Pigs immunized with a novel E2 subunit vaccine are protected from subgenotype heterologous classical swine fever virus challenge.

BACKGROUND: Classical swine fever (CSF) or hog cholera is a highly contagious swine viral disease. CSF endemic countries have to use routine vaccination with modified live virus (MLV) vaccines to prevent and control CSF. However, it is impossible to serologically differentiate MLV vaccinated pigs from those infected with CSF virus (CSFV). The aim of this study is to develop a one-dose E2-subunit vaccine that can provide protection against CSFV challenge. We hypothesize that a vaccine consisting of a suitable adjuvant and recombinant E2 with natural conformation may induce a similar level of protection as the MLV vaccine. RESULTS: Our experimental vaccine KNB-E2 was formulated with the recombinant E2 protein (Genotype 1.1) expressed by insect cells and an oil-in-water emulsion based adjuvant. 10 pigs (3 weeks old, 5 pigs/group) were immunized intramuscularly with one dose or two doses (3 weeks apart) KNB-E2, and 10 more control pigs were administered normal saline solution only. Two weeks after the second vaccination, all KNB-E2 vaccinated pigs and 5 control pigs were challenged with 5 × 10(5) TCID50 CSFV Honduras/1997 (Genotype 1.3, 1 ml intramuscular, 1 ml intranasal). It was found that while control pigs infected with CSFV stopped growing and developed high fever (>40 °C), high level CSFV load in blood and nasal fluid, and severe leukopenia 3-14 days post challenge, all KNB-E2 vaccinated pigs continued to grow as control pigs without CSFV exposure, did not show any fever, had low or undetectable level of CSFV in blood and nasal fluid. At the time of CSFV challenge, only pigs immunized with KNB-E2 developed high levels of E2-specific antibodies and anti-CSFV neutralizing antibodies. CONCLUSIONS: Our studies provide direct evidence that pigs immunized with one dose KNB-E2 can be protected clinically from CSFV challenge. This protection is likely mediated by high levels of E2-specific and anti-CSFV neutralizing antibodies.

Pigs immunized with Chinese highly pathogenic PRRS virus modified live vaccine are protected from challenge with North American PRRSV strain NADC-20.

Modified live virus (MLV) vaccines developed to protect against PRRSV circulating in North America (NA) offer limited protection to highly pathogenic (HP) PRRSV strains that are emerging in Asia. MLV vaccines specific to HP-PRRSV strains commercially available in China provide protection to HP-PRRSV; however, the efficacy of these HP-PRRSV vaccines to current circulating NA PRRS viruses has not been reported. The aim of this study is to investigate whether pigs vaccinated with attenuated Chinese HP-PRRSV vaccine (JXA1-R) are protected from infection by NA PRRSV strain NADC-20. We found that pigs vaccinated with JXA1-R were protected from challenges with HV-PRRSV or NADC-20 as shown by fewer days of clinical fever, reduced lung pathology scores, and lower PRRS virus load in the blood. PRRSV-specific antibodies, as measured by IDEXX ELISA, appeared one week after vaccination and virus neutralizing antibodies were detected four weeks post vaccination. Pigs vaccinated with JXA1-R developed broadly neutralizing antibodies with high titers to NADC-20, JXA1-R, and HV-PRRSV. In addition, we also found that IFN-α and IFN-ß occurred at higher levels in the lungs of pigs vaccinated with JXA1-R. Taken together, our studies provide the first evidence that JXA1-R can confer protection in pigs against the heterologous NA PRRSV strain NADC-20.

Targeted inactivation of ß1 integrin induces ß3 integrin switching, which drives breast cancer metastasis by TGF-ß.

Mammary tumorigenesis and epithelial-mesenchymal transition (EMT) programs cooperate in converting transforming growth factor-ß (TGF-ß) from a suppressor to a promoter of breast cancer metastasis. Although previous reports associated ß1 and ß3 integrins with TGF-ß stimulation of EMT and metastasis, the functional interplay and plasticity exhibited by these adhesion molecules in shaping the oncogenic activities of TGF-ß remain unknown. We demonstrate that inactivation of ß1 integrin impairs TGF-ß from stimulating the motility of normal and malignant mammary epithelial cells (MECs) and elicits robust compensatory expression of ß3 integrin solely in malignant MECs, but not in their normal counterparts. Compensatory ß3 integrin expression also 1) enhances the growth of malignant MECs in rigid and compliant three-dimensional organotypic cultures and 2) restores the induction of the EMT phenotypes by TGF-ß. Of importance, compensatory expression of ß3 integrin rescues the growth and pulmonary metastasis of ß1 integrin-deficient 4T1 tumors in mice, a process that is prevented by genetic depletion or functional inactivation of ß3 integrin. Collectively our findings demonstrate that inactivation of ß1 integrin elicits metastatic progression via a ß3 integrin-specific mechanism, indicating that dual ß1 and ß3 integrin targeting is necessary to alleviate metastatic disease in breast cancer patients.

