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.     

Effects of mycophenolic acid on inosine monophosphate dehydrogenase I and II mRNA expression in white blood cells and various tissues in sheep

Mycophenolic acid (MPA) is a mycotoxin commonly found as Penicillium genus secondary metabolite in feedstuffs and silages. Feeding with MPA contaminated silages may modulate the immune system in the farm animals and can cause appetite lost, ketosis, paralysis and abortion. The aim of the present study was to characterize the long-term MPA effect on both the inosine monophosphate dehydrogenase (IMPDH) isoforms I and II mRNA expression in white blood cells (WBC) and various tissue of healthy sheep. In treated animals 300 mg MPA/day/sheep was applied. In all investigated tissues the IMPDH I and II mRNA was abundant: WBC, spleen, thymus, ileum, jejunum, kidney, liver, pharyngeal and mesenterial lymph node. An efficiency-corrected relative quantification of the IMPDH types I and II isoforms mRNA were performed by normalizing with the constant reference gene expression of beta-actin. High IMPDH I mRNA expression levels were seen in kidney > mesenterial lymph node > jejunum > spleen > pharyngeal lymph node. Medium and low abundance was found in ileum > WBC > liver > thymus. Type II mRNA was highly expressed in liver > thymus > jejunum. In pharyngeal lymph node > spleen > ileum > mesenterial lymph node > kidney > WBC medium to low IMPDH II mRNA concentrations were detected. Under MPA treatment the IMPDH I mRNA expression was not significantly regulated in WBC, only trends of down- and upregulation were observed. Surprisingly in jejunum an upregulation could be observed (P < 0.05). In pharyngeal lymph node a tendency to downregulation was shown. This may be due to frequent ruminant activities and frequent exposition of MPA to the pharyngeal lymph nodes. In contrast to type I mRNA expression, IMPDH II mRNA was significantly downregulated in ileum (3.4-fold, P < 0.01) and tendencies in downregulation could be seen in jejunum (5.1-fold, P = 0.14). In addition, significant downregulation of IMPDH II gene expression over the entire feeding experiment could be shown in WBC of MPA-treated animals compared with untreated animals (P < 0.05). In conclusion, the recent study demonstrates that feeding sheep with MPA-contaminated silage did not induce IMPDH I mRNA expression in various tissues and blood, except in jejunum, but has suppressive effects on IMPDH II mRNA expression in WBC and ileum.     

Cytochemical identification of lymphocytes and other mononuclear cells in ovine and bovine hemal nodes

Cytochemical and immunocytochemical studies were carried out with specific markers for B lymphocytes, dendritic reticulum cells (ATPase, 5'Nase, SIg) and T lymphocytes (ANAE, A.P.) in an attempt to identify the mononuclear cells present in bovine and ovine hemal nodes. The results show that primary nodes and mantle of secundary nodes are composed of B cells, whereas T cells are mainly localized in the interfollicular cords. Since such an arrangement resembles the picture in normal lymph nodes, but the direct contact between lymphatic tissue and blood is more reminiscent of the spleen, hemal nodes probably perform immunological functions similar to those of both normal lymph nodes and spleen.     

Comparative studies on the distribution and population of immunocompetent cells in bovine hemal node, lymph node and spleen

The distribution and population of immunocompetent cells in bovine hemal node, mesenteric lymph node and spleen were analyzed comparatively by immunohistochemistry and flow cytometry. Many CD8(+) cells, CD172a(+) cells and γδ T cells were found in the lymphatic cord along the sinus of the hemal node and the splenic red pulp. A few CD8(+) cells and γδ T cells were distributed diffusely in the paracortex and medullary cord of the mesenteric lymph node. Many germinal centers were recognized in the lymphatic regions such as the cortex and white pulp of these lymphoid organs. The populations of CD8(+) cells and γδ T cells in the hemal node and the spleen were higher than those of the mesenteric lymph node. In addition, the populations of CD21(+) cells and MHC class II(+) cells in the hemal node and the mesenteric lymph node were higher than those of the spleen. The results suggest that the hemal node has an important role in both cellular and humoral immunity as well as the lymph node and the spleen in cattle.     

