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.     

Influence of bestatin, an inhibitor of aminopeptidases, on T and B lymphocyte subsets in mice

Bestatin, a low-molecular weight dipeptide, is a potent inhibitor of aminopeptidase N which has been demonstrated to have antitumor and immunomodulatory effects. The effects of bestatin (10, 1 and 0.1 mg/kg) administered intraperitoneally once, five or ten times to mice on the total number of lymphocytes in the thymus, spleen and mesenteric lymph nodes and the percentage and the absolute number of T cell subsets (CD4+CD8+, CD4-CD8-, CD4+, CD8+) in the thymus and T (CD3+, CD4+, CD8+) and B (CD19+) lymphocytes in the spleen and mesenteric lymph nodes were studied. It has been found that bestatin administered ten times at doses of 10, 1 and 0.1 mg/kg increased the total number of thymocytes, splenocytes and lymphocytes of mesenteric lymph nodes. Bestatin also changed the percentage and the absolute number of T cell subsets in the thymus and T and B lymphocytes in the peripheral lymphatic organs. Five and ten exposures to bestatin (10, 1 and 0.1 mg/kg) increased the absolute count of both immature CD4+CD8+ and CD4-CD8- thymic cells. Moreover, both a single and multiple administration of bestatin (1 and 0.1 mg/kg) decreased the percentage and absolute count of CD3+ splenocytes and mesenteric lymph node cells with corresponding decreases in the percentage and absolute count of CD4+ and CD8+ cells. Both a single and multiple administration of bestatin at all the doses under investigation augmented the percentage and the absolute count of CD19+ (B lymphocytes) in the peripheral lymphatic organs. The results of the study show that there is a relationship between the effect induced by bestatin and the dose of the drug as well as the number of doses applied. The strongest effect on the T and B lymphocyte subsets was noted after five injections of bestatin at doses of 1 and 0.1 mg/kg.     

Identification and phenotypic characterization of γδ T cells in rat lymph

γδ T cells represent an unconventional subset of T lymphocytes that are abundant in epithelial tissues and serve as an early immune defense against microbes. We have, for the first time, identified γδ T cells in steady-state thoracic duct lymph (TDL) from rats. The lymph contains γδ T cells expressing CD8 but not CD4, CD25, MHC-II or CD103. The percentage of TDL γδ T cells in rats does not change when the mesenteric lymph nodes (MLN) are surgically removed. Our data suggest that a proportion of γδ T cells migrate from the intestine into rat TDL, under steady-state conditions.     

Modulatory effects of chitosan adipate on the T and B lymphocyte subsets in mice

This study examined the subsets of T lymphocytes in the thymus, spleen and mesenteric lymph nodes as well as the subsets of B lymphocytes in the spleen and mesenteric lymph nodes in mice administered chitosan adipate (20 mg/kg) intraperitoneally once or four times at 24 h intervals. The results showed that chitosan adipate decreased the percentage of immature CD4⁺CD8⁺ thymic T cells and increased the percentage of mature CD4⁺ and CD8⁺ thymocytes. The most significant stimulating effect was observed after four injections. A single exposure to chitosan adipate increased the percentage of CD4⁺ mesenteric lymph node cells, but four injections of the drug increased the percentage of CD4⁺ and CD8⁺ mesenteric lymph node cells. Chitosan adipate had no effect on the subset of splenic T cells. In contrast, chitosan adipate administered either once or four times increased the percentage of CD19⁺ splenocytes but had no effect on the percentage of CD19⁺ mesenteric lymph node cells. Overall, chitosan adipate induces the maturation and differentiation of thymocytes, and regulates the number of B splenic cells and lymph node T cells irrespective of the number of doses.     

