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Biomedical subjects

M P Murtaugh

Publications and source records attributed to M P Murtaugh.

At least 37 records · Page 2Linked to original sources
PubMed1999

Mucosal immunogenicity and adjuvanticity of cholera toxin in swine.

The oral immunogenic and adjuvant properties of cholera toxin (CT) and its nontoxic B subunit (CT-B) were assessed in swine. Both whole CT and CT-B are oral immunogens in swine and CT is relatively more potent. Oral administration of 100 microg of CT resulted in a greater immune response than 1 mg of CT-B as measured by anti-CT-B IgA, IgG and IgM in local (jejunum) and distant (oral cavity) mucosal sites, and in systemic sites including blood and spleen. Lower doses of CT were potent adjuvants for the response to CT-B, but did not induce detectable immunity alone. The predominant response to oral CT-B administered with CT was intestinally produced and secreted IgA, with about 2500 per 10(6) jejunal lamina propria cells producing anti-CT-B IgA in immunized animals. While CT is a potent adjuvant for CT-B, its ability to act as adjuvant for heterologous proteins is more restricted. 50 microg of CT in combination with 1 mg of CT-B did not induce antibodies to 25 mg of coadministered KLH. However, chemical linking of ovalbumin to CT-B and coadministration with CT resulted in a detectable antibody response to ovalbumin. These results suggest that CT is immunogenic and is a potent adjuvant for CT-B in swine and that the induction of mucosal immunity to heterologous antigens may require specific targeting to the gut-associated lymphoid tissues.

Adjuvants, Immunologic↗
PubMed1999

Cloning, expression and functional role of a nociceptin/orphanin FQ receptor in the porcine gastrointestinal tract.

The heptadecapeptide nociceptin/orphanin FQ is the cognate ligand for the opioid receptor-like orphanin FQ (OFQ) receptor, a member of the G protein-coupled receptor superfamily. The gastrointestinal tract is a major site of opioid action, and preliminary evidence suggests that an OFQ receptor may be expressed in rat small intestine. We addressed the hypothesis that this receptor is expressed in the gastrointestinal tract of the pig, a model for the human digestive system. A 1205-bp cDNA was isolated from porcine forebrain which contained the 370 amino acid open reading frame encoding the OFQ receptor. The receptor mRNA is likely to arise from a single gene, as determined by Southern blotting of porcine genomic DNA restriction digests using a porcine OFQ receptor cDNA probe. A semi-nested reverse transcriptase-polymerase chain reaction survey of receptor mRNA indicates that it is expressed in the porcine cerebral cortex and kidney, and along the length of the gastrointestinal tract. OFQ decreased initial contractile responses of porcine ileal smooth muscle strips to trains of electrical field stimulation with an IC50 value of 1.3 nM; its effects were resistant to the opioid antagonist, naloxone. The peptide, at concentrations > or =3 nM, also attenuated Isc elevations evoked by electrical transmural stimulation of mucosa-submucosa sheets from porcine ileum. The actions of OFQ appeared to differ from those previously reported for opioid receptor agonists in these tissue preparations. These results indicate that an OFQ receptor is expressed in the porcine intestine which modulates the neural control of intestinal smooth muscle contractility and mucosal transport.

Amino Acid Sequence↗
PubMed1999

Role of macrophage cytokines in mucosal adjuvanticity.

Delivery of protein antigens to the GALT can result in immunity or oral tolerance depending on the circumstances of the encounter. One mechanism by which mucosal adjuvants can affect these circumstances is by the induction of macrophage cytokines, including IL-1 and IL-12. These cytokines can directly affect the immune response by their effects on antigen-specific T cells and by the induction of IFN-gamma by T cells or NK cells. This IFN-gamma also activates macrophages to up-regulate MHC or costimulatory molecules and by further inducing IL-1 and IL-12. In effect, mucosal adjuvants function both directly and indirectly as activators of antigen presenting cells, resulting in stimulation of the immune response to coincidental antigens. Our studies in swine have shown CT is a potent mucosal adjuvant for CT-B. CT also increased IL-1 and IL-12 mRNA in cultured macrophages, especially after activation with IFN-gamma. The effect of CT on the secretion of bioactive IL-12 protein is currently being investigated. While the mucosal adjuvanticity of CT involves a variety of mechanisms, these findings suggest a role for the induction of the macrophage cytokines IL-1 and IL-12.