Peptide nanofiber hydrogel adjuvanted live virus vaccine enhances cross-protective immunity to porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is prevalent in swine farms worldwide and is a major source of economic loss and animal suffering. Rapid genetic variation of PRRSV makes it difficult for current vaccines to confer protection against newly emerging strains. We recently demonstrated that a novel peptide nanofiber hydrogel (H9e) could act as a potent adjuvant for killed H1N1 vaccines. Therefore, the objective of this study was to evaluate H9e as an adjuvant for PRRSV modified live virus (MLV) vaccines. Pigs were vaccinated with Ingelvac PRRSV MLV with or without H9e adjuvant before being challenged with the VR-2332 (parental vaccine strain) or MN184A (genetically diverse strain) PRRSV. Pigs vaccinated with MLV+H9e had higher levels of circulating vaccine virus. More importantly, pigs vaccinated with MLV+H9e had improved protection against challenge by both PRRSV strains, as demonstrated by reduced challenge-induced viremia compared with pigs vaccinated with MLV alone. Pigs vaccinated with MLV+H9e had lower frequency of T-regulatory cells and IL-10 production but higher frequency of Th/memory cells and IFN-γ secretion than that in pigs vaccinated with MLV alone. Taken together, our studies suggest that the peptide nanofiber hydrogel H9e, when combined with the PRRSV MLV vaccine, can enhance vaccine efficacy against two different PRRSV strains by modulating both host humoral and cellular immune responses.

Caspase-1 activation and mature interleukin-1ß release are uncoupled events in monocytes.

AIM: To investigate whether caspase-1 activation/intracellular processing of pro-interleukin-1ß (pro-IL-1ß) and extracellular release of mature IL-1ß from activated monocytes are separable events. METHODS: All experiments were performed on fresh or overnight cultured human peripheral blood monocytes (PBMCs) that were isolated from healthy donors. PBMCs were activated by lipopolysaccharide (LPS) stimulation before being treated with Adenosine triphosphate (ATP, 1 mmol/L), human α-defensin-5 (HD-5, 50 µg/mL), and/or nigericin (Nig, 30 µmol/L). For each experiment, the culture supernatants were collected separately from the cells. Cell lysates and supernatants were both subject to immunoprecipitation with anti-IL-1ß antibodies followed by western blot analysis with anti-caspase-1 and anti-IL-1ß antibodies. RESULTS: We found that pro-IL-1ß was processed to mature IL-1ß in LPS-activated fresh and overnight cultured human monocytes in response to ATP stimulation. In the presence of HD-5, this release of IL-1ß, but not the processing of pro-IL-1ß to IL-1ß, was completely inhibited. Similarly, in the presence of HD-5, the release of IL-1ß, but not the processing of IL-1ß, was significantly inhibited from LPS-activated monocytes stimulated with Nig. Finally, we treated LPS-activated monocytes with ATP and Nig and collected the supernatants. We found that both ATP and Nig stimulation could activate and release cleaved caspase-1 from the monocytes. Interestingly, and contrary to IL-1ß processing and release, caspase-1 cleavage and release was not blocked by HD-5. All images are representative of three independent experiments. CONCLUSION: These data suggest that caspase-1 activation/processing of pro-IL-1ß by caspase-1 and the release of mature IL-1ß from human monocytes are distinct and separable events.

Structural and functional analysis of the pro-domain of human cathelicidin, LL-37.

