Enhanced replication of porcine circovirus type 2 (PCV2) in a homogeneous subpopulation of PK15 cell line

Post-weaning multi-systemic wasting syndrome (PMWS) has emerged as a major disease that poses a significant threat to the economics of global swine industry. Porcine circovirus type 2 (PCV2) is the causal agent of PMWS in pigs. Currently, the prevention of PCV2 infection based on vaccines is limited, and the available vaccines are either killed viral vaccines or recombinant protein based vaccines and not cost effective. The PK-15 cells, which is widely used for PCV2 propagation, is not efficient and heterogeneous in terms of permissivity to viral infection. In order to acquire a homogeneous porcine kidney cell line that can reliably produce PCV2 in high titers, cell clones that show high- (PK15-C1) or low-permissive (PK15-A2) phenotype to PCV2 infection were derived from heterogeneous PK15 parent cells by limiting dilution and cell cloning. Maximum virus titers in PK15-C1, PK15-A2 and PK15 parent cells were 10(8), 10(2) and 10(5) tissue culture infective dose 50 (TCID 50)/ml, respectively. The viral proteins of PCV2 were produced and accumulated faster in PK15-C1 cells than those in PK15 parent cells. These results indicate that PK15-C1 cell clone is more permissive to PCV2 infection than PK15 parent cells and thus will be useful for PCV2 replication in vitro, as well as, vaccines, diagnostic and research applications on PCV2.     

Inhibition of porcine circovirus type 2 replication in mice by RNA interference

Porcine circovirus type 2 (PCV2) is the primary causative agent of an emerging swine disease, postweaning multisystemic wasting syndrome (PMWS) for which no antiviral treatment is available. To exploit the possibility of using RNA interference (RNAi) as a therapeutic approach against the disease, plasmid-borne short hairpin RNAs (shRNAs) were generated to target the PCV2 genome. Transfection of these shRNAs into cultured PK15 cells caused a significant reduction in viral RNA production that was accompanied by inhibiting viral DNA replication and protein synthesis in infected cells. The effect was further tested in vivo in a mouse model that has been developed for PCV2 infection. Mice injected with shRNA before PCV2 infection showed substantially decreased microscopic lesions in inguinal lymph nodes compared to controls. In situ hybridization and immunohistochemical analyses showed that shRNA caused a significant inhibition in the level of viral DNA and protein synthesis detected in the lymph nodes of the treated mice relative to the controls. Taken together, these results indicate that shRNAs are capable of inhibiting PCV2 infection in vitro as well as in vivo and thus may constitute an effective therapeutic strategy for PCV2 infection.     

Genetic characterization and phylogenetic analysis of porcine circovirus type 2 (PCV2) strains from cases presenting various clinical conditions

Porcine circovirus type 2 (PCV2) has been associated with a newly identified and described disease in swine called the postweaning multisystemic wasting syndrome (PMWS). An association of PCV2 with various other clinical conditions in pigs are also increasingly being reported. To date molecular studies to determine the extent of genetic variations of this virus have been limited. To more fully understand the extent of genetic diversity we report the sequencing of 34 PCV2 strains from Eastern Canada originating from a variety of clinical conditions spanning years 1990–2001 along with the phylogenetic analysis of these strains and that of 36 PCV2 sequences published in GenBank. Sequence analysis of the complete genome indicated that the Canadian PCV2 strains analyzed in the present study are closely related to each other but also to other PCV2 strains originating from Western Canada, US, Europe and Asia. Sequence analysis of ORF1 and ORF2 genes of the 34 strains revealed that the extent of nucleotide variation was greater for the ORF2 than for the ORF1. The amino acid sequences alignment of the PCV2 capsid protein identified three major regions of amino acid heterogeneity, two of which correspond with dominant immunoreactive areas. No repeatable amino acid motifs for these two regions could be associated with PCV2 strains identified from cases of PMWS or other clinical conditions. Phylogenetic analysis of all 70 strains revealed one large cluster composed of strains from Europe, Taiwan, China and Canada. This large cluster could be divided in several sub clusters, in two of which Canadian strains were found closely related to strains from Germany. The remaining strains from Canada and the US were spread in small groupings along the phylogenetic tree and an association with geographic origin could not be established. The genomic characterizations performed in this study indicate that PCV2 strains associated with PMWS are scattered throughout the phylogenetic tree often in groupings including PCV2 strains identified from cases other that PMWS such as, porcine reproductive and respiratory syndrome (PRRS), generalized tremors, porcine dermatitis and nephropathy syndrome (PDNS), arthritis, nervous signs, erysipelas and even healthy pigs.     

