Effects of lipopolysaccharide exposure in primary bovine ruminal epithelial cells.

The objective of this study was to investigate whether cultured ruminal epithelial cells (REC) responded to lipopolysaccharide (LPS) stimulation and determine whether LPS induced a proinflammatory response. Primary bovine REC were isolated and grown in culture for 2 studies. In study 1, REC were isolated from Holstein bull calves (n = 8) and grown in culture for 10 to 12 d. Cells were then exposed to 0, 10,000, 50,000, or 200,000 endotoxin (E)U/mL of LPS (Escherichia coli O55:B5) for either 6 or 24 h. The effect of LPS exposure on cell viability was analyzed by flow cytometry using a propidium iodide stain. In study 2, cells were isolated from Holstein bull calves (n = 5) and yearling beef heifers (n = 4). Cells were exposed to either 1,000 or 50,000 EU/mL of LPS using the following conditions: (1) medium alone time-matched controls, (2) 12-h LPS exposure, (3) 24 h of LPS exposure, (4) 36 h of LPS exposure, (5) 12 h of LPS exposure followed by LPS removal for 24 h before restimulating with LPS for an additional 12 h (RPT), and (6) 12 h of LPS exposure followed by LPS removal for 36 (RVY). For both experiments, total RNA was extracted from REC and real-time quantitative PCR was performed to determine relative expression of genes for toll-like receptors (TLR2 and TLR4), proinflammatory cytokines (TNF and IL1B), chemokines (CXCL2 and CXCL8), a lipid mediator (PTGS2), and growth factor-like cytokines (CSF2 and IL7). In study 1, LPS exposure did not negatively affect cell viability. Treatment of cells with LPS resulted in increased transcript abundance for all genes analyzed. The TLR2, IL7, and TLR4 had a greater magnitude of change at 6 h compared with 24 h. Quadratic expression patterns were detected for TNF, IL1B, CXCL2, CXCL8, and CSF2. These results suggested that REC increase expression of proinflammatory genes following exposure to LPS. In study 2, all genes analyzed were upregulated in a quadratic manner following exposure to LPS for different time intervals. The TLR4, TNF, CXCL2, CXCL8, CSF2, and IL7 gene expression was significantly greater after a single 12 h of LPS exposure than after RPT exposure, suggesting repeated exposure of REC to LPS may induce a tolerogenic effect. When LPS was removed from the medium (RVY), transcript abundance for all genes analyzed decreased and expression of TLR2, TLR4, and IL7 returned to baseline levels, suggesting REC recovered following exposure to LPS. Overall, the data suggest cultured REC respond to LPS stimulation by increasing transcription of proinflammatory genes and this transcriptional response was influenced by the dose, duration, and frequency of LPS exposure.

IL-10 secreting CD21⁺ B cells are present in jejunal Peyer's patches of sheep during fetal development.

We recently reported a novel interleukin-10 (IL-10)-secreting CD21(+) B cell population in jejunal Peyer's patches (JPP) of sheep with a regulatory function (Bregs) suppressing Toll-like receptor 9 (TLR9)-induced cytokine responses. However, little is known about the development of these cells. Therefore, we investigate their existence in JPP cells from fetal and newborn lambs. CD21(+) B cells were purified from JPP cells by magnetic cell sorting and subsequently stimulated with the TLR9 agonist, CpG ODN (CpG oligodeoxynucleotide). Lymphocyte proliferative responses, cytokine production (IL-10, IL-12 and interferon-γ [INF-γ]) and antibody secretion were assayed. We found that fetal and neonatal CD21(+) B cells spontaneously secreted high levels of IL-10 regardless of CpG stimulation but that these cells did not produce any IL-12 or INF-γ upon stimulation with CpG. The observed responses are consistent with those previously reported for Bregs characterized in JPP of older lambs. Surprisingly, unlike in older lambs, fetal and neonatal JPP CD21(+) B cells proliferated in response to CpG stimulation. Our investigations of fetal and neonatal lambs provide evidence for the development of IL-10-secreting CD21(+) B cells in PPs prior to antigen exposure.