Adjuvants, Immunologic↗
PubMed1999

Recombination between North American strains of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV), a recently discovered arterivirus swine pathogen, was shown to undergo homologous recombination. Co-infection of MA-104 cells with two culture-adapted North American PRRSV strains resulted in recombinant viral particles containing chimeric ORF 3 and ORF 4 proteins. Nucleotide sequence analysis of cloned recombinant PCR products, encompassing 1182 bases of the 15.4 kb viral genome, revealed six independent recombination events. Recombinant products persisted in culture for at least three passages, indicating continuous formation of recombinant viruses, growth of recombinant viruses in competition with parental viruses, or both. The frequency of recombination was estimated from <2% up to 10% in the 1182 b fragment analyzed, which is similar to recombination frequencies observed in coronaviruses. An apparent example of natural ORF 5 recombination between naturally occurring wild type viruses was also found, indicating that recombination is likely an important genetic mechanism contributing to PRRSV evolution.

Amino Acid Sequence↗
PubMed1999

In vitro and in vivo bioactivity of single-chain interleukin-12.

Interleukin-12 is a heterodimeric cytokine with potent immunoregulatory properties, making it a potential vaccine adjuvant and an immune response modulator. The study of its function is confounded by its heterodimeric structure. In order to facilitate the study of interleukin-12 in both in vitro and in vivo models, we constructed a single-chain porcine interleukin-12 gene and expressed the recombinant protein in Pichia pastoris. Single-chain porcine interleukin-12 was bioactive in vitro on both human and porcine cells as measured by its ability to induce proliferation of lymphoblasts and interferon-gamma secretion by lymph node cells. In contrast, the p40 subunit of porcine interleukin-12 alone did not induce proliferation or inhibit the activity of the single-chain porcine interkeukin-12. The in vivo bioactivity of single-chain porcine interleukin-12 was demonstrated in an oral immunization model where it increased antigen-specific IgA and IgG in jejunal mucus. These results indicate that binding of interleukin-12 to its receptor and transduction of intracellular signals requires both p40 and p35 subunits. The bioactivity of interleukin-12 expressed as a single polypeptide will facilitate its in vivo delivery and study of its structure and function.

Adjuvants, Immunologic↗
PubMed1999

Differential regulation of macrophage interleukin-1 (IL-1), IL-12, and CD80-CD86 by two bacterial toxins.

The ability of innate immune cells to differentially respond to various bacterial components provides a mechanism by which the acquired immune response may be tailored to specific pathogens. The response of innate immune cells to bacterial components provides regulatory signals to cognate immune cells. These signals include secreted cytokines and costimulatory molecules, and to a large extent they determine the quantitative and qualitative nature of the immune response. In order to determine if innate immune cells can differentially respond to bacterial components, we compared the responses of macrophages to two bacterially derived molecules, cholera toxin (CT) and lipopolysaccharide (LPS). We found that CT and LPS differentially regulated the expression of interleukin-12 (IL-12) and CD80-CD86 but not that of IL-1beta. LPS and CT each induced IL-1beta expression in macrophages, while only LPS induced IL-12 and only CT induced CD80-CD86. These differences were markedly potentiated in gamma interferon (IFN-gamma)-treated macrophages, in which LPS potently induced IL-12 and CD80-CD86 expression. In contrast, IFN-gamma treatment had no effect on the expression of IL-1beta. These results define a molecular basis for the differential pathogenicities of bacterial toxins and are relevant to the design of vaccine adjuvants able to selectively induce desired types of immunity.

