serum cholesterol composition and that GHES inhibits hypertrophy in vasculature as well.

Effects of long-term administration of hesperidin and glucosyl hesperidin to spontaneously hypertensive rats.

Hesperidin (HES) is a flavonoid contained in citrus fruit peel. We investigated the effects of long-term administration of HES and its newly developed water soluble analogue, glucosyl hesperidin (GHES), to spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Animals were fed with diets containing HES or GHES (30 mg/d/kg body weight) for 25 wk. While the daily food intake and the body weight of administered rats were not different from those of the non-administered control rats in both SHR and WKY through the experimental period, the blood pressure and heart rate of SHR administered HES or GHES for longer than 15 wk decreased as compared to the control group. The blood pressure and heart rate of WKY were not changed by the long-term administration of HES or GHES. These results suggest that HES and GHES have anti-hypertensive effects on hypertensive animals.