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Biomedical subjects

M S Abrahamsen

Publications and source records attributed to M S Abrahamsen.

At least 37 records · Page 2Linked to original sources
PubMed1998

Bovine T cell responses to Cryptosporidium parvum infection.

To better understand the immune mechanisms important for clearing of the primary infection and the subsequent development of resistance to Cryptosporidium parvum infection, several groups have recently characterised changes within the lymphoid cell population of the intestinal mucosa and associated lymphoid tissue in calves with cryptosporidiosis. In naive animals, infection results in a significant increase in the number of CD4+ and CD8+ T cells present within the intraepithelial lymphocyte population, lamina propria and Peyer's patch of the ileum. This is accompanied by a rapid and transient increase in the number of gamma/delta T cells present within the intestinal villi. In response to a challenge infection in immune calves, there is a substantial increase in the number of CD4+ T cells present in the Peyer's patch of the ileum and a specific localization of CD8+ T cells to the epithelium of the intestinal villi. Together, these data demonstrate that C, parvum elicits a strong cell-mediated response following both primary and secondary infections in calves, and that CD8+ T cells may play an important role in the bovine immune response to C. parvum infection.

Animals↗
PubMed1998

The biphasic mRNA expression pattern of bovine interleukin-8 in Pasteurella haemolytica lipopolysaccharide-stimulated alveolar macrophages is primarily due to tumor necrosis factor alpha.

Pasteurella haemolytica serotype 1 is the bacterial agent responsible for the pathophysiological events associated with bovine pneumonic pasteurellosis. Our previous studies support a role for the lipopolysaccharide (LPS) from P. haemolytica in the induction of proinflammatory cytokines. One of the pathological hallmarks of bovine pneumonic pasteurellosis is an influx of neutrophils into the alveolar spaces. This pronounced influx suggests the local production of a chemotactic factor(s) such as interleukin-8 (IL-8). In the context of the lung, the alveolar macrophage appears to be the major producer of IL-8, a proinflammatory cytokine with potent neutrophil chemotactic activity. By using Northern blot analysis, we have examined the kinetics of IL-8 mRNA expression in P. haemolytica LPS-stimulated bovine alveolar macrophages and found that 1 ng of LPS per ml induces maximal expression of IL-8 mRNA. The results also indicate a biphasic time course expression pattern in which IL-8 mRNA levels peak between 1 and 2 h in the first phase and between 16 and 24 h in the second phase (P < 0.01). In addition, monospecific polyclonal antibodies were used to demonstrate the role of tumor necrosis factor alpha (TNF-alpha) in the second phase of IL-8 mRNA expression. Our findings support a role for P. haemolytica LPS and TNF-alpha in the induction of IL-8 from bovine alveolar macrophages.

Animals↗
PubMed1997

Localization of alpha/beta and gamma/delta T lymphocytes in Cryptosporidium parvum-infected tissues in naive and immune calves.

The nature of the host's T-lymphocyte population within the intestinal villi following Cryptosporidium parvum infection was characterized with a bovine model of cryptosporidiosis. In naive animals, infection with C. parvum resulted in substantial increases in the numbers of alpha/beta T cells, both CD4+ (150%) and CD8+ (60%), and of gamma/delta T cells (70%) present within the intestinal villi of the infected ileum. In immune animals, the host T-lymphocyte response to a challenge infection with C. parvum was restricted to alpha/beta T cells. The number of CD4+ T cells within the Peyer's patch of the ileum increased dramatically; however, there was little change in the number or localization of CD4+ T cells within the intestinal villi. In contrast, the number of CD8+ T cells within the intestinal villi increased following a challenge infection. In addition, the CD8+ T cells were found to be intimately associated with the epithelial cells of the intestinal villi. The precise correlation between the accumulation of CD8+ T cells and the normal site of parasite development suggests an important role for CD8+ T cells in the immune animal.

Animals↗
PubMed1995

Expression of granulocyte colony stimulating factor (G-CSF) and granulocyte/macrophage colony stimulating factor (GM-CSF) mRNA upon stimulation with phorbol ester.

