71 research outputs found
Construction of a Retroviral Vector for Production of Transgenic Cells Expressing Feline Immunodeficiency Virus (FIV) Viral Protein
No full textProgram year: 1994/1995Digitized from print original stored in HDRRetroviral vectors are being used as vehicles for transporting foreign genes into mammalian cells for the purpose of human gene therapy to treat viral disease like human immunodeficiency virus (HIV). In efforts to develop a preventative vaccine against a lentiviral infection, research is centering on developing a protective vaccine that will identify antigens which induce protective cell mediated immunity, especially cytotoxic T lymphocytes (CTL). Retroviral vectors provide the required endogenous expression of a retrovirus antigen for identificatoin of CTL responses against that individual antigen. Feline immunodeficiency virus (FIV) is an animal lentivirus that shares common clinical properties with HIV. The commonalities shared between HIV and FIV allows the use of FIV as an animal model for developing a protective vaccine that will identify antigens which induce protective cell mediated immunity or CTL responses. The FIV gag nucleocapsid (p10) protein will be cloned into the retroviral vector pLXSN for production of transgenic cells expressing the p10 protein for study of specific T-cell responses to this FIV antigen.
PCR will be used to amplify the nucleocapsid from the pUC119 plasmid containing the FIV genome. The amplified p10 PCR product will be EcoRI and BamHI digested for directional insertion into the EcoRI and BamHI digested retroviral vector pLXSN. Transformation of competent E. coli cells will provide colonies containing the recombinant retroviral vector DNA. Analysis of the recombinant retroviral vector DNA will be carried out by PCR.
Amplification by PCR of the nucleocapsid protein provided the p10 DNA sequence. Subsequent EcoRI and BamHI digestion revealed a natural EcoRI site at base pair 1871, therefore cutting off 160 bp from the p10 sequence. The upstream primer was reconstructed with an XhoI cut site for reamplification of the p10 protein from the pUC119 plasmid and will be inserted into the retroviral vector by XhoI and BamHI digestion. The 204 bp sequence generated from the EcoRI and BamHI digestion was inserted into the retroviral vector and awaits analysis of the idetification of recombinant retroviral vector colonies
Molecular characterization of oct4-expressing yolk sac endoderm stem cell lines.
No full textThe extraembryonic endoderm (XEN) defines the yolk sac, a set of membranes that
provide essential support for mammalian embryos. Recently, the committed XENprecursor
was identified in the embryonic Inner Cell Mass (ICM) as a group of cells that
intermingles with the closely related, anatomically indistinguishable epiblast (EPI)-
precursor that gives rise to the fetus. In vitro, the EPI-precursor is represented by the
well-known embryonic stem (ES) cell lines, but cell lines representing the XENprecursor
are not known. Furthermore, since the XEN-precursor cells were discovered
only very recently, the unexpected fact that they express the key pluripotency marker
Oct4 has not been explored. Recently, however, our laboratory has isolated rat XEN cell
lines that express Oct4, leading to the following two questions: (i) Do these new XEN
cell lines represent XEN-precursor cells? (ii) Is their Oct4 expression regulated similarly
as previously known from ES cells? These two questions are addressed here by lineage
marker and reporter gene analyses. Whole culture analyses showed that rat XEN cell lines expressed markers of all
XEN stages including XEN-precursor, primitive endoderm (PrE) and/or visceral
endoderm (VE), and parietal endoderm (PE) but trophoectoderm and EPI-precursor
markers were missing. In line with this, immunocytochemistry demonstrated
heterogeneity and directly visualized the XEN-precursor, PrE/VE, and PE
subpopulations. Low-density plating and time-dependent immunocytochemistry on
resulting colonies strongly suggested that XEN-precursor cells generate the other XEN
stages. Moreover, by analyzing single-cell derived clones, it was shown that culture
heterogeneity results from the self-renewal and differentiation of a single cell. Reporter
gene analyses using the 5��� regulatory region of the mouse Oct4 gene revealed that a
DNA fragment containing the previously described distal enhancer drove reporter gene
expression only in ES cells whereas inclusion of an upstream fragment led to high
expression in both mouse ES and rat XEN cells.
