Glucose,epithelium, and enteric nervous system: dialogue in the dark

The gastrointestinal epithelium is in close contact with the various components of the chymus, including nutrients, bacteria and toxins. The epithelial barrier has to decide which components are effectively absorbed and which components are extruded. In the small intestine, a nutrient like glucose is mainly absorbed by the sodium linked glucose cotransporter 1 (SGLT1) and the glucose transporter 2 (GLUT2). The expression and activity of both transport proteins is directly linked to the amount of intraluminal glucose. Besides the direct interaction between glucose and the enterocytes, glucose also stimulates different sensory mechanisms within the intestinal wall. The most important types of cells involved in the sensing of intraluminal contents are enteroendocrine cells and neurones of the enteric nervous system. Regarding glucosensing, a distinct type of enteroendocrine cells, the enterochromaffine (EC) cells are involved. Excitation of EC cells by intraluminal glucose results in the release of serotonin (5‐HT), which modulates epithelial functions and activates enteric secretomotorneurones. Enteric neurones are not only activated by 5‐HT, but also directly by glucose. The activation of different cell types and the subsequent crosstalk between these cells may trigger appropriate absorptive and secretory processes within the intestine.     

嗅鞘细胞生物学特性及其在脊髓再生中应用的研究

近年来研究证明嗅球及嗅神经组织可以分离出嗅神经鞘细胞(olfactory ensheathing cells,OECs)，OECs具有中枢神经系统（CNS）星形胶质细胞和外周雪旺式细胞的特性，移植后的OCEs能够在宿主损伤的脊髓组织进行迁移，能够引导支持损伤的神经元突起再生、生长、延伸。而且还可以通过转基因技术使其携带某些促进神经元再生的外源性基因，表达一些促神经再生的分子，从而改善神经损伤的内环境，诱导损伤的脊髓神经元再生，使得受损脊髓再生的潜力得以发挥，达到脊髓再生和功能恢复的目的。本文就OECs的生物学特性及其在脊髓损伤和CNS的基因治疗进行综述。     

Gene expression signatures in neutrophils exposed to glucocorticoids: a new paradigm to help explain "neutrophil dysfunction" in parturient dairy cows

Neutrophils are the first line of immunity against most pathogens that infect cattle. These normally short-lived white blood cells develop from myeloid-lineage cells in bone marrow. Upon maturation, bone marrow neutrophils are released into the circulation where they marginate on inflamed blood vessel endothelial cells and migrate through them into the area of infection. Once migrated, neutrophils do not reenter the circulation, but rather, perform their bactericidal functions and die by apoptosis in the tissue. The cytokine and hormonal milieu of the blood and extracellular tissue fluid can influence neutrophil development and immunity-related activities, but the molecular basis of these phenotypic changes and physiological benefits or drawbacks of them are poorly understood. In the current paper, we review new gene expression information that resulted from two of our functional genomics studies designed to evaluate effects of glucocorticoid hormones on bovine neutrophils. This work provides one model to describe complex changes that occur in neutrophils as the cells respond to glucocorticoids, which might act to alter the cells' functional priorities and tip the delicate balance between health and disease during stress, including at parturition. A bovine immunobiology microarray and real time RT-PCR were used to study blood neutrophils collected during the natural surge of endogenous glucocorticoid (cortisol) in parturient dairy cows and bone marrow neutrophils collected from glucocorticoid (dexamethasone)-treated dairy steers. The gene expression signatures we observed led us to perform additional phenotyping of the neutrophils and correlation analyses, which together painted a picture suggesting that glucocorticoids have key roles in modulating neutrophil development, life span, and tissue defense functions during parturition and hormone therapy. Based on these observations, we postulate that glucocorticoids orchestrate adaptive changes in the entire neutrophil system that support increased cell numbers and longevity in blood and heightened remodeling activity in tissues, while at the same time decreasing some important antimicrobial defense activities of the cells. Thus, our functional genomics studies have enabled us to elucidate multiple consequences of neutrophil exposure to glucocorticoids, highlighting a probable role for this interaction in the induction of parturition and partly explaining why some parturient dairy cows may experience heightened incidence and severity of inflammatory diseases like mastitis.     