Stimulated human peripheral blood mononuclear cells (MNC) have been shown to express both G-CSF and GM-CSF, Furthermore, G-CSF is expressed by monocytes but not lymphocytes, whereas GM-CSF is expressed largely by T lymphocytes and at low levels in monocytes/macrophages, Here we present the effect of TPA (120-O-tetradecanoyl phorbol-13-acetate) on G-CSF and GM-CSF expression in stimulated human MNCs and T lymphocytes. We observed that TPA (30nM) decreased G-CSF mRNA levels in MNCs, while ionomycin increased G-CSF in a dose-dependent manner. TPA and ionomycin individually increased GM-CSF mRNA levels in T-lymphocytes and MNCs. Further, GM-CSF was induced synergistically by TPA plus ionomycin, whereas this combination markedly decreased G-CSF mRNA levels in MNCs. These data suggest at least two signaling pathway by which G-CSF and GM-CSF and GM-CSF mRNA levels are modulated in a mixed population of monocytes and T lymphocytes, namely protein kinase C (PKC) and calcium. These signals seems to act synergistically in lymphocytes to increase GM-CSF, and not G-CSF mRNA levels specifically. It would also appear these signals act on MNCs in an opposing manner to decrease G-CSF mRNA levels, indicating that activation of PKC and the calcium signaling pathway lead to a cell-type specific modulation of individual cytokines and precise regulation of granulocyte production.

Adult↗
PubMed1995

Identification of Eimeria bovis merozoite cDNAs using differential mRNA display.

Differences in gene expression between Eimeria bovis sporozoites and first-generation merozoites were analyzed using the technique of differential mRNA display. Approx. 5% of the sequences detected in first-generation merozoites appear to be unique relative to sporozoites. Several of the bands corresponding to merozoite-specific gene expression were isolated and cloned. Northern blot analysis revealed that the cDNA fragments DMZ-7, DMZ-8 and NMZ-6 hybridized to mRNAs expressed at > 50-fold higher levels in merozoites relative to sporozoites. A fourth cDNA fragment, NMZ-4, hybridized to a mRNA expressed at 3-fold higher levels in merozoites. Further characterization demonstrated that expression of DMZ-8 in E. bovis-infected bovine cells begins as early as 12 h after sporozoite invasion and continues throughout the entire 14 days of first-generation schizogony. Sequence analysis of each of the four merozoite cDNAs failed to identify any significant similarity to any entries in the GenBank database, suggesting that these developmentally regulated genes may be unique to coccidian parasites.

Animals↗
PubMed1995

An improved method for isolating RNA from coccidian oocysts.

A method, based on the disruption of eimerian oocysts in a French pressure cell in the presence of guanidine isothiocyanate, has been developed to isolate large quantities of high quality total RNA efficiently. This procedure results in a 12.5-fold greater number of oocysts broken, and a 22-fold greater yield of total RNA than from disruption by conventional grinding in liquid N2. In addition, the RNA isolated by the French pressure cell method was of equal or superior quality when compared to RNA isolated by grinding. This procedure provides a significant improvement in RNA extraction from eimerian oocysts and will greatly facilitate the study of gene expression in this important group of parasites.

Animals↗
PubMed1994

Complex interactions at a GC-rich domain regulate cell type-dependent activity of the ornithine decarboxylase promoter.

Regulation of ornithine decarboxylase (ODC) is critical to the control of cellular growth, differentiation, and carcinogenesis. A GC-rich region in the ODC promoter contains two overlapping protein binding sites that interact to regulate basal level expression in some cell types. A perfect binding motif for transcription factor Sp1 (CCCCGCCCC) is located at nucleotides -114 to -106 relative to the site of transcriptional initiation, binds strongly to purified Sp1 protein, and forms several complexes when incubated with nuclear extracts. Only one of these complexes is recognized by Sp1-specific antibody. A new protein-binding motif (GCCCCTCCCC, located at -110 to -100) partially overlaps with the Sp1 site and analyses by DNase I protection showed that a new protein ("NF-ODC1") and the Sp1-like proteins interact with the ODC promoter in a mutually exclusive manner. Mutation of the NF-ODC1 binding motif strongly enhanced ODC promoter strength in some cell types, but had little or no influence in others. The effect of mutating the Sp1 site also varied with cell type. These cell type specificities did not correlate with the levels of Sp1 and NF-ODC1 binding activities in nuclear extracts. These results show that regulation of the ODC promoter by the Sp1 family is cell type-specific and modulated by a negative effector that we have termed NF-ODC1.

