Toll-like receptor (TLR) 2 and TLR4 gene expression in canine heart

Toll-like receptors (TLRs) are archetypal pattern recognition receptors of immediate importance for an efficacious innate immune response. TLRs exhibit marked differential tissue activity and their levels within a discrete cell type can be highly dynamic. Of 13 known mammalian paralogues, three TLRs have been identified in the dog. Although cardiac TLR expression has been reported in other species, this study is the first to present evidence that these innate immune receptors are expressed in the canine heart. Heart tissue samples from all four chambers were collected from healthy dogs immediately after euthanasia and stored at -80°C until analysis. Total RNA was extracted with TRI Regent. Specific primers were designed for amplification of canine TLR2 and TLR4 based on previously reported sequences for these genes. Reverse transcription was performed with M-MLV reverse transcriptase. PCR amplification was performed and PCR products analyzed by agarose gel electrophoresis. Bands were excised from the gel and the DNA isolated and cloned using the TA Cloning® Kit. The correct sequence for each product was verified by nucleotide sequencing. TLR4 expression was detected in the left ventricle and right atrium; TLR2 was detectable at low levels in the right atrium only. Identity of the RT-PCR products was confirmed by sequencing. Our findings show that at least two TLR paralogues- namely TLR2 and TLR4 - are expressed in the canine heart. Additional studies are warranted to determine these immune receptors' potential implication in the development of naturally occurring heart disease in the dog

Palomar 13: a velocity dispersion inflated by binaries ?

Recently, combining radial velocities from Keck/HIRES echelle spectra with published proper motion membership probabilities, Cote et al (2002) observed a sample of 21 stars, probable members of Palomar 13, a globular cluster in the Galactic halo. Their projected velocity dispersion sigma_p = 2.2 +/-0.4 km/s gives a mass-to-light ratio M/L_V = 40 +24/-17, about one order of magnitude larger than the usual estimate for globular clusters. We present here radial velocities measured from three different CCD frames of commissioning observations obtained with the new ESO/VLT instrument FLAMES (Fibre Large Array Multi Element Spectrograph). From these data, now publicly available, we measure the homogeneous radial velocities of eight probable members of this globular cluster. A new projected velocity dispersion sigma_p = 0.6-0.9 +/-0.3 km/s implies Palomar 13 mass-to-light ratio M/L_V = 3-7, similar to the usual value for globular clusters. We discuss briefly the two most obvious reasons for the previous unusual mass-to-light ratio finding: binaries, now clearly detected, and more homogeneous data from the multi-fibre FLAMES spectrograph.Comment: 9 pages, 2 Postscript figure

Molecular evolution of the porcine type I interferon family: subtype-specific expression and antiviral activity

Type I interferons (IFNs), key antiviral cytokines, evolve to adapt with ever-changing viral threats during vertebrate speciation. Due to novel pathogenic pressure associated with Suidae speciation and domestication, porcine IFNs evolutionarily engender both molecular and functional diversification, which have not been well addressed in pigs, an important livestock species and animal model for biomedical sciences. Annotation of current swine genome assembly Sscrofa10.2 reveals 57 functional genes and 16 pseudogenes of type I IFNs. Subfamilies of multiple IFNA, IFNW and porcine-specific IFND genes are separated into four clusters with ~60 kb intervals within the IFNB/IFNE bordered region in SSC1, and each cluster contains mingled subtypes of IFNA, IFNW and IFND. Further curation of the 57 functional IFN genes indicates that they include 18 potential artifactual duplicates. We performed phylogenetic construction as well as analyses of gene duplication/conversion and natural selection and showed that porcine type I IFN genes have been undergoing active diversification through both gene duplication and conversion. Extensive analyses of the non-coding sequences proximal to all IFN coding regions identified several genomic repetitive elements significantly associated with different IFN subtypes. Family-wide studies further revealed their molecular diversity with respect to differential expression and restrictive activity on the resurgence of a porcine endogenous retrovirus. Based on predicted 3-D structures of representative animal IFNs and inferred activity, we categorized the general functional propensity underlying the structure-activity relationship. Evidence indicates gene expansion of porcine type I IFNs. Genomic repetitive elements that associated with IFN subtypes may serve as molecular signatures of respective IFN subtypes and genomic mechanisms to mediate IFN gene evolution and expression. In summary, the porcine type I IFN profile has been phylogenetically defined family-wide and linked to diverse expression and antiviral activity, which is important information for further biological studies across the porcine type I IFN family

Antiviral regulation in porcine monocytic cells at different activation states

Monocytic cells, including macrophages and dendritic cells, exist in different activation states that are critical to the regulation of antimicrobial immunity. Many pandemic viruses are monocytotropic, including porcine reproductive and respiratory syndrome virus (PRRSV), which directly infects subsets of monocytic cells and interferes with antiviral responses. To study antiviral responses in PRRSV-infected monocytic cells, we characterized inflammatory cytokine responses and genome-wide profiled signature genes to investigate response pathways in uninfected and PRRSV-infected monocytic cells at different activation states. Our findings showed suppressed interferon (IFN) production in macrophages in non-antiviral states and an arrest of lipid metabolic pathways in macrophages at antiviral states. Importantly, porcine monocytic cells at different activation states were susceptible to PRRSV and responded differently to viral infection. Based on Gene Ontology (GO) analysis, two approaches were used to potentiate antiviral activity: (i) pharmaceutical modulation of cellular lipid metabolism and (ii) in situ PRRSV replication-competent expression of interferon alpha (IFN-α). Both approaches significantly suppressed exogenous viral infection in monocytic cells. In particular, the engineered IFN-expressing PRRSV strain eliminated exogenous virus infection and sustained cell viability at 4 days postinfection in macrophages. These findings suggest an intricate interaction of viral infection with the activation status of porcine monocytic cells. An understanding and integration of antiviral infection with activation status of monocytic cells may provide a means of potentiating antiviral immunity