In conclusion, our rat XEN cell lines contain XEN-precursor cells that differentiate
extensively, providing for the first time an in vitro model that mimics the natural process
of early XEN differentiation. In addition, they regulate Oct4 gene transcription
differently than ES cells suggesting heterogeneous Oct4 regulation within the
mammalian ICM
First Characterization of Avian Memory T Lymphocyte Responses to Avian Influenza Virus Proteins
No full textAlthough wild birds are natural hosts of avian influenza viruses (AIVs), these
viruses can be highly contagious to poultry and a zoonotic threat to humans. The
propensity of AIV for genetic variation through genetic shift and drift allows virus to
evade vaccine mediated humoral immunity. An alternative approach to current vaccine
development is induction of CD8+ T cells which responds to more conserved epitopes
than humoral immunity and targets a broader spectrum of viruses. Since the memory
CD8+ T lymphocyte responses in chickens to individual AIV proteins have not been
defined, the modulation of responses of the memory CD8+ T lymphocytes to H5N9 AIV
hemagglutinin (HA) and nucleocapsid (NP) proteins over a time course were evaluated.
CD8+ T lymphocyte responses induced by intramuscular inoculation of chickens with
AIV HA and NP expressing cDNA plasmids or a non-replicating human adenovirus
vector were identified through ex vivo stimulation with virus infected, major
histocompatibility complex (MHC) matched antigen presenting cells (APCs). The IFN?
production by activated lymphocytes was evaluated by macrophage production of nitric
oxide and ELISA. MHC-I restricted memory T lymphocyte responses were determined at 10 days and 3, 5, 7 and 9 weeks post-inoculation (p.i). The use of non-professional
APCs and APC driven proliferation of cells with CD8+ phenotype correlated with the
activation of CD8+ T lymphocytes. The responses specific to nucleocapsid protein (NP)
were consistently greater than those to the hemagglutinin (HA) at 5 weeks when the
CD8+ T cell responses were maximum. By 8 to 9 weeks p.i., responses to either protein
were undetectable. The T lymphocytes also responded to stimulation with a heterologous
H7N2 AIV infected APCs. Administration of booster dose induced secondary effector
cell mediated immune responses which had greater magnitudes than primary effector
responses at 10 days p.i. Flow cytometric analysis (FACS) of the T lymphocytes
demonstrated that memory CD8+ T lymphocytes of chickens can be distinguished from
naive lymphocytes by their higher expression of CD44 and CD45 surface antigens.
CD45 expression of memory lymphocytes further increases upon ex vivo stimulation
with APCs expressing AIV. This is the first characterization of avian memory responses
following both primary and secondary expression of any individual viral protein
Intracellular trafficking and plasma membrane microdomain distribution of the NSP4 enterotoxin during rotavirus infection in epithelial cells
No full textRotavirus (RV) nonstructural protein 4 (NSP4) is a multifunctional glycoprotein
that induces secretory diarrhea in mouse pups in the absence of other viral proteins. The
intracellular transport route(s) and functional mechanism(s) of NSP4 are poorly
understood; however, the recent association of the enterotoxin with cellular caveolin-1
may provide a link between NSP4 transport and function. To determine if NSP4 traffics
to a specific subset of lipid rafts at the plasma membrane (PM), we isolated caveolae
from a PM-enriched fraction with a new method that yielded endoplasmic reticulum
(ER)-free caveolae membranes with a unique membrane structure and composition.
