Development and characterization of Rift Valley fever virus-like particles.

Rift Valley fever (RVF) is an acute, febrile zoonotic disease that is caused by the RVF virus (RVFV) and spread by arthropod vectors. RVF is currently prevalent in Africa and the Arabian Peninsula, and causes substantial economic losses. Furthermore, this disease poses a serious threat to animal and human health in regions worldwide, making it a serious public health concern. However, RVFV vaccines for human use are still unavailable, and hence there is an urgent need for novel efficient vaccines against RVFV. Vaccine preparation techniques have become a crucial factor in developing new vaccines. In the current study, the N and G protein genes of RVFV were inserted into the pFastBacDual baculovirus expression vector downstream of the pP10 and pPH promoters. The resultant recombinant vector, pFastBacDual-S-M, was transfected into Sf9 insect cells by lipofection. The recombinant baculovirus, named rBac-N-G, was retrieved and infected into Sf9 insect cells to generate RVFV virus-like particles (VLPs). Using polyclonal antibodies against RVFV proteins in immunofluorescence and western blot analyses, we positively identified the presence of the RVFV proteins in VLP preparations. Sucrose density gradient centrifugation and transmission electron microscopy revealed that the morphology of the RVFV VLPs was consistent with previous reports of RVFV virions. This study describes a technique for efficient production of RVFV VLPs, and has laid the foundation for future VLP-based RVFV vaccines.

Bovine Mx1 enables resistance against foot-and-mouth disease virus in naturally susceptible cells by inhibiting the replication of viral RNA.

Innate immunity, especially the anti-viral genes, exerts an important barrier function in preventing viral infections. Myxovirus-resistant (Mx) gene take an anti-viral role, whereas its effects on foot-and-mouth disease virus (FMDV) in naturally susceptible cells are still unclear. The bovine primary fetal tracheal epithelial cell line BPTE-siMx1, in which bovine Mx1 gene was silenced, was established and treated with IFN alpha for 6 hr before FMDV infection. The copy numbers of the negative and positive strand viral RNA were determined by strand-specific real-time fluorescence quantitative RT-PCR. The TCID50 of BPTE-siMx1 cells increased at least 17-fold as compared to control cells BPTE-LacZ at 8 hr post infection, thus silencing of bovine Mx1 could promote the replication of FMDV. The amount of both the negative and positive strand viral RNA in BPTE-siMx1 cells significantly increased as compared to BPTE-LacZ cells, indicating that the replication levels of viral RNA were promoted by silencing bovine Mx1. The bovine Mx1 gene could provide resistance against FMDV in the bovine primary fetal tracheal epithelial cells via suppressing the replication of viral RNA.

Potential role of high-mobility group box 1 protein in the pathogenesis of influenza H5N1 virus infection.

During influenza A virus (IAV) (H5N1) infection, the levels of inflammatory cytokines are markedly elevated in the lungs of infected hosts. One of them, high-mobility group box 1 protein (HMGB1) functions in regulation of cellular transcription and activation of proinflammatory responses, but little is known about its role in viral infection. In this study, we attempted to address this question. Using an IAV (H5N1) - mouse model, lung tissues were analyzed for virus titer, expression of HMGB1 and other inflammatory cytokines and histopathological changes. Moreover, the effect of administration of HMGB1-specific antibody to the infected mice on these parameters was investigated. The results showed that the HMGB1 expression was induced on days 3-7 post infection (p.i.) and primarily localized to epithelial cells of alveoli and bronchioles. The HMGB1-specific antibody reduced the levels of inflammatory cytokines and chemokines and the survival rate, but did not influence the virus titer. Summing up, these data suggest that HMGB1 contributes to the pathogenesis of IAV (H5N1) infection in mice by inducing extensive inflammatory responses and severe pneumonia.

Pharmacology of AMG 181, a human anti-α4 ß7 antibody that specifically alters trafficking of gut-homing T cells.

