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Paul M Coussens

Publications and source records attributed to Paul M Coussens.

At least 19 recordsLinked to original sources

Comparison of gene expression by co-cultured WC1+ gammadelta and CD4+ alphabeta T cells exhibiting a recall response to bacterial antigen.

Immunization of cattle with a Leptospira borgpetersenii serovar hardjo-bovis vaccine results in the development of a recall response by WC1(+) gammadelta T cells and CD4(+) alphabeta T cells characterized by proliferation and interferon-gamma production. It was hypothesized that these two T cell subpopulations had largely redundant effector functions, principally differing in their requirements for activation. To test this, gene expression in cells proliferating to antigen were compared utilizing RT-PCR and bovine microarrays. Both T cell populations had similar transcript profiles for effector molecules, including IFN-gamma, FasL and granzyme B. In contrast, transcripts for costimulatory receptors and ligands were notably different following activation, as WC1(+) T cells expressed no or lower levels of transcripts for CD28 and CD40L, while CD4(+) T cells expressed substantial levels of both. However, both cell types had high levels of CTLA-4 transcript suggesting the cells may be regulated similarly following activation but differ in their need for and ability to provide costimulation. Microarray analyses to extend the number of genes examined revealed that while both subpopulations upregulated anti-apoptotic genes as well as those involved in cell activation and protein biosynthesis, overall there were limited differences between the two antigen-activated cell populations. Those genes that did differ were involved in cell signaling, protein production and intracellular protein trafficking. These results strengthen the hypothesis that these particular activated WC1(+) and CD4(+) T cells have overlapping effector functions and therefore may differ principally with regard to how they are recruited into immune responses.

Animals↗

Gene expression profiling of monocyte-derived macrophages following infection with Mycobacterium avium subspecies avium and Mycobacterium avium subspecies paratuberculosis.

Mycobacterium avium subspecies paratuberculosis (MAP) and Mycobacterium avium subspecies avium (MAA) represent two closely related intracellular bacteria with vastly different associated pathologies. MAA can cause severe respiratory infections in immune compromised humans but is nonpathogenic in ruminants and is more readily controlled by the bovine immune system than MAP. MAP causes a fatal wasting syndrome in ruminants, typified by granulomatous enteritis localized in the small intestine. MAP has also been cited as a potential cause of human Crohn's disease. We used a bovine immune-specific microarray (BOTL-5) to compare the response of mature bovine monocyte-derived macrophages (MDM cells) to MAP and MAA. Statistical analysis of microarray data revealed 21 genes not appreciably expressed in resting MDM cells that were activated following infection with either MAA or MAP. Further analysis revealed 144 genes differentially expressed in MDM cells following infection with MAA and 99 genes differentially expressed following infection with MAP. Of these genes, 37 were affected by both types of mycobacteria, with three being affected in opposite directions. Over 41% of the differentially expressed genes in MAA and MAP infected MDM cells were members of, regulated by, or regulators of the MAPK pathways. Expression of selected genes was validated by quantitative real-time reverse transcriptase PCR and in several key genes (i.e., IL-2 receptor, tissue inhibitor of matrix metalloproteinases-1, and Fas-ligand) MAA was found to be a stronger activating factor than MAP. These gene expression patterns were correlated with prolonged activation of p38 MAPK and ERK1/2 by MAA, relative to MAP.

Animals↗

Gene expression profiling of peripheral blood mononuclear cells (PBMC) from Mycobacterium bovis infected cattle after in vitro antigenic stimulation with purified protein derivative of tuberculin (PPD).