Comparative effects of fluoroquinolones on subsets of T lymphocytes in normothermic and hyperthermic mice

The subsets of T lymphocytes in thymus, spleen and mesenteric lymph nodes were investigated in normothermic and hyperthermic mice treated with fluoroquinolones administered orally six times at 24 h intervals at doses of 15 or 75 mg/kg (flumequine, norfloxacin and ciprofloxacin) and 5 or 25 mg/kg (enrofloxacin). It has been found that fluoroquinolones can modulate CD3+, CD4+ and CD8+ marker expression on thymocytes, splenocytes and lymphocytes of mesenteric lymph nodes. Flumequine (15 mg/kg) decreased the percentage of immature CD4+CD8+ thymic cells and increased the percentage of mature CD4+ and CD8+. When the dose of flumequine was increased to 75 mg/kg a reduction in the maturation of thymocytes was observed. Administration of flumequine, norfloxacin and ciprofloxacin, irrespective of doses applied, increased the percentages of CD3+ splenocytes of CD4+ spleen cells. Exposure to enrofloxacin decreased the percentage of T helper-inducer cells. Flumequine and ciprofloxacin augmented the percentage of CD3+ mesenteric lymph node cells and increased the percentage of CD8+ cells. In contrast, norfloxacin and enrofloxacin decreased the percentage of CD3+ mesenteric lymph node cells and the percentage of CD4+ cells. Lipopolysaccharide (LPS) from E. coli (25 micro g/mouse) increased the percentage of single-positive CD4+ thymocytes, but did not affect the percentage of CD3+, CD4+ and CD8+ splenocytes and mesenteric lymph node cells. Flumequine and ciprofloxacin administered to mice pior to LPS potentiated its stimulant effect on the maturation of thymic cells ( increased percentage of mature CD4+ and CD8+ thymocytes). Pre-treatment with norfloxacin or enrofloxacin either reduced or did not modify the stimulant effect of LPS on maturation of thymic cells. Flumequine, norfloxacin, enrofloxacin and ciprofloxacin administered prior to LPS decreased the percentage of CD8+ splenocytes and increased the percentage of CD4+ spleen cells. Norfloxacin and ciprofloxacin at a dose of 75 mg/kg reduced the percentage of CD8+ mesenteric lymph node cells in hyperthermic mice. Pretreatment with norfloxacin at a dose of 15 mg/kg augmented the percentage of mesenteric lymph node cells. It was concluded that the modulating effects of fluoroquinolones depends on the chemial structure of drugs, dose administered as well as immunologic status.     

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.     

Development of γδ T cell subset responses in gnotobiotic pigs infected with human rotaviruses and colonized with probiotic lactobacilli

γδ T cell responses are induced by various viral and bacterial infections. Different γδ T cells contribute to activation and regulation of the inflammatory response and to epithelial repair. How γδ T cells respond to rotavirus infection and how the colonization of probiotics influences the γδ T cell response were unknown. In this study, we evaluated by multicolor flow cytometry the frequencies and distribution of total γδ T cells and three major subsets (CD2-CD8-, CD2+CD8- and CD2+CD8+) in ileum, spleen and blood of gnotobiotic (Gn) pigs at early (3-5 days) and late phases (28 days) after rotavirus infection. The Gn pigs were inoculated with the virulent human rotavirus Wa strain and colonized with a mixture of two strains of probiotics Lactobacillus acidophilus and Lactobacillus reuteri. In na?ve pigs, the highest frequency of total γδ T cells was found in blood, followed by spleen and ileum at the early age (8-10 days old) whereas in older pigs (32 days of age) the highest frequency of total γδ T cells was found in ileum and spleen followed by blood. Rotavirus infection significantly increased frequencies of intestinal total γδ T cells and the putatively regulatory CD2+CD8+ γδ T cell subset and decreased frequencies of the putatively proinflammatory CD8- subsets in ileum, spleen and blood at post-infection days (PID) 3 or 5. The three γδ T cell subsets distributed and responded differently after rotavirus infection and/or lactobacilli colonization. The CD2+CD8+ subset contributed the most to the expansion of total γδ T cells after rotavirus infection in ileum because more than 77% of the total γδ T cells there were CD2+CD8+ cells. There was an additive effect between lactobacilli and rotavirus in inducing total γδ T cell expansion in ileum at PID 5. The overall effect of lactobacilli colonization versus rotavirus infection on frequencies of the CD2+CD8+ γδ T cell subset in ileum was similar; however, rotavirus-infected pigs maintained significantly higher frequencies of CD8- subsets in ileum than lactobacilli-colonized pigs. The dynamic γδ T cell responses suggest that γδ T cell subsets may play important roles in different stages of immune responses after rotavirus infection and probiotic colonization. The knowledge on the kinetics and distribution patterns of γδ T cell subsets in na?ve pigs and after rotavirus infection or lactobacilli colonization provides the foundation for further mechanistic studies of their functions.     