Animals↗
PubMed1994

Developmental regulation of an Eimeria bovis mRNA encoding refractile body-associated proteins.

Eimeria bovis antigens defined by the monoclonal antibody (mAb) 2.4 are associated with the refractile bodies of sporozoites and are found in the parasitophorous vacuole and host cell cytoplasm during schizogony. Screening of an E. bovis oocyst cDNA library with mAb 2.4 resulted in the identification of a single unique cDNA sequence (Eb-25/50). Comparison of the predicted protein sequence of Eb-25/50 revealed a high degree of identity to an Eimeria tenella refractile body protein and mAb 2.4 was found to cross-react with refractile bodies from Eimeria acervulina, demonstrating that these proteins are highly conserved among eimerian species. Measurements of Eb-25/50 mRNA showed that the multiple proteins recognized by mAb 2.4 are encoded by a single mRNA species whose kinetics of expression during sporulation and schizogony closely correlated with protein expression. Consistent with multiple Eb-25/50 proteins arising from a single polypeptide, results from a Southern analysis of E. bovis genomic DNA indicated that Eb-25/50 mRNA is derived from a single copy gene. The presence of Eb-25/50 proteins in the host cytoplasm during schizogony, the high degree of conservation of these proteins, and the apparent complex post-translational modification raises interesting questions about the biochemistry of these proteins during eimerian development.

Amino Acid Sequence↗
PubMed1993

Developmental gene expression in Eimeria bovis.

By differential screening of stage-specific cDNA libraries of Eimeria bovis, we have identified and isolated a large set of genes that are regulated during development of the sporozoites and merozoites. Duplicate lifts of cDNA libraries constructed from partially sporulated oocysts and merozoites were probed with radioactively labeled first-strand cDNA prepared from partially sporulated oocyst and merozoite mRNA. Out of 60,000 plaques screened in each case, over 250 plaques from the partially sporulated oocyst library preferentially hybridized with the oocyst cDNA probe and 67 plaques from the merozoite library preferentially hybridized with the merozoite cDNA probe. Three of the oocyst phage and 7 of the merozoite phage were selected for further characterization. Northern analysis revealed a common pattern of mRNA expression for the oocyst cDNA clones. Consistent with the results of the differential screen, no hybridization to merozoite RNA was detected with any of these 3 oocyst cDNA clones. The expression of the merozoite cDNA clones was more complex, with 3 different classes of merozoite genes being identified based on their pattern of developmental regulation. Although each of the merozoite clones was expressed to some extent during sporulation, in all cases, expression was higher in merozoites than in partially sporulated oocysts, consistent with the restriction of expression defined by the differential screen. Sequence analysis revealed that 2 of the merozoite cDNA clones encode elongation factor 1 alpha and the ubiquitin/ribosomal protein fusion, and 1 of the sporozoite cDNAs displays a significant identity to insulin-degrading enzyme. The developmental expression of E. bovis genes involved in protein synthesis and degradation provides additional evidence for the importance of regulation of protein metabolism during parasite development.

Amino Acid Sequence↗
PubMed1992

Multiple DNA elements responsible for transcriptional regulation of the ornithine decarboxylase gene by protein kinase A.

Transcription of the ornithine decarboxylase (ODC) gene is rapidly elevated by activation of protein kinase A (PKA). The additive influence of three cis-acting elements is responsible for this regulation in an adrenal carcinoma cell line. Two sites, CRE2 at -48 base pairs (bp) relative to the start of transcription and CRE3 at +95 bp, are identical to the core motif of the cAMP-responsive element (CRE) of the somatostatin gene and are conserved in the mouse, rat, and human ODC genes. Mutation of CRE2 resulted in a substantial decrease in basal promoter activity, as well as a 5-fold decrease in inducibility of the ODC promoter by PKA. CRE3 did not contribute to the basal activity of the ODC promoter, but mutation of this site resulted in a 2-fold decrease in inducibility by PKA. Deletion of a 45-bp sequence (GC-box) located 5' of CRE2, also resulted in a 2-fold decrease in inducibility of the ODC promoter. DNase I protection revealed the presence of protein binding at CRE2, the TATA box, and the GC-box of the ODC promoter. Mutation of CRE2 resulted in loss of protection of this sequence, as well as the 3' extension of the footprint over the TATA box, without affecting interactions at the GC box. Antibodies to the well characterized CRE-binding protein CREB recognized proteins binding to CRE2, suggesting that binding of CREB, or an antigenically related protein, is important for the activity of CRE2. Additionally, recombinant CREB bound to a DNA probe containing the CRE2 sequence.