Animals↗
PubMed1999

Porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a recently described arterivirus responsible for disease in swine worldwide. Comparative sequence analysis of 3'-terminal structural genes of the single-stranded RNA viral genome revealed the presence of two genotypic classes of PRRSV, represented by the prototype North American and European strains, VR-2332 and Lelystad virus (LV), respectively. To better understand the evolution and pathogenicity of PRRSV, we obtained the 12,066-base 5'-terminal nucleotide sequence of VR-2332, encoding the viral replication activities, and compared it to those of LV and other arteriviruses. VR-2332 and LV differ markedly in the 5' leader and sections of the open reading frame (ORF) 1a region. The ORF 1b sequence was nearly colinear but varied in similarity of proteins encoded in identified regions. Furthermore, molecular and biochemical analysis of subgenomic mRNA (sgmRNA) processing revealed extensive variation in the number of sgmRNAs which may be generated during infection and in the lengths of noncoding sequence between leader-body junctions and the translation-initiating codon AUG. In addition, VR-2332 and LV select different leader-body junction sites from a pool of similar candidate sites to produce sgmRNA 7, encoding the viral nucleocapsid protein. The presence of substantial variations across the entire genome and in sgmRNA processing indicates that PRRSV has evolved independently on separate continents. The near-simultaneous global emergence of a new swine disease caused by divergently evolved viruses suggests that changes in swine husbandry and management may have contributed to the emergence of PRRS.

5' Untranslated Regions↗
PubMed1999

Detection of porcine reproductive and respiratory syndrome virus by reverse transcription-polymerase chain reaction using different regions of the viral genome.

Serologic studies have revealed strain variability between American and European isolates and among American isolates of porcine reproductive and respiratory syndrome virus (PRRSV). The objective of this study was to develop an assay for the routine diagnosis of PRRSV in field specimens using reverse transcription-polymerase chain reaction (RT-PCR) amplification of conserved genomic regions. Twenty-four field isolates of PRRSV from different regions of the USA were analyzed in the study. Six primer pairs from open reading frames (ORFs) 4, 6, and 7 of the American strain (ATCC VR-2332) and from ORF 1b of the Lelystad strain were used for the amplification of the viral genome by PCR. Amplification products of the expected sizes were obtained from all isolates by PCR amplification of ORF 7, the gene encoding the nucleocapsid protein. Oligonucleotide primers designed to amplify ORFs 4 and 6 detected 92% and 96% of the isolates, respectively, whereas primers for the amplification of ORF 1b detected 88% of all isolates. The specificity of the amplified products of ORF 7 from 7 field isolates and 2 reference strains was confirmed by chemiluminescent hybridization using an internal digoxigenin-labeled DNA probe. Sequence analysis of this region indicated variation in the nucleotide sequence of 2 isolates that did not hybridize with the internal probe. These results indicate that ORF 7 may serve as a potential target for the detection of PRRSV strains by RT-PCR and that genomic variability should be considered when nucleic acid hybridization is used to confirm the specificity of PCR amplification for diagnostic purposes.

Animals↗
PubMed1998

The porcine mu opioid receptor: molecular cloning and mRNA distribution in lymphoid tissues.

The porcine mu opioid receptor (pMOR), was cloned from cerebral cortex RNA using PCR methodologies. Porcine MOR is 96% identical with human MOR in amino acid sequence. An RT-PCR survey for pMOR mRNA indicated that pMOR is widely distributed in the gut, and is present in thymus and Peyer's patches but absent in other immune tissues and in isolated immune cells. Based on these findings, it appears that opioids do not exert an immunosuppressive effect through direct interaction with the mu-opioid receptor on immune cells. In certain tissues, however, opioids may modulate immune function indirectly through neuronal MOR.

Animals↗
PubMed1998

Construction of internal cDNA competitors for measuring IL-10 and IL-12 cytokine gene expression in swine.