Cathelicidins form a family of small host defense peptides distinct from another class of cationic antimicrobial peptides, the defensins. They are expressed as large precursor molecules with a highly conserved pro-domain known as the cathelin-like domain (CLD). CLDs have high degrees of sequence homology to cathelin, a protein isolated from pig leukocytes and belonging to the cystatin family of cysteine protease inhibitors. In this report, we describe for the first time the X-ray crystal structure of the human CLD (hCLD) of the sole human cathelicidin, LL-37. The structure of the hCLD, determined at 1.93 Å resolution, shows the cystatin-like fold and is highly similar to the structure of the CLD of the pig cathelicidin, protegrin-3. We assayed the in vitro antibacterial activities of the hCLD, LL-37, and the precursor form, pro-cathelicidin (also known as hCAP18), and we found that the unprocessed protein inhibited the growth of Gram-negative bacteria with efficiencies comparable to that of the mature peptide, LL-37. In addition, the antibacterial activity of LL-37 was not inhibited by the hCLD intermolecularly, because exogenously added hCLD had no effect on the bactericidal activity of the mature peptide. The hCLD itself lacked antimicrobial function and did not inhibit the cysteine protease, cathepsin L. Our results contrast with previous reports of hCLD activity. A comparative structural analysis between the hCLD and the cysteine protease inhibitor stefin A showed why the hCLD is unable to function as an inhibitor of cysteine proteases. In this respect, the cystatin scaffold represents an ancestral structural platform from which proteins evolved divergently, with some losing inhibitory functions.

IL-1ß promotes stemness and invasiveness of colon cancer cells through Zeb1 activation.

BACKGROUND: IL-1ß is a pleiotropic pro-inflammatory cytokine and its up-regulation is closely associated with various cancers including gastrointestinal tumors. However, it remains unclear how IL-1ß may contribute to the initiation and development of these inflammation-associated cancers. Here we investigated the role of IL-1ß in colon cancer stem cell (CSC) development. METHODS: Using self-renewal assay, soft-agar assay, invasion assay, real-time PCR analysis, immunoblot assay and shRNA knockdown, we determined the effects of IL-1ß on cancer stem cell development and epithelial-mesenchymal transition (EMT) in human primary colon cancer cells and colon cancer cell line HCT-116. RESULTS: We found that IL-1ß can increase sphere-forming capability of colon cancer cells in serum-free medium. IL-1ß-induced spheres displayed an up-regulation of stemness factor genes (Bmi1 and Nestin) and increased drug resistance, hallmarks of CSCs. Importantly, expression of EMT activator Zeb1 was increased in IL-1ß-induced spheres, indicating that there might be a close association between EMT and IL-1ß-induced CSC self-renewal. Indeed, IL-1ß treatment led to EMT of colon cancer cells with loss of E-cadherin, up-regulation of Zeb1, and gain of the mesenchymal phenotype. Furthermore, shRNA-mediated knockdown of Zeb1 in HCT-116 cells reversed IL-1ß-induced EMT and stem cell formation. CONCLUSION: Our findings indicate that IL-1ß may promote colon tumor growth and invasion through activation of CSC self-renewal and EMT, and Zeb1 plays a critical role in these two processes. Thus, IL-1ß and Zeb1 might be new therapeutic targets against colon cancer stem cells.

Proinflammatory actions of glucocorticoids: glucocorticoids and TNFα coregulate gene expression in vitro and in vivo.

Synthetic glucocorticoids are widely used for treatment of many inflammatory diseases. However, long-term glucocorticoid treatment can cause a variety of negative side effects. A genome-wide microarray analysis was performed in human lung A549 cells to identify genes regulated by both the antiinflammatory steroid dexamethasone (Dex) and the proinflammatory cytokine TNFα. Unexpectedly, we discovered that numerous genes were coregulated by treatment with both Dex and TNFα. We evaluated the mechanism of coregulation of one of these genes, serpinA3 (α-1 antichymotrypsin), a secreted, acute phase protein strongly associated with numerous inflammatory diseases. Up-regulation of serpinA3 requires the presence of both the glucocorticoid receptor and TNFα soluble receptor 1. Treatment with Dex or TNFα resulted in a 10- to 25-fold increase of serpinA3 mRNA, whereas coadministration of Dex and TNFα led to a synergistic increase in serpinA3 mRNA. The naturally occurring glucocorticoid, cortisol, also resulted in a synergistic increase in serpinA3 mRNA levels in A549 cells. Furthermore, in vivo treatment of C57BL/6 mice with Dex and TNFα resulted in coregulation of serpinA3 mRNA levels in both lung and liver tissues. Finally, chromatin immunoprecipitation analyses suggest that glucocorticoid receptor binding to the serpinA3 transcriptional start site can be enhanced by the combination of Dex plus TNFα treatment of A549 cells. These studies demonstrate that glucocorticoids and proinflammatory compounds can coregulate genes associated with human disease. This discovery may underlie the basis of some of the adverse effects associated with long-term glucocorticoid therapy.