IFN gamma and IL-4 production by CD4, CD8 and WC1 gamma delta TCR(+) T cells from cattle lymph nodes and blood

The synthesis of IFN gamma and IL-4 by CD4, CD8 and WC1 gamma delta TCR(+) T cell sub-populations, and T cells stained with activation/memory-sub-set markers has been examined by flow cytometric analysis. Cells from blood, prescapular, bronchial and mesenteric lymph nodes and Peyer's patches were incubated with phorbol 12-myristate 13-acetate (PMA), ionomycin and brefeldin-A before staining. Lymphocytes that stained for cytoplasmic IFN gamma were evident within the CD4 and CD8 populations from all tissues and also in the WC1 population from lymph nodes. IL-4 producing cells were primarily evident within the CD4 population. IFN gamma synthesis was evident within both CD45RO(+) and CD45RB(+) populations, but IL-4 synthesis was predominantly by cells that were CD45RO(+)/CD45RB(-). Expression of CD62L is not related to functional memory in CD4(+) T cells from cattle and CD62L(+) cells, particularly from the lymph nodes draining the skin and the lungs, stained with mAb to IFN gamma and IL-4. The findings indicate that at least for CD4(+) T cells, where CD45 isoform expression is related to functional memory, these two cytokines are produced predominantly by cells with a memory phenotype. The observation that some WC1(+) cells produce IFN gamma implies the presence of distinct sub-sets of this gamma delta TCR(+) population cattle and suggests a functional role.     

Characterization of efferent lymph cells and their function following immunization of cattle with an allogenic Theileria annulata infected cell line

Immunization of cattle with in vitro propagated bovine mononuclear cells infected with Theileria annulata induces a protective immune response. Activation and effector function of T cells exiting the lymph node draining the site of cell line immunization were investigated to understand the mechanisms involved in the generation of immunity. Immunized animals exhibited a biphasic immune response in efferent lymph as well as peripheral blood. The first phase corresponded to allogenic responses against MHC antigens of the immunizing cell line and the second was associated with parasite specific responses. An increase in the output of CD2(+) cells and MHC class II(+) cells in efferent lymph was observed after cell line immunization with a corresponding decrease in WC1(+) cells. Although the percentage of CD4(+) T cells did not change significantly over the course of the experiment, they became activated. Both CD25 and MHC class II expressing CD4(+) T cells were detected from day 7 onwards, peaking around day 13. Efferent lymph leukocytes (ELL) exhibited sustained responses to IL-2 in vitro following cell line immunization. Antigen specific proliferation was also detected first to the immunizing cell line and then to parasite antigens. The two peaks of CD2(+) cells were observed, which corresponded to similar peaks of CD8(+) cells. The increase in CD8(+) cells was more pronounced during the second parasite specific phase than the first allogenic phase. Activated CD8(+) T cells mainly expressed MHC class II and some expressed CD25. Significantly the peak of activated CD4(+) T cells preceded the peak of activated CD8(+) T cells, highlighting the role of T. annulata specific CD4(+) T cells in inducing parasite specific CD8(+) cytotoxic responses. A biphasic cytotoxic response also appeared in efferent lymph and peripheral blood, the first directed against MHC antigens of the immunizing cell line followed by MHC class I restricted parasite specific cytotoxicity. The cytotoxic responses in efferent lymph appeared earlier than peripheral blood, suggesting that activated CD8(+) cells exiting the draining lymph node following immunization with T. annulata infected schizonts play an important role in the development of protective immune responses.     

Morphological and immunological characteristics of the bovine temporal lymph node and hemal node

Anatomical dissection of the temporal regions of 62 cattle demonstrated that lymph nodes and hemal nodes are present in 89% of the animals (bilaterally in 65% and unilaterally in 24% of the cases). Lymph nodes accounted for 60% and hemal nodes for 40% of all examined nodes. They are nearly always round with diameters ranging from 1 to 9mm. Injections of India ink showed that their drainage area consists of the forehead, the upper eyelid, the base of the horn and the temporal muscle. Immunohistochemistry and flow cytometry revealed that the distribution and percentages of the different cell populations in the lymph nodes and hemal nodes are similar to other cranial lymph nodes. Based on its anatomical location the name temporal lymph node (lymphonodus temporalis) is proposed.     