Human and ovine lentiviral infections compared

Maedi-visna virus (MVV) of sheep was the first lentivirus to be isolated. The genomic organization of MVV is very similar to that of human immunodeficiency virus (HIV) with several genes regulating the expression of the viral genome. Viral replication is severely restricted in the host and some cells apparently contain the genetic information in a DNA provirus form with little or no expression of viral antigens. This seems to be a major factor in causing the “slowness” of lentiviral infections and the persistence of the virus in the host since the immune system may not recognize the provirus-containing cells. The target cells for HIV and MVV are similar although T4 lymphocytes are not specifically destroyed in maedi-visna. There are also certain similarities in the pathological changes in both diseases, both in the central nervous system, the lungs and the lymphatic system. Although the severe final immunodeficiency state characteristic of AIDS has not been observed in maedi-visna, the basic biological features of the MVV and its interaction with host cells are so similar to HIV infection, that we consider ovine maedi-visna useful animal model for the human lentivirus infections.     

Differentiation and characterization of rat adipose tissue mesenchymal stem cells into endothelial‐like cells

In this study, mesenchymal stem cells were isolated from rat adipose tissue (AD‐MSCs) to characterize and differentiate them into endothelial‐like cells. AD‐MSCs were isolated by mechanical and enzymatic treatments, and their identity was verified by colony‐forming units (CFU) test and by differentiation into cells of mesodermal lineages. The endothelial differentiation was induced by plating another aliquot of cells in EGM‐2 medium, enriched with specific endothelial growth factors. Five subcultures were performed. The expression of stemness genes (OCT4, SOX2 and NANOG) was investigated. The presence of CD90 and the absence of the CD45 were evaluated by flow cytometry. The endothelial‐like cells were characterized by the evaluation of morphological changes and gene expression analysis for endothelial markers (CD31, CD144, CD146). Characterization of AD‐MSCs showed their ability to form clones, to differentiate in vitro and the OCT‐4, SOX‐2, NANOG genes expression. Immunophenotypic characterization showed the CD90 presence and the CD45 absence. The endothelial‐like cells showed morphological changes, the expression of CD31, CD144, CD146 genes and the presence of CD31 membrane receptor. Matrigel assay showed their ability to form network and vessels‐like structures. This study lays the foundations for future evaluation of the potential AD‐MSCs pro‐angiogenic and therapeutic role.     

Expression of Genes in the Canine Pre‐implantation Uterus and Embryo: Implications for an Active Role of the Embryo Before and During Invasion

The aim of the present study was to assess genes expressed in maternal uterine tissue and pre‐implantation embryos which are presumably involved in maternal recognition and establishment of canine pregnancy. For this purpose, 10 pregnant bitches were ovariohysterectomized between days 10 and 12 after mating. Four non‐pregnant bitches served as controls. Early pregnancy was verified by flushing the uterine horns with PBS solution. The collected embryos (n = 60) were stored deep‐frozen (?80°C). Uterine tissue was excised, snaps frozen in liquid nitrogen and homogenized using TRI Reagent. All embryos from one litter were thawed together and also homogenized in TRI Reagent. RT‐PCR was performed to prove mRNA expression of progesterone receptor, key enzymes of the prostaglandin synthesis pathway, selected growth factors, cytokines, immune cell receptors, major histocompatibility complex (MHC) and matrix‐metalloproteinases (MMP). Only pregnant uteri revealed the presence of mRNA for interferon (IFN)‐γ, IL‐4 and CD‐8, which resembles the milieu in humans and other mammalians. Similarly, in day 10 embryos, mRNA for transforming growth factor‐β, insulin‐like growth factor‐1,‐2, hepatocyte growth factor, leukaemia inhibitor factor, tumour necrosis factor‐α, interleukin‐1β,‐6,‐8, cyclooxygenase‐2, CD4⁺ cells, and MMP‐2 and ‐9 were detected, but not MHC‐I or ‐II. We therefore suppose that the canine embryo, like its human counterpart, actively initiates measures to prevent attacks from the maternal immune system to prepare its own adhesion, nidation, growth and further development.     