Fetal immunization by a DNA vaccine delivered into the oral cavity.

Infectious diseases are the main cause of neonatal morbidity and mortality in humans. The World Health Organization estimated that in 1995 approximately 8 million infants died within the first year of life from infectious diseases, including 5 million during the first week of life. Some of the salient pathogens involved include herpes simplex virus, human immunodeficiency virus, hepatitis B virus, human cytomegalovirus, group B streptococcus, hemophilus and chlamydia. Infection with these pathogens usually occurs at the end of pregnancy, during birth or by breastfeeding. To reduce the risk of disease transmission, caesarian sections, prophylactic treatment with antibiotics or maternal antiviral therapy during the last trimester are used where available, together with improved neonatal care. None of these approaches, however, completely eliminates the risk of neonatal infection. Therefore, active or passive immunization of the fetus might represent an effective approach to reduce the high risk of neonatal diseases. Here, we demonstrate that a single immunization with a DNA vaccine delivered into the amniotic fluid in the oral cavity induces high serum antibody titers and a cell-mediated immune response, combined with induction of local immunity in the oral cavities of fetal lambs.

Natural and inducible regulatory B cells are widely distributed in ovine lymphoid tissues.

Regulatory B cells that produce IL-10 are now recognized as an important component of the immune system. We previously confirmed that IL-10 secreting CD21+ regulatory B cells (Breg cells) were present in ovine jejunal Peyer's patches (JPP) and this IL-10 production suppressed IL-12 and IFN-γ secretion. It is not known, however, whether ovine Breg cells are restricted to JPP or are present in other lymphoid tissues. Therefore, CD21+ B cells were purified from sheep JPP and from a variety of mucosal and systemic lymphoid tissues using magnetic cell sorting. Purified CD21+ B cells were stimulated with a TLR9-agonist, CpG oligodeoxynucleotide (CpG ODN), and the frequency of spontaneous and inducible (i) IL-10-secreting B cells was evaluated by ELISPOT. Spontaneous IL-10 secreting CD21+ B cells were present in mucosal (jejunal PP, parabronchial lymph nodes (LN), mesesnteric LN, and palatine tonsils) and systemic (spleen and blood) lymphoid tissues. Mucosal lymphoid tissues (parabronchial and mesenteric LNs and JPP) had the highest frequency of cells spontaneously secreting IL-10 while tonsils had the lowest. The frequency of B cells spontaneously secreting IL-10 was lowest in blood and spleen. There was large inter-animal variation in the frequency of CD21+ B cells spontaneously secreting IL-10 and no significant difference was detected following CpG ODN stimulation. When comparing within individual animals there was, however, a consistent increase in the frequency of CD21+ cells secreting IL-10 following CpG ODN stimulation versus stimulation with GpC control ODN. The presence of inducible (i)Breg cells in ovine mucosal tissues supports previous evidence from mice indicating that B cells have the capacity to modulate inflammatory responses. The presence of iBreg cells in ruminants may also provide a novel therapeutic target for both immunomodulatory drugs and vaccines designed to control antigen-specific mucosal inflammation.

Knowledge gaps that hamper prevention and control of Mycobacterium avium subspecies paratuberculosis infection.

In the last decades, many regional and country-wide control programmes for Johne's disease (JD) were developed due to associated economic losses, or because of a possible association with Crohn's disease. These control programmes were often not successful, partly because management protocols were not followed, including the introduction of infected replacement cattle, because tests to identify infected animals were unreliable, and uptake by farmers was not high enough because of a perceived low return on investment. In the absence of a cure or effective commercial vaccines, control of JD is currently primarily based on herd management strategies to avoid infection of cattle and restrict within-farm and farm-to-farm transmission. Although JD control programmes have been implemented in most developed countries, lessons learned from JD prevention and control programmes are underreported. Also, JD control programmes are typically evaluated in a limited number of herds and the duration of the study is less than 5 year, making it difficult to adequately assess the efficacy of control programmes. In this manuscript, we identify the most important gaps in knowledge hampering JD prevention and control programmes, including vaccination and diagnostics. Secondly, we discuss directions that research should take to address those knowledge gaps.