A multiplex PCR for rapid and simultaneous detection of porcine circovirus type 2, porcine parvovirus,porcine pseudorabies virus,and porcine reproductive and respiratory syndrome virus in clinical specimens

A multiplex PCR (mPCR) assay was developed and evaluated for its ability to simultaneously detect multiple viral infections of swine. Specific primers were designed for each of the following four DNA or RNA viruses: porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), pseudorabies virus (PRV), and porcine reproductive and respiratory syndrome virus (PRRSV). Each target produced a specific amplicon with a size of 353 bp (PCV2), 271 bp (PPV), 194 bp (PRV), or 434 bp (PRRSV). The assay was sensitive and specific in detecting each target agent in composite cell cultures and clinical specimens. Results from mPCR were confirmed by PCR for individual viruses and by virus isolation. In conclusion, the mPCR has the potential to be useful for routine molecular diagnosis and epidemiology.     

Arginine vasopressin prevents amyloid β protein-induced impairment of long-term potentiation in rat hippocampus in vivo

Amyloid β protein (Aβ) is thought to be responsible for the loss of memory in Alzheimer's disease (AD). A significant decrease in [Arg⁸]-vasopressin (AVP) in the AD brain has been found. However, it is unclear whether the decrease in AVP is involved in Aβ-induced impairment of memory and whether AVP can protect against Aβ-induced neurotoxicity. The present study examines the effects of intracerebroventricular (i.c.v.) injection of AVP on hippocampal long-term potentiation (LTP), a synaptic model of memory, and investigates the potential protective function of AVP in Aβ-induced LTP impairment. The results showed that (1) i.c.v. injection of different concentrations of AVP or Aβ_25–35 did not affect the baseline field excitatory postsynaptic potentials (fEPSPs); (2) AVP administration alone induced a significant increase in HFS-induced LTP, while Aβ_25–35 significantly suppressed HFS-induced LTP; (3) Aβ_25–35-induced LTP suppression was significantly prevented by the pretreatment with AVP; (4) paired-pulse facilitation did not change after separate application or co-application of AVP and Aβ_25–35. These results indicate that AVP can potentiate hippocampal synaptic plasticity and dose-dependently prevent Aβ_25–35-induced LTP impairment. Thus, the present study provides further insight into the mechanisms by which Aβ impairs synaptic plasticity and suggests an important approach in the treatment of AD.     

IFITM proteins drive type 2 T helper cell differentiation and exacerbate allergic airway inflammation

The interferon‐inducible transmembrane (Ifitm/Fragilis) genes encode homologous proteins that are induced by IFNs. Here, we show that IFITM proteins regulate murine CD4⁺ Th cell differentiation. Ifitm2 and Ifitm3 are expressed in wild‐type (WT) CD4⁺ T cells. On activation, Ifitm3 was downregulated and Ifitm2 was upregulated. Resting Ifitm‐family‐deficient CD4⁺ T cells had higher expression of Th1‐associated genes than WT and purified naive Ifitm‐family‐deficient CD4⁺ T cells differentiated more efficiently to Th1, whereas Th2 differentiation was inhibited. Ifitm‐family‐deficient mice, but not Ifitm3‐deficient mice, were less susceptible than WT to induction of allergic airways disease, with a weaker Th2 response and less severe disease and lower Il4 but higher Ifng expression and IL‐27 secretion. Thus, the Ifitm family is important in adaptive immunity, influencing Th1/Th2 polarization, and Th2 immunopathology.     