Bovine γδ T cells: cells with multiple functions and important roles in immunity

The γδ T-cell receptor (TCR)-positive lymphocytes are a major circulating lymphocyte population in cattle, especially in young calves. In contrast, human and mice have low levels of circulating γδ TCR(+) T cells (γδ T cells). The majority of the circulating γδ T cells in ruminants express the workshop cluster 1 (WC1) molecule and are of the phenotype WC1(+) CD2(-) CD4(-) CD8(-). WC1 is a 220000 molecular weight glycoprotein with homology to the scavenger receptor cysteine-rich (SRCR) family, closely related to CD163. The existence of 13 members in the bovine WC1 gene family has recently been demonstrated and although murine and human orthologues to WC1 genes exist, functional gene products have not been identified in species other than ruminants and pigs. Highly diverse TCRδ usage has been reported, with expanded variable genes in cattle compared to humans and mice. Differential γ chain usage is evident between populations of bovine γδ T cells, this may have implications for functionality. There is a growing body of evidence that WC1(+) γδ T cells are important in immune responses to mycobacteria and may have important roles in T cell regulation and antigen presentation. In this review, we will summarize recent observations in γδ T cell biology and the importance of γδ T cells in immune responses to mycobacterial infections in cattle.     

Total and Differential Leucocyte Counts and Lymphocyte Subpopulations in Lymph, Afferent and Efferent to the Supramammary Lymph Node, During Endotoxin-Induced Bovine Mastitis

Leucocyte trafficking in afferent and efferent mammary lymph and the supramammary lymph node in cows was examined during 4 h after intramammary infusion of endotoxin from Escherichia coli. Total and differential leucocyte counts were measured in milk, blood and lymph. The proportions of CD4(+), CD8(+), major histocompatibility complex (MHC) class II(+) and IgM(+) lymphocytes were examined in the lymph and lymph node. At post-infusion hour (PIH) 4, the flow rates of both lymph fluids had increased approximately eightfold. Total leucocyte concentration increased in afferent lymph, but decreased in efferent lymph. Neutrophils increased in afferent lymph at PIH 2 and in efferent lymph and milk at PIH 4. The predominant cell type in afferent lymph shifted from lymphocyte to neutrophil while lymphocyte was still at PIH 4 the predominant type in efferent lymph. Among the lymphocytes, B cells were predominant in afferent lymph and lymph node at PIH 4 while T cells, mainly CD4(+) cells, were predominant in efferent lymph both at PIH 0 and PIH 4. The CD4 : CD8 ratio was higher in efferent lymph and the challenged lymph node than in afferent lymph and the control node, respectively. There was a significant difference in proportions of each lymphocyte subpopulation except for IgM(+) cells, between afferent and efferent lymph after infusion. According to the results, there was already during the first hours of the immune response, a non-random trafficking of neutrophils and lymphocyte subpopulations resulting in a changed distribution of cells in afferent and efferent lymph and a difference in lymphocyte reactivity between the two lymph fluids.     

The effects of florfenicol on lymphocyte subsets and humoral immune response in mice

Florfenicol is a broad-spectrum bacteriostatic antibiotic used in domestic animals. The aim of the study was to determine the effect of florfenicol on the total number of lymphocytes in the thymus, spleen and mesenteric lymph nodes and the percentage and the absolute number of T cell subsets (CD4+CD8+, CD4-CD8-, CD4+, CD8+) in the thymus and T (CD3+, CD4+, CD8+) and B (CD19+) lymphocytes in the peripheral lymphatic organs in non-immunized mice and humoral immune response in sheep red blood cells (SRBC)-immunized mice. Florfenicol was administered orally at a dose of 30 mg/kg six times at 24 h intervals to non-immunized mice and four or seven times at 24 h intervals to SRBC-immunized mice. SRBC was injected 2 hours prior to the first dose of the drug. Florfenicol increased the percentage of CD4CD8- thymocytes and the absolute number of CD4+ and CD8+ thymocytes on day 7. The increased percentage and absolute number of CD3+, CD4+ and CD8+ lymphocytes in mesenteric lymph nodes and decreased percentage of lymphocytes B were also observed 24 hours from the last administration of florfenicol. Florfenicol administered after SRBC immunization reduced the number of plaque forming cells (PFC) and the production of anti-SRBC antibodies on days 4 and 7 after priming.     