Microarray analysis of messenger RNA (mRNA) abundance was used to investigate the gene expression program of peripheral blood mononuclear cells (PBMC) from cattle infected with Mycobacterium bovis, the causative agent of bovine tuberculosis. An immunospecific bovine microarray platform (BOTL-4) with spot features representing 1336 genes was used for transcriptional profiling of PBMC from six M. bovis-infected cattle stimulated in vitro with bovine purified protein derivative of tuberculin (PPD-bovine). Cells were harvested at four time points (3 h, 6 h, 12 h and 24 h post-stimulation) and a split-plot design with pooled samples was used for the microarray experiment to compare gene expression between PPD-bovine stimulated PBMC and unstimulated controls for each time point. Statistical analyses of these data revealed 224 genes (approximately 17% of transcripts on the array) differentially expressed between stimulated and unstimulated PBMC across the 24 h time course (P<0.05). Of the 224 genes, 87 genes were significantly upregulated and 137 genes were significantly downregulated in M. bovis-infected PBMC stimulated with PPD-bovine across the 24 h time course. However, perturbation of the PBMC transcriptome was most apparent at time points 3 h and 12 h post-stimulation, with 81 and 84 genes differentially expressed, respectively. In addition, a more stringent statistical threshold (P<0.01) revealed 35 genes (approximately 3%) that were differentially expressed across the time course. Real-time quantitative reverse transcription PCR (qRT-PCR) of selected genes validated the microarray results and demonstrated a wide range of differentially expressed genes in PPD-bovine-, PPD-avian- and Concanavalin A (ConA) stimulated PBMC, including the interferon-gamma gene (IFNG), which was upregulated in PBMC stimulated with PPD-bovine (40-fold), PPD-avian (10-fold) and ConA (8-fold) after in vitro culture for 12 h. The pattern of expression of these genes in PPD-bovine stimulated PBMC provides the first description of an M. bovis-specific signature of infection that may provide insights into the molecular basis of the host response to infection. Although the present study was carried out with mixed PBMC cell populations, it will guide future studies to dissect immune cell-specific gene expression patterns in response to M. bovis infection.

Animals↗

Differential expression of genes encoding CD30L and P-selectin in cattle with Johne's disease: progress toward a diagnostic gene expression signature.

Mycobacterium avium subspecies paratuberculosis (Mycobacterium paratuberculosis), the causative agent of paratuberculosis (paraTB) or Johne's disease in ruminants, is a health problem for the global cattle industry with significant economic losses related to decreased milk production and reduced fertility. Commonly paraTB in cattle is diagnosed by antibody detection by serum enzyme-linked immunosorbent assay (ELISA), by detection of the pathogen by cultivation of individual faecal samples, or by in vitro measurement of cell mediated immune responses using the IFN-gamma test. There is an ongoing need for developing new diagnostic approaches as all currently available diagnostic tests for paraTB may fail to detect sub-clinical infection. We used cDNA microarrays to simultaneously measure expression of over 1300 host genes to help identify a subset of gene expression changes that might provide a unique gene expression signature for paraTB infection. In the present study, non-stimulated leukocytes isolated from 10 sub-clinical paraTB infected cows were examined for genes being expressed at significantly different levels than in similar cells from control cows with the same herd background. We included cattle (Holstein) from two locations (Denmark and USA) for the microarray experiment. Our results indicate that expression profiles of at least 52 genes are different in leukocytes from M. paratuberculosis infected cattle compared to control cattle. Gene expression differences were verified by quantitative real-time reverse transcriptase polymerase chain reactions (qRT-PCR) on the same group of cattle (Holstein) used for the microarray experiment. In order to assess the generality of the observed gene expression, a second and different group of cattle (Jersey) was also examined using qRT-PCR. Out of the seven genes selected for qRT-PCR, CD30 ligand (CD30L) and P-selectin were consistently differentially expressed in freshly isolated leukocytes from paraTB infected and control animals of both breeds of cattle. Although further work is clearly needed to develop a more complete gene expression signature specific for paraTB, our results demonstrate that a subset of genes in leukocytes are consistently expressed at different levels, depending upon M. paratuberculosis infection status.

Animals↗

Understanding bovine trypanosomiasis and trypanotolerance: the promise of functional genomics.

African bovine trypanosomiasis, caused by the protozoan parasite Trypanosoma congolense, is endemic throughout sub-Saharan Africa and is a major constraint on livestock production. A promising approach to disease control is to understand and exploit naturally evolved trypanotolerance. We describe the first attempt to investigate the transcriptional response of susceptible Boran (Bos indicus) cattle to trypanosome infection via a functional genomics approach using a bovine total leukocyte (BOTL) cDNA microarray platform. Four male Boran cattle were experimentally infected with T. congolense and peripheral blood mononuclear cells (PBMC) were collected before infection and 13, 17, 23 and 30 days post-infection (dpi). A reference experimental design was employed using a universal bovine reference RNA pool. Data were normalised to the median of a set of invariant genes (GAPDH) and BRB-Array tools was used to search for statistically significant differentially expressed genes between each time-point. Using a set of 20 microarray hybridisations, we have made a significant contribution to understand the temporal transcriptional response of bovine PBMC in vivo to a controlled trypanosome infection. The greatest changes were evident 13 dpi after parasites were first detected in the blood. Significant differences were observed in clusters of protein kinase C subunits and MHC class I/II related molecules.