BACKGROUND AND PURPOSE: AMG 181 is a human anti-α4 ß7 antibody currently in phase 1 and 2 trials in subjects with inflammatory bowel diseases. AMG 181 specifically targets the α4 ß7 integrin heterodimer, blocking its interaction with mucosal addressin cell adhesion molecule-1 (MAdCAM-1), the principal ligand that mediates α4 ß7 T cell gut-homing. EXPERIMENTAL APPROACH: We studied the in vitro pharmacology of AMG 181, and the pharmacokinetics and pharmacodynamics of AMG 181 after single or weekly i.v. or s.c. administration in cynomolgus monkeys for up to 13 weeks. KEY RESULTS: AMG 181 bound to α4 ß7 , but not α4 ß1 or αE ß7 , and potently inhibited α4 ß7 binding to MAdCAM-1 (but not vascular cell adhesion molecule-1) and thus inhibited T cell adhesion. Following single i.v. administration, AMG 181 Cmax was dose proportional from 0.01 to 80 mg·kg(-1) , while AUC increased more than dose proportionally. Following s.c. administration, dose-proportional exposure was observed with single dose ranging from 5 to 80 mg·kg(-1) and after 13 weekly doses at levels between 20 and 80 mg·kg(-1) . AMG 181 accumulated two- to threefold after 13 weekly 80 mg·kg(-1) i.v. or s.c. doses. AMG 181 had an s.c. bioavailability of 80%. The linear elimination half-life was 12 days, with a volume of distribution close to the intravascular plasma space. The mean trend for the magnitude and duration of AMG 181 exposure, immunogenicity, α4 ß7 receptor occupancy and elevation in gut-homing CD4+ central memory T cell count displayed apparent correlations. CONCLUSIONS AND IMPLICATIONS: AMG 181 has in vitro pharmacology, and pharmacokinetic/pharmacodynamic and safety characteristics in cynomolgus monkeys that are suitable for further investigation in humans.

Role of macrophage migration inhibitory factor in influenza H5N1 virus pneumonia.

The severe and often fatal disease in humans and birds caused by H5N1 influenza viruses has been attributed to aberrant pulmonary inflammatory responses. We investigated the role of macrophage migration inhibitory factor (MIF), a proinflammatory cytokine and a pivotal regulator of innate immunity, in H5N1 influenza virus pneumonia in murine model. We found increased MIF mRNA levels in the lungs and MIF protein levels in the serum of infected mice. Although the inhibition of MIF action by isoxazolone-1 (ISO-1) did not render mice more resistant to the lethality of infection, it caused a significant reduction in pulmonary inflammatory cytokines interleukin-1 beta (IL-1beta), IL-6 and tumor necrosis factor alpha (TNF-alphalfa) and chemokine interferon-inducible protein-10 (IP-10). These results indicate the involvement of MIF in inflammatory responses to H5N1 influenza virus infections by induction of pulmonary inflammatory cytokines and chemokines, and suggest that pharmacotherapeutic approaches targeting MIF may hold promise for the treatment of H5N1 influenza virus pneumonia.

Detection and differentiation of CAV-1 and CAV-2 by polymerase chain reaction.

Canine adenovirus type 1 (CAV-1) and type 2 (CAV-2) can be categorized in the laboratory by haemagglutination and neutralization tests, but they are difficult to differentiate from each other in specimens, especially when infection occurs in the digestive tract. The object of this study was to develop a simple method of detecting and differentiating them. One pair of common primers was designed and synthesized according to the sequences of the E3 and flanking regions and a polymerase chain reaction (PCR) assay was established using these two primers to amplify the virus-specific DNA fragment from clinical specimens as well as from cell cultures. After elecctrophoresis, under the same amplification conditions, 508 bp and 1030 bp PCR products were observed for CAV-1 and CAV-2, respectively. These were further shown to be adenovirus specific by dot hybridization and sequencing. As only one pair of primers was involved in the PCR procedure, it was faster and easier to perform than any of the other assays used for detecting canine adenovirus, making it applicable in the rapid confirmation of diagnosis and differentiation of the two types of canine adenoviruses.

TACI is a TRAF-interacting receptor for TALL-1, a tumor necrosis factor family member involved in B cell regulation.