A competitive PCR assay (cPCR) was used to quantify swine cytokine responses to parasite infection. Internal standards (deleted cDNA competitor molecules [DcDNA mimics]) were produced and tested for swine interleukin-12 (IL-12), interleukin-10 (IL-10) and hypoxanthine phosphoribosyltransferase (HPRT) from PCR generated cDNA cloned in plasmid vectors. Deletion clones for the cDNA competitor molecules (DcDNA mimics) were generated for IL-10, IL-12 and HPRT by PCR in a single step and verified by (1) amplification of the expected smaller PCR product with the original primers, (2) appropriate fragment size released by restriction digestion of the deleted clone, and (3) correct sequence of the new DcDNA insert. DcDNA mimics were used to quantitate cytokine gene mRNA production during experimental and natural infections of swine with the gastrointestinal nematode parasite Trichuris suis. Mesenteric lymph node cells were collected from control and infected pigs at the time of maximal pathogenicity (35 days after infection) and snap frozen. After RNA extraction, samples were reverse transcribed (RT) to cDNA. cPCR was performed using the housekeeping gene HPRT DcDNA mimic and HPRT specific primers to insure RNA integrity and concentration. Cytokine cDNA content in these samples was then quantitated using cytokine mimics and gene specific primers. IL-10 gene expression in MLN draining the colon of pigs experimentally infected with T. suis increased 10-20 fold at day 35 compared to control pigs. IL-12 gene expression was not detectable in MLN of these pigs, but was detectable in MLN of pigs exposed naturally to T. suis on a contaminated dirt lot that also exhibited signs of secondary bacterial invasion. Swine IL-10 and IL-12 gene expression can be quantitated in local mesenteric tissues. This cPCR assay will enable scientists to quantitate cytokine gene expression in swine and determine the nature of immune responses to important infectious diseases.

Animals↗
PubMed1998

Inducible nitric oxide synthase expression in porcine immune cells.

Porcine immune cells were examined for the ability to produce inducible nitric oxide synthase following in vitro or in vivo stimulation. Enzyme activity and product formation were not detected following stimulation of porcine peripheral blood mononuclear cells (PBMC), splenocytes, or alveolar macrophages with a combination of ConA and lipopolysaccharide (LPS) or recombinant porcine interferon gamma and LPS. In vitro engulfment of Haemophilus parasuis by macrophages also failed to induce inducible nitric oxide synthase (iNOS) activity or nitrite formation. Swine Herpes Virus infection led to a small but significant increase in level of nitrite detected in lung lavage fluid, whereas the infection of pigs with Porcine Respiratory and Reproductive Syndrome Virus did not alter the lavage fluid nitrite levels. iNOS mRNA was detected in both stimulated and unstimulated porcine immune cells and in macrophages from both control and infected animals suggesting that it is constitutively expressed with little or no upregulation following cellular stimulation. The results presented in this paper indicate that the reactive nitrogen intermediate pathway is not an vital innate immune response in the pig.

Animals↗
PubMed1998

Monoclonal antibodies putatively identifying porcine B cells.

Comparison was made of the binding of 38 test and three standard monoclonal antibodies (mAbs) to B cells from various pig lymphoid tissues by flow cytometry (FCM) and immunohistochemistry. Some mAbs were also tested on B cells from foetal pig tissues. Twenty of the new mAbs bound, though to variable degrees, to porcine B cells but only three were given cluster assignations: C35 (#147) and BB6-11C9 (#167) were assigned to wCD21 and 2F6/8 (#057) was assigned to SWC7.

Animals↗
PubMed1998

Workshop studies with monoclonal antibodies identifying a novel porcine differentiation antigen, SWC9.

Two monoclonal antibodies (mAb) within cluster M4 of the myeloid section of the Second International Swine CD Workshop, C4 (No. 144) and PM18-7 (No. 192), showed reactivity with thymocytes and among cells of myelomonocytic origin with mature macrophages but not with monocytes and granulocytes. Both mAb recognize a protein showing two bands of 205 kDa and 130 kDa under both reducing and non-reducing conditions. Although epitope mapping with these mAb could not be performed, this cluster received the SWC9 designation.

Animals↗
PubMed1998

Genetic variation in the PRRS virus.

Porcine reproductive and respiratory syndrome (PRRS) is characterized by late-term abortions and stillbirths in sows and respiratory difficulties in nursery pigs. The disease appeared in Europe and North America at approximately the same time between 1985 and 1990. The PRRS virus was isolated shortly thereafter and demonstrated unexpectedly profound differences between European (Lelystad) and North American (VR2332) isolates as measured by serological crossreactivity and nucleotide sequence similarity. In order to determine the amount of genetic variation in the PRRS virus and to understand the molecular mechanisms of viral evolution, nucleotide sequences of PRRS virus strains were determined. Comparisons among ten U.S. strains showed that variation in primary nucleotide sequence between isolates ranged from 2.5% to 7.9% for ORFs 2-7. In contrast, Lelystad virus was, on average, 35% different from US clones. These results provided direct molecular evidence that US and European PRRSV isolates represented genetically distinct groups of the same viral family. A further analysis of more than 150 isolates in the United States and Canada demonstrated that the PRRS virus in North America represents a single large and diverse genetic group that is distinct from European forms of the virus.