Characterization and Distribution of B Cells in the Lymphoid Organs of Goats

The distribution of B cells in the lymphoid organs of the goat was studied using a panel of 13 monoclonal antibodies (mAbs) developed against different markers for bovine B cells. Samples of mesenteric lymph nodes, jejunal and ileal Peyer's patches, duodenum, jejunum, ileum, colon, caecum and rectum were taken from four 7-month-old male Murciano-granadina goats using the avidin-biotin-peroxidase (ABC) method on frozen sections as described by Hsu et al. (1981). The mAbs against immunoglobulins (Ig) recognized a large number of cells, particularly in the light zones of the germinative centres of the lymphoid follicles, regardless of the isotype against which they were directed. However, the greatest numbers of B cells in the germinative centres and outer coronas of the lymphoid follicles of the lymph node, spleen and Peyer's patches were recognized by mAbs against the L lambda chain of Ig and against IgM. This was also the case in other locations where B cells were abundant, such as the medulla of the lymph node and the dome of the Peyer's patches. These mAbs recognized not only B lymphocytes but also plasma cells, showing an intracytoplasmatic reaction (numerous in the spleen red pulp and the intestinal lamina propria when mAbs were used against the L lambda chain of the Ig, scarce in the intestinal lamina propria when used against IgM and scarce in spleen red pulp and numerous in the intestinal lamina propria when mAbs against IgA were used). The mAbs BAQ44 A, GC65 A and GB25 A are of interest because, besides marking cells in the B areas where lymphocytes show surface Ig, they give a positive reaction in areas where there are Ig-cells (the dark zone of the germinative centre) and do not immunostain plasma cells. Thus, these mAbs recognize a surface marker which is not an Ig.     

Monoclonal antibodies against porcine macrophages

Two mouse monoclonal antibodies (MAB) (clones 2G6 and 2B10) directed against porcine macrophages are described that are suitable for use in immunohistochemistry, FACS analysis and western blot. As immunogen, porcine cells from bronchoalveolar lavage (BAL) were used. The MABs obtained belonged to the mouse IgG1 subclass. The molecular weights of the corresponding antigens were detected by western blot under non-reducing conditions (2G6: 140-150kDa; 2B10: 140-145kDa). For specificity screening, porcine snap-frozen tissues of lung, lung lymph node, tonsil, spleen, thymus, brain, liver, gut and kidney were used. The MABs were able to identify cell populations of the mononuclear phagocytic system in these organs. While MAB 2G6 detected tissue macrophages (sinusoidal lymph node macrophages, red pulp spleen macrophages, Kupffer cells, Langerhans cells, thymus macrophages, macrophages of lung and macrophages of kidney), MAB 2B10 stained cells scattered in the lymph node (subsinusoidal, interfollicular and follicular macrophages) and in the lung interstitium. Additionally, it showed reactivity with Kupffer cells, spleen and kidney macrophages. An immunoreactivity of the MABs could be established also for human but not for bovine and avian macrophages. By flow cytometric analysis, MAB 2B10 reacted with a subpopulation of BAL and peritoneal cells. Antibody 2G6 detected macrophages of the BAL and the peritoneal fluid as well as peripheral blood monocytes.     

NODs/RIP2信号通路关键基因在断奶仔猪不同组织中的mRNA表达

本试验旨在研究核苷酸结合寡聚化结构域蛋白/受体相互作用蛋白2(NODs/RIP2)信号通路关键基因在断奶仔猪不同组织的分布情况。选择12头杜×长×大断奶仔猪,屠宰,取脾脏、胸腺、肠道淋巴结、下丘脑、垂体、肾上腺、肝脏、腓肠肌、皮下脂肪、空肠和回肠组织。应用实时荧光定量PCR技术测定NODs/RIP2信号通路关键基因,包括NOD1、NOD2和RIP2在各组织的mRNA表达水平。结果表明:NOD1、NOD2和RIP2在所检测的11个组织中均有表达。NOD1 mRNA在肠道淋巴结和下丘脑表达量较高;NOD2 mRNA在肠道淋巴结、脾脏、下丘脑和胸腺表达量较高;RIP2 mRNA在肠道淋巴结、胸腺和脾脏表达量较高。NODs/RIP2信号通路关键基因在不同组织中表达差异较大,可能与仔猪各组织对病原的识别和抵抗能力有关。     

Relation between phenotype of tumor cells and clinicopathology in bovine leukosis

