Requirement for MHC class II positive accessory cells in an antigen specific bovine T cell response

Purified populations of bovine antigen presenting cells (APCs) and T cells have been isolated from peripheral blood and characterised using various monoclonal antibodies (mAbs) for cell surface markers. Bovine APCs were found in an adherent cell fraction and were non-specific esterase positive, phagocytic and expressed bovine major histocompatibility complex (MHC) class II determinants, all of which are typical macrophage characteristics. T cells were rigorously depleted of accessory cell function before being used in an antigen presenting cell assay. The generation of T helper cells in response to the soluble antigen, ovalbumin, was entirely dependent upon a critical number of APCs. Further the proliferative response was inhibited by several mAbs to bovine MHC class II molecules. Thus the interaction between bovine APCs and helper/inducer T lymphocytes (TH/I) appears to be similar to that in other species.     

奶牛乳腺上皮细胞的克隆与特性分析

用组织块接种法分离培养奶牛乳腺上皮细胞,进行形态学观察、生长曲线的绘制和核型分析等生物学性状的检测,并进行酪蛋白基因的RT-PCR鉴定。结果显示,体外培养的奶牛乳腺上皮细胞生长旺盛,具有典型的上皮细胞形态特征;染色体数目为60;能够正常表达奶牛乳腺上皮细胞的2种特异性酪蛋白基因,即as1-酪蛋白基因和β-酪蛋白基因。表明成功获得了奶牛乳腺上皮细胞克隆。     

Evaluation of a replacement method for mammary gland biopsies by comparing gene expression in udder tissue and mammary epithelial cells isolated from milk

Somatic cells isolated from milk offer an attractive non-invasive replacement of invasive udder biopsies for monitoring bovine mammary gland metabolism. However, for metabolic gene expression studies the mammary gland epithelial cells (MEC) isolated from milk have to be purified from the non-epithelial leukocyte fraction in milk samples. In our study, enrichment of MEC by using anti-cytokeratin peptide 18 (KRT18) antibody coated magnetic beads was evaluated. MEC showed a substantially increased expression of the epithelial-cell-specific KRT18 gene compared to udder tissue. The expression levels of genes specific for mammary gland epithelial cells (CSN3 and LALBA) showed a significant positive correlation in MEC and also in udder tissue. However, no significant correlation of the expression of a specific gene was found between udder and MEC samples. Therefore, MEC isolated from total milk samples via KRT18 antibodies probably do not reflect the true metabolic situation of the bovine udder. Thus, quantitative gene expression profiling of MEC isolated via KRT18 antibodies has to be interpreted carefully with respect to the situation in the udder.     

Immortalized goat milk epithelial cell lines replicate CAEV at high level

Primary milk epithelial cells were isolated from CAEV-uninfected goats and three cell lines designated TIGMEC-1, TIGMEC-2 and TIGMEC-3 were established. The three cell lines retained their morphological characteristics of epithelial cells and expressed specific epithelial cytokeratin marker as well as the immortalizing SV40 large T antigen. The kinetics of growth of TIGMEC1, TIGMEC2 and TIGMEC3 cell lines showed a doubling time of 24-48 hours while the parental cell lines became senescent after the passage 6 in cell culture. Like the parental primary cells, the three cell lines were found to be highly sensitive to CAEV-pBSCA, an infectious molecular clone of CAEV-CO strain, and to a French isolate CAEV-3112. TIGMEC cell lines infected with CAEV-pBSCA became chronically infected producing high virus titers in absence of cytopathic effects. These cell lines may be useful for study of the possible physiological alterations in mammary epithelial cells infected with small ruminant lentiviruses and their consequences on milk quality. On an other hand, these cell lines can be used to study their role in virus transmission and pathogenesis.     

Porcine IPEC-J2 intestinal epithelial cells in microbiological investigations

IPEC-J2 cells are porcine intestinal columnar epithelial cells that were isolated from neonatal piglet mid-jejunum. This cell line forms polarized monolayers with high transepithelial electrical resistance when cultured on 0.4 μm pore-size filters. The cell line is unique in that it is derived from small intestinal tissue (compared to the common human colon-derived lines HT-29, T84, and Caco-2) and is not transformed (compared to the porcine small intestinal line, IPI-2I). Porcine intestinal epithelial cells more closely mimic human physiology than analogous rodent cell lines (e.g. IEC-6 or IEC-18), which is important in studies of zoonotic infections; in addition, they provide specificity to study porcine-derived infections. IPEC-J2 cells are increasingly being used in microbiological studies to examine the interactions of various animal and human pathogens, including Salmonella enterica and pathogenic Escherichia coli, with intestinal epithelial cells. The IPEC-J2 cell line has also been employed in some probiotic studies, in which the cells have been used as an initial screening tool for adhesiveness and anti-inflammatory properties of the potential probiotic microorganisms. The validity of these studies is not clear as follow-up studies to assess the efficacy of the probiotics in vivo have not been published to date. The aims of this review are to provide a comprehensive overview of the microbiological studies that have been conducted with IPEC-J2 cells and a reference guide of key cellular and immune markers that have been identified in this cell line that may prove to be useful in future studies.     