Relationship between diarrhea and peripheral leukocyte population in neonatal Japanese black calves

Neonatal Japanese Black (JB) calves show a high incidence of diarrhea. The objective of this study was to analyze the immune cell populations of neonatal JB calves in detail and examine its correlation with the incidence of diarrhea immediately after birth. Understanding the immune cell populations is helpful in clinics in order to determine the condition of the immune system for prevention of diseases. Blood samples were obtained from JB calves on the day of birth. The peripheral leukocyte populations were analyzed separately for calves that had diarrhea within 2 weeks after birth (diarrhea group; n = 26) and for calves without diarrhea (control group; n = 74). The numbers of the peripheral blood CD3(+)TcR1-N12(+) and CD8(+) T cells were significantly lower in the diarrhea group compared with the control group. These findings suggest that the congenital lower peripheral γδ and CD8(+) T cells results in a high risk of diarrhea in neonatal JB calves.     

Cellular profiles in the abomasal mucosa and lymph node during primary infection with Haemonchus contortus in sheep

Cellular changes in the abomasal tissue and draining abomasal lymph nodes were examined after primary infection of lambs with Haemonchus contortus for 3, 5 or 27-36 days.Infection with H. contortus larvae resulted in a rapid and selective increase in the percentage of CD4(+) T-cells in the abomasal lymph node at 3 days post-infection (PI). By 5 days PI, the lymph node weight had increased two-fold; however, the percentage of lymphocyte populations in the abomasal lymph node resembled that seen in uninfected sheep. Lymph node weights remained at increased levels in the adult nematode infected sheep and down-regulation of B-cell surface markers (sIg and MHC Class II) was apparent in this group. Significant increases in the percentage of CD4(+) T-cells co-expressing MHC Class II, but not CD25, were observed in the larval infected groups except in adult nematode infected sheep. Increased numbers of eosinophils, CD4(+), gamma delta(+) T-cells and B-cells were found in the abomasal tissue by 5 days PI, but no further increases in these cell populations were observed in the adult nematode infected group. In contrast, the level of both lamina propria and intraepithelial mast cells observed in the abomasal mucosa was highest in the sheep carrying an adult nematode burden. These findings indicate that sheep are able to generate an early immune response to infection with H. contortus larvae, characterised by the activation of CD4 T-cells and B-cells in the draining lymph nodes and recruitment of eosinophils, CD4(+) and gamma delta-TCR,WC1(+) T-cells and B-cells in larval infected tissues. However, these changes do not seem to be maintained during infection with the adult parasite where increases in mast cell numbers dominate the local response, indicating that different parasite stages may induce distinct and possibly counteractive immune responses.     

Apoptosis in normal lymphoid organs from healthy normal, conventional pigs at different ages detected by TUNEL and cleaved caspase-3 immunohistochemistry in paraffin-embedded tissues

The frequency and the distribution of apoptotic cells were investigated in formalin-fixed paraffin-embedded lymphoid tissues from healthy conventional pigs at four different ages (6 days, 2 months, 3.5 months and 5 months). Samples of tonsil, mesenteric lymph node, spleen, thymus and Peyer's patches were histologically processed and apoptosis evaluated with the TUNEL reaction and cleaved caspase-3 immunohistochemistry. In each technique, quantification of positive labelling was done for each particular lymphoid tissue area. The labelling pattern and distribution were similar for TUNEL and cleaved caspase-3. TUNEL stained mainly apoptotic bodies inside macrophages, but signal was also seen in free apoptotic bodies and in the nuclei of lymphocyte-like cells. The anti-cleaved caspase-3 antibody labelled mainly nuclei of lymphocyte-like cells. All tissues presented a similar distribution pattern of apoptosis, except for the 6-day-old group. In this group, a scattered distribution of positive cells was detected in tonsil, lymph node and spleen. In the tonsil and mesenteric lymph nodes from the older pigs, follicular areas presented higher amounts of positive cells than interfollicular areas. Moreover, the splenic white pulp showed more positive reaction than the red pulp, especially when they included germinal centres. In all groups, the follicular areas of ileal Peyer's patches presented more labelled cells than the dome and the lamina propria. In the thymus, the higher apoptotic rates were found in the cortex. In general, TUNEL yielded higher rates of positive cells than cleaved caspase-3 immunolabelling. A good correlation between the two techniques was found for thymus, tonsil and mesenteric lymph node, but not for Peyer's patches and spleen. This study describes a detailed histochemical characterization of apoptosis in pig lymphoid tissues using TUNEL and a cleaved caspase-3 immunolabelling at different ages. Moreover, our results indicate that TUNEL and cleaved caspase-3 techniques can be equivalent only when tissues have a high or low levels of apoptosis, since a considerable discrepancy was found in intermediate situations. Data from this study should be useful for future comparative studies under disease conditions.     