Animals↗

Johne's disease in cattle is associated with enhanced expression of genes encoding IL-5, GATA-3, tissue inhibitors of matrix metalloproteinases 1 and 2, and factors promoting apoptosis in peripheral blood mononuclear cells.

Infection of ruminants with Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) leads to a chronic and often fatal granulomatous enteritis known as Johne's disease. Most infections with M. paratuberculosis occur during the first 6 months of life, and there is some evidence for transmission in utero. Once established, infections typically exist in a subclinical state for several years. Recent gene-expression profiling studies suggested the hypothesis that inherent gene-expression profiles in peripheral blood mononuclear cells (PBMCs) from M. paratuberculosis-infected cattle may be different than expression profiles in PBMCs from uninfected controls. If true, this would suggest that it is possible to identify an M. paratuberculosis infection "signature" through transcriptional profiling of peripheral immune cells. In addition, identification of groups or classes of genes showing inherently different expression in PBMCs from M. paratuberculosis-infected cattle relative to PBMCs from uninfected controls might highlight important interactions between this pathogen and the host immune system. In this report, we describe studies aimed at testing this hypothesis. Our novel results indicate that, indeed expression profiles of at least 42 genes are inherently different in freshly isolated PBMCs from M. paratuberculosis-infected cattle when compared to similar cells from uninfected controls. Gene-expression differences observed following microarray analysis were verified and expanded upon by quantitative real-time PCR (Q-RT-PCR). Our results indicate that T cells within PBMCs from M. paratuberculosis-infected cows have adopted a predominant Th 2-like phenotype (enhanced expression of IL-5, GATA 3, and possibly IL-4 mRNA), that cells within infected cow PBMCs may exhibit tissue remodeling deficiencies through higher expression of tissue inhibitor of matrix metalloproteinase (TIMP) 1 and TIMP2 RNA and lower expression of matrix metalloproteinase (MMP) 14 RNA than similar cells from healthy controls, and that cells within the PBMC population of M. paratuberculosis-infected cows are likely poised for rapid apoptosis (upregulation of CIDE-A, Bad, TNFRI, and Fas).

Animals↗

Validation and application of a high fidelity mRNA linear amplification procedure for profiling gene expression.

The need for microgram quantities of RNA for microarray experiments has hindered application of this novel technology in cell types/tissue samples with limited abundance of RNA. In this study, potential application of T7-based linear RNA amplification was investigated for use in gene expression profiling experiments where starting material is limited. Yield and integrity of amplified antisense RNA (aaRNA), microarray hybridization intensities, and fidelity of differential gene expression detected were determined for arrays generated for unamplified versus amplified RNA from the same homogenous starting pools. Total RNA was extracted from bovine spleen and fetal ovary, serially diluted to concentrations ranging from 2 microg to 500 pg and amplified. Quality and quantity of total input RNA and aaRNA were assessed by spectrophotometry, gel electrophoresis and bioanalyzer. In experiment 1, we determined the optimal amounts of aaRNA generated from 20, 40, 200 ng and 2 microg input total RNA for use in cDNA synthesis, labeling and array hybridization that would yield robust and consistent hybridization signals on a bovine oocyte cDNA microarray. In experiment 2, comparison of microarray hybridization intensities and fidelity of differential gene expression between aaRNA generated from 2, 20 and 40 ng input total RNA versus unamplified RNA (uRNA) were conducted. The hybridization intensities for each of the 7000 spots per slide for microarrays conducted using aaRNA versus uRNA were highly correlated (2 ng = 0.84, 20 ng = 0.88, 40 ng = 0.90; P < 0.01). The false positive rate was low and similar (4.0% versus 4.4%) for arrays done with uRNA and aaRNA. Ninety-seven ESTs were detected as differentially expressed in the fetal ovary versus spleen at > 1.5- or < 0.5-fold using uRNA (P < 0.05). However, the number of genes detected in arrays using aaRNA was approximately 1.5-2.5 times greater than with uRNA. Approximately, 65-70% of differentially expressed genes were common between uRNA and aaRNA arrays. Relative fold-expression (Cy3/Cy5 ratios) for 25 overlapping abundant genes was comparable for uRNA versus aaRNA arrays with 2 and 20 ng total RNA as input. Results demonstrate that T7-based linear amplification of small amounts of input RNA and use of aaRNA in microarray experiments retains fidelity of detection of differential gene expression that is relatively comparable to experiments done with uRNA and provides a potentially viable approach to facilitate gene expression profiling using limited amounts of starting material.

