Ke-Mei Peng

Molecular characterization and expression analysis of the IFN-gamma related gene (IFN-γrel) in grass carp Ctenopharyngodon idella

Interferon gamma (IFN-gamma), the only member of the type II class of interferons, has been identified in teleost fish. In addition to the IFN-gamma gene, fish possess an IFN-gamma related gene (IFN-gammarel) neighbouring the authentic IFN-gamma gene in the genome. In the present study, the cDNA sequence encoding 167 amino acids of IFN-gammarel and its genomic organization were identified in grass carp Ctenopharyngodon idella. The predicted protein sequence of grass carp IFN-gammarel (gcIFN-gammarel) showed 63% and 50% identities to zebrafish and common carp IFN-gammarel (previously termed as IFN-gamma1), respectively. The IFN-gammarel gene consists of 4 exons, with 3 intervening introns, spanning approximately 2kb of genomic sequence. The gcIFN-gammarel gene did not contain any polymorphic DNA repeats in the introns. Realtime PCR analysis showed that grass carp reovirus induced a high and long lasting (from days 1 to 7) expression of gcIFN-gammarel in spleen. The expression of gcIFN-gammarel in blood, head kidney, trunk kidney and spleen was also increased by bacterial peptidoglycan (PGN), lipopolysaccharide (LPS) and the interferon inducer polyI:C. The highest induction of gcIFN-gammarel expression by PGN was observed in spleen, then in blood and head kidney. Further analysis of the expression patterns of gcIFN-gammarel and PGN receptors, nucleotide oligomerization domains (NOD) 1 and 2, may suggest that IFN-gammarel was possibly activated in a NOD2-dependent mechanism.

The morphological study on the adrenal gland of African ostrich chicks

The morphology of the adrenal gland has been studied for a number of animal species all over the world, yet the detailed data about ostrich chick has not been reported. In the present study, the morphological features of the adrenal gland in African ostrich chicks were investigated by means of gross anatomy, light and electron microscope. Differences between the left and right adrenal glands were found in shape, size and location. The interrenal tissue and chromaffin cell interdigitated irregularly. The interrenal tissue was divided into a peripheral zone (PZ) and a central inner zone (CZ), and the PZ was further distinguished into an outer area (subcapsular zone, SCZ) and an inner area (IZ). The cellular arrangement in these zones showed evident zonation that resembled the mammalian. This phenomenon had been previously described only for the pelicanus. The cytoplasm of interrenal cells in SCZ was stained lightly than in IZ and CZ, and contained several vacuoles. Additionally, unlike CZ cells, SCZ cells appeared to contain more mitochondria and less lipid droplets. Two types of chromaffin cells: epinephrine cells and norepinephrine cells could be detected. The type 1 granules possessed a central core and a variable distance between membrane and core; the type 2 granules had an eccentric core, which leant to one side of granule and sticked to the membrane, giving a lager lacouna appearance in another side of the granule.

Effect of boric acid supplementation of ostrich water on the expression of Foxn1 in thymus.

Foxn1 is essential for thymus development. The relationship between boric acid and thymus development, optimal dose of boric acid in ostrich diets, and the effects of boric acid on the expression of Foxn1 were investigated in the present study. Thirty healthy ostriches were randomly divided into six groups: Group I, II, III, IV, V, VI, and supplemented with boric acid at the concentration of 0 mg/L, 40 mg/L, 80 mg/L, 160 mg/L, 320 mg/L, 640 mg/L, respectively. The histological changes in thymus were observed by HE staining, and the expression of Foxn1 analyzed by immunohistochemistry and western blot. TUNEL method was used to label the apoptotic cells. Ostrich Foxn1 was sequenced by Race method. The results were as following: Apoptosis in ostrich thymus was closely related with boric acid concentrations. Low boric acid concentration inhibited apoptosis in thymus, but high boric acid concentration promoted apoptosis. Foxn1-positive cells were mainly distributed in thymic medulla and rarely in cortex. Foxn1 is closely related to thymus growth and development. The nucleotide sequence and the encoded protein of Foxn1 were 2736 bases and 654 amino acids in length. It is highly conserved as compared with other species. These results demonstrated that the appropriate boric acid supplementation in water would produce positive effects on the growth development of ostrich thymus by promoting Foxn1 expression, especially at 80 mg/L, and the microstructure of the thymus of ostrich fed 80 mg/L boric acid was well developed. The supplementation of high dose boron (>320 mg/L) damaged the microstructure of thymus and inhibited the immune function by inhibiting Foxn1 expression, particularly at 640 mg/L. The optimal dose of boric acid supplementation in ostrich diets is 80 mg/L boric acid. The genomic full-length of African ostrich Foxn1 was cloned for the first time in the study.

The role of visfatin on the regulation of inflammation and apoptosis in the spleen of LPS-treated rats.