Prostaglandin F2α can modulate the growth and the differentiation of bovine corneal epithelial cells cultured in vitro

The effects of PGF_2α on the growth, morphology, morphometry and keratinization pattern of bovine corneal epithelial cells cultured in vitro were studied. The cells were grown with a basal medium or, in the presence of keratocytes and/or their products, using a keratocyte-conditioning medium. Cell growth was evaluated by MTT assay. Daily treatments with exogenous PGF_2α at concentrations equal to or lower than 10⁻⁶ M induced significant increases in cell proliferation when the epithelial cells were cultured on a keratocyte feeder-layer or with the conditioning medium. No variations were observed in cultures grown with the basal medium. 10⁻⁵ M PGF_2α induced a decrease in cell growth under all culturing conditions. PGF_2α did not affect cell morphology and modified only nuclear dimensions among the cells grown under different culturing conditions. No variations of any parameters were observed between cells cultured on feeder-layer, with conditioning or basal medium and the corresponding cultures supplemented with the autacoid. Moreover, PGF_2α induced only the disappearance of 43 kDa keratin in cells grown on basal medium, while the keratin pattern of epithelial cells cultured on feeder-layer or with the conditioning medium was not modified by the autacoid. From these data we can suppose that a cooperation could exist between PGF_2α and fibroblasts and their products for the modulation of cell growth. Finally, it was observed that the autacoid had no effect on cell morphology and morphometry, except for nuclear dimensions, despite the presence of other prostaglandins, such as PGE₂.     

Induction of mRNA for tumor necrosis factor-α and interleukin 1β in cultured rat smooth muscle cells and in rat aortic strips by Escherichia coli lipopolysaccharide: effects of cycloheximide

The expression of mRNA for TNFa and IL-1β was studied in cell cultures of rat aortic smooth muscle cells (SMCs), and in organ cultures of rat aortic strips, representing different phenotypes, synthetic vs. contractile. The SMCs and aortic strips were exposed to Escherichia coli lipopolysaccharide (LPS) alone, or with the addition of the protein synthesis inhibitor cycloheximide. TNFα and IL-1β mRNA were induced after exposure to LPS, in both cultured SMCs and aortic strips. This induction was more pronounced in the aortic strips than in cultured SMCs. Cycloheximide alone induced a weak expression of TNFα mRNA and a pronounced induction IL-1β mRNA in cultured SMCs. LPS together with cycloheximide potentiated the induction of TNFα and IL-1β mRNA compared to these substances alone in the cultured SMCs. In contrast, cycloheximide did not affect the levels of either TNFα or IL-1β mRNA in the strips of aorta. The results show that LPS induces mRNA for TNFα and IL-1β in both cultured SMCs and SMCs in aortic strips. The results also indicate that a difference in expression of short-lived repressor proteins and/or RNase exists between cultured SMCs and aortic strips.     

Modulation of Apo-1/Fas (CD95)-induced programmed cell death in myeloma cells by interferon-α2

The Apo-1/Fas (CD95) antigen is known to be involved in the process of T cell-mediated target cell killing and has recently been shown to be expressed on myeloma cell lines and native malignant plasma cells. Several cytokines have been reported to interfere with spontaneous and even Apo-1/Fas-induced apoptosis, but no attempt has been made yet to investigate these interactions and the possible underlying mechanisms in myeloma cells. Since in myeloma patients Interferon (IFN)-α₂ displays a profound therapeutic effect in vivo, which is usually attributed to its growth inhibitory and/or immunomodulatory capacity, we set out to study the potential interference of IFN-α₂ with Apo-1/Fas-induced apoptosis. Contrary to expectations, IFN-α₂ reduced the degree of apoptosis caused by the treatment of five Apo-1/Fas-sensitive myeloma cell lines with a Fas monoclonal antibody (mAb). Simultaneous application of IFN-α₂ and Fas mAb was superior to the prolonged (i.e. > 8 h) preincubation with the cytokine as far as inhibition of Apo-1/Fas-induced apoptosis was concerned. This effect of IFN-α₂ was neither explained by a down-regulation of the Apo-1/Fas receptor nor caused by modulation of the expression levels of c-myc, bcl-2-, bcl-x_L, bax- or p53 genes. IFN-α₂ did not alter the Apo-1/Fas-induced activity of Mitogen-activated protein kinase (MAPK) 1 and did not inhibit the Apo-1/Fas-mediated proteolytic cleavage of ADP-ribosyltransferase, a substrate of Interleukin-β1 converting enzyme (ICE) and homologues. However, activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) mimicked the effects of IFN-α₂. Furthermore, the bis-indolylmaleimide GF 109203X, a specific inhibitor of PKC, inhibited the effect of PMA as well as that of IFN-α₂ on Apo-1/Fas-induced apoptosis. These results point to a PKC-dependent mechanism of transient interaction between the intracellular signaling along the IFN-α₂ and the Apo-1/Fas pathway (downstream of MAPK signaling as well as of ICE homologues), which becomes exhausted by prolonged stimulation with the cytokine. According to our data IFN-α₂, applied continuously and in high doses resembling the therapeutic situation in vivo, inhibits myeloma growth. However, based on the observed inhibitory effect of IFN-α₂ on Apo-1/Fas-induced apoptosis, a partial inhibition of the natural immune surveillance on myeloma cells by endog-genous IFN-α₂ present in the bone marrow microenvironment of this malignancy should be investigated.     