Thirty-three cases of enzootic bovine leukosis (EBL) and 14 cases of sporadic bovine leukosis (SBL) were examined by immunohistochemistry using 6 monoclonal antibodies against leukocyte differentiation molecules of bovine leukocytes. There were 17 cases of B-1a cell type, 10 cases of B-1b cell type and 6 cases of B-2 cell type in EBL, and 5 cases originating from B cells (B-2 cell type) and 9 cases originating from immature T cells in SBL. The average age for the EBL cases of B-1a cell type was 8.6 years, B-1b cell type was 6.5 years, and of B-2 cell type was 4.5 years. In cases of SBL, immature T cell type patients were younger than B-2 cell type ones. The lymphoma originating from B cells differed from that originating from T cells in morphology. In T cell tumors, the nucleus of tumor cells was round, the edge of the cytoplasm obvious, and tumor cells were sporadically present and proliferated. When compared with T cells, the region among B cells was obscure. But, there was no relation between phenotype and the histologic classification of tumor cells. In EBL, beyond the lymph node, tumors of B-1a and B-1b types had developed in the heart and abomasum, and those of the B-2 type tended to occur in liver. In SBL, B-2 type and T type cells formed tumors in the liver, kidney, thymus, and one case of T-cell type tumor formed on the skin. We would like to propose a new classification of bovine leukosis as EBL, calf type B-cell lymphoma, juvenile T-cell lymphoma and skin type T-cell lymphoma.     

Theileria parva infection in calves causes massive lymphocyte death in the thymus, spleen and lymph nodes without initial proliferation

In a study of trends and magnitudes of lymphocytes proliferation, destruction or apoptosis eleven 3-month-old healthy calves were experimentally infected with the protozoan parasite Theileria parva, which is reported to cause lymphocyte proliferation. Four control calves were not infected. Infected and non-infected calves were sacrificed on days 9, 12, 16, 19, 23, 24 and 25 to examine lymphoid tissue changes and lymphocyte proliferation, apoptosis or necrosis in the thymus, spleen and lymph nodes. All infected calves developed severe East Coast fever, with enlargement of lymph nodes, dyspnoea, high fever and pulmonary oedema. Lymphocyte proliferation was not observed in lymph nodes, thymus and spleen; instead there were massive deaths of lymphocytes and other cells. The terminal severe disease caused massive lymphoid parenchyma coagulation terminating with caseation, organs and cells being undeterminable histologically. Tissues surrounding the lymph nodes were oedematous. Lymph node and thymus parenchyma were caseated and cortices and medulla indistinguishable because of severe lymphocyte and accessory cell deaths. The lymph node fibrous reticular stroma was necrotic and caseated. Lymphoid follicles in lymph nodes degenerated and lacked germinal centres. Lymph nodes, spleen and thymus were grossly enlarged, hardened, potato or cheese like, but microscopically very hypocellular and in the terminal disease acellular because of massive lymphocytes destruction. In the thymus there was extensive thymocyte and epithelioid cell necrosis and loss of distinction between cortex and medulla. The spleen white and red pulps were indistinguishable because of extensive lymphoid cell necrosis. The white pulp degenerated more than the red pulp. The massive lymphocyte deaths in the lymph nodes, thymus and spleen, without lymphocyte proliferation in this T. parva infection in calves leads to a conclusion that this parasite is lympho-destructive and lympho-degenerative in vivo rather than lympho-proliferative.     

Effects of spray-dried porcine plasma and plant extracts on intestinal morphology and on leukocyte cell subsets of weaned pigs

We evaluated the effects of a 6% spray-dried porcine plasma (SDPP) and a plant extracts mixture (XT; 5% carvacrol, 3% cinnamaldehyde, and 2% capsicum oleoresin) on the productive performance, intestinal morphology, and leukocyte cell subsets of early-weaned pigs compared with a control group. Morphometry of the jejunum, ileum, and colon, and immune cell analysis of blood, ileocolic lymph node (LN), and ileal Peyer's patches were done in 24 weaned pigs (20 +/- 2 d) at 19 or 21 d postweaning. Although SDPP and XT treatments did not increase ADG or ADFI, SDPP improved the G:F ratio (P = 0.024) compared with the control group. Dietary SDPP reduced the percentages of blood monocytes (P = 0.006) and macrophages in ileal Peyer's patches and LN (P = 0.04), of B lymphocytes (P = 0.04) and gammadelta+ T cells in LN (P = 0.009), and of intraepithelial lymphocytes (P = 0.026) as well as the density of lamina propria cells in the colon (P < 0.01). Dietary XT reduced intraepithelial lymphocyte numbers in jejunum (P = 0.034) and the percentages of blood cytotoxic cells (P = 0.07) and B lymphocytes in LN (P = 0.03); however, XT increased blood monocytes (P = 0.038) and the density of lamina propria lymphocytes in the colon (P = 0.003). These results indicate that dietary SDPP and plant extracts can affect intestinal morphology and immune cell subsets of gut tissues and blood in weaned pigs. Furthermore, the effects of SDPP suggest lower activation of the immune system of the piglets.     