Effect of dipeptide on intestinal peptide transporter 1 gene expression: An evaluation using primary cultured chicken intestinal epithelial cells

Peptide transporter 1 (PepT1) is a transporter responsible for absorbing dipeptide and tripeptide in enterocytes and is upregulated by dipeptide in mammals. It has not been certain whether intestinal PepT1 expression is responsive to dipeptides in chickens because of the lack of in vitro study using the cultured enterocytes. This study established a primary culture model of chicken intestinal epithelial cells (IECs) in two-dimensional monolayer culture using collagen gel by which the response of chicken PepT1 gene expression to dipeptide stimuli was evaluated. The cultured chicken IECs showed the epithelial-like morphology attached in a patch-manner and exhibited positive expression of cytokeratin and epithelial cadherin, specific marker proteins of epithelial cells. Moreover, the chicken IECs exhibited the gene expression of intestinal cell type-specific marker, villin1, mucin 2, and chromogranin A, suggesting that the cultured IECs were composed of enterocytes as well as goblet and enteroendocrine cells. PepT1 gene expression was significantly upregulated by synthetic dipeptide, glycyl-l-glutamine, in the cultured IECs. From the results, we herein suggested that dipeptide is a factor upregulating PepT1 gene expression in chicken IECs.     

Bovine T lymphocyte response to bovine herpesvirus-1: cell phenotypes, viral recognition and acid-labile interferon production

The phenotype of bovine cells that proliferate to bovine herpesvirus (BHV-1) were identified by peanut agglutinin (PNA) and monoclonal antibodies with specificity for Pan T cells (B29), a T cell subset (B24), and an MHC class II (Ia-like) antigen (R-1). PNA+ cells but not PNA- cells separated by fluorescent activated cell sorting responded to BHV-1. Virally-activated T cells expressed MHC class II antigens, and possibly antigen specific receptors for virus. Binding of radiolabeled virus increased six-fold beyond the expected value for activated cells suggesting specificity of the lymphocytes for the virus. Finally, cells that responded to BHV-1 produced high levels of acid sensitive interferon. Taken together these results characterize the phenotypes of bovine lymphocytes that interact with BHV-1.     

Theileria annulata-transformed cell lines are efficient antigen-presenting cells for in vitro analysis of CD8 T cell responses to bovine herpesvirus-1

ABSTRACT: Continuously growing cell lines infected with the protozoan parasite Theileria annulata can readily be established by in vitro infection of leukocytes with the sporozoite stage of the parasite. The aim of the current study was to determine whether such transformed cell lines could be used as antigen presenting cells to analyse the antigenic specificity of bovine CD8 T cell responses to viral infections. Bovine herpes virus 1 (BHV-1), which is known to induce CD8 T cell responses, was used as a model. T. annulata- transformed cells were shown to express high levels of CD40 and CD80 and were susceptible to infection with BHV-1, vaccinia and canarypox viruses. The capacity of the cells to generate antigen-specific CD8 T cell lines was initially validated using a recombinant canarypox virus expressing a defined immunodominant T. parva antigen (Tp1). Autologous T. annulata-transformed cells infected with BHV-1 were then used successfully to generate specific CD8 T cell lines and clones from memory T cell populations of BHV-1-immune animals. These lines were BHV-1-specific and class I MHC-restricted. In contrast to previous studies, which reported recognition of the glycoproteins gB and gD, the CD8 T cell lines generated in this study did not recognise these glycoproteins. Given the ease with which T. annulata-transformed cell lines can be established and maintained in vitro and their susceptibility to infection with poxvirus vectors, these cell lines offer a convenient and efficient in vitro system to analyse the fine specificity of virus-specific CD8 T cell responses in cattle.     