Comparison of these caveolae with other detergent- and non-detergent-extracted
membranes revealed that each caveolae/raft fraction contained caveolae markers;
however, only our PM caveolae fraction mimicked the membrane structure and sterol
exchange dynamics of intact PM without ER or non-raft PM contaminants. When these
PM caveolae were isolated from RV-infected cells, full-length, high-mannose
glycosylated NSP4 was present. Confocal imaging showed association of NSP4 with
caveolin-1 moving from perinuclear and cytoplasmic sites toward the PM as the
infection progressed. Fluorescent imaging also indicated exposure of the NSP4 Cterminus
at the exofacial PM surface without transport of the enterotoxin through the
Golgi apparatus. Surface-specific biotinylation was used to confirm NSP4 exposure at
the surface of infected MDCK cells and to determine that the exposed protein was fulllength
and high-mannose glycosylated. This study presents an ER contaminant-free PM
caveolae isolation methodology, identifies the presence of full-length, high-mannose glycosylated NSP4 in both PM caveolae and exposed at the cell surface, and confirms
the Golgi-bypassing nature of NSP4 ER to PM transport in RV-infected MDCK cells
The effect of stress on the neuropathogenesis of Theiler's virus-induced demyelination as an animal model of multiple sclerosis
No full textStressful life events have been associated with the onset and/or exacerbation of
multiple sclerosis (MS). To investigate the effects of stress on the pathogenesis of MS,
we employed restraint stress (RST) in the Theiler��������s virus-induced demyelination
(TVID) model, an animal model for human MS. Intracerebral inoculation of
susceptible strain of mice with Theiler��������s murine encephalomyelitis virus (TMEV)
results in a biphasic disease �������� an acute encephalomyelitis and chronic demyelination.
The establishment of persistent viral infection is critical in inducing immune-mediated
demyelination during the chronic disease. The exposure of mice to RST prior to viral
infection produced a stress response as evidenced by elevated circulating corticosterone
(CORT). To further study the effect of stress on the immune response to TMEV
infection and demyelination, we first examined the cytokine and chemokine response
during the acute TMEV infection. We demonstrated that RST down-regulated the
virus-induced expression of chemokines, Ltn, IP-10, RANTES, and pro-inflammatory
cytokines, TNF, IFN and LT in both the brain and spleen during early infection.
Histologically, a decreased pattern of inflammation was observed in the brain of
restrained mice as compared to non-restrained mice. The increased viral titer was noted in the CNS of restrained mice and was correlated with the decreased production
of pro-inflammatory cytokine, suggesting an impaired immune response by RST.
Secondly, the duration of stress on the late demyelination was investigated. Repeated
and chronically stressed SJL/J mice developed an early onset of clinical signs and a
delayed onset was observed in acutely stressed mice. Both acute and chronic RST
suppressed the antibody response to TMEV and stressed displayed a higher incidence
of demyelination than non-restrained mice. Axonal loss was also noted in chronic
stressed mice. Additionally, RST caused an increased systemic viral infection in
extraneural organs during the acute infection and cardiotropic TMEV was isolated
from the heart of stressed mice. Taken together, stress resulted in profound
immunsuppression during acute infection, which may consequently increase the
incidence of demyelination. The present study may be generalized in human MS
which is potentially triggered by viral infection
Differential cytokine mRNA expression induced by binding of virulent and avirulent molecularly cloned equine infectious anemia viruses to equine macrophages
No full textEquine infectious anemia virus (EIAV) causes rapid development of acute disease followed by recurring episodes of fever, thrombocytopenia and viremia, termed chronic EIA. Most infected horses control the virus by immune mechanisms and become inapparent carriers. To further our understanding of the equine immune response to EIAV, a multi-probe ribonuclease protection assay (RPA) was developed to quantitate equine-specific cytokine mRNAs. Eleven template plasmids specific to ten equine cytokine genes and the ?