Adrenal Gland Neoplasms↗
PubMed1992

Cyclic AMP-dependent protein kinase controls basal gene activity and steroidogenesis in Y1 adrenal tumor cells.

Transfection of mouse Y1 adrenal tumor cells with DNA encoding mutant type I regulatory subunit generated stable transformants in which the basal activity of cAMP-dependent protein kinase was repressed. As expected, steroidogenesis in these kinase-deficient cells was no longer stimulated by corticotropin or cAMP analogues, and the expression of three cAMP-regulated genes (ornithine decarboxylase, urokinase-type plasminogen activator, and P450 side-chain cleavage) could no longer be induced. However, in addition to the loss of hormone responsiveness, the basal level of steroidogenesis and the constitutive expression of these cAMP-inducible genes was also repressed in kinase-defective mutant clones. To verify that functional cA-PK would revert this repressed phenotype, we transfected a cA-PK defective subclone of Y1 cells, Kin 8, with DNA encoding the C alpha and C beta subunits of cAMP-dependent protein kinase. Basal levels of steroid production were restored to normal in stable transformants, and the elevation of kinase activity following induction of the C-subunit expression vectors elicited a steroidogenic response. Gene transcription was also shown to be regulated by either C alpha or C beta as measured by the induction of plasminogen activator and ornithine decarboxylase mRNA levels and transcription rates. The dominant role played by cAMP-dependent protein kinase in these adrenal cells was demonstrated by experiments showing the regulation of ornithine decarboxylase gene expression by protein kinase C requires basal cAMP-dependent protein kinase activity.

Adrenal Cortex↗
PubMed1992

A nucleic acid-based test for detection of Fasciola hepatica.

The use of nucleic acid techniques in the diagnosis of parasitic infection has become increasingly widespread. An oligonucleotide probe derived from a rRNA sequence was developed for the detection of Fasciola hepatica in its intermediate snail host Pseudosuccinea columella. Total RNA obtained from whole adult liver flukes was used in a polymerase chain reaction to isolate and amplify a region of approximately 650 base pairs in the small subunit rRNA. This portion of the ribosomal cDNA, which contains highly conserved regions as well as variable regions, was subcloned and sequenced. In comparison to known small subunit rRNA sequences, a sequence unique to F. hepatica was identified and an oligonucleotide probe (CS4) for detection of F. hepatica was developed. A northern blot analysis using CS4 successfully identified small subunit rRNA from F. hepatica. Slot-blot analysis determined that RNA derived from 5 miracidia can be detected with CS4. Moreover, a slot blot utilizing CS4 distinguished RNA derived from snails infected with F. hepatica from RNA of uninfected snails.

Animals↗
PubMed1990

Cell type-specific mechanisms of regulating expression of the ornithine decarboxylase gene after growth stimulation.

Ornithine decarboxylase (ODC) mRNA is strongly induced by mitogenic activation of resting Swiss 3T3 fibroblasts and T lymphocytes. Nuclear run-on analysis revealed a low level of nascent transcripts in resting fibroblasts that was elevated upon activation. In contrast, there was a high level of transcription across the entire ODC gene in resting T cells, which remained unchanged upon activation. The stability of the mature ODC message was found to be unaffected by mitogenic stimulation. These results indicate that ODC mRNA levels are regulated transcriptionally in Swiss 3T3 cells and posttranscriptionally within the nucleus of T lymphocytes in response to mitogenic stimuli. In this unique situation, the mitogenic induction of a single gene, ODC, is regulated by two very distinct, cell-specific mechanisms.

Animals↗