Conversion of Goat Fibroblasts into Lineage‐Specific Cells Using a Direct Reprogramming Strategy

Direct reprogramming is an efficient strategy to convert one cell type to another. In this study, due to the failure of maintaining the undifferentiated state of goat embryotic stem‐ and induced pluripotent stem‐like cells in vitro, we explored an alternative way to directly convert goat fibroblasts to lineage‐specific cells. The ‘Yamanaka factors’ was ectopically expressed in fibroblasts for a short term to situate cells in a metastable state. By culturing with lineage‐specific media for 1–2 weeks, the cardiomyocyte‐like cells and neurocyte‐like cells were generated and confirmed by the quantitative RT‐PCR and immunocytochemical staining. The metastable‐state cells could also be converted into oocyte‐like cells (OLCs) after culturing in media with retinoic acid (RA) and bovine follicular fluid (bFF) for 2–3 weeks. The generated OLCs were surrounded by cumulus granulosa cell‐like cells and formed a structure resembling goat cumulus‐oocyte complex from ovaries. This primary follicular structure could be developed further in oocyte mature medium and expressed germ cell‐specific markers. In addition, we found that the induction efficiency was higher and OLC cell size was bigger in bFF than in RA treatment. Altogether, the direct reprogramming of goat fibroblasts into lineage‐specific cells can facilitate stem cell research in domestic animals.     

Analysis of preference for domestic grass‐fed beef in Japanese youths

A questionnaire based on sensory evaluation of completely domestic grass‐fed beef was carried out on 157 Japanese undergraduate students aged between 18 and 22 years in Kitasato University. The sensory evaluation sheet consisted of 10 questions concerning preference for domestic grass‐fed beef, and three demographic/lifestyle questions. Using principal component analysis and cluster analysis, the respondents were divided into four groups (G1–G4). G1 accepted almost all properties. G2 accepted most properties but disliked chewiness. G3 accepted juiciness and flavor but disliked the color and texture of the meat. G4 tended to dislike almost all properties. According to chi‐square test, most G2‐people statistically liked other commercial beef and G4‐people had neutral and negative impressions. G1‐ or G3‐people did not have any significant tendency as regards beef preference. These results indicate that most of the young respondents who preferred domestic grass‐fed beef could not accept its texture, and some respondents could accept its juiciness and flavor. It is also suggested that a part of the people who like commercial beef do not prefer chewiness of grass‐fed beef. Such information will aid grass‐fed beef cattle breeders, producers and packers to improve the quality of beef and its evaluation.     

神经干细胞的研究进展

近年来的研究表明胚胎期和成年期动物的神经组织及人脑中可以分离出神经干细胞。神经干细胞具有增殖并分化成神经元及神经胶质细胞的潜能。本文综述了神经干细胞的分布、生物学特性、识别及其在治疗中枢神经系统疾病中的应用前景。     

How the sheep's brain controls the visual recognition of animals and humans.

Visual recognition of objects and individuals is important for humans and many animal species. How does the brain process such visual information and how does experience modify such processing? To answer these questions we have used single-cell, electrophysiological recording techniques to investigate the responses of single neurons in the temporal cortex of the brain of the conscious sheep to visual images of animals and humans. Results show that a small population of these cells responds (latencies less than 180 ms) specifically to projected images of animal and human faces. Different cells respond to 1) faces with horns, and how large the horns are; 2) faces of sheep of the same breed, particularly to socially familiar individuals, and 3) faces of humans or dogs. In general, frontal views of faces (i.e., direct eye contact) are more effective stimuli than profiles or views of the back of the head. Some other cells in the temporal cortex respond to the sight of a human shape, rather than to the face. These latter cells are specialized for visual recognition of the human shape dependent on what actions are displayed (approaching or withdrawing figure), posture adopted (bipedal or quadrupedal), and view presented (front or side). These results provide important information on the complex neural processing of visual recognition of individuals by sheep and suggest that experience may modify sensory processing. Thus, behaviorally important, distinguishing features (such as horns), preferred individuals (socially familiar animals), and potentially threatening individuals (humans and sheep dogs) are specifically coded for, at a sensory level, so that appropriate behavioral or emotional responses can be made with the minimum delay.     