Evidence for the existence of regulatory and effector B cell populations in Peyer's patches of sheep.

IL-10 secreting CD21(+) B cells exist in sheep Peyer's patches (PP). It's not known however, whether all PP B cells are regulatory or whether an effector population also exists in this tissue. To further characterize the subpopulations of B cells in PP's, highly purified B cells were negatively sorted from jejunal PP and fractionated according to co-expression of CD72(+)CD21(+)or CD72(+)CD21(-) molecules and then stimulated with the TLR9-agonist, CpG ODN. IL-10, IL-12, IFN-γ, and IgM production were then assayed. We observed that only highly purified CD72(+)CD21(+) B cells spontaneously secreted high levels of IL-10, but they did not produce any IL-12, IFN-γ or IgM suggesting that this cell population contains regulatory B cells. In contrast, CD72(+)CD21(-) B cells did not secrete IL-10, but secreted IL-12, IFN-γ, and IgM, suggesting they include effector cells. In addition, B cells expressing surface IgA, IgM and IgG1 all secreted similar levels of IL-10. We further confirmed that only B cells produce IL-10, while other cells in the PP including DCs and T cells do not. Our investigations may provide evidence for the existence of two sub-populations in sheep PP; IL-10 secreting regulatory (CD72(+)CD21(+)) cells, and IL-12/IFN-γ/IgM-secreting effector (CD72(+)CD21(-)) cells.

Cloning non-transformed sheep B cells.

The capacity to clone B cells and establish permanent B cell lines has greatly facilitated a wide variety of studies characterising the growth, differentiation, and gene expression of murine and human B cells. Similar investigations of B cell biology for other species have been severely restricted by an inability to culture or clone B cells. This is the first report of a method to clone non-transformed sheep B cells using a culture system based on murine CD154 and a combination of human gamma chain-common cytokines. Sheep Peyer's patch B cells were cultured for 120 days and then cloned by limiting dilution culture. The parental B cell culture contained both surface immunoglobulin (sIg)M(+) and sIgG1(+) B cells and both types of B cell were cloned. Clonality was confirmed by PCR analysis of Ig heavy chain (HC) and light chain (LC) expression and DNA sequencing of HC V genes. There was agreement between the PCR and flow cytometric analyses of HC isotype expression on the B cell clones but the available monoclonal antibodies specific for sheep lambda and kappa LC did not react with all clones. Soluble Ig was detected in the culture supernatant of sIgG1(+) clones but not sIgM(+) clones. The B cell clones remained dependent upon CD154 and gamma chain-common cytokine co-stimulation for sustained growth and maintained stable Ig expression. The cloning of non-transformed sheep B cells should provide a valuable tool for studying sheep B cell biology, establishing Ig HC- and LC-specific monoclonal antibodies, analysing the B cell Ig repertoire, and may be used to produce sheep monoclonal antibodies.

Multiple intestinal 'loops' provide an in vivo model to analyse multiple mucosal immune responses.