Pro-inflammatory cytokine interleukin-1ß promotes the development of intestinal stem cells.

OBJECTIVE: We investigated the effect of IL-1ß on the development of intestinal epithelial stem cells. MATERIALS AND METHODS: Normal intestinal epithelial cell line IEC-18 cells were cultured in the presence or absence of 200 pM of IL-1ß in serum-free medium (SFM) for various time periods. The effects of IL-1ß on intestinal stem cell self-renewal and IEC-18 cell proliferation were evaluated by a colony formation assay, MTT assay, and a focus formation assay. The expression of stemness genes including Bmi-1, Lgr-5, c-myc, Nanog, and ß-catenin in IEC-18 cells were measured by quantitative PCR and western blot analysis. RESULTS: IEC-18 cells grew as a monolayer in SFM in the absence of IL-1ß. Cellular spheres were formed when IEC-18 cells were grown in SFM in the presence of IL-1ß. IL-1ß induced the development of large colonies in the soft-agar as well as the formation of foci when IEC-18 cells were cultured in type-I collagen-coated plates. The expression of Bmi-1, Lgr-5, c-myc, Nanog, and ß-catenin were significantly increased in IEC-18 cells treated with IL-1ß. CONCLUSION: Our studies provide direct evidence the IL-1ß may play an important role in the self-renewal of intestinal epithelial stem cells and the development of cancer stem cells.

Complete genome sequence of two novel Chinese virulent porcine reproductive and respiratory syndrome virus variants.

A highly pathogenic strain of porcine reproductive and respiratory syndrome virus (PRRSV), characterized by a discontinuous 30-amino-acid deletion in its Nsp2-coding region, has been emerging in China since 2006. Here, we report the complete genomic sequence of two novel Chinese virulent PRRSV variants with additional NSP2-gene deletions, which will help us understand the molecular and evolutionary characteristics of PRRSV in Asia.

Ligand-independent phosphorylation of the glucocorticoid receptor integrates cellular stress pathways with nuclear receptor signaling.

Glucocorticoids are stress hormones that maintain homeostasis through gene regulation mediated by nuclear receptors. We have discovered that other cellular stressors are integrated with glucocorticoid signaling through a new hormone-independent phosphorylation site, Ser134, on the human glucocorticoid receptor (GR). Ser134 phosphorylation is induced by a variety of stress-activating stimuli in a p38 mitogen-activated protein kinase (MAPK)-dependent manner. Cells expressing a mutant glucocorticoid receptor incapable of phosphorylation at Ser134 (S134A-GR) had significantly altered hormone-dependent genome-wide transcriptional responses and associated hormone-mediated cellular functions. The phosphorylation of Ser134 significantly increased the association of the GR with the zeta isoform of the 14-3-3 class of signaling proteins (14-3-3zeta) on chromatin promoter regions, resulting in a blunted hormone-dependent transcriptional response of select genes. These data argue that the phosphorylation of Ser134 acts as a molecular sensor on the GR, monitoring the level of cellular stress to redirect glucocorticoid-regulated signaling through altered 14-3-3zeta cofactor binding and promoter recruitment. This posttranslational modification allows prior cellular stress signals to dictate the transcriptional response to glucocorticoids.

Emerging roles of glucocorticoid receptor phosphorylation in modulating glucocorticoid hormone action in health and disease.

Glucocorticoids (GCs) are hormones naturally released when the body perceives stress and function to return homeostatic balance within various tissues. Synthetic GCs are widely prescribed therapeutics for the treatment of numerous inflammatory disorders and cancers. The effects of GCs are mediated by their binding and activation of the GC receptor (GR), a transcription factor that is subject to hormone-dependent and -independent phosphorylation on several serine and threonine residues. The GR is phosphorylated by kinases such as MAPKs, CDKs, and GSK-3beta, and these modifications modulate the transcriptional activity of the GR within cells. Here, we discuss the phosphorylation status of the GR as a mechanism to dictate how cells will ultimately respond to GCs. In doing so, we will review current knowledge about each phosphorylated residue within the GR and their contributions to modulating GC signaling in normal homeostatic physiology and during the progression of disease.

Activated Abl kinase inhibits oncogenic transforming growth factor-beta signaling and tumorigenesis in mammary tumors.