We and others recently reported tumor necrosis factor (TNF) and apoptosis ligand-related leukocyte-expressed ligand 1 (TALL-1) as a novel member of the TNF ligand family that is functionally involved in B cell proliferation. Transgenic mice overexpressing TALL-1 have severe B cell hyperplasia and lupus-like autoimmune disease. Here, we describe expression cloning of a cell surface receptor for TALL-1 from a human Burkitt's lymphoma RAJI cell library. The cloned receptor is identical to the previously reported TNF receptor (TNFR) homologue transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI). Murine TACI was subsequently isolated from the mouse B lymphoma A20 cells. Human and murine TACI share 54% identity overall. Human TACI exhibits high binding affinities to both human and murine TALL-1. Soluble TACI extracellular domain protein specifically blocks TALL-1-mediated B cell proliferation without affecting CD40- or lipopolysaccharide-mediated B cell proliferation in vitro. In addition, when injected into mice, soluble TACI inhibits antibody production to both T cell-dependent and -independent antigens. By yeast two-hybrid screening of a B cell library with TACI intracellular domain, we identified that, like many other TNFR family members, TACI intracellular domain interacts with TNFR-associated factor (TRAF)2, 5, and 6. Correspondingly, TACI activation in a B cell line results in nuclear factor kappaB and c-Jun NH(2)-terminal kinase activation. The identification and characterization of the receptor for TALL-1 provides useful information for the development of a treatment for B cell-mediated autoimmune diseases such as systemic lupus erythematosus.

Severe B cell hyperplasia and autoimmune disease in TALL-1 transgenic mice.

TALL-1/Blys/BAFF is a member of the tumor necrosis factor (TNF) ligand superfamily that is functionally involved in B cell proliferation. Here, we describe B cell hyperplasia and autoimmune lupus-like changes in transgenic mice expressing TALL-1 under the control of a beta-actin promoter. The TALL-1 transgenic mice showed severe enlargement of spleen, lymph nodes, and Peyer's patches because of an increased number of B220+ cells. The transgenic mice also had hypergammaglobulinemia contributed by elevations of serum IgM, IgG, IgA, and IgE. In addition, a phenotype similar to autoimmune lupus-like disease was also seen in TALL-1 transgenic mice, characterized by the presence of autoantibodies to nuclear antigens and immune complex deposits in the kidney. Prolonged survival and hyperactivity of transgenic B cells may contribute to the autoimmune lupus-like phenotype in these animals. Our studies further confirm TALL-1 as a stimulator of B cells that affect Ig production. Thus, TALL-1 may be a primary mediator in B cell-associated autoimmune diseases.

APRIL and TALL-I and receptors BCMA and TACI: system for regulating humoral immunity.

We report that the tumor neurosis factor homolog APRIL (a proliferation-inducing ligand) stimulates in vitro proliferation of primary B and T cells and increases spleen weight due to accumulation of B cells in vivo. APRIL functions via binding to BCMA (B cell maturation antigen) and TACI (transmembrane activator and CAML-interactor) and competes with TALL-I (also called BLyS or BAFF) for receptor binding. Soluble BCMA and TACI specifically prevent binding of APRIL and block APRIL-stimulated proliferation of primary B cells. BCMA-Fc also inhibits production of antibodies against keyhole limpet hemocyanin and Pneumovax in mice, indicating that APRIL and/or TALL-I signaling via BCMA and/or TACI are required for generation of humoral immunity. Thus, APRIL-TALL-I and BCMA-TACI form a two ligands-two receptors pathway involved in stimulation of B and T cell function.

[The development of an intellectualized dynamic strain device for three-dimensional cell culture].

The design principles of an intellectualized dynamic strain device for three-dimensional cell cultures based on microcomputer are mainly introduced here, which includes principles of hardware and software design of controlled unit, principle and structure of mechanical unit, and construction of three dimensional scaffold and culture unit. The main technical index and advantages of the device have been also discussed in the paper.

Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand.