Animals↗
PubMed1998

Subgenomic RNA7 is transcribed with different leader-body junction sites in PRRSV (strain VR2332) infection of CL2621 cells.

Porcine reproductive and respiratory syndrome virus (PRRSV), like all members of the order Nidoviridae, is expressed in the infected cell as a nested set of subgenomic (sg) RNAs with a common 5'-leader sequence. We have determined that the 5'-leader sequence for the US prototype strain (VR2332, Collins, et al., 1992) is distinct from the European prototype strain [Lelystad (LV); Wensvoort, et al., 1991, Meulenberg et al., 1993a], yet these two strains use almost the same sequence for downstream sites of 5'-leader-body junction formation. Analysis of VR2332 genomic sequence identified several potential 5'-leader-body junction sequences upstream of open reading frame (ORF) 7, coding for the nucleocapsid protein, that could be used for generation of VR2332 sgRNA7 transcripts. Sequence determinations of RT-PCR-generated cDNA clones of sgRNA7 identified two species of RNA7 transcripts in infected cells, one utilizing a leader-body junction sequence (AUAACC) 123 nucleotides upstream of the AUG start site and one utilizing a sequence (UAAACC) 9 nucleotides upstream of the AUG start site for ORF7 translation.

5' Untranslated Regions↗
PubMed1998

Delta-opioid receptor mRNA expression and immunohistochemical localization in porcine ileum.

Opiates have potent antidiarrheal actions that are mediated in part by delta-opioid receptors (DOR). We examined DOR localization within subregions of porcine ileum, a tissue analogous to human small bowel. A partial cDNA sequence for porcine DOR was obtained after reverse transcription-polymerase chain reaction cloning of forebrain RNA; it encoded the end of transmembrane domain 1 through the beginning of transmembrane domain 7 and exhibited 93% nucleotide identity with human DOR. Positive signals for DOR mRNA were found in all subregions of the porcine ileal wall. With an antiserum recognizing an N-terminal epitope in murine DOR, DOR-like immunoreactivity was localized in neurons within myenteric and submucous ganglia, longitudinal and circular smooth muscle, and villous lamina propria. The DOR agonist [D-Ser2, Leu5, Thr6]enkephalin (DSLET) attenuated circular smooth muscle contractions in porcine ileum that were evoked by electrical stimulation of myenteric cholinergic neurons. These results are consistent with previous reports of the DOR-mediated neuromodulation that underlies the antipropulsive and antisecretory effects of opioids in the intestinal tract.

Animals↗
PubMed1998

Regulation of hypoxanthine phosphoribosyltransferase, glyceraldehyde-3-phosphate dehydrogenase and beta-actin mRNA expression in porcine immune cells and tissues.

Various "housekeeping" genes are often used as endogenous controls in gene expression experiments. We have cloned from swine, three genes commonly used as endogenous controls in other species and have characterized their relative levels of expression in various porcine tissues and their response to various cell activators. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin were readily detected by northern hybridization in various tissues and in alveolar macrophages. The expression of hypoxanthine phosphoribosyltransferase (HPRT) was detected only by northern hybridization of poly-A+ enriched RNA and by reverse transcriptase-polymerase chain reaction (RT-PCR), making it more suitable for highly sensitive detection methods. Expression of GAPDH varied less among tissues than did beta-actin, making it more useful control for comparisons of gene expression between tissues with northern hybridizations. Various treatments of cultured alveolar macrophages differentially affected levels of beta-actin and GAPDH, while HPRT expression was unchanged in alveolar macrophages or spleen cells similarly treated. Therefore, while HPRT can be used as the endogenous control with sensitive detection methods such as RT-PCR, less sensitive detection methods require a more abundant gene such as GAPDH.

Actins↗