Transforming growth factor-beta (TGF-beta) is a ubiquitous cytokine with dual roles in tumor suppression and promotion, and these dichotomous functions have frustrated the development of therapies targeting oncogenic signaling by TGF-beta. In comparison, Abl is well established as an initiator of hematopoietic cancers; however, a clear role for Abl in regulating solid tumor development remains elusive. Here, we investigated the role of Abl in TGF-beta-mediated epithelial-mesenchymal transition (EMT) in normal and metastatic mammary epithelial cells (MECs). In doing so, we identified Abl as an essential regulator of MEC morphology and showed that Abl inactivation was sufficient to induce phenotypic and transcriptional EMT in normal MECs. Increasing Abl activity in metastatic MECs resulted in their complete morphological reversion, restored their cytostatic response to TGF-beta, and blocked their secretion of matrix metalloproteinases induced by TGF-beta. Constitutively active Abl expression blocked TGF-beta-responsive mammary tumor growth in mice, while Imatinib therapy afforded no clinical benefit in mice bearing mammary tumors. Collectively, this investigation establishes Abl as a potent mediator of MEC identity, and as a suppressor of oncogenic TGF-beta signaling during mammary tumorigenesis. Notably, our findings strongly caution against the use of pharmacological Abl antagonists in the treatment of developing and progressing mammary tumors.

Glycogen synthase kinase 3beta-mediated serine phosphorylation of the human glucocorticoid receptor redirects gene expression profiles.

Aberrant glycogen synthase kinase 3beta (GSK-3beta) activity is associated with the progression of several pathological conditions such as diabetes, Alzheimer's, and cancer. GSK-3beta regulates cellular processes by directly phosphorylating metabolic enzymes and transcription factors. Here, we discovered a new target for GSK-3beta phosphorylation: the human glucocorticoid receptor (GR). Glucocorticoid signaling is essential for life and regulates diverse biological functions from cell growth to metabolism to apoptosis. Specifically, we found hormone-dependent GR phosphorylation on serine 404 by GSK-3beta. Cells expressing a GR that is incapable of GSK-3beta phosphorylation had a redirection of the global transcriptional response to hormone, including the activation of additional signaling pathways, in part due to the altered ability of unphosphorylatable GR to recruit transcriptional cofactors CBP/p300 and the p65 (RelA) subunit of NF-kappaB. Furthermore, GSK-3beta-mediated GR phosphorylation inhibited glucocorticoid-dependent NF-kappaB transrepression and attenuated the glucocorticoid-dependent cell death of osteoblasts. Collectively, our results describe a novel convergence point of the GSK-3beta and the GR pathways, resulting in altered hormone-regulated signaling. Our results also provide a mechanism by which GSK-3beta activity can dictate how cells will ultimately respond to glucocorticoids.

Grb2 binding to Tyr284 in TbetaR-II is essential for mammary tumor growth and metastasis stimulated by TGF-beta.

We demonstrated previously that growth factor receptor-bound protein 2 (Grb2) associates with the transforming growth factor-beta (TGF-beta) type II receptor [TbetaR-II] upon its phosphorylation on Tyr284 by Src. Although this phosphotransferase reaction is critical in mediating TGF-beta stimulation of epithelial-mesenchymal transition (EMT) and invasion in mammary epithelial cells (MECs), the necessity of Grb2 in promoting these TGF-beta-dependent events remain purely correlative. Herein, we further evaluated the role of Grb2 in mediating the oncogenic activities of TGF-beta and show that the binding of Grb2 to TbetaR-II paralleled the induction of EMT in MECs stimulated by TGF-beta. Introducing siRNAs against Grb2 or expression of a TbetaR-II mutant that cannot bind Grb2 (i.e. Y284F-TbetaR-II) had no effect on the ability of TGF-beta to activate Smad3, but significantly impaired its stimulation of p38 mitogen-activated protein kinase (MAPK) in MECs. Importantly, these same cellular conditions also prevented the ability of MECs to undergo EMT in response to TGF-beta, and to invade synthetic basement membranes when stimulated by beta3 integrin and TGF-beta. Finally, we show that the ability of TGF-beta to stimulate breast cancer growth and pulmonary metastasis in mice required TbetaR-II to be phosphorylated on Tyr284, which activated p38 MAPK in developing and progressing mammary tumors. Collectively, our findings have established the necessity of Grb2 in mediating TGF-beta stimulation of EMT and invasion in MECs, as well as demonstrated the essential function of the alphavbeta3 integrin:Src:phospho-Y284-TbetaR-II:Grb2:p38 MAPK signaling axis to promote breast cancer growth and metastasis in vivo.