Animals↗

Using human microarrays to identify differentially expressed genes associated with increased steroidogenesis in boars.

Human microarrays are readily available, and it would be advantageous if they could be used to study gene expression in other species, such as pigs. The objectives of this research were to validate the use of human microarrays in the analysis of porcine gene expression, to assess the variability of the data generated, and to compare gene expression in boars with different levels of steroidogenesis. Cytochrome b5 (CYB5) expression was used to assess array detection sensitivity. Samples having high or low CYB5 RNA levels were hybridized to microarrays to determine if the known expression difference could be detected. Six hybridizations were conducted using human microarrays containing 3840 total spots representing 1718 characterized human ESTs. To analyze gene expression in boars with different levels of steroidogenesis, testis RNA from four boars with high levels of plasma estrone sulphate was hybridized to testis RNA from four boars with lower levels. Eight microarray hybridizations were conducted including fluor-flips. Self-self hybridizations were also conducted to assess the variability of array experiments. The Cy5 and Cy3 intensity values for each array were normalized using a locally weighted linear regression (LOESS). Statistical significance was assessed using a Student's t-test followed by the Benjamini and Hochberg multiple testing correction procedure. Quantitative real-time PCR (Q-RT-PCR) was used to verify select gene expression differences. The results show that CYB5 was significantly overexpressed in the high CYB5 sample by 1.8 fold (P < 0.05), verifying the known expression difference. The average log2 ratio of the majority of genes (1643) falls within one standard deviation of the mean, indicating the data were reproducible. In the high versus low steroidogenesis experiment, seven genes were significantly overexpressed in the high group (P < 0.05). Quantitative real-time PCR was used to validate five genes with the highest fold change, and the results corroborated those found by the microarray experiments. The results of the self-self hybridizations showed that no genes were significantly differentially expressed following the application of the Benjamini and Hochberg multiple testing correction procedure. The results presented in this report show that human arrays can be used for gene expression analysis in pigs.

Animals↗

Effects of glucosamine and chondroitin sulfate on mediators of osteoarthritis in cultured equine chondrocytes stimulated by use of recombinant equine interleukin-1beta.

OBJECTIVE: To determine whether glucosamine and chondroitin sulfate (CS) at concentrations approximating those achieved in plasma by oral administration would influence gene expression of selected mediators of osteoarthritis in cytokine-stimulated equine articular chondrocytes. SAMPLE POPULATION: Samples of grossly normal articular cartilage obtained from the metacarpophalangeal joint of 13 horses. PROCEDURE: Equine chondrocytes in pellet culture were stimulated with a subsaturating dose of recombinant equine interleukin (reIL)-1beta. Effects of prior incubation with glucosamine (2.5 to 10.0 microg/mL) and CS (5.0 to 50.0 microg/mL) on gene expression of matrix metalloproteinase (MMP)-1, -2, -3, -9, and -13; aggrecanase 1 and 2; inducible nitric oxide synthase (iNOS); cyclooxygenase (COX)-2; nuclear factor kappaB; and c-Jun-N-terminal kinase (JNK) were assessed by use of a quantitative real-time polymerase chain reaction assay. RESULTS: Glucosamine at a concentration of 10 microg/mL significantly reduced reIL-1beta-induced mRNA expression of MMP-13, aggrecanase 1, and JNK. Reductions in cytokine-induced expression were also observed for iNOS and COX-2. Chondroitin sulfate had no effect on gene expression at the concentrations tested. CONCLUSIONS AND CLINICAL RELEVANCE: Concentrations of glucosamine similar to those achieved in plasma after oral administration in horses exerted pretranslational regulation of some mediators of osteoarthritis, an effect that may contribute to the cartilage-sparing properties of this aminomonosaccharide. Analysis of results of this study indicated that the influence of CS on pretranslational regulation of these selected genes is limited or lacking.

ADAM Proteins↗

Generation of a bovine oocyte cDNA library and microarray: resources for identification of genes important for follicular development and early embryogenesis.