OCT4 Expression in Outgrowth Colonies Derived from Porcine Inner Cell Masses and Epiblasts

The present study was conducted to test different methods for porcine inner cell mass (ICM) and epiblast isolation and to evaluate the morphology and expression of pluripotency genes in ICM‐ and epiblast‐derived outgrowth colonies (OCs) and passages thereof with particular attention on the relationship between OCT4 expression and embryonic stem cell (ESC)‐like morphology. A total of 104 zona pellucida‐enclosed and 101 hatched blastocysts were subjected to four different methods of ICM and epiblast isolation, respectively: Manual isolation, immunosurgery, immunosurgery with manual cleaning, or whole blastocyst culture. OCs were established on mouse embryonic fibroblast (MEF) cells and categorized according to morphology and OCT4 staining. Although all isolation methods resulted in ESC‐like OCs, immunosurgery with manual cleaning yielded significantly higher rates of ICM/epiblast attachment and subsequent ESC‐like morphology, whereas no significant difference was found between ICM and epiblasts with respect to these characteristics. All ESC‐like OCs showed nuclear OCT4 staining and expression of OCT4, NANOG and SOX2 as evaluated by RT‐PCR. Upon initial passages, the expression of pluripotency markers was, however, gradually lost in spite of maintained ESC‐like morphology. In conclusion, we have established a robust system for derivation of ESC‐like OCs from porcine ICM and epiblasts and we have shown that localization of OCT4 is associated with an ESC‐like morphology although this relationship is lost during early passages.     

Characteristics of testicular cell development of 5‐day‐old mice in culture in vitro

The crude testicular cells (CTCs) contain many cell types, such as Sertoli cells, leydig cells, spermatogonial stem cells (SSCs), spermatocytes, and other somatic testicular cells, that secrete various growth factors needed in spermatogenesis. The objective of this study was to characterize development of 5‐day‐old mice testicular cells cultured. Crude testicular cells prepared from the testes of 5‐day‐old male mice were cultured in Dulbecco's Modified Eagle Medium and incubated at 37°C in a 5% CO₂ atmosphere for 6 days. The results demonstrated that the testicular cells developed rapidly with a population doubling time (PDT) of 0.63 days and more than 90% of cells were viable after being cultured for 3 days. The number of Sertoli‐like cells increased significantly over days 1, 3, and 6 to 22.1%, 34.6%, and 50.1%, respectively. A significant increase was also observed in fibroblast‐like cells (15.5% on day 1 to 28.8% on day 3 and to 26.6% on day 6). In contrast, the number of spermatogonia‐like cells decreased significantly (54.3%, 30.4%, and 18.7%, on days 1, 3, and 6, respectively). These data indicated that the developmental pattern of the testicular cell in this study might be affected by the niche provided by the cultured testicular cells.     

Homologies between the major histocompatibility complex of man and cattle: Consequences for disease resistance and susceptibility

Summary

The major histocompatibility complex (MHC) of mammals number of mostly duplicated contains a large genes. In the HLA system (the MHC of man), which is by far the best‐studied major histocompatibility system so far, roughly 20 genes have been defined and mapped. They code for three classes of proteins: HLA‐A, ‐B and ‐C (Class I), HLA‐DP, ‐DQ and ‐DR (Class II) and serum complement components C2, C4 and Bf (Class III). Furthermore, the region contains genes for 21‐hydroxylase (21‐OH) and tumor necrosis factor (TNF).

The MHC thus forms a chromosomal segment containing seva‐al clusters of genes of only partially defined biological significance, but ondoubtedly playing a role in disease suscepti‐ bility. In view of the recently obtained structural information on BoLA, the MHC of cattle, it is hypothesized that susceptibility to diseases in cattle is associated with BoLA in thesame way as human diseases.

Finally, new technical and conceptual developments in the field of MHC research and their application to the BoLA system are discussed.     