Cytosolic phospholipase A2α has a crucial role in the pathogenesis of DSS‐induced colitis in mice

Colitis, an inflammation of the colon, is a well‐characterized massive tissue injury. Cytosolic phospholipase A₂α (cPLA₂α) upregulation plays an important role in the development of several inflammatory diseases. The aim of the present study was to define the role of cPLA₂α upregulation in the development of colitis. We used a mouse model of dextran sulfate sodium induced colitis. Immunoblotting analysis showed that cPLA₂α and NF‐κB were upregulated and activated in the colon from day 2 of colitis induction. This molecular event preceded the development of the disease, as determined by Disease Activity Index score, body weight, colon length, and the expression of colonic inflammatory markers, including neutrophil infiltration detected by myeloperoxidase and by NIMP‐R14, ICAM‐1, COX‐2, iNOS upregulation and LTB₄ and TNF‐α secretion. Prevention of cPLA₂α upregulation and activity in the colon by i.v. administration of specific antisense oligonucleotides against cPLA₂α 1 day prior and every day of exposure to dextran sulfate sodium significantly impeded the development of the disease and prevented NF‐κB activation, neutrophils infiltration into the colonic mucosa, and expression of proinflammatory proteins in the colon. Our results demonstrate a critical role of cPLA₂α upregulation in inflammation and development of murine colitis.     

Cerebrovascular miRNAs correlate with the clearance of Aβ through perivascular route in younger 3xTg‐AD mice

The “two‐hit vascular hypothesis for Alzheimer's disease (AD)” and amyloid‐β (Aβ) oligomer hypothesis suggest that impaired soluble Aβ oligomers clearance through the cerebral vasculature may be an initial step of the AD process. Soluble Aβ oligomers are driven into perivascular spaces from the brain parenchyma and toward peripheral blood flow. The underlying vascular‐based mechanism, however, has not been defined. Given that microRNAs (miRNAs), emerging as novel modulators, are involved in numerous physiological and pathological processes, we hypothesized that cerebrovascular miRNAs may regulate the activities of brain blood vessels, which further affects the concentration of Aβ in the AD brain. In this study, perivascular Aβ deposits, higher vascular activation, increased pericyte coverage and up‐regulated capillaries miRNAs at 6 months old (6 mo) were found to correlate with the lower Aβ levels of middle AD stage (9 mo) in 3xTg‐AD (3xTg) mice. It is implicated that at the early stage of AD when intracellular Aβ appeared, higher expression of vessel‐specific miRNAs, elevated pericyte coverage, and activated endothelium facilitate Aβ oligomer clearance through the perivascular route, resulting in a transient reduction of Aβ oligomers at 9 mo. Additionally, ghrelin‐induced upregulation of capillary miRNAs and increased pericyte coverage attenuated Aβ burden at 9 mo, in further support of the relationship between vascular miRNAs and Aβ clearance. This work suggests a cerebral microvessel miRNA may boost endothelial highly activated phenotypes to promote elimination of Aβ oligomers through the perivascular drainage pathway and contribute to AD progression. The targeting of brain vessel‐specific miRNAs may provide a new rationale for the development of innovative therapeutic strategies for AD treatment.     