Mucosal immunity plays an important role in preventing disease but the induction of protective mucosal immune responses remains a significant challenge. We describe a novel in vivo model to analyze the induction of multiple mucosal immune responses in the small intestine. A sterile segment of intestine ('intestinal-segment'; 2-3 m long) was surgically prepared in the jejunum of 4-6-month-old lambs. This 'intestinal-segment' was then subdivided into consecutive segments, designated as 'loops' (15-20 cm long), that included a Peyer's patch (PP), or 'interspaces' (15-70 cm long), that lacked a visible PP. All 'loops' were sterile when collected 1-4 weeks post-surgery and there was no macroscopic or histological evidence of altered lymph or blood flow. Flow cytometric analysis of cells isolated from PP, mucosal epithelium (IEL) and the lamina propria (LPL) revealed no significant alterations in the cell populations present in 'loop' tissues. The functional integrity of M-cell antigen uptake in sterile intestinal 'loops' was evaluated by comparing the immune response induced by varying doses of soluble versus particulate porcine serum albumin (PSA formulated in alginate microspheres). A dose-dependent, PSA-specific antibody-secreting cell response was restricted to PP present in 'loops' injected with particulate PSA. These observations suggested that PP present in sterile 'loops' were functional and this conclusion was confirmed by detecting cholera toxin-specific antibody-secreting cells and secreted antibody in PP and intestinal contents, respectively, of immunized 'loops.' Thus, each 'loop' provided an independent site to analyze antigen-uptake and the induction of mucosal immune responses by a variety of antigen or vaccine formulations.

Immunocytochemical identification of bovine Langerhans cells by use of a monoclonal antibody directed against class II MHC antigens.

We undertook a study to develop a reliable light microscopic technique for identifying Langerhans cells (LC) in bovine epidermis. Monoclonal antibodies (MCA) detecting bovine class II MHC antigens were used in conjunction with an avidin-biotin-peroxidase complex (ABC) immunocytochemical staining method. The specificity of the MCA for LC was confirmed ultrastructurally by use of gold-labeled second antibody. Epidermal sheets and epidermal single-cell suspensions examined by light microscopy confirm that bovine epidermal LC express class II antigens. Anti-bovine class II MCA is a dependable reagent for identification of LC in normal bovine epidermis.

Bovine peripheral blood leukocyte subpopulation dynamics following a primary bovine herpesvirus-1 infection.

Population dynamics of bovine peripheral blood leukocyte subpopulations were quantitated following a primary bovine herpesvirus-1 (BHV-1) infection. Percoll isolated peripheral blood mononuclear cell (PBMC) subpopulations were analyzed using flow cytometry (FC) and cytochemical stains. Between days two to eight post-infection (PI) there was a significant decrease in the percentage of T-cells and nonT/nonB cells which was accompanied by an increased percentage of B-cells and monocytes. These percentages were extrapolated to the number of Percoll isolated PBMC during this period. A decrease in the T-cell population was the primary cause of the observed lymphopenia and a relative increase in the percentage of B-cells. The increased percentage of monocytes was caused by an increased number of circulating monocytes. These monocytes were characterized by an increase in Fc receptor expression, a decrease in plastic and Sephadex-G10 adherence and no apparent change in the level of class II MHC antigen (Ia) expression. Serum cortisol was significantly elevated on day 2 PI and may have been responsible for both the reduction in circulating T-cells and a decrease in the in vitro viability of peripheral blood lymphocytes. The percentage of Ia positive PBMC was increased significantly on day 4 PI. However, on days 4 and 6 PI the summated percentages of monocytes and B-cells (total Ia expressing population) exceeded significantly the actual percentage of Ia positive cells. This apparent suppression of Ia expression did not coincide with the elevated serum prostaglandin E2 concentrations on days 8 and 10 PI.

T lymphocyte population dynamics and function following a primary bovine herpesvirus type-1 infection.

Following a primary bovine herpesvirus-1 (BHV-1) infection the concanavalin A (Con A) induced proliferative responses of peripheral blood T lymphocytes were suppressed. This suppression occurred in the absence of detectible serum suppressor factors, suppressor cell activity or decreased accessory cell function. However, regression analysis demonstrated a significant correlation between the percentage of T lymphocytes present within the peripheral blood mononuclear cell (PBMC) population and the amplitude of Con-A-induced lymphocyte proliferative responses (LPR). Direct evidence that a numerical deficit of responder T lymphocytes was limiting LPR was obtained by using an immunomagnetic microsphere (IMM) negative enrichment protocol to produce a PBMC population with a constant percentage (75 +/- 6%) of T lymphocytes. The Con-A-induced LPR of these enriched T lymphocytes remained constant following BHV-1 infection. Flow cytometric (FC) analysis of PBMC indicated that the decreased percentage of circulating T lymphocytes, associated with BHV-1 infection, was caused primarily by a selective depletion of the BoT8 subset. These FC data were consistent with the indirect evidence of increased TH activity, as indicated by elevated Con A-induced IL-2 production. Thus, 2 to 5 days following viral infection, the circulating T lymphocytes were activated as shown by elevated IL-2 production, increased recombinant bovine IL-2 (rBo