-actin gene were generated, from which radiolabeled anti-sense RNA probes were produced. The RPA simultaneously quantitated mRNA levels of interleukin (IL)-1���, IL-1���, IL-6, IL-8, IL-10, IL-12 p35, IL-12 p40, interferon (IFN)-���, transforming growth factor (TGF)-1��� and tumor necrosis factor (TNF)-��� in equine peripheral blood mononuclear cells and equine monocyte-derived macrophages (EMDM). The assay detected as few as 5���105 RNA molecules and displayed coefficients of variation of 0.03-0.08 when normalized to ���-actin expression. Using this RPA, cytokine expression in EMDM infected with 2 molecularly cloned viruses (EIAV17 and EIAV19) was determined. EIAV17 varies from EIAV19 only in env, rev and LTR and causes fatal disease in Shetland ponies. When added to EMDM cultures, virulent EIAV17 stimulated expression of IL-1���, IL-1���, IL-6, IL-10 and TNF-���. These cytokine mRNAs were significantly elevated by 0.5 to 1 hr post infection (hpi) and returned to basal levels by 12 to 24 hpi, indicating modulation by early event(s), such as receptor binding. In contrast to EIAV17, EIAV19 is avirulent in vivo and failed to induce any of the tested cytokines in EMDM. These data show a direct correlation between the virulence of the EIAV clone and the induction of cytokines. The cytokines stimulated by EIAV17 may contribute to EIA-associated symptoms, enhance viral replication in the host, and regulate the host immune response. To determine whether cytokine induction requires EIAV17 replication, EMDM cultures were exposed to UV-inactivated EIAV17 and cytokine induction was monitored. UV-inactivation did not block cytokine induction by EIAV17, suggesting dispensability of viral replication. Given that EIAV17 induces cytokines in a rapid and replication-independent manner, the activation of cytokine expression is likely mediated by binding of EIAV17 to equine macrophage receptor(s)
Promoters, enhancers and insulators for improved mosquito transgenesis
No full textLow level and variable transgene expression plague efforts to produce and
characterize transgenic lines in many species. When transformation efficiency is high,
productive transgenic lines can be generated with reasonable effort. However, most
efforts to date in mosquitoes have resulted in suboptimal levels of transformation. This,
coupled with the large space and intensive labor requirements of mosquito colony
maintenance makes the optimization of transformation in mosquitoes a research priority.
This study proposes two strategies for improving transgene expression and
transformation efficiency. The first is to explore exogenous promoter/enhancer
combinations to direct expression of either the transgene itself, or the transposase
required for insertion of the transgene into the genome. An extension of this strategy is
to investigate the use of a powerful viral transactivating protein and its cognate enhancer
to further increase expression of these targets. The second strategy involves the
identification of an endogenous boundary element for use in insulating transgenes and
their associated regulatory elements. This would mitigate the inappropriate expression
or silencing of many transgenes inserted into ��������unfavorable�������� genomic environments as a consequence of an inability to specifically target the integration of transposons currently
used in mosquito transgenesis.
The IE1 transactivating protein and its cognate enhancer from a baculovirus were
shown to significantly increase expression of a reporter gene from three different
promoters in cultured mosquito cells. Other heterologous enhancer/promoter
combinations resulted in minimal increases or insignificant changes in expression.
Orthologues of the vertebrate insulator-binding factor, CTCF, were cloned and
characterized in two mosquito species, Aedes aegypti and Anopheles gambiae. The
expression profile of mosquito CTCF is consistent with its role as a putative insulatorbinding
protein. Preliminary binding site studies reveal a C/G-rich binding site
consistent with that known in vertebrates and indicate that CTCF may bind widespread
sites within mosquito genomes
CD8+ T cell antiviral activity: mechanism of induction and the suppression of emerging feline immunodeficiency virus strains
No full textIn the present studies, the essential role of inducer cells for the induction of soluble anti-viral activity against feline immunodeficiency virus (FIV) was investigated. Induction of suppression of FIV replication was found to not strictly require autologous cells and was probably not FIV specific. Suppression was maximum when the inducer cells and the effector CD8+ T cells were in contact with each other, suggesting a potential role for membrane antigen interactions and/or cytokines in the induction process. Additionally, flow cytometry analysis demonstrated a significant increase in the percentage of CD8+ B7-1+ T cells in the peripheral blood of chronically FIV infected cats as compared with uninfected cats. Examination of the FIV V3-V4 envelope sequences from PBMC, lymph nodes and spleen from six cats chronically infected from three to six years with the molecular clone of FIV-PPR did not demonstrate viral variants specific for the tissues examined, emphasizing the critical role of the initial diversity and virulence of the infecting virus inoculum. Additionally, in vitro CD8+ T cell antiviral activity demonstrated by four of the six cats could have led to the control of virus replication in vivo, resulting in the uniform viral variants observed. Infection of specific pathogen free cats