The oocyte is a key regulator of ovarian folliculogenesis and early embryonic development. However, the composition of the oocyte transcriptome and identities and functions of key oocyte-specific genes involved in the above processes are relatively unknown. Using a PCR-based cDNA amplification method (SMART technology), we constructed a bovine oocyte cDNA library. Analysis of 230 expressed sequence tags (ESTs) from this library identified 102 unique sequences. Although some correspond to housekeeping genes (e.g., ribosomal protein L15) and some represent genes previously known to be expressed in oocytes and other tissues, most encode for genes whose expression in mammalian oocytes has not been reported previously (e.g., cocaine- and amphetamine-regulated transcript) or genes of unknown function. Sixteen did not show significant sequence similarity to any entries in the GenBank database and were classified as novel. Using over 2,000 unsequenced, randomly selected cDNA clones from the library, we constructed an oocyte microarray and performed experiments to identify genes preferentially expressed in fetal ovary (an enriched source of oocytes) relative to somatic tissues. Eleven clones were identified by microarray analysis with consistently higher expression in fetal ovaries (collected from animals at days 210-260 of gestation) compared with spleen and liver. DNA sequence analysis of these clones revealed that two correspond to JY-1, a novel bovine oocyte-specific gene. The remaining nine clones represent five identified genes and one additional completely novel gene. Increased abundance of mRNA in fetal ovary for five of the six genes identified was confirmed by real-time PCR. Results demonstrate the potential utility of these unique resources for identification of oocyte-expressed genes potentially important for reproductive function.

Animals↗

Evidence that cocaine- and amphetamine-regulated transcript is a novel intraovarian regulator of follicular atresia.

We recently obtained evidence that cocaine- and amphetamine-regulated transcript (CART), a potent anorectic neuropeptide, is expressed in the bovine ovary. The objectives of this study were to characterize bovine ovarian CART and determine its localization, regulation, and regulatory role during follicular development. CART mRNA was detected in stroma of adult ovaries and in large follicles, but was undetectable in several peripheral tissues, fetal ovaries, and corpora lutea. Within the ovary, CART mRNA and peptide were localized to the granulosal layer of some, but not all, antral follicles, with low, but detectable, expression in oocytes and cumulus cells. CART mRNA was undetectable in granulosal cells of dominant ovulatory follicles collected before and after the preovulatory gonadotropin surge, but was detected in the granulosal layer of adjacent subordinate follicles. In addition, amounts of CART mRNA and follicular fluid concentrations of CART peptide were greater in subordinate follicles vs. dominant follicles of the first follicular wave. Furthermore, CART treatment inhibited basal estradiol production, but not progesterone production, by granulosal cells in a dose-dependent fashion, and the effect was dependent on stage of cell differentiation. We conclude that granulosal cell CART expression is temporally regulated and potentially associated with follicle health status, and CART can inhibit granulosal cell estradiol production. Thus, CART may be a novel local regulator of follicular atresia in the bovine ovary.

Amino Acid Sequence↗

Microarray analysis of gene expression in blood neutrophils of parturient cows.

It is well documented that blood neutrophils from parturient dairy cows do not perform as well as neutrophils from nonparturient cows in laboratory assays of adhesion, migration, or phagocytosis-induced respiratory burst. However, little is known about the possible molecular basis for parturition-induced changes in neutrophils. cDNA microarray analysis was used in the current study to explore parturition-induced changes in gene expression profiles in bovine blood neutrophils. Total RNA from isolated blood neutrophils of four parturient Holstein cows was obtained before, during, and after parturition, reverse transcribed into cDNA, and sequentially labeled with Cy3 or Cy5 dyes prior to paired hybridizations to 1,056 member bovine total leukocyte (BOTL-3) microarrays in a loop design. Resulting gene expression data were LOWESS normalized by array and analyzed using a mixed model approach. Results showed that expression profiles for 302 BOTL-3 genes were influenced by parturition. BLASTn analysis and preliminary clustering of affected genes by biological function indicated that the largest proportion (14%) of changed genes encode proteins critical to regulation of apoptosis. Independent confirmation of altered expression for 16 of these genes was achieved using quantitative real-time RT-PCR (Q-RT-PCR). A predominantly survival phenotype inferred from the microarray and Q-RT-PCR results was substantiated by monitoring apoptosis status of blood neutrophils from castrated male cattle cultured in the presence of sera from parturient cows. Thus our combined gene expression and apoptosis phenotyping results suggest that bovine parturition may induce prolonged survival in normally short-lived blood neutrophils.

Animals↗

Rapid and transient activation of gene expression in peripheral blood mononuclear cells from Johne's disease positive cows exposed to Mycobacterium paratuberculosis in vitro.