Immunization of neonates with DNA encoding a bovine herpesvirus glycoprotein is effective in the presence of maternal antibodies.

Neonates generally display low immune responsiveness to conventional vaccines, which may be due to the immaturity of their immune system and interference by maternal antibodies. Because of the unique capacity of plasmid DNA for the production of low doses of antigen over extended periods of time, we used DNA immunization as an approach to induce immunity in neonates. Previously, we demonstrated that a plasmid encoding a truncated secreted version of bovine herpesvirus-1 gD (tgD) induces protective immunity in adult animals. For the present study, 3-day-old lambs were immunized intradermally with the tgD-expressing plasmid. The lambs developed antibody as well as T-cell responses to the tgD glycoprotein, which clearly demonstrates the ability of the animals to respond to vaccination at this age. Furthermore, lambs born to tgD-hyperimmunized ewes, thus containing high levels of passively acquired serum antibodies, responded to the tgD DNA vaccine in a similar manner, which shows that the maternal antibodies did not inhibit the development of an immune response. These results indicate that DNA immunization might be a useful approach to vaccinate neonates that possess high levels of maternal antibodies.

Negative enrichment of bovine T lymphocytes with monoclonal antibodies and magnetic microspheres.

A highly enriched population of bovine T lymphocytes was produced from peripheral blood leukocytes following the depletion of monoclonal antibody-labelled B lymphocytes and monocytes with magnetic microspheres. This negative-enrichment protocol was simple, rapid, and specific. Also, it had a high recovery efficiency and was consistently reproducible. The enriched T lymphocytes proliferated in response to recombinant bovine interleukin 2 and, following the addition of monocytes, to concanavalin A. This methodology made it possible to determine the proliferative responses of peripheral blood lymphocytes utilizing a constant number of T lymphocytes within each assay. In this way, the in vitro T lymphocyte responses were determined independent of changes in the number of responder cells within peripheral blood.

In vivo immunostimulatory effects of CpG oligodeoxynucleotide in cattle and sheep.

Oligodeoxynucleotides (ODN) containing cytosine-phosphate-guanosine (CpG) motifs have been shown to activate the innate immune system and protect mice and chicken from bacterial and viral infections. Unfortunately, similar studies in other veterinary species are lacking. In this study we assessed the in vivo immunostimulatory effects of CpG ODN 2007, an ODN with previously demonstrated in vitro biological activity. The in vivo effects of ODN 2007 were compared in two closely related outbred species, sheep and cattle, to determine if there were common biological responses. We demonstrated that subcutaneous (s.c.) injection of the CpG ODN induces an acute phase response in the form of a transient fever, a mild transient increase in circulating neutrophils and elevated serum haptoglobin in both sheep and cattle. Sheep injected with CpG ODN also exhibited increased serum 2'5'-oligoadenylate (2'5'-A) synthetase activity, but no increase in serum 2'5'-A synthetase was detected in cattle. The ODN-induced responses were stronger in animals injected with CpG ODN formulated in 30% emulsigen than phosphate buffer saline (PBS) alone. These in vivo data demonstrate for the first time that a CpG ODN induces acute phase immunostimulatory responses in sheep and cattle. However, CpG ODN-induced antiviral effector molecule 2'5'-A synthetase was detected only in sheep but not in cattle.

Fusion of C3d molecule with bovine rotavirus VP7 or bovine herpesvirus type 1 glycoprotein D inhibits immune responses following DNA immunization.