The use of haemostatic gelatin sponges in veterinary surgery

Objectives : To describe the use of absorbable gelatin sponges as haemostatic implants in clinical veterinary surgical cases and to document any related postoperative complications. Methods : Practice databases were searched for the product names “Gelfoam” and “Spongostan”. Patient records were retrieved and data regarding patient signalment, surgical procedure, National Resource Council (NRC) wound classification, source of haemorrhage, pre‐ and postoperative body temperature, postoperative complications, time to discharge and details of any postoperative imaging were recorded and reviewed. Follow‐up information was obtained by repeat clinical examination or telephone interview with either the owner or referring veterinary surgeon. Cases with incomplete surgical records or those which were not recovered from anaesthesia were excluded from the analysis. Results : Fifty cases (44 dogs and 6 cats) satisfied the inclusion criteria. Satisfactory haemostasis was achieved in 49 cases with one case requiring reoperation during which a second gelatin sponge was used. There were no detected hypersensitivity responses or confirmed postoperative complications relating to the use of gelatin sponges during the follow‐up period (median 13 months). Clinical Significance : This is the first review of the use of gelatin sponges in clinical veterinary surgery and suggests that gelatin sponges are safe to use in cats and dogs.     

Dissociated and Explant Cultures of Myenteric Neurons of Pig Small Intestine

The gastrointestinal tract of pig is a good and available (slaughterhouse material) model to study the enteric nervous system in relation to nutrition, inflammation, development and disease in general. In order to investigate the responses of the enteric nervous network to a variety of stimuli, e.g. growth factors, hormones, extracellular matrix components, but also noxious compounds in a controlled manner, an in vitro experimental set‐up is most appropriate. Methods to obtain in vitro cultures of enteric neurons of rodents, chicken and human are well described. This study attempted to use these methods on pig material. The muscle layer containing the myenteric plexus was dissected from pieces of fetal, neonatal and adult pig jejunum. They were rinsed in Hanks basal salt solution (BSS) in which antibiotics were added. Pieces of ±25 mm² were transferred to BSS with 1 mg/ml collagenase and 1 mg/ml trypsin inhibitor and incubated for 60 min (5% CO₂, 90% RH). Subsequently, they were vortexed for 20 s and ganglia were selected. The procedure was repeated ±4 times. Myenteric ganglia could then be plated or further dissociated in 1 mg/ml trypsin in BSS for 30 min in the incubator. Afterwards, the dissociated ganglia were centrifuged (5 min 1500 rpm) and the trypsin‐solution was replaced with Dulbecco's minimal essential medium (DMEM). The explants or dissociated myenteric ganglia were transferred on non‐coated or coated (poly‐L‐lysine, laminin, fibronectin, collagen or extracellular matrix gel) cover slips. After incubating for 45 min they were topped with 1 ml of DMEM with antibiotics and with or without fetal calf serum (5%). Medium was replaced twice a week. Using immunohistochemistry, both neurons (PGP9.5) and glial cells (S100) could be identified in both culture types. When cultivated under harsh conditions, the dissociated cultures gave rise to neurosphere‐like bodies, containing neurons and glial cells. Thus, the digestion and dissociation technique is applicable to pig material.     

SC1 sustains the self‐renewal capacity and pluripotency of chicken blastodermal cells by inhibiting the phosphorylation of ERK1 and promoting the phosphorylation of Akt

Small molecules discovered during the recent years can be used to regulate the growth of embryonic stem cells (ES cells). Chicken blastodermal cells (cBCs) play an important role in both basic and transgenic researches as an important ES cell. However, the regulatory mechanism of small molecules involved in the self‐renewal and pluripotency of cBCs remains unknown. This study revealed that the small molecule, SC1, can maintain cBCs in an undifferentiated, pluripotent state in serum‐ and feeder‐free E8 media without leukaemia inhibitory factor. Furthermore, SC1 inhibits downregulation of pluripotency‐related genes caused by retinoic acid and promotes the proliferation of cBCs. Furthermore, the results of this study indicated that SC1 functions by inhibiting ERK1 phosphorylation and promoting Akt phosphorylation, thus promoting the expression of pluripotency‐related genes and maintaining the pluripotency of cBCs. The results also demonstrated that SC1 sustains the self‐renewal capacity and pluripotency of cBCs cells by inhibiting ERK1 phosphorylation and promoting Akt phosphorylation. This kind of regulatory mechanism might be conserved in avian ES cells. Other molecules, similar to SC1, might provide insights into the molecular mechanisms that control the fate of stem cells and ultimately help in‐vivo stem cell biology and therapy.