Monoclonal antibodies identify phenotypically and functionally distinct cell types in the bovine lymphoid system

Monoclonal antibodies were produced against bovine lymphoid cells. The reactivities of the antibodies for membrane determinants were examined on both cell suspensions and cryostat tissue sections prepared from bovine blood, thymus, spleen and lymph nodes. The antibodies were putatively grouped into sets which reacted with monomorphic and polymorphic determinants associated with bovine class I and class II major histocompatibility complex (MHC) antigens (MAbs P12 and P3, and R1 and P2 respectively), or associated with differentiation antigens expressed on T cells and monocytes (MAb P5) or exclusively on monocytes (MAb P8). The antibodies were used to identify the surface phenotypes of cells which stimulate (R1+ P5+ P8+) and proliferate (R1- P5+ P8-) in the bovine mixed leukocyte cultures, and cells which proliferate in response to the mitogen, concanavalin A (R1- P5+).     

Preparation and characterization of a monoclonal antibody against bovine CD5 lymphocyte surface antigen.

The M1 monoclonal antibody (mAb) was proved to recognize 51-70% of Bovine peripheral blood lymphocytes (PBL). The M1+ cells were SIg-. In spleen and lymph nodes, the M1 positive lymphocytes were located within the T cell areas. All the lymphoid follicles remained negative. In the thymus, 10% of thymocytes were M1+, most of them were located in the medulla. The M1 mAb did not inhibit spontaneous rosette formation by sheep erythrocytes and bovine lymphocytes. On the other hand, biochemical analysis of membrane antigen with bovine thymic tumor cell line LB203 gave a molecular weight of 75 kDa. Despite a slight difference in biochemical results (75 vs 67-69 kDa). Our data permit us to consider M1 mAb as a possible homologous of human anti-CD5 mAb. Finally, M1 cross-reacted with sheep peripheral blood T lymphocytes.     

Toll-like receptor expression in feline lymphoid tissues

Toll-like receptors (TLRs) are germline-encoded pattern recognition receptors (PRRs) that activate the innate immune system. While it is clear that TLRs are important in the immune response against pathogens, they may also be exploited by some pathogens. Our objective is to determine whether feline immunodeficiency virus (FIV) infection affects TLR expression or function thereby resulting in innate immune dysfunction. To this end, we cloned partial sequences for feline TLRs 1--3, 5--8, and developed real-time PCR assays to quantify feline TLRs 1--9. TLR expression was quantified in normal cat lymphoid tissues, purified lymphocyte subsets, and FIV-infected cell lines. Different expression patterns of TLRs were found in spleen, mesenteric lymph node, retropharyngeal lymph node, thymus, intestinal intraepithelial lymphocytes, and lamina propria lymphocytes. B lymphocytes, CD4+ T cells, and CD8+ T cells all expressed TLRs 2--5, 7--9; however, the relative levels of expression varied among lymphocyte phenotypes. Infection of cell lines with FIV resulted in altered TLR expression levels that differed depending on cell type. These results demonstrate that tissue distribution of TLRs is associated with the immunological role of a particular tissue, that lymphocytes may also express these 'innate immune' receptors, and that FIV infection can alter TLR expression.     

Influence of betulinic acid on lymphocyte subsets and humoral immune response in mice

Betulinic acid is a pentacyclic triterpene found in many plant species, among others, in the bark of white birch Betula alba. Betulinic acid was reported to display a wide range of biological effects, including antiviral, antiparasitic, antibacterial, anticancer and anti-inflammatory activities. The effects of betulinic acid (50, 5, 0.5 mg/kg) administered orally five times at 24 hours intervals to non-immunized and red blood cells (SRBC)-immunized mice were determined. The present study examined the total number of lymphocytes in the thymus, spleen and mesenteric lymph nodes, and the percentage of subsets of T cells (CD4+CD8+, CD4CD8, CD4+, CD8+) in thymus,T (CD3+, CD4+, CD8+) and B (CD19+) lymphocytes in the spleen and mesenteric lymph nodes, as well as white blood cell (WBC) and differential leukocyte counts in non-immunized mice, and humoral immune response in SRBC-immunized mice. SRBC was injected 24 hours after administration of the last dose of betulinic acid. It was found that betulinic acid administered orally five times at the dose of 0.5 mg/kg increased the total number of thymocytes, splenocytes, lymphocytes of mesenteric lymph node cells, and the weight ratio of the spleen and mesenteric lymph nodes in non-immunized mice. Betulinic acid also changed the percentage of T cell subsets in the thymus and T and B lymphocytes in peripheral lymphatic organs. The effects of betulinic acid on T and B cell subpopulations depended on the dose applied. The strongest stimulating effect of betulinic acid was observed when the drug was administered at the dose of 0.5 mg/kg. Five exposures to betulinic acid (0.5 mg/kg) decreased the percentage of immature CD4+CD8+ thymic cells with corresponding increases in the percentage and absolute count of mature, single-positive CD4+ thymocytes and decreased the percentage and total count of CD3+ splenocytes and mesenteric lymph node cells with corresponding decreases in the percentage and absolute count of CD4+ and CD8+ cells. Multiple administration of betulinic acid at the investigated doses augmented the percentage and absolute count of CD19+ cells in the peripheral lymphatic organs. Moreover, betulinic acid at the dose of 5 mg/kg administered prior to SRBC immunization increased the number of plaque forming cells (PFC) but decreased the production of anti-SRBC antibodies on day 4 after priming. Thus, betulinic acid is a potential biological response modifier and may strengthen the immune response of its host.     