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the causative agent of Johne's disease in ruminants. M. paratuberculosis is a slow-growing intracellular bacterium and infections with M. paratuberculosis can persist in a subclinical state for several years. An early and appropriate T cell-mediated cytotoxic response (Th1-like) to M. paratuberculosis infection is often replaced with an antibody or Th 2-like response as infected animals move toward a progressively more clinical state. The reasons for this shift in immune response are unknown. Recent studies suggest that in vitro exposure of peripheral blood mononuclear cells (PBMCs) from Johne's disease positive cows to M. paratuberculosis for 18-24 h results in suppressed expression of numerous immune cell genes. This effect appears at odds with the notion that immune cells from infected cows would respond to M. paratuberculosis-specific antigens in a vigorous and positive manner. In this report, we detail experiments designed to test the hypothesis that many positive changes in PBMC gene expression induced by M. paratuberculosis in vitro are transient, being rapidly suppressed by as yet unknown mechanisms. Our results demonstrate that, indeed, in vitro stimulation with M. paratuberculosis induces rapid changes in infected cow PBMC gene expression (within 2-4 h of exposure) and that many of these changes are no longer evident by 8-16 h of exposure to M. paratuberculosis. Although precise mechanisms responsible for rapid M. paratuberculosis-mediated activation of PBMC gene expression and the loss thereof remain to be determined, our novel results suggest that PBMCs from Johne's disease positive cows are indeed capable of vigorously responding to M. paratuberculosis and that, for many genes, this response is tempered within 8 h of exposure.

Animals↗

Structure of the bovine natural resistance associated macrophage protein (NRAMP 1) gene and identification of a novel polymorphism.

The NRAMP 1 gene is a major candidate gene influencing the outcome of infections with intracellular pathogens in numerous species. NRAMP 1 is highly conserved in many mammalian species and the NRAMP 1 gene shows considerable conservation in structure between mice and humans. The association of NRAMP 1 gene polymorphisms with disease in cattle has been limited to a single microsatellite located within the 3'-non coding region of the bovine NRAMP 1 gene. In order to facilitate further studies on this important gene, we now report the nearly complete structure of the bovine NRAMP 1 gene, including sizes and positions of 13 introns relative to the bovine NRAMP 1 gene coding sequence and the DNA sequence of intron-exon junctions. Comparison of the bovine, murine and human NRAMP 1 gene structures revealed a high degree of conservation in intron placement, though the lengths of several introns were less-well conserved. In general, the greatest divergence in intron lengths occurred in regions of the NRAMP 1 gene displaying the lowest coding sequence conservation. In addition, mutations near intron-exon junctions could account for 25 of the 75 total amino acid differences between murine and bovine NRAMP 1. Using information gained through this study, it was possible to rapidly identify a novel polymorphism within the bovine NRAMP 1 gene intron X. This polymorphism was shown by direct DNA sequence analysis to consist of insertion of three guanine nucleotides at positions 37,40 and 98 relative to the intron X start point. Initial scans of several cattle breeds suggest that the two intron X alleles identified here are stable and widespread in the Bos taurus population.

Animals↗

Cytokine gene expression in peripheral blood mononuclear cells and tissues of cattle infected with Mycobacterium avium subsp. paratuberculosis: evidence for an inherent proinflammatory gene expression pattern.