The binding of the complement C3d molecule with receptors on B cells and/or follicular dendritic cells (FDCs) influences the induction of humoral immune responses. For example, C3d fused to an antigen has been shown to have a strong adjuvant effect on antibody production. We investigated the possibility that co-expression of antigen and C3d as a fusion protein could enhance antigen-specific immune responses, following plasmid immunization. One or two copies of murine C3d-cDNA, C3d or (C3d)(2), respectively, were cloned together with bovine rotavirus (BRV) VP7 or bovine herpesvirus type 1 (BHV-1) glycoprotein D (gD) genes. All constructs contained a signal peptide that resulted in the secretion of the expressed proteins. In vitro, the characterization of the chimeric proteins indicated that both VP7 and gD retained their antigenicity and the C3d remained biologically active. However, immunization with plasmids encoding VP7-C3d chimeras did not enhance rotavirus-specific antibody responses and the frequency of BRV-specific IFN-gamma secreting cells in the spleens were significantly lower in mice immunized with pVP7-(C3d)(2) when compared with mice immunized with plasmid encoding VP7. The same pattern of immune responses was observed for plasmids encoding gD-C3d. Both gD-specific antibody responses and the frequency of gD-specific IFN-gamma secreting cells were significantly lower in mice immunized with plasmid expressing gD-C3d chimeras when compared with mice immunized with plasmid encoding gD alone. These results indicate that co-expression of C3d with an antigen actually inhibit both humoral and cell-mediated antigen-specific immune responses.

Monocytes are required for optimum in vitro stimulation of bovine peripheral blood mononuclear cells by non-methylated CpG motifs.

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs within certain flanking base pairs are recognized as a danger signal by the innate immune system of vertebrates. Using lymphocyte proliferative response (LPR) and IFN-gamma secretion assays, a panel of 38 ODN was screened for immunostimulatory activity on bovine peripheral blood mononuclear cells. ODN composed of a nuclease resistant phosphorothioate backbone and a leading 5'-TCGTCGTT-3' motif with two 5'-GTCGTT-3' motifs were highly stimulatory in both assays. Flow cytometric analysis and cell-specific surface marker labeling determined that B-cells (surface IgM(+)) were the primary cell population responding in the LPR assay. Depletion of T cells (CD3(+)) from the PBMC population did not affect IFN-gamma secretion or B-cell proliferation when cultured with CpG-ODN. However, depletion of monocytes (DH59B(+)) completely abrogated the ability of CpG-ODN to stimulate IFN-gamma secretion, and significantly reduced the B-cell proliferative response. These data establish the identity of an optimal immunostimulatory CpG motif for cattle and demonstrate that monocytes play a pivotal role in the ability of cell populations to respond to CpG-ODN. These data provide insight for future studies investigating the mechanism of CpG-ODN bioactivity and its application in novel vaccine formulations and immunotherapy.

Ontogeny of the immune response: effect of protein energy malnutrition in neonatal calves.

Immunocompetence of neonatal, Holstein bull calves fed for maximal growth (Control; n = 4) or protein energy malnutrition feeding (PEM; n = 4) for four weeks was assayed in vitro and in vivo. All calves exhibited elevated cortisol levels for ten days postnatally. At this time calves also were neutrophilic and lymphopenic. In addition lymphocyte function, as measured by lymphocyte proliferation and interleukin-2 activity, was reduced at this time as compared to older calves. After two weeks of protein energy malnutrition feeding, calves had significantly lower body weight, lymphocyte interleukin-2 activity and lymphocyte proliferation when compared with age-matched controls. Two weeks after protein energy malnutrition ration reversal, interleukin-2 activity and lymphocyte proliferation was comparable for both groups. There was no significant difference in serum cortisol concentration between control and protein energy malnutrition calves. The kinetics of the protein energy malnutrition group's primary humoral immune response was retarded, thus significantly lower antibody levels to K99 antigen were observed 8 to 12 days postimmunization. There was no significant difference between groups when comparing secondary response to K99 antigen.