A receptor that mediates osteoprotegerin ligand (OPGL)-induced osteoclast differentiation and activation has been identified via genomic analysis of a primary osteoclast precursor cell cDNA library and is identical to the tumor necrosis factor receptor (TNFR) family member RANK. The RANK mRNA was highly expressed by isolated bone marrow-derived osteoclast progenitors and by mature osteoclasts in vivo. Recombinant OPGL binds specifically to RANK expressed by transfected cell lines and purified osteoclast progenitors. Transgenic mice expressing a soluble RANK-Fc fusion protein have severe osteopetrosis because of a reduction in osteoclasts, similar to OPG transgenic mice. Recombinant RANK-Fc binds with high affinity to OPGL in vitro and blocks osteoclast differentiation and activation in vitro and in vivo. Furthermore, polyclonal Ab against the RANK extracellular domain promotes osteoclastogenesis in bone marrow cultures suggesting that RANK activation mediates the effects of OPGL on the osteoclast pathway. These data indicate that OPGL-induced osteoclastogenesis is directly mediated through RANK on osteoclast precursor cells.

The cellular level of prostatic acid phosphatase and the growth of human prostate carcinoma cells.

Prostatic acid phosphatase (PAcP) is a prostate epithelium-specific differentiation antigen. It has been demonstrated that human PAcP exhibits endogenous protein tyrosine phosphatase (PYP) activity, and that it represents the major PYP activity in normal prostate cells. Thus, it has been postulated that cellular PAcP may play a role in the tyrosine phosphorylation-mediated signal transduction. In this paper, we used LNCaP human prostate carcinoma cells, which express the endogenous PAcP, to study changes in cellular PAcP activity during cell growth. Our results demonstrated that PAcP activity increased when the cells reached confluence. Stimulation of cell growth by fresh culture medium or 5 alpha-dihydrotestosterone (DHT), a classical stimulator of prostate epithelial growth, resulted in a decline in PAcP activity. Moreover, transfection of PC-3 cells, which do not express PAcP, with a PAcP-expressing vector led to diminished cellular growth rate. These data established an inverse relationship between the cellular level of PAcP and the cell growth rate, suggesting that PAcP may be involved in regulating the growth of human prostate carcinoma cells.

Regulation of prostatic acid phosphatase expression and secretion by androgen in LNCaP human prostate carcinoma cells.

The expression and the secretion of human prostatic acid phosphatase (PAcP), a differentiation antigen which is the major acid phosphatase in prostate epithelial cells, are thought to be regulated by an androgen. We investigated this regulatory mechanism at the post-transcriptional level in LNCaP human prostate carcinoma cells using a cDNA clone for the secretory form of PAcP. 5 alpha-Dihydrotestosterone (DHT, an active form of endogenous androgen) stimulated the secretion of PAcP from cells grown in a steroid-reduced medium and in a defined serum-free medium, respectively. The secreted PAcP activity was increased following a DHT dose in a dose-dependent fashion at concentrations of up to 1 microM. Further, the stimulation of PAcP secretion occurred following the period of exposure to DHT. During a 5-day treatment period, with 10 nM of DHT in the steroid-reduced medium, the secretion of PAcP was stimulated approximately 150% over that from control cells. Nevertheless, PAcP was secreted from cells grown in the absence of added DHT. First, the androgen dependency of PAcP expression was examined. The expression and the secretion of PAcP were observed in cells that were grown in a defined serum-free medium and grown in a steroid-reduced medium, in the absence of DHT. The increased secretion by DHT was further demonstrated to be in part due to an increase in PAcP mRNA level, as evidenced by Northern blot analysis. PAcP mRNA levels were elevated approximately 2-fold and corresponded to an increase of approximately 2.5-fold in the secreted level of newly synthesized 35S-PAcP. Then, the effect of DHT on the secretory process was investigated. Results of pulse-chase labeling experiments indicated that the secretory rate of PAcP was stimulated by about 50% on average by DHT. In conclusion, our data demonstrated that, in LNCaP cells, the expression and the secretion of PAcP regulated by androgen are apparently hormone-responsive processes. Further, DHT stimulation of PAcP secretion operates within at least two levels: increased PAcP mRNA and stimulation of the secretory pathway.