In cattle and other ruminants, infection with the intracellular pathogen Mycobacterium avium subsp. paratuberculosis results in a granulomatous enteritis (Johne's disease) that is often fatal. The key features of host immunity to M. avium subsp. paratuberculosis infection include an appropriate early proinflammatory and cytotoxic response (Th1-like) that eventually gives way to a predominant antibody-based response (Th2-like). Clinical disease symptoms often appear subsequent to waning of the Th1-like immune response. Understanding why this shift in the immune response occurs and the underlying molecular mechanisms involved is critical to future control measures and diagnosis. Previous studies have suggested that M. avium subsp. paratuberculosis may suppress gene expression in peripheral blood mononuclear cells (PBMCs) from infected cows, despite a continued inflammatory reaction at sites of infection. In the present study, we tested the hypothesis that exposure to M. avium subsp. paratuberculosis suppresses a proinflammatory gene expression pattern in PBMCs from infected cows. To do this, we examined expression of genes encoding interleukin-1alpha (IL-1alpha), IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p35, IL-16, and IL-18, as well as genes encoding gamma interferon (IFN-gamma), transforming growth factor beta (TGF-beta), and tumor necrosis factor alpha (TNF-alpha), in PBMCs, intestinal lesions, and mesenteric lymph nodes of cattle naturally infected with M. avium subsp. paratuberculosis. Cytokine gene expression in these cells and tissues was compared to expression in similar cells and tissues from control uninfected cattle. Our comprehensive results demonstrate that for most cytokine genes, including the genes encoding IFN-gamma, TGF-beta, TNF-alpha, IL-1alpha, IL-4, IL-6, IL-8, and IL-12p35, differential expression in PBMCs from infected and control cattle did not require stimulation with M. avium subsp. paratuberculosis. In fact, stimulation with M. avium subsp. paratuberculosis tended to reduce the differential expression observed in infected and uninfected cows for genes encoding IFN-gamma, IL-1alpha, and IL-6. Only IL-10 gene expression was consistently enhanced by M. avium subsp. paratuberculosis stimulation of PBMCs from subclinically infected cattle. In ileal tissues from M. avium subsp. paratuberculosis-infected cattle, expression of the genes encoding IFN-gamma, TGF-beta, IL-5, and IL-8 was greater than the expression in comparable tissues from control uninfected cattle, while expression of the gene encoding IL-16 was lower in tissues from infected cattle than in control tissues. Mesenteric lymph nodes draining sites of M. avium subsp. paratuberculosis infection expressed higher levels of IL-1alpha, IL-8, IL-2, and IL-10 mRNA than similar tissues from control uninfected cattle expressed. In contrast, the genes encoding TGF-beta and IL-16 were expressed at lower levels in lymph nodes from infected cattle than in tissues from uninfected cattle. Taken together, our results suggest that cells or other mechanisms capable of limiting proinflammatory responses to M. avium subsp. paratuberculosis develop in infected cattle and that a likely place for development and expansion of these cell populations is the mesenteric lymph nodes draining sites of infection.

Animals↗

Bovine mammary gene expression profiling using a cDNA microarray enhanced for mammary-specific transcripts.

A cDNA microarray resource enhanced for transcripts specific to the bovine mammary gland (BMAM) has been developed and used in pilot studies to examine gene expression profiles in the mammary gland. One goal driving development of this resource was to shed some light on the pathways and mechanisms specifically related to bovine mammary gland growth and development. To accomplish this, gene expression patterns from bovine adipose, liver, adrenal, lymph, spleen, thymus, gut, and developing mammary tissue were compared using the BMAM microarray. We have thus identified a putative set of 16 genes being preferentially expressed in developing mammary gland. Another of our long-term goals is to elucidate the genes and pathways associated with bovine lactation and involution and to use these as a model for human mammary gland development as it relates to human breast cancer risks. To begin this process, we conducted a pilot study, comparing gene expression profiles of lactating bovine mammary tissue against nonlactating tissue on the BMAM microarray. Our results have yielded many novel and interesting genes exhibiting differential expression in lactating mammary tissue, including oncogenes (VAV3, C-myc), mediators of apoptosis (Caspase 8), and cell cycle regulators (LASP1).

Animals↗

Development of a porcine brain cDNA library, EST database, and microarray resource.

Recent developments in expressed sequence tag (EST) and cDNA microarray technology have had a dramatic impact on the ability of scientists to study responses of thousands of genes to internal and external stimuli. In neurobiology, studies of the human brain have been expanding rapidly by use of functional genomics techniques. To enhance these studies and allow use of a porcine brain model, a normalized porcine brain cDNA library (PBL) has been generated and used as a base for EST discovery and microarray generation. In this report, we discuss initial sequence analysis of 965 clones from this resource. Our data revealed that library normalization successfully reduced the number of clones representing highly abundant cDNA species and overall clone redundancy. Cluster analysis revealed over 800 unique cDNA species representing a redundancy rate for the normalized library of 6.9% compared with 29.4% before normalization. Sequence information, BLAST results, and TIGR cluster matches for these ESTs are publicly available via a web-accessible database (http://nbfgc.msu.edu). A cDNA microarray was created using 877 unique porcine brain EST amplicons spotted in triplicate on glass slides. This microarray was assessed by performing a series of experiments designed to test hybridization efficiency and false-positive rate. Our results indicate that the PBL cDNA microarray is a robust tool for studies of brain gene expression using swine as a model system.

Animals↗