Preparation and characterization of monoclonal antibodies against bovine B lymphocyte surface antigens

Two monoclonal antibodies (MoAbs; BLMo-4 and BLMo-10) were prepared by immunizing with a cell line established from peripheral blood mononuclear cells (PBMC) of enzootic bovine leukosis (EBL) cattle. The specificities of these MoAbs were assayed using bovine PBMC. BLMo-4 reacted with all surface immunoglobulin-positive cells (SIg+ cells; B lymphocytes) and also recognized monocytes, but did not react with T lymphocytes. BLMo-10 recognized a majority, although not all, B lymphocytes, but did not react with either T lymphocytes or monocytes. The antigens recognized by BLMo-4 and BLMo-10 were not Ig, Fc or C3 receptors on the surface of B lymphocytes. The reactivity of the MoAbs with mononuclear cells from the lymphoid organs of adult cattle was studied. BLMo-4 and BLMo-10 did not react with any bone marrow cells. BLMo-10 reacted with 7.4% of thymocytes, and stained the medulla of the thymus in the immunoperoxidase assay. In the case of PBMC, spleen and lymph node cells, the percentage of cells positive for BLMo-4 was slightly higher than that of SIg+ cells, but BLMo-10 showed a slightly lower value.     

Engraftment of severe combined immune deficient/beige mice with bovine foetal lymphoid tissues.

To develop a model of bovine thymus and lymph node growth in vivo, we have implanted bovine foetal tissues (16-23 weeks gestation) under the renal capsule of severe combined immune deficient (SCID)/beige (BG) mice and assayed for graft growth and characteristics 2-18 weeks after engraftment. Bovine foetal thymus and lymph node grew considerably following engraftment of SCID/BG mice. Growth was optimal if bovine foetal tissues were used before gestation Week 17. Bovine-mouse chimerism was confirmed using glucose phosphate isomerase analysis. Bovine thymus grew during the entire 18 weeks of study. Growth of bovine lymph node was initially rapid, reaching a maximum at 2 weeks after transplantation followed by a progressive decrease in size. Transplanted bovine lymph node and thymus were morphologically similar to age-matched bovine foetal tissue for a limited time period. Fibrosis, degeneration and depletion of lymphocytes were evident 6 weeks after engraftment; changes were more severe in lymph node than in thymus whereas increases in lymphocytes, lymphopoiesis and follicle formation were evident in age-matched bovine foetal tissue. Despite growth and morphological similarities of the transplanted tissue, blood counts suggested there was no peripheralization of bovine leucocytes. Bovine immunoglobulins (IgG1 and IgG2) were detected in serum of some SCID/BG chimeric mice for a limited time. The appearance of bovine immunoglobulins at 2 weeks in SCID/BG chimeric mice depended on the age of the foetal donor (> 18 weeks) and coincided with the appearance of morphologically mature lymphocytes in the donor foetus lymph nodes. The ability to produce bovine immunoglobulins decreased 8 weeks after engraftment, coinciding with the depletion of lymphocytes in the engrafted lymph node. Lymphocyte depletion and loss of function of engrafted tissues appear the result of a lack of lymphoid progenitors normally derived from hematopoietic stem cells in the bone marrow.     

Ontogeny of T cells in the pig fetus

Mononuclear cells isolated from thymus, spleen and cord blood of pig fetuses ranging in age from 48 to 112 days were examined for the presence of sheep red blood cell rosette-forming cells (SRBC-RFC). After an initial increase from 77 % (mean) at 48 days of gestation to 88 % at 60 days, the proportion of SRBC-RFC in thymus remained constant throughout the gestational period. In spleen and cord blood, the proportion of SRBC-RFC increased with age, from occasional rosette-forming cells at 48 days of gestation to 21 % and 30 %, respectively, at 112 days. The demonstrated development of SRBC-RFC in the thymus, spleen and cord blood is considered to reflect the ontogeny of T cells in these fetal pig tissues.