Uterine mast cells and immunoglobulin-E antibody responses during clearance of Tritrichomonas foetus

We showed earlier that Tritrichomonas foetus-specific bovine immunoglobulin (Ig)G1 and IgA antibodies in uterine and vaginal secretions are correlated with clearance of this sexually transmitted infection. Eosinophils have been noted in previous studies of bovine trichomoniasis but the role of mast cells and IgE responses have not been reported. The hypothesis that IgE and mast cell degranulation play a role in clearance was tested in 25 virgin heifers inseminated experimentally and infected intravaginally with T. foetus strain D1 at estrus and cultured weekly. Groups were euthanatized at 3, 6, 9, or 12 weeks, when tissues were fixed and secretions were collected for culture and antibody analysis. Immunohistochemistry using a monoclonal antibody to a soluble lipophosphoglycan (LPG)-containing surface antigen (TF1.17) demonstrated antigen uptake by uterine epithelial cells. Lymphoid nodules were detected below antigen-positive epithelium. Little IgG2 antibody was detected but IgG1, IgA, IgM, and IgE T. foetus-specific antibodies increased in uterine secretions at weeks 6 and 9 after infection. This was inversely proportional to subepithelial mast cells numbers and most animals cleared the infection by the sampling time after the lowest mast cell count. Furthermore, soluble antigen was found in uterine epithelium above inductive sites (lymphoid nodules). Cross-linking of IgE on mast cells by antigen and perhaps LPG triggering appears to have resulted in degranulation. Released cytokines may account for production of predominantly Th2 (IgG1 and IgE) and IgA antibody responses, which are related to clearance of the infection.     

Two monoclonal antibodies (CC17, CC29) recognizing an antigen (Bo5) on bovine T lymphocytes, analogous to human CD5

Two new monoclonal antibodies (CC17 and CC29) raised against bovine thymocytes are described. The antibodies, both of which were IgG1, recognize a molecule of approximately 67,000 molecular weight on bovine T cells. They react T cells in peripheral blood, the lymph node paracortex and the periateriolar lymphoid sheath in the spleen. Both the cortex and medulla of the thymus are stained but the medulla reacts more intensely. They do not stain B cells in peripheral blood, the ileal Peyer's patch, the cortex or the primary follicles in lymph nodes. No activity was found on cells outside the lymphoid system, i.e. monocytes, alveolar macrophages or endothelial and epithelial tissue. The antigen recognized is considered to be the bovine homologue of CD5 (T1) in humans and Lyt1 in mice. The mAbs appear to be particularly useful for detecting cells in the peripheral blood of young calves which are of the T cell lineage but do not express BoT2 or the mature pan T cell antigen recognized by mAb IL-A27 and may thus allow identification of a population of bovine lymphocytes previously described as null cells.     

A newly established bovine intestinal epithelial cell line is effective for in vitro screening of potential antiviral immunobiotic microorganisms for cattle

We evaluated whether a bovine intestinal epithelial (BIE) cell line could serve as a useful in vitro model system for studying antiviral immune responses in bovine intestinal epithelial cells (IECs) and for the primary screening of immunobiotic microorganisms with antiviral protective capabilities. Immunofluorescent analyses revealed that toll-like receptor 3 (TLR3) was expressed in BIE cells, and the results of real-time quantitative PCR showed that these cells respond to stimulation with poly(I:C) by up-regulating pro-inflammatory cytokines and type I interferons. In addition, we demonstrated that BIE cells are useful for the primary screening of immunobiotic lactic acid bacteria strains which are able to beneficially modulate antiviral immune responses triggered by TLR3 activation in bovine IECs. The characterization of BIE cells performed in the present study represents an important step towards the establishment of a valuable bovine in vitro system that could be used for the development of immunomodulatory feed for bovine hosts.     

荷斯坦奶牛乳腺上皮细胞的分离培养

目的：建立荷斯坦奶牛乳腺上皮细胞的分离培养方法。方法：采用组织块种植法培养奶牛乳腺上皮细胞,利用胰蛋白酶差时消化法分离、纯化上皮细胞。结果：成功培养出奶牛乳腺上皮细胞,显微镜下观察,纯化的乳腺上皮细胞呈典型上皮细胞形态,细胞之间排列紧密,呈鹅卵石铺路样,形态均一,多角形的单层聚集。通过荧光免疫细胞染色方法对细胞骨架蛋白-角蛋白18进行鉴定,呈现阳性反应。乳腺上皮细胞增殖旺盛,经25次以上传代后长势仍然良好。结论：采用组织块种植法结合胰酶差时消化法成功获得纯化的奶牛乳腺上皮细胞。     