The purpose of the present study is to determine if visfatin is involved in inflammation or apoptosis induced by LPS in rat. Forty Wistar rats were divided into four groups: saline group, LPS group, visfatin group and Visfatin + LPS co-stimulated group. Spleen samples from each group of rats were collected for study. The spleen structure was examined by histological imaging. Apoptosis was evaluated with TUNEL reaction. Caspase-3 was detected with immunohistochemistry and western blot. The apoptosis-related genes were detected by qPCR and inflammatory cytokines were tested by ELISA. Our main findings were as follows. (1) Macrophages were markedly increased in the visfatin group compared with the saline group. This finding was confirmed when spleen samples were examined with western blot using CD68 antibody. (2) Visfatin promoted the expression of CD68 and caspase-3 in rat spleen, whereas visfatin could inhibit the expression of CD68 and activated caspase-3 in spleen of LPS-induced acute inflammation. (3) Visfatin had a pro-apoptotic effect on normal rat spleen, whereas it exerted an anti-apoptotic effect during LPS-induced lymphocytes apoptosis in rat spleen. Moreover, the effect of visfatin on cell apoptosis was mediated by the mitochondrial pathway. (4) Visfatin could modulate both the anti-inflammatory cytokines and pro-inflammatory cytokines in rat spleen, such as IL-10, IL-4, IL-6, TNF-α and IL-1β. Taken together, we demonstrate that visfatin could participate in the inflammatory process in rat spleen by modulating the macrophages and inflammatory cytokines. Also, visfatin plays a dual role in the apoptosis in rat spleen, which is mediated by the mitochondrial pathway.

Effects of lipopolysaccharide on the histomorphology and expression of toll-like receptor 4 in the chicken trachea and lung

ABSTRACT Endotoxin or lipopolysaccharide (LPS) exposure can cause injury to the respiratory airways and in response, the respiratory epithelia express toll-like receptors (TLRs) in many species. However, its role in the innate immunity in the avian respiratory system is poorly understood. The aim of the present study was to evaluate the effects of LPS on the chicken trachea and lung. After intraperitoneal LPS or saline injection, the trachea and lungs were harvested at 0, 12, 36 and 72 h (n = 6 at each time point) and histopathologically analysed using haematoxylin and eosin and periodic acid-Schiff staining, while TLR4 expression was determined by immunohistochemistry and secretory Immunoglobulin A (SIgA) levels by enzyme-linked immunosorbent assay. After LPS stimulation, we observed a remarkable decrease in the number of goblet cells along with obvious disruption and desquamation of the ciliated epithelium in the trachea, blurring of the boundary between pulmonary lobules, narrowed or indistinguishable lumen of the pulmonary atria and leukostasis in the lungs. Following LPS stimulation, TLR4 protein expression was up-regulated in both the trachea and the lungs and was found on the ciliated columnar cells as well as in the submucosa of the trachea, and in the lungs on parenchymal and immune cells. However, SIgA levels were only up-regulated in the trachea at 12 h following LPS stimulation. Hence, this report provides novel information about the effects of LPS on the microstructure of the lower respiratory tract and it is concluded that its intra-peritoneal administration leads to TLR4-mediated destruction of the tracheal epithelium and pulmonary inflammation along with increased SIgA expression in the tracheal mucosa.

Lipopolysaccharide stimulation upregulated Toll-like receptor 4 expression in chicken cerebellum.

Toll-like receptors (TLRs) play crucial roles in innate and adaptive immune responses to invading pathogens. TLR4 is responsible for the recognition of bacterial lipopolysaccharide (LPS) in different parts of central nervous system of many vertebrates. To better understand the functions of TLR4 in cerebellum of chicken, present study was designed to identify the cell types that express TLR4 during postnatal stages as well as the changes in its expression in response to LPS challenge. For this purpose, cerebella were collected from chicken aged 1, 14 and 40 days (n=7 in each group) to analyze TLR4 distribution pattern. The cerebella from 14 chickens injected with LPS or sterilizing saline were also collected at Day 14 (n=7 in each group) to investigate changes in TLR4 expression. This expression was analyzed by immunohistochemistry using an anti-TLR4 antibody. TLR4 was constitutively expressed in the Purkinje cell layer, pia mater, neurons in medulla and blood vessels in the cerebellum and LPS stimulation significantly up-regulated TLR4 expression on Day 14 in the chicken cerebellum. This study provides evidence that neurons in chicken cerebellum can express TLR4 in vivo and suggests that these neurons may play an important role in initiating a defense reaction via activation of TLR4.

Effects of supplemental boron on growth performance and meat quality in African ostrich chicks.