2'-5' oligo-A-synthetase activity in bovine peripheral blood leukocytes and alveolar macrophages exposed to recombinant interferons and tumor necrosis factor-alpha.

In vitro treatment of bovine peripheral blood mononuclear leukocytes, polymorphonuclear neutrophilic granulocytes and alveolar macrophages with recombinant bovine interferons -alpha 1 1, -beta 2 or -gamma induced an immediate increase in the intracellular level of 2'-5' oligoadenylate synthetase activity. The induction was dose-dependent, with interferon -alpha 1 1 and -beta 2 being more potent than interferon-gamma. Maximal levels were reached within 10-12 h with IFN-alpha 1 1, which corresponded well with findings in vivo. In contrast to what has been found in nonlymphoid bovine cells, tumour necrosis factor-alpha did not potentiate the induction of 2-5A synthetase by interferons, neither did it by itself induce the enzyme.

Proinflammatory cytokine gene expression in endometrial cytobrush samples harvested from cows with and without subclinical endometritis.

Thirty postpartum cows (28 to 41 days in milk) without signs of clinical endometritis were categorized as inflammation-negative (N = 18) or subclinical endometritis-positive (N = 12) based on endometrial cytobrush cytology (> 18% polymorphonuclear cells; PMNs). Slides for cytology were prepared before the same cytobrush was transferred to a tube containing 1 mL Trizol reagent. Total RNA was extracted from each cytobrush sample and analysis of il6, il8, tnfα, and ßactin gene expression was performed using quantitative real-time polymerase chain reaction. Cytobrush sampling provided sufficient material to prepare cytosmears and extract high quality endometrial mRNA (mean = 0.96 µg RNA per sample). Cytokine expression varied between experimental groups with a 20-fold higher tnfα (P = 0.001), a 30-fold higher il6 (P = 0.01), and a greater than 50-fold higher il8 mRNA expression level (P = 0.0001) in subclinical endometritis-positive versus disease-negative cows. Regression analysis of gene expression levels (cycle threshold) versus PMN frequency showed that the frequency of PMNs in the cytosmear decreased by 3.3% (P = 0.000 01), 2.3% (P = 0.015), and 2.4% (P = 0.05) for each additional cycle threshold required to detect il8, il6, and tnfα gene expression, respectively. Expression of the individual cytokines was positively associated: il8 and il6 (P = 0.0001); il8 and tnfα (P = 0.000 01); and il6 and tnfα (P = 0.0002). In conclusion, the endometrial cytobrush technique was successfully used to obtain material for both cytology and RNA extraction, and il8 gene expression may be useful to predict endometrial inflammation.

A novel regulatory B-cell population in sheep Peyer's patches spontaneously secretes IL-10 and downregulates TLR9-induced IFNalpha responses.

Peyer's patches (PPs) play an important role in the induction of immune responses in the intestine, but regulation of Toll-like receptor (TLR)-induced innate immune responses in PPs is not well understood. We investigated the responses of PPs and other immune cells to the TLR9 agonist, CpG oligodeoxynucleotide (ODN). Peripheral blood mononuclear cells and lymph node cells secreted significant amounts of interferon (IFN)-alpha, IFNgamma, and interleukin (IL)-12 following stimulation with CpG ODN. In contrast, PP cells exhibited poor cytokine responses, despite abundant expression of TLR9 mRNA. PP cells spontaneously secreted high levels of IL-10, and the primary source of the IL-10 was resting CD5(-)CD11c(-)CD21(+) B cells. Neutralization of the IL-10 or depletion of CD21(+) B cells resulted in a significant increase in CpG-induced IFNalpha-response in PPs, suggesting that IL-10 from B cells regulate innate responses in PPs. These IL-10-secreting PP B cells may represent a novel subset of the recently proposed regulatory B cells (B(regs)) in the intestine.