Bovine T cell responses to recombinant thioredoxin of Fasciola hepatica

Fasciolosis is an economically significant disease of ruminants, caused by infection with the digenetic trematodes, Fasciola hepatica and F. gigantica. Some vaccination trials using irradiated metacercariae or isolated proteins have been shown to afford significant protection. However, the mechanisms of specific immunity against this pathogen have not been elucidated. We have identified thioredoxin, a tegument antigen of F. hepatica, among several proteins that are common to both the juvenile and adult fluke within the mammalian host and have undertaken studies to characterize bovine T cell responses to recombinant thioredoxin protein (FH 2020). Peripheral blood mononuclear cells from immune cattle proliferated specifically to crude F. hepatica antigenic extract but not to FH 2020. However, after repeated stimulation of lymphocytes by alternating crude extract and FH 2020, FH 2020-specific proliferation by T cell lines was observed. T cell clones were subsequently generated and found to respond specifically but weakly to both crude antigen and FH 2020. Thioredoxin appears to be only weakly antigenic for bovine T cells and is, therefore, an unpromising candidate for inducing resistance to F. hepatica.     

Indirect fluorescent antibody test for experimental and epizootiological studies on East coast fever (Theileria parva infection in cattle). Evaluation of a cell culture schizont antigen fixed and stored in suspension

A schizont antigen for the indirect fluorescent antibody test against Theileria parva was prepared from a T parva-infected bovine lymphoblastoid cell line by fixing the cells in suspension with a mixture of acetone and formaldehyde. The antigen was stored in suspension in phosphate buffered saline for one and a half years at -60 degrees C without loss of activity; the antigen could also be lyophilised. The fluorescence of the intracellular schizonts was bright and specific with T parva positive bovine control serum and absent with negative bovine control serum and Theileria mutans positive bovine control serum. Fluorescence of the lymphoblastoid cell itself was observed with Trypanosoma brucei positive control serum and some bovine test sera: this fluorescence, which masked the intracellular schizonts, was eliminated by absorbing the sera in the supernatant of sonicated lymphocytes obtained from bovine lymph nodes. The antigen was evaluated with sera from cattle experimentally infected with T parva. In an epizootiological study on East Coast fever in the Coast Province of Kenya, there was good correlation between the serological responses of cattle to T parva schizont antigen and the distribution of Rhipicephalus appendiculatus ticks.     

Regulatory T cells in cattle and their potential role in bovine paratuberculosis

The intracellular bacterium Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease in wild and domestic ruminants. Johne's disease presents as a chronic enteritis with severe inflammation of intestinal tissues, characterized by widespread infiltration of macrophages, the target cell of MAP. Clinical signs of Johne's disease are typically accompanied by a loss of peripheral CD4+ T cell responses to MAP antigens and an increase in anti-MAP serum IgG levels. Recently, it was proposed that regulatory T cells might develop over the lengthy course of subclinical MAP infection. In the past five years, significant progress in defining bovine regulatory T cells has been made. These studies grew out of observations that IL-10 is produced by PBMCs in response to MAP antigen stimulation and that neutralization of this IL-10 could enhance IFN-γ production from MAP-antigen reactive effector T cells. Depletion studies revealed that MAP responsive cell populations producing IL-10 were largely CD4+ and CD25+, although monocytes have also been shown to produce IL-10 in response to MAP. In addition, evidence for a regulatory population of γδ T cells has also begun to accumulate. We summarize current thinking regarding regulatory T cells in MAP infection and provide data suggesting a potential link between regulatory T cells, bovine leukemia virus, and MAP.     

Persorption of bovine lactoferrin from the intestinal lumen into the systemic circulation via the portal vein and the mesenteric lymphatics in growing pigs

The absorption and the transportation of intestinally administrated bovine lactoferrin (LF) were immunohistochemically and physiochemically investigated in the small intestine of growing pigs. At the apical halves of the small intestinal villi, bovine LF was absorbed by transcytosis as small vesicles through villous columnar epithelial cells. The presence of bovine LF-positive membranes of transcytotic vesicles suggests that the absorption was mediated by LF-binding factors on the epithelial cell membranes. Almost all of the absorbed bovine LF was demonstrated to be transported via the lymphatics and the portal vein into the systemic circulation. The LF-concentration in systemic circulation was significantly higher at 1 hr following intestinal administration of bovine LF. Bovine LF-positive lymphocytes also were transferred into the systemic circulation from intestine via the lymphatics and the portal vein.