VIP inhibition of vascular smooth muscle: complementary to β2-adrenoceptor mediated relaxation in the isolated rat portal vein

Exogenous VIP caused a concentration dependent inhibition of the spontaneous mechanical activity in the isolated rat mesenteric-portal vein preparation via a mechanism which was completely independent of the propranolol-blocked β-adrenoceptor, of high K⁺ in the medium and of exogenous bovine pancreatic polypeptide, neurotensin and opioids. The potency of VIP ((pD₂=7.52±0.18, n=6) was about 30 times higher than that of isoprenaline in the atropine and phentolamine-blocked preparation. The isoprenaline inhibition was mediated via a β₂-type of adrenoceptor with low apparent affinity for noradrenaline (intrinsic activity (a) = 0.27±0.01, n=8). Opposite effects of exogenous VIP and noradrenaline were on the other hand observed in the atropinized and β-blocked preparation. These results suggest that in the rat portal vein neuronal VIP and circulating adrenaline may be complementary in their antagonism of the α-adrenoceptor mediated increase in contractility.     

Association of the −31C/T functional polymorphism in the interleukin‐1β gene with the intractability of Graves' disease and the proportion of T helper type 17 cells

Interleukin (IL)-1β is a proinflammatory cytokine and has been implicated in the pathogenesis of several autoimmune diseases. To evaluate the hypothesis that the functional −31C/T polymorphism (rs1143627) in the gene encoding IL-1β is associated with the intractability and the severity of autoimmune thyroid diseases, we genotyped this polymorphism in 64 patients with intractable Graves'' disease (GD), 28 GD patients in remission, 49 patients with Hashimoto''s disease (HD) who developed hypothyroidism (severe HD), 28 untreated euthyroid HD patients (mild HD) and 59 healthy volunteers. The −31T allele, which is related to the high producibility of IL-1β, was significantly more frequent in patients with intractable GD than in those with GD in remission (P = 0·0017; odds ratio 2·8; 95% confidence interval 1·5-5·3), although there was no difference in this frequency between two groups of HD patients. We showed additionally that the proportion of IL-17-producing T helper type 17 (Th17) cells, whose differentiation and proliferation are promoted by IL-1β, was higher in autoimmune thyroid disease patients with the T allele than in those with CC genotypes. In conclusion, our data indicated that the T allele of −31C/T polymorphism in the IL1B gene was involved in the intractability of GD, and this involvement may arise through the differentiation and proliferation of Th17 cells.     

Galectin‐3 is an amplifier of the interleukin‐1β‐mediated inflammatory response in corneal keratinocytes

Interleukin‐1β (IL‐1β) is a potent mediator of innate immunity commonly up‐regulated in a broad spectrum of inflammatory diseases. When bound to its cell surface receptor, IL‐1β initiates a signalling cascade that cooperatively induces the expression of canonical IL‐1 target genes such as IL‐8 and IL‐6. Here, we present galectin‐3 as a novel regulator of IL‐1β responses in corneal keratinocytes. Using the SNAP‐tag system and digitonin semi‐permeabilization, we show that recombinant exogenous galectin‐3 binds to the plasma membrane of keratinocytes and is internalized into cytoplasmic compartments. We find that exogenous galectin‐3, but not a dominant negative inhibitor of galectin‐3 polymerization lacking the N‐terminal domain, exacerbates the response to IL‐1β by stimulating the secretion of inflammatory cytokines. The activity of galectin‐3 could be reduced by a novel d ‐galactopyranoside derivative targeting the conserved galactoside‐binding site of galectins and did not involve interaction with IL‐1 receptor 1 or the induction of endogenous IL‐1β. Consistent with these observations, we demonstrate that small interfering RNA‐mediated suppression of endogenous galectin‐3 expression is sufficient to impair the IL‐1β‐induced secretion of IL‐8 and IL‐6 in a p38 mitogen‐activated protein kinase‐independent manner. Collectively, our findings provide a novel role for galectin‐3 as an amplifier of IL‐1β responses during epithelial inflammation through an as yet unidentified mechanism.