To investigate the effects of boron on growth performance and meat quality, 10-day-old Africa ostrich chicks were randomly divided into 6 groups with 6 replicates in each group. For 80 days, birds in the treatments were fed the same basal diet but given different concentrations of boron-supplemented water. The highest final BW (33.4 ± 0.30 kg), ADFI (376 ± 1.83 g), and ADG (224 ± 1.01 g) appeared in the group receiving 160 mg/L boron (group 4). 160 mg/L boron also decreased drip loss (2.20 ± 0.59), cooking loss (35.3 ± 1.14), and elevated pH value (6.13 ± 0.28) of meat (P < 0.05). Ostrich chicks in the 640 mg/L treatment group (group 6) had the lowest final BW (30.8 ± 1.05 kg) and ADG (208 ± 0.74 g) (P < 0.05). The highest ash (1.35 ± 0.01%) and pH (6.18 ± 0.03) and the lowest protein (20.4 ± 1.74%), drip loss (2.10 ± 0.76%), cooking loss (35.0 ± 0.41%), C18:1 (28.2 ± 0.65%), and C18:3ω3 (2.60 ± 0.51%) appeared in group 6 (P < 0.05) as well. Overall, the optimum concentration of 160 mg/L supplemental boron improved ostrich growth performance and meat quality; however, high concentrations of boron decreased both performance and meat quality.

TLR4 is constitutively expressed in chick thymic epithelial cells

Toll-like receptor 4 (TLR4) has been suggested to play a regulatory role in immune cell development; however, studies regarding the role of TLR4 in the development of the chick thymus are scarce. In this study, we investigated the distribution and expression pattern of TLR4 in normal chick thymi at different stages of development, in order to better understand the role of TLR4 in chick thymus development. We studied the thymi from 15 chicks, collected at days 7, 21 and 35 of age. The relative change in TLR4 mRNA expression in the chick thymus at different ages was determined by quantitative real-time PCR, and changes in protein expression were analyzed by immunohistochemistry and Western blotting. Furthermore, the distribution of TLR4 in the chick thymus was analyzed by immunohistochemistry, and compared with the distribution of TLR4 expression in juvenile female pigs (gilts). Our results indicated that TLR4 was constitutively expressed in the chick thymus. TLR4 was primarily expressed in the thymic cortico-medullary junction and the medulla, particularly in the epithelial cells of Hassalls corpuscles. The mRNA and protein expression level of TLR4 increased in the thymus with increasing age (p<0.05). Taken together, these results indicate that TLR4 is constitutively expressed by epithelial cells in the chick thymus, suggesting it may participate in thymic development by inducing factors affecting its development.

Molecular cloning, characterization and tissue distribution of two ostrich β-defensins: AvBD2 and AvBD7.

Avian β-defensins (AvBDs) are a family of small antimicrobial peptides that play important roles in the innate immunity of birds. Herein, we report on two new ostrich AvBD genes, AvBD2 and AvBD7, which were isolated from the bone marrow of ostriches (Struthio camelus). The coding regions of ostrich AvBD2 and AvBD7 comprised 195 bp and 201bp, which encoded 64 and 66 amino acids, respectively. Homology analysis showed that ostrich AvBD2 had the highest similarity (up to 86%) with the swan goose (Anser cygnoides) AvBD2, while ostrich AvBD7 shared the highest similarity (up to 81%) with chicken AvBD7. Analysis of the codon-usage bias showed that the two ostrich AvBDs had different codon-usage patterns from other AvBDs. The two synthetic AvBD peptides exhibited antibacterial activities against both Gram-positive and Gram-negative bacteria, and these activities decreased significantly in the presence of 100mM NaCl (P<0.01). Real-time reverse transcription-polymerase chain reaction analysis showed that AvBD2 and AvBD7 were widely expressed at different levels in 17 different tissues. This is the first report of the nucleotide sequences of ostrich AvBDs. Further investigations of these two AvBDs may help us to gain new insights into the immune defense system of the ostrich and to make subsequent therapeutic use of ostrich defensins.

Strategic Localization of Toll‐like Receptor 4 in the Digestive Tract of Blunt Snout Bream (Megalobrama amblycephala)

This study was performed to determine the localization strategies of Toll‐like Receptor 4 (TLR4) in digestive tract (oesophagus, bulbodium, foregut, midgut and hindgut) of Blunt snout bream (Megalobrama amblycephala) using immunohistochemical staining method. TLR4 positive cells were observed throughout the digestive tract. In the oesophagus, some positive reactions in lamina propria were found around small blood vessels and there were also some positive cells within the stratified squamous epithelium. Lots of positive cells were observed in the muscular layer of the oesophagus. In bulbodium, foregut and hindgut, the expression of TLR4 was mainly restricted to the apical surface of epithelial cells located at the bottom of the mucosal folds and the mesenchymal cells in lamina propria. It was very interesting that epithelial cells in the midgut, but none in other parts, had many TLR4 positive cytoplasmic granular structures which were also periodic acid Schiff positive. These findings suggested that TLR4 was expressed in a compartmentalized manner in the Blunt snout bream (M. amblycephala) digestive tract and provided novel information about the in vivo localization of pattern recognition receptors.