Lymphatic organ development in dogs: major lymphocyte subsets and activity

In the present study, we have characterized lymphocyte subsets and activity in peripheral blood, spleen, mesenteric and popliteal lymph nodes in pups from birth till the age of one month and compared the results with the situation in the group of three adult dogs. In neonatal pups, lower numbers of CD3(+) T-cells were detected in both the spleen and peripheral blood than in lymph nodes. In contrast to the other compartments, CD21(+) B-cells prevailed in the spleen, which resulted in low values (<1) of the CD3(+)/CD21(+) ratio. Low numbers of CD8(+) lymphocytes were characteristic in all compartments immediately after birth; consequently a high CD4(+)/CD8(+) ratio has been calculated. Postnatal development was characterized by an increasing frequency of CD8(+) lymphocytes in all organs studied. Another typical feature of the early period of life was a relative decrease of B-cell numbers, which was compensated by an increasing proportion of T-lymphocytes, particularly in the peripheral blood and spleen. DNA synthesis in newborn pups' cells as measured by in vitro thymidine incorporation was surprisingly high in non-stimulated control samples, notably in the spleen. Further development of lymphocyte activity was characterized by the decline in spontaneous activity in all organs. Stimulation indices upon mitogen-induced proliferation increased proportionally to the decrease in spontaneous activity. Based on our experimental data, we have concluded that pups are born with a relatively competent immune system the structure of which, however, markedly develops during a few postnatal weeks.     

Mesenteric lymph node cells from neonates present a prominent IL-12 response to CpG oligodeoxynucleotide via an IL-15 feedback loop of amplification

ABSTRACT: At birth, the immune system is still in development making neonates more susceptible to infections. The recognition of microbial ligands is a key step in the initiation of immune responses. It can be mimicked to stimulate the immune system by the use of synthetic ligands recognising pattern recognition receptors. In human and mouse, it has been found that neonatal cytokine responses to toll-like receptor (TLR) ligands differ in many ways from those of adults but the relevant studies have been limited to cord blood and spleen cells. In this study, we compared the responses in neonate and adult sheep to CpG oligodeoxynucleotides (ODN), a TLR9 ligand, in both a mucosal and a systemic organ. We observed that in response to CpG-ODN more IL-12 was produced by neonatal than adult sheep cells from mesenteric lymph nodes (MLN) and spleen. This higher IL-12 response was limited to the first 20 days after birth for MLN cells but persisted for a longer period for spleen cells. The major IL-12-producing cells were identified as CD14+CD11b+. These cells were poor producers of IL-12 in response to direct stimulation with CpG-ODN and required the cooperation of other MLN cells. The difference in response to CpG-ODN between neonates and adults can be attributed to both a higher proportion of CD14+CD11b+ cells in neonate lambs and their higher capacity to produce IL-15. The IL-15 increases IL-12 production by an amplifying feedback loop involving CD40.     

Use of Monoclonal Antibodies for Immunohistochemical Study of Bovine Spleen on Frozen Sections

Many monoclonal antibodies reactive with bovine leukocyte differentiation antigens are now available. Immunohistochemical staining on frozen sections using these monoclonal antibodies permits study of the functional morphology of bovine spleen. This study confirms accepted notions (B and T dependent-zones) and supplies complementary data about the repartition of CD4 and CD8 cells. γδ cells. MHC (Major Histocompatibility Complex) II expression, and macrophages.