with FIV-TX53, an FIV isolate that belongs to an emerging subtype more closely related to FIV clade B, demonstrated an acute stage infection characterized by lymphoadenopathy and a viral dose dependent decline of CD4+/CD8+ T cell ratios below 1 by 11 weeks post infection. Interestingly, an expansion of CD8 low population of CD8+ T cells was observed in the infected cats. The soluble antiviral activity generated from inducer T cell stimulated CD8+ T cells from FIV-A-PPR infected cats also suppressed in vitro replication of the emerging FIV-TX53 and FIV-TX078 isolates. This is the first report demonstrating that the CD8+ T cell antiviral activity is inter-clade effective among FIV strains. As the success of a FIV vaccine could be hampered by occurrence of highly divergent viral variants in the fields, the exploitation of this innate, soluble anti-FIV activity could contribute to the design of novel, safe and complementary anti-FIV therapeutic strategies
In vitro assembly of an infectious cDNA clone of infectious bronchitis virus and its application as a gene transfer vector
No full textAn infectious cDNA clone of Vero cell adapted Beaudette strain of IBV was constructed using in vitro assembly of cDNA fragments. The entire genome of IBV was RT-PCR amplified into seven fragments, with each piece overlapping about 10 nucleotides. The fragments were ligated and transcribed to synthesize RNA, which was transfected into BHK-21 cells. These cells were then overlaid onto IBV susceptible Vero cells. After five days transfection, the virus was successfully rescued from the transfected cells. The cDNA clone from our laboratory strain has a five nucleotide insertion not present in the originally sequenced virus, resulting in total genome size of 27,613 nucleotides. The infectious cDNA clone was further manipulated to demonstrate its potential as a gene transfer vector, by replacing the ORF5a open reading frame with enhanced green fluorescent protein. The recombinant infectious cDNA clone was also successfully rescued after three days transfection of BHK-21 cells followed by co-culturing with Vero cells. This study showed that the 5a protein, whose function is not known, is not necessary for in vitro IBV replication. This study also showed that the 5a ORF is a good candidate for an insertion site of recombinant genes for the development of IBV infectious cDNA clone as a gene transfer vector
The Effects of Chronic Restraint Stress on Innate and Adaptive Immune Responses to Acute Theiler?s Murine Encephalomyelitis Virus Infection ? An Animal Model of Human Multiple Sclerosis
No full textMultiple sclerosis (MS) is an immune-mediated prevalent chronic demyelinating and neurodegenerative disease of the central nervous system that begins with an abrupt onset during early adulthood. MS is idiopathic, but many factors are thought to influence the pathogenesis of the disease, which include genetic, gender and environmental factors. To date, there is much evidence that suggest that both the onset and progression of MS is facilitated by both viral infections and stress. Theiler���s murine encephalomyelitis virus (TMEV) is a picornavirus that upon inoculation into susceptible strains of mice (i.e. SJL and CBA) causes a persistent infection which, in turn, results in an early acute encephalomyelitis followed by a late chronic immune-mediated demyelinating and neurodegenerative disease that pathologically resembles MS. In contrast, resistant mice (i.e C57BL/6 and BALB/c) are able to clear the virus from the CNS, and consequently do not develop chronic demyelination. Previous studies indicated that stress during early infection of susceptible mice can increase CNS viral titers and alter dissemination of TMEV, decrease early cytokine and chemokine expression in the spleen and CNS, and result in an exacerbated late demyelinating disease. The studies herein, focused on the hypothesis that chronic stress during early infection with TMEV infection would lead to drastic immunosuppression of both innate and adaptive arms of immunity, and that this immunosuppression may overcome the genetically controlled resistance of C57BL/6 mice to Theiler���s virus-induced demyelination. In these series of studies, we were able to show that stress, regardless of mouse strain susceptibility, decreases NK cell activity, and increased viral titers at day 1 p.i. Furthermore, after seven days of stress, susceptible mice demonstrated decreased virus specific T-cell effector function in both the CNS and spleens as indicated by a globalized reduction in type 1 and type 2 cytokines, as well as transcription factors. Importantly, these decreased responses were, in part, attributable to the actions of glucocorticoids. However, stress during early infection of C57BL/6 mice did not alter resistance to demyelination. These results begin to shed light on how stress, infection, and